Phytic acid (InsP_6) activates HDAC3 epigenetic axis to maintain intestinal barrier function

Domain: neuroscience
Published: 2026-02-09
Editorial outcome: major_revision

Abstract
HDAC inhibition shows promise in cancer treatment, but pan-HDAC inhibitors cause gastrointestinal issues in 48% of patients. Understanding HDAC activation mechanisms is crucial to treating diverse diseases beyond cancer. Our study reveals that inositol polyphosphate multikinase (IPMK) and inositol hexakisphosphate (InsP6 or phytic acid), enriched in vegan diets, play essential roles in activating the HDAC3 epigenetic axis and maintaining intestinal barrier integrity. IPMK binds to HDAC3 and drives InsP6 synthesis, which selectively activates HDAC3 at a 10 nM concentration by recruiting the DAD domain of its corepressor protein. IPMK deletion diminishes HDAC3 activation, leading to histone hyperacetylation and MMP gene transcription that compromise intestinal barrier integrity. InsP6 treatment is sufficient to rescue these effects. In inflammatory bowel disease, diminished IPMK levels exacerbate intestinal permeability, while oral InsP6 treatment mitigates leaky gut effects by restoring the HDAC3 epigenetic axis, highlighting the clinical significance of the IPMK-HDAC3 pathway and the therapeutic potential of phytic acid.

Introduction
HDAC3, a key regulator of histone acetylation1, plays a crucial role in epigenetic modification1. While nonspecific HDAC inhibitors show promise in cancer treatment 2, their side effects3 highlight the importance of understanding individual HDAC functions. HDAC3, a prominent class I HDAC family member1, has emerged as a critical regulator of various physiological functions. Preclinical mouse model studies have demonstrated that HDAC3 is pivotal in maintaining metabolic homeostasis and circadian rhythms, particularly in the liver4. Furthermore, HDAC3 loss has been observed in inflammatory bowel disease (IBD) patients5, and its deletion in mouse intestinal epithelial cells induces Crohn's disease-like symptoms, including increased intestinal permeability5. Despite its physiological importance, our understanding of the underlying mechanisms regulating HDAC3's enzymatic activity remains limited.

What is known about the HDAC3 activation mechanism is mostly related to protein-protein interaction, suggesting HDAC3 binds to the corepressor complex silencing mediator for retinoid and thyroid hormone receptors (SMRT/NCoR2)1 and nuclear receptor co-repressor 1 (NCoR1)1, essential for its enzymatic activity1. Recent studies have illuminated the interaction between HDAC3 and inositol phosphates, particularly InsP4, through crystal structure analysis1. However, significant gaps in our understanding persist. The physiological basis of InsP's role in activating HDAC3 remains unclear. The precise mechanism and cellular location (cytoplasm, nucleoplasm, or chromatin) of InsP-mediated HDAC3 activation are unknown. Most crucially, the downstream effects of InsP-activated HDAC3 on the epigenetic axis, gene regulation, and overall cell function remain largely unclear.

While negatively charged InsP binding to HDAC3 was charge-dependent1,6, in a cellular environment, it is produced in very small amounts7. In contrast, InsP6, which is more negatively charged than InsP4, is produced in cells at higher micromolar concentrations and is more abundant8. Although the crystal structure did not observe InsP6 binding to HDAC31, biochemical in vitro studies identified high-affinity binding of InsP6 to HDAC36. While in vitro studies show InsP4, InsP5, and InsP6 can activate purified HDAC3 complexes from wild-type cells, these experiments involved dissociating InsPs in high-salt conditions6,9. Although this suggests InsPs regulate HDAC3 outside the cellular environment, it doesn't confirm that physiological InsPs influences HDAC3 function. Thus, the biological significance of in vivo InsP-mediated HDAC3 regulation remains unclear.

At the physiological level, intracellular InsPs production is regulated by several enzymes, with IPMK (inositol polyphosphate multikinase) playing a critical role8, as its deletion diminishes InsP4, InsP5, and InsP6 levels7. Here, we investigated the effect of IPMK loss of function on HDAC3 activation to determine the importance of InsPs in regulating HDAC3 activation and the precise molecular mechanism. We discovered that IPMK physically interacts with HDAC3, and the interaction occurs specifically in chromatin but not in other cellular compartments. This finding is particularly intriguing given the known dynamic behavior of HDAC3, which shuttles between the cytoplasm and nucleus and requires recruitment to chromatin for its deacetylation function10. This chromatin-specific binding of IPMK to HDAC3 suggests a targeted regulatory mechanism, potentially influencing HDAC3's activity precisely where it exerts its epigenetic effects.

Our research further uncovered a critical role for InsP6 in the activation of HDAC3. Notably, treatment with InsP6 to IPMK knockout cells and mouse models completely restored HDAC3 activity to levels comparable to those observed in wild-type cells, firmly establishing InsP6 as a physiological small molecule activator of HDAC3.

Notably, InsP6, also known as phytic acid, is abundant in plant-based diets, particularly in rice and legumes11. This dietary connection becomes significant in light of our findings. We discovered that in a mouse model of inflammatory bowel disease (IBD), there was a notable loss of IPMK protein expression. This loss disrupted the IPMK-HDAC3 epigenetic axis, leading to the acetylated histone lysine 16 (H4K16) promoter enrichment and transcriptional upregulation of matrix metalloproteinase (MMP) genes elevating gut permeability. MMPs are proteolytic enzymes that play a crucial role in degrading cell-cell junctions, consequently exacerbating intestinal permeability, a hallmark feature of IBD12. This aligns with genome-wide association studies (GWAS) and our preclinical study that have identified IPMK as a risk gene for IBD13. While transcriptional upregulation of MMPs in IBD is well-established12,14, the mechanism of epigenetic regulation of their expression was previously obscure. Our findings suggest that disruption of the IPMK- HDAC3 epigenetic axis influence the transcriptional upregulation of MMP family genes in IBD. Notably, we demonstrate that oral InsP6 (Phytic acid) treatment is effective in reducing intestinal permeability.

In conclusion, our study reveals that IPMK physically interacts with chromatin-bound HDAC3 and identifies InsP6 as a crucial physiological small molecule activator of HDAC3. InsP6 functions by recruiting the DAD domain of the corepressor to HDAC3, thereby activating it and leading to the deacetylation of specific histone marks. This mechanism plays a crucial role in modulating the epigenetic landscape, specifically by repressing MMPs transcription. Consequently, it helps maintain cell-cell junctions and preserve intestinal barrier integrity, highlighting its importance in gut homeostasis and potentially in preventing or intercepting inflammatory bowel diseases.

Results
IPMK binds to the nuclear HDAC3 and is essential for HDAC3 activation regulating histone acetylation.
To investigate the physiological impact of HOIPs on HDAC3 activation, IPMK was deleted in HCT116 (colon), A549 (lung), and MEF (mouse embryonic fibroblast cell lines (Supplementary Fig 1 A). As the rate-limiting enzyme in the HOIP pathway (Fig 1A), IPMK deletion is expected to cause a profound loss in the levels of all downstream HOIP pathway products8. Consistent with this expectation, we observed a significant reduction in the levels of InsP5 and InsP6 in IPMK knockout (KO) cells, with only a modest decline in InsP4 (Fig 1B). Additionally, HPLC data from the wild-type (WT) cells indicated that the overall concentrations of InsP5 and InsP6 are 3 to 4 times higher than that of InsP4 (Fig 1B). To investigate the impact of IPMK deletion on HDAC3’s enzymatic activity, we immunopurified endogenous HDAC3 from respective WT and IPMK KO cells, followed by HDAC3 activity assay (Fig 1C). We optimized the HDAC3 activity assay by immunoprecipitating HDAC3 from various protein concentrations ranging from 5μg, 20μg, 100μg and 500μg. Our results showed that HDAC3 purified from 20μg of protein exhibited strong deacetylase activity, with only modest increments in activity observed up to 500μg (Supplementary Fig 1B). Based on these findings, we selected 20μg as the minimum protein concentration for all subsequent HDAC3 pulldown followed by enzymatic assays. This choice allowed us to achieve better resolution in detecting activity differences.
Notably, IPMK deletion dramatically reduced HDAC3's enzymatic activity by over 90% in all tested cell lines compared to their WT counterparts (Fig 1 D), however, without affecting HDAC3 or corepressor protein levels (Supplementary Fig 1C). Strikingly, this reduction in HDAC3 activity was comparable to that observed in HDAC3-KO cells (Supplementary Fig 1D) or WT cells treated with the HDAC3-specific inhibitor RGFP966 (Fig 1 E). The selectivity of RGFP966 for HDAC3 is precise, as it shows 200-fold selectivity for HDAC3 compared to other HDACs and binds to HDAC3’s enzymatic site, inhibiting its deacetylase activity15. These findings demonstrate that at the physiological level, IPMK deletion impairs HDAC3 activation to an extent similar to complete HDAC3 deletion or pharmacological inhibition, highlighting IPMK's crucial role in maintaining HDAC3 function.
Given HDAC3's role as a deacetylase, the loss of its activity in IPMK KO cells was expected to increase histone acetylation. Our findings confirmed that IPMK deletion enhanced acetylation levels of specific histone residues, particularly H3K9/27 and H4K16 (Supplementary Fig 1E, F). Notably, the extent of H3K9/27 and H4K16 acetylation in IPMK KO cells was comparable to that observed in HDAC3 KO cells and wild-type cells treated with the HDAC3 inhibitor RGFP966 (Fig 1 F, G). To determine whether this effect in IPMK deleted cells was due to the loss of HDAC3 activity, we treated IPMK KO cells with the HDAC3-specific inhibitor RGFP966. Interestingly, the inhibitor failed to induce any further increase in histone acetylation either in IPMK or HDAC3 KO cells (Fig 1 F, G) while profoundly increasing H3K9/27 and H4K16 acetylation when treated to WT cells (Fig 1 F, G). This differential response strongly suggests that HDAC3 activity was already substantially compromised in IPMK-deficient cells; consequently, RGFP966 was unable to enhance histone acetylation in IPMK KO cells further.
Next, we explored the potential direct interaction between IPMK and HDAC3 under physiological conditions. Considering HDAC3's known shuttling between the nucleus and cytoplasm10 and its requirement to bind chromatin for histone deacetylation in the nucleus, we performed cell fractionation and chromatin immunopurification, revealing that IPMK specifically associates with chromatin-bound HDAC3 (Fig 1 H, I). We did not find the interaction between IPMK and HDAC3 in the cytoplasm; however, it was detected when chromatin-bound HDAC3 was immunopurified (Fig 1H, I). To further validate this interaction, we conducted an in vitro binding assay using recombinant HDAC3 and IPMK proteins, which confirmed a direct physical interaction between these two proteins (Supplementary Fig 1G).
Overall, the study demonstrated that IPMK binds to nuclear HDAC3 and is crucial for its enzymatic activity.

IPMK’s inositol kinase activity is essential for HDAC3 activation.
To elucidate the role of IPMK's inositol kinase activity in regulating HDAC3's enzymatic activity, we conducted experiments using IPMK knockout (KO) cells. We transfected these cells with either wild-type (WT) or kinase-dead (KD) IPMK7. Our results showed that rescuing IPMK KO cells with WT-IPMK restored HDAC3 deacetylase activity, while KD-IPMK failed to do so (Fig 2A). Interestingly, immunoprecipitation studies revealed that both WT-IPMK and KD-IPMK could interact with HDAC3 complex proteins (Fig 2B).
These findings suggest a crucial distinction: while IPMK's physical binding to HDAC3 is insufficient for rescuing HDAC3’s enzymatic activity, the kinase activity of IPMK is required for HDAC3 activation.
Next, we tested the possibility of rescuing HDAC3’s enzymatic activity with InsP4, InsP5, and InsP6 in vitro test tubes after immunopurification of HDAC3 from IPMK KO cells. Biochemical analysis revealed that InsP6, in a dose-dependent manner, rescued the enzymatic activity of HDAC3 (Fig 2 C). While 10 nM (0.01μM) InsP6 restored >50% of HDAC3 activity, 1 μM InsP6 [InsP (1,2,3,4,5,6) P6] fully restored activity comparable to the WT (Fig 2 C). In IPMK KO cells, 5 or 10 μM InsP6 did not further increase HDAC3 activity, suggesting InsP6 saturation and a threshold effect at 1μM (Fig 2C). Although InsP4 [InsP (1,4,5,6) P4] and InsP5 [InsP (1,3,4,5,6) P5] also restored HDAC3's activity at 1 μM (Fig 2D), InsP6 showed the highest effect compared to all other HOIPs. Furthermore, while 10 nM of InsP5 and InsP6 both restored around 50% of HDAC3 activity compared to WT cells (SupplementaryFig 2A), InsP4 (10 nM) did not affect HDAC3 activation and was similar to IPMK KO cells. Interestingly, a second InsP4 isomer [InsP (1,3,4,5) P4] and InsP3 [InsP (1,4,5) P3] completely failed to rescue HDAC3 activity, even at 10 μM dose (Fig 2 D). While Wu et al.'s16 previous study indicated InsP3 activating HDAC3, our experiments showed that InsP3 could not influence HDAC3 activity at either 1 μM or 10 μM concentrations (Fig 2 D). This finding is supported by previous crystal structure analysis6, indicating the necessity of a phosphate group at the 6th position of higher order inositolphosphate (HOIP) activating HDAC3. This crucial phosphate is missing in both InsP3 and the InsP4 isomer [InsP(1,3,4,5)P4], explaining their inability to activate HDAC3 in our study. As IPMK is a physiological PI3 kinase 13that also generates PIP3, we further tested the role of PIP3 in rescuing HDAC3 activity when purified from IPMK KO cells. We found that even at 10 μM, PIP3 failed to restore HDAC3 activity and was similar to the IPMK KO (Fig 2 D). Based on the dose-dependent study (Fig 2 C, D) and previous research demonstrating InsP6's health benefits11, we opted to utilize InsP6 for our subsequent experiments.
Next, we explored the possibilities of rescuing HDAC3 activity in vivo by treating cells with cell-permeable InsP6 (cell-permeable InsP4 and InsP5 have not been successfully synthesized). InsP6 exhibits poor cell permeability in cultured cells, as cations present in cell culture media (such as calcium, magnesium, sodium, and potassium) may chelate InsP6, diminishing its availability for cellular uptake. Consequently, experiments often require the use of high, non-physiological concentrations of InsP6 to achieve desired effects. To overcome this, we used a cell-permeable InsP6 precursor molecule, where the negative charges of InsP6 are temporarily masked with biolabile groups, allowing it to pass through the cell membrane and then releasing active InsP6 after removal of the biolabile groups by enzymatic digestion17. We treated IPMK KO cells with this cell-permeable InsP6 and measured HDAC3 activity. Remarkably, 24 hours of cell-permeable InsP6 treatment rescued HDAC3 activity in IPMK KO cells to levels comparable to those of WT cells (Fig 2 E), followed by a substantial decrease in histone acetylation, which was also comparable to the WT (Fig 2 F). However, CP-InsP6 did not exert any effect when treated to wild type cells (Supplementary Fig 2B). Notably, the treatment of HDAC3 inhibitor RGFP966 inhibited the rescue effect of cell-permeable InsP6 (Fig 2 F), indicating that InsP6 deacetylates acetylated H3K9/27 and H4K16 by activating HDAC3.
Lastly, we evaluated the ability of CP-InsP6 to enhance intracellular InsP6 levels. Following 24 hours of treatment with 50 μM CP-InsP6, mass spectrometric analysis revealed a significant increase in intracellular InsP6 compared to wild-type cells (Supplementary Fig 2C). We also observed a parallel rise in InsP5 and InsP4 levels, suggesting dephosphorylation of InsP6 by phosphatases into lower-order inositol phosphates. Nevertheless, InsP6 remained the most abundant among the detected species.

IPMK deletion disrupts HDAC3's interaction with the DAD domain of its corepressor proteins, diminishing the activity of chromatin-bound HDAC3.
Next, we wondered about the mechanism underlying the loss of HDAC3’s enzymatic activity in IPMK-deleted cells. As mentioned earlier, the HDAC3 protein level remains unaltered in IPMK KO cells (Supplementary Fig 1B); hence, we wondered whether IPMK deletion disrupts HDAC3 binding to its corepressor protein, which is essential for HDAC3 function1. Immunoaffinity purification (Supplementary Fig 3A) and proximity ligation assay (PLA) (Fig 3A, B) demonstrated that IPMK deletion did not disrupt the association between HDAC3 and its corepressor protein, maintaining the overall integrity of the complex. While the SANT domain of the corepressor protein maintains the integrity of the HDAC3:corepressor complex1,9, the corepressor’s DAD domain binding to HDAC3 induces structural changes1,9,that are crucial for HDAC3's enzymatic activity, although it is not necessary for preserving the complex's integrity (Fig 3C). Given this, it's plausible that IPMK deletion specifically compromised the interaction between the DAD domain and HDAC3, thereby hindering HDAC3's enzymatic activity without disrupting the overall complex integrity. To investigate whether IPMK deletion interferes with DAD domain interaction with HDAC3, we overexpressed DAD-Flag in IPMK KO cells and subsequently performed a pull-down assay to isolate endogenous HDAC3. DAD-Flag pulled down endogenous HDAC3 from WT cells but, interestingly, failed when pulled down from IPMK KO cells (Fig 3 D). Notably, the impaired DAD binding to HDAC3 observed in IPMK KO cells was successfully rescued by treating the cells with cell-permeable InsP6 (Fig 3 E). This observation aligns with previous crystal structure analyses, which revealed HOIP's role as a molecular "glue" facilitating the interaction between the DAD domain of the corepressor protein and HDAC31,6,9.
Given that HDAC3’s chromatin recruitment is crucial for HDAC3-mediated histone deacetylation, next, we wondered whether IPMK deletion impaired HDAC3’s chromatin recruitment. Using HDAC3-to-histone H3 PLAs, or immunopurification of H3 or H4 chromatin fraction followed by HDAC3 western blot, we found a comparable amount of HDAC3 chromatin recruitment in IPMK KO and WT cells ( Fig 3 F, G, Supplementary Fig 3B), indicating that IPMK deletion did not impair HDAC3's chromatin recruitment.
Our data collectively support a functional model where HDAC3 recruitment to chromatin and its binding to the corepressor complex occur independently of IPMK. However, InsP6 is crucial for the DAD domain's interaction with HDAC3, which triggers its enzymatic activity and subsequent histone deacetylation (Fig 3H).
To investigate whether InsP6 serves as the final rate-limiting step in HDAC3-mediated histone deacetylation, we employed a simplified cell-free system. We treated cell-free, chromatin fraction in reaction buffer from IPMK KO cells with InsP6 for one hour, a context where any observed effects would likely result from InsP6's direct action on chromatin-bound HDAC3, independent of complex multistep processes of HDAC3 chromatin recruitment involving other proteins. Strikingly, InsP6 treatment fully suppressed histone acetylation in IPMK KO nuclear lysate (Fig 3I), restoring it to wild-type levels. The specificity of this effect was confirmed by co-treatment with the HDAC3-selective inhibitor RGFP966, which negated InsP6's impact (Fig 3I). However, the overall integrity of the HDAC3 chromatin protein complex (including NCoR1/2, TBL1, GPS2)18, remains intact in IPMK KO cells and is comparable to the WT (Supplementary Fig 3C). These results provide strong evidence that InsP6 directly activates chromatin-bound HDAC3 by recruiting the DAD domain of the corepressor to HDAC3, functioning as the critical final step in the histone deacetylation process.
IPMK generates InsP5, while IPPK produces InsP6. Given that IPMK interacts with HDAC3, we examined whether IPPK also associates with HDAC3, potentially contributing to a localized HOIP signaling event within the HDAC3 complex. Our biochemical and microscopy analyses revealed that IPPK indeed interacts with nuclear HDAC3 (Supplementary Fig. 4, A, B).

Disruption of the IPMK-HDAC3 epigenetic axis hypertranscribes MMP gene family.
We aimed to investigate the role of the IPMK-HDAC3 epigenetic axis in transcriptional regulation. Our bulk RNA sequencing analysis of IPMK-deleted HCT116 cells revealed significant transcriptional changes compared to wild-type cells, with 4,014 genes upregulated and 3,271 downregulated (Fig 4A). Pathway analysis indicated that the genes related to the extracellular matrix degradation, which includes the collagenase gene family, were notably upregulated in IPMK KO cells (Fig 4B). Specifically, archetypal matrix metalloproteinases (MMPs), including MMP1, MMP10, MMP12, and MMP13, were significantly upregulated in the RNA-seq data (Fig 4A). We validated these findings at the protein level using western blot analysis for MMP1, MMP10, and MMP13, confirming the RNA-seq results (Fig 4C). Due to a lack of high-quality antibodies, we were unable to test MMP12.
Given that IPMK is recognized as a risk gene for Inflammatory bowel disease (IBD)13, we also analyzed transcriptomic data from human patients using the Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBDTaMMA19, https://ibd-meta-analysis.herokuapp.com/)19 (Supplementary Fig 5 A). This analysis revealed significant transcriptional upregulation of MMP1, MMP3, MMP10, MMP12, and MMP13, in IBD patient cells, similar to the findings in IPMK KO HCT116- intestinal cells. Furthermore, separate analyses of Crohn’s disease (CD) and ulcerative colitis (UC) patients revealed a striking increase in major MMPs—such as MMP3, MMP13, and MMP10—that were consistently elevated in patient tissues and similarly upregulated in IPMK-depleted cells (Supplementary Fig 6 A,B).
Interestingly, IPMK KO cells rescued with wild-type IPMK showed a reduction in MMP expression, whereas cells with kinase-dead IPMK did not exhibit this effect (Supplementary Fig. 5B). Importantly, cell-permeable InsP6 treatment to IPMK KO cells suppressed MMP expression as well, making it comparable to that of the WT cells (Fig 4D), indicating that the loss of IPMK’s inositol kinase activity upregulates MMP gene expression. To determine whether InsP6 treatment repressed MMP gene expression by activating HDAC3, we co-treated HDAC3 inhibitor RGFP966, which abrogated the rescue effect of InsP6 (Fig 4D).
Moreover, in the HDAC3 KO cells, MMP genes (Supplementary Fig 5C) and protein expression were markedly increased (Fig 4E) and comparable to the IPMK KO cells (Fig 4E). In our second approach to determine whether identified MMP genes are regulated via the IPMK-HDAC3 epigenetic axis, we examined the effects of the HDAC3 inhibitor RGFP966 on WT, IPMK KO, and HDAC3 KO cells. We hypothesized that RGFP966 would increase MMP expression in WT cells but not in IPMK or HDAC3 KO cells, as these cells lack HDAC3 activity. However, if MMP expression in IPMK KO cells was influenced by pathways independent of HDAC3, RGFP966 treatment might further increase MMP expression. As anticipated, RGFP966 significantly increased MMP expression in WT cells but failed to induce further expression in IPMK and HDAC3 KO cells (Fig 4F, G). Taken together, the results confirm that the disruption of the IPMK-HDAC3 epigenetic axis is responsible for the transcriptional upregulation of MMPs, ruling out the involvement of HDAC3-independent pathways in this process.
Next, we examined whether the promoters of matrix metalloproteinases (MMPs) exhibit enrichment of histone acetylation. We selected MMP13 as a representative example for this analysis. Our biochemical studies confirmed that IPMK deletion selectively upregulates H3K9/27 and H4K16 acetylation at the protein level, an effect also observed in HDAC3 KO cells and those treated with HDAC3 inhibitors (Fig 1F). Intriguingly, ChIP-qPCR targeting the MMP13 promoter region (Fig 4H) revealed a selective enrichment of acetylated H4K16 in the promoters of MMP13 in IPMK KO cells, while H3K9/27 acetylation showed no significant change (Fig 4 H). The ChIP data also indicated an enrichment of HDAC3 as tested using MMP13 promoter regions (Supplementary Fig 5 D).
A particularly fascinating finding emerged when we treated IPMK KO cells with cell-permeable InsP6. This treatment substantially reduced the enrichment of acetylated H4K16 at MMP promoters (Fig 4I). However, this effect was nullified when cells were co-treated with the HDAC3 inhibitor RGFP966 (Fig 4I). Notably, InsP6 treatment did not affect HDAC3 chromatin recruitment (Supplementary Fig 5 D).
Given the known upregulation of MMP3 in IBD20, we revisited our IPMK KO HCT116 RNA-seq data to understand the MMP3 log fold change. Although we found a 2.5-Log 2-fold increase in MMP3 expression in HCT116 cells, the p-value of 0.3 exceeded our significance threshold of p<0.05. Raw data analysis revealed zero reads in all three WT samples and a maximum of two reads in KO samples for MMP3, likely contributing to the lack of statistical significance. While deeper sequencing might yield statistically significant results, our qPCR (Supplementary Fig 5 E) and protein-level data (Supplementary Fig 5 F - H) clearly demonstrated a substantial increase in MMP3 levels in both IPMK and HDAC3 KO cells, which was reversed by InsP6 treatment (Supplementary Fig 5 I). Furthermore, studies using HDAC3 inhibitors and knockout models confirmed that disrupting the IPMK-HDAC3 epigenetic axis substantially elevated MMP3 transcriptional expression, providing additional validation of our findings.
Finally, to assess the overlap between the global transcripts regulated by HDAC3 and IPMK, we first identified differentially expressed genes in HDAC3 KO cells and compared this dataset with the expression profile of IPMK KO cells. The results showed that approximately 44% of HDAC3-regulated genes were also affected in IPMK KO cells (Fig 4J). Importantly, HDAC3 exhibits a dual regulatory role in transcription: it functions not only through its deacetylase activity but also as a cotranscriptional activator independent of this enzymatic function16. This dual functionality likely explains why certain genes are affected by HDAC3 deletion but remain unaltered in IPMK KO cells. Specifically, the genes impacted in HDAC3 KO cells but not in IPMK KO cells may be influenced by HDAC3's non-enzymatic roles, such as its function as a cotranscriptional activator.
IPMK deletion is associated with increased cell and intestinal permeability in mice.
The upregulation of MMP is associated with compromised intestinal barrier function by disrupting tight junctions, leading to a "leaky gut”, which is commonly observed in IBD patients12. Given these associations, we investigated the consequences of IPMK deletion stimulating increased MMP production in cell permeability. To assess that, we employed a transwell assay using Caco2 cells, which is classically used for in vitro cell permeability assay21. We cultured these cells in a confluent layer on a trans well insert and treated them with 4kDa FITC-Dextran. Cell permeability would increase the leakage of 4kDa FITC-Dextran from the upper cell chamber into the bottom chamber (Fig 5 A). Notably, conditioned media from IPMK KO or HDAC3 KO cells elevated Caco2 cell permeability (Fig 5 B), while media transferred from WT cells failed to induce cell permeability. The observed effect may be attributed to the secretory nature of MMPs. In IPMK KO HCT116 cells, the conditioned media contained elevated levels of secreted MMPs (Supplementary Fig 8 A). When this MMP-enriched medium was transferred to wild-type Caco2 cells, it increased cellular permeability by disrupting the cell-cell junction. This hypothesis was corroborated by the western blot study that identified the loss of tight junction proteins ZO-1 and Occludin in Caco2 cells treated with either IPMK KO cell-conditioned media (Supplementary Fig 7A). Remarkably, IPMK or HDAC3 KO cells pretreated with Pan-MMP inhibitor (Ilomastat) followed by media transfer completely blocked the Caco2 cell permeability (Fig 5 B), confirming that the effect was due to MMP in the conditioned media.
Furthermore, IPMK KO cells treated with just cell permeable InsP6 followed by media transfer to Caco2 cells prevented the Caco2 cell permeability, but the RGFP966 co-treatment abrogated InsP6’s rescue effect (Fig 5 B). This confirms that disruption of the IPMK-HDAC3 epigenetic axis leads to MMP upregulation and consequent increased cell permeability, effects successfully rescued by InsP6 treatment.
In vivo, mouse studies further supported the cell-level findings. We created intestine-specific IPMK KO mice (IPMK∆IEC) (Supplementary Fig 7 B-D) and tested gut permeability (Fig 5C) by orally gavaging mice with FITC-dextran (4kD) and Rhodamine-dextran (70 kD). The disruption of the tight junction only allows 4kD dextran but restricts 70 kD22-24, activating the leaky pathway, while significant destruction of the epithelial cell layer allows both 4 kD and 70 kD dextran (Fig 5 C). Notably, the IPMK∆IEC mouse showed a marked increase in the leak pathway, allowing 4 kD dextran but without allowing 70 kD to pass through (Fig 5 D), indicating intestinal loss of IPMK selectively activates the leak pathway expected due to loss of tight junction disruption.
To determine the hyper-transcription of MMPs in IPMK∆IEC mice, we flow purified (Supplementary Fig 5 B-E) pure intestinal epithelial cells (IEC) from IPMKF/F and IPMK∆IEC mice followed by RNA-seq. Data indicated a profound increase in MMP transcription (Fig 5 E), also validated by qPCR, tested in two representative MMP genes (MMP3 and MMP13) (Fig 5 F). Pathway analysis using Metascape (Supplementary Fig. 8 G-I) and GSEA (Supplementary Fig. 8 J) confirmed enrichment of gene sets associated with MMP activity and extracellular matrix organization. Protein level analysis by performing western blots of crypts (Fig 5G, Supplementary Fig 8 B,C) and immunohistochemistry (Fig 5 H) reconfirmed transcriptomic data showing elevated MMPs in IPMK∆IEC mice. Fascinatingly, oral administration of InsP6 solution normalized to pH 7.0, treated to IPMK∆IEC mice completely reduced MMP levels, making them comparable to those observed in IPMKF/F mice (Fig 5 G,H, Supplementary Fig 8 B,C). Next, we studied the levels of major tight junction proteins like ZO-1, Occludin, and claudin 2. Notably, ZO-1 and Occludin levels were significantly reduced in IPMK∆IEC mice as tested in Western blot and IHC studies (Fig 5 G,I,J, Supplementary Fig 8 D,E). Interestingly, claudin 2 levels were rather increased in IPMK∆IEC (Supplementary Fig 8 F), which agrees with previous studies indicating increased claudin 2 is linked with elevated intestinal permeability23. Importantly, oral InsP6 treatment rescued all tight junction protein levels. Moreover, the HDAC3 activity assay indicated a marked decline in HDAC3 activation in IPMK∆IEC (Fig 5 K). Notably, oral InsP6 treatment restored HDAC3 activity to the level of IPMKF/F mice (Fig 5 K). Protein level analysis showed that in IPMK∆IEC mice intestine H4K16 acetylation was markedly increased which oral InsP6 restored back to the IPMKF/F level; however, the IPMK protein was unaffected by InsP6 treatment (Fig 5 L, Supplementary Fig 7 F,G), indicating oral InsP6 treatment alone could restore the epigenetic change of H4K16 acetylation in IPMK∆IEC mice in absence of IPMK (Fig 5 L, Supplementary Fig 7 F,G). The protein level epigenetic change was reconfirmed at the chromatin level by ChIP-qPCR analysis of MMP3, and 13, indicating in IPMK∆IEC mice marked increase of H4K16ac in MMP3, 13 promoters, which were reduced and reversed back to the level of IPMKF/F mice by oral InsP6 treatment (Fig 5 M,N). As expected, MMP promoter recruitment of HDAC3 was evident in IPMK∆IEC mice and comparable to the IPMKF/F mice, and it was unaffected by oral InsP6 treatment (Supplementary Fig 7 H). While InsP6 cellular uptake is poor in cell culture systems, in our animal studies, a 2% body weight, InsP6 treatment was sufficient to demonstrate its effects. This observation aligns with previous research showing intestinal absorption25-27 and therapeutic efficacy of oral InsP628 in mice and rats.
To determine whether the above finding has any clinical significance in relation to IBD, we found that both CD and UC patient samples showed diminished HDAC3 activity (Fig 5 O, P). Remarkably, IPMK protein levels were markedly reduced in both CD and UC patient samples (Fig 5Q,R), but this did not affect IPPK protein levels, indicating specificity. Moreover, we found a loss of ZO-1 and Occludin levels in CD and UC patient samples, along with increased H4K16 acetylation (Fig 5Q,R).
The collective findings suggest that the deletion of intestinal IPMK leads to a reduction in HDAC3 activity, which in turn promotes the expression of (MMPs). This phenomenon is evident in IBD patient samples, showing a pronounced loss of IPMK and its downstream effects, as observed in IPMK∆IEC mice. This cascade of events disrupts cell junctions, ultimately resulting in a leaky gut condition. Notably, oral administration of InsP6 effectively reversed leaky gut effects.

In a mouse model of IBD, IPMK expression and HDAC3 activity are reduced, and oral InsP6 treatment restores intestinal permeability.
We used a DSS (dextran sodium sulfate)-induced IBD mice model 29 to test the leaky gut, where , marked elevation in intestinal permeability for 4kD dextran was evident as early as 24h, not allowing 70kD (Fig 6A). Remarkably, pre-treatment with oral InsP6 dosed at 2% of the mouse body weight 72h prior to the DSS treatment, prevented leaky gut comparable to the untreated mice (Fig 6 A). Since InsP6’s bioavailability is modest25, we pretreated mice for 72 hours. Notably, the 24h of DSS treatment selectively reduced IPMK protein expression in the HOIP pathway (Fig. 1 A) while keeping IPPK protein expression unaltered (Fig 6B, Supplementary Fig. 9B ), with a substantial loss of HDAC3 activity, which was restored in DSS-treated mice by oral InsP6 pre-treatment (Fig 6C). Moreover, the western blot of colonic crypts indicated a substantial increase in MMP 3 and 13 expression after DSS treatment, which oral InsP6 pretreatment attenuated and restored to the level of untreated mice (Fig 6D). We reconfirmed the western blot data using immunohistochemistry for MMP3 in the colon and the ileum, showing that both the small and large intestines exhibited increased MMP3 expression which oral InsP6 diminished (Fig 6E,F). Though acute DSS treatment mostly shows ulcerative colitis-like phenotype in mice colon, the previous study also indicated its pathological effects in the ileum30; hence, we also included the ileum to show that the molecular effect is unrestricted throughout the intestine after DSS treatment.
At the epigenetic level, DSS treatment markedly increased H4K16 acetylation at the protein level (Fig 6 G) with increased H4K16 acetylation enriched MMP3 and MMP13 promoter (Fig 6 H) and transcriptional upregulation of MMP 3 and 13 (Fig 6 I). These were restored to the levels of untreated mice by oral InsP6 pretreatment (Fig 6G-I). While HDAC3 promoter enrichment on the MMP3 and 13 promoter was evident in DSS-treated mice, it was unaltered by oral InsP6 pretreatment (Supplementary Fig 9 A). Its important to mention that oral InsP6 also has no effects on IPMK protein expression so works indepdnet of IPMK (Supplementary Fig 9 B) .
This finding highlights that disruption of the IPMK-HDAC3 epigenetic axis in a preclinical mouse model of IBD exacerbates intestinal permeability; however, oral InsP6 (phytic acid) treatment mitigates the effects of the leaky gut.

Oral InsP6 treatment increased intestinal epithelial cell-specific InsP6 levels and prevented DSS-induced intestinal injury.
To assess the preventative effect of InsP6 on intestinal inflammation, wild-type mice were exposed to 2% dextran sodium sulfate (DSS) in drinking water for 7 days, with or without 2% oral InsP6 co-treatment (Fig 7A). For the InsP6 group, mice were pretreated with oral InsP6 for 2 days prior to DSS exposure to enhance the protective effect (Fig. 7A). To induce colitis, mice were given 2% DSS in drinking water continuously for 7 days, followed by sacrifice on the morning of the 8th day (Fig 7A). Each day, mice were also gavaged with 200 µl of DSS to ensure uniform exposure.
As expected, mice treated with DSS alone developed severe colitis, characterized by body weight loss starting around the 6th day (Supplementary Fig. 10 A), reduced colon length (Fig. 7B), and mucosal erosion and tissue damage observed in H&E-stained colonic sections (Supplementary Fig. 10 B). This inflammatory phenotype was further confirmed by markedly elevated colonic IL-1β (Fig. 7C) and myeloperoxidase (MPO) activity (Fig. 7D), indicating robust inflammatory cytokine production and neutrophil infiltration, respectively.
Notably, oral co-treatment with InsP6 completely prevented these DSS-induced pathologies. Mice receiving InsP6 maintained their body weight (Supplementary Fig. 10 A) and exhibited no reduction in colon length (Fig. 7B) compared to untreated controls. Histological analysis revealed preserved tissue integrity with minimal damage (Supplementary Fig. 10 B), and both IL-1β (Fig. 7C) and MPO levels (Fig. 7D) remained at baseline , comparable to those of untreated mice. Collectively, these data demonstrate that oral InsP6 provides robust protection against the development of DSS-induced colitis.
Notably, oral co-treatment with InsP6 completely prevented these DSS-induced pathologies. Mice receiving InsP6 maintained their body weight (Supplementary Fig. 10 A) and exhibited no reduction in colon length (Fig. 7B) compared to untreated controls. Histological analysis revealed preserved tissue integrity with minimal damage (Supplementary Fig. 10 B), and both IL-1β (Fig. 7C) and MPO levels (Fig. 7D) remained at baseline , comparable to those of untreated mice. Collectively, these data demonstrate that oral InsP6 provides robust protection against the development of DSS-induced colitis.
Lastly, we assessed intestinal permeability. Both 4 kD and 70 kD dextran assays showed increased permeability following DSS treatment (Fig 7E), with the greatest increase observed for 4 kDa dextran (Fig 7E). Remarkably, oral InsP6 treatment robustly reduced intestinal permeability (Fig 7E) and effectively preserved barrier integrity.
We next sought to determine if mice with an intestinal epithelial cell-specific deletion of IPMK (IPMK∆IEC) exhibit heightened susceptibility to DSS-induced inflammation and whether oral InsP6 could rescue this phenotype. Three groups of mice were studied: IPMK F/F (controls), IPMK∆IEC, and IPMK∆IEC + InsP6. All groups were treated with 2% DSS for five days and euthanized on day six (Fig 7F). The IPMK∆IEC + InsP6 group was pre-treated with oral InsP6 for three days before DSS co-treatment (Fig 7F), a strategy intended to replenish the depleted endogenous InsP6 pools resulting from IPMK loss.
Consistent with a critical role for IPMK in maintaining barrier function, IPMK∆IEC mice displayed a markedly exacerbated inflammatory response to DSS compared to IPMK F/F controls, as evidenced by more severe tissue damage in H&E-stained sections (Supplementary Fig 10 E) and significantly elevated levels of IL-1β (Fig 7G) and MPO (Fig 7H). Furthermore, intestinal permeability to both 4 kD and 70 kD dextran was increased in IPMK∆IEC mice after DSS treatment (Fig 7I). Remarkably, oral InsP6 treatment almost completely abrogated these effects (Fig 7I). The InsP6-treated IPMK∆IEC mice exhibited robust protection, with inflammation markers and intestinal permeability comparable to those of the uninjured IPMKF/F controls. In this experiment, the duration of 6 daysof DSS treatment failed to induce any weight loss or colon size (Supplementary Fig 10 C,D).
While our data established that oral InsP6 protects against intestinal inflammation, we sought to determine if this effect stems from direct elevation of intestinal epithelial cell-specific InsP6 levels. Mass spectrometry of HOIP metabolites from isolated ileal crypts (Fig 7J) revealed that InsP4, InsP5, and InsP6 were markedly reduced in IPMK∆IEC mice relative to IPMKF/F controls, with InsP6 levels diminished by approximately sevenfold (Fig 7K). Remarkably, a single 24-hour oral dose of pH-calibrated (7.2) InsP6 treated to IPMK∆IEC mice restored InsP6 to levels comparable to controls (Fig 7K), while also increasing InsP4 and InsP5, mirroring observations with cell-permeable InsP6 treated to cell lines (Supplementary Fig 2C). These findings support a model of direct epithelial uptake of orally administered InsP6, which is then partially dephosphorylated. Although gut microbial phytases can generate lower-order inositol phosphates and myoinositol for uptake via transporters like SMIT2/SGLT634, the lack of IPMK in IPMK∆IEC mice would prevent the subsequent re-phosphorylation to InsP5 and InsP6, thereby implying the existence of a specific epithelial transport mechanism for InsP6 itself.
As we found that incorporation of InsP6 at both cellular and tissue levels not only increased InsP6 concentration but also elevated InsP5 and InsP4, we sought to determine which HOIP is physiologically critical for HDAC3 activation and MMP expression. To specifically deplete InsP6, we examined HDAC3 activity and MMP expression in IPPK knockout cells. IPPK catalyzes the conversion of InsP5 to InsP6; therefore, the loss of IPPK selectively reduces InsP6 without affecting upstream polyphosphates such as InsP5 or InsP4.
Intriguingly, HDAC3 activity was markedly reduced in IPPK knockout cells (Fig 7L), accompanied by elevated MMP levels (Fig 7M), however, without influencing HDAC3 expression itself. These findings indicate that InsP6 plays a physiologically significant role in HDAC3 activation compared to other HOIPs. In the clinical context, the IPMK depletion model better reflects disease conditions such as inflammatory bowel disease (IBD), where IPMK levels are reduced while IPPK remains unchanged (Fig 5Q,R), leading to lowered InsP6 concentrations.
Finally, we performed MiniMUGA background analysis to confirm that the IPMKF/F mice possess a 98% resemblance to the C57BL/6 background (Supplementary Fig 10F), thereby ruling out significant off-target genetic effects.

Methods
Cell culture.
Human Colon Tumor 116 (HCT116), Mouse Embryonic Fibroblast (MEF), A549, HEK and CaCo2 cells were cultured in McCoy's 5A medium, Dulbecco's Modified Eagle's Medium (DMEM) or F12/K respectively. DMEM, F12/K media was supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin was used for MEF or A549 cells. While McCoy's 5A medium was supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. IPMK wild-type (WT) and knockout (KO) HCT116 and A549 cells were generated using CRISPR/Cas9 technology, which partially deleted exon 5 and the coding region of exon 6. Afterward, clonal populations were isolated, purified, and screened for IPMK expression.
For the generation of IPMKI KO MEF cells, floxed MEFs were immortalized through transfection with pSG5-Large T (a plasmid provided by William Hahn to Addgene) using the Polyfect transfection reagent (Qiagen) according to the manufacturer’s instructions. After serial passaging five times to select against non-transformed cells, IPMK KO cells were produced by transducing floxed cells with an adenovirus carrying the Cre recombinase gene (obtained from the University of Iowa Gene Transfer Vector Core). The virus was combined with Gene Jammer (Stratagene) transfection reagent to improve transduction efficiency. Clonal populations were then purified and screened for IPMK expression.

Animals.
All protocols were approved by the Institutional Animal Care and Use Committee (IACUC, UNLV, IACUC-01204, IACUC-01206), University of Nevada, Las Vegas. Mice were housed according to institutional guidelines, in a controlled environment at a temperature of 22°C ± 1°C, under a 12-h dark-light period and provided with standard chow diet and water ad libitum. Male and female IPMK F/F, IPMK∆IEC (between eight to 16 weeks) were used. Specifically, intestinal epithelial cells from male mice were used for biochemical and transcriptomic analysis. All mice were maintained in CE57BL/6 background.

Generation of intestine-specific IPMK knockout mice.
IpmkF/F mice were generated as previously described13. IpmkF/F mice were crossed with C57BL/6J mice carrying Cre expressed under the control of the murine villin promoter as described previously to create intestinal-specific Ipmk knockout mice (IpmkΔIEC). Homozygous IpmkF/F mice were crossed with the IpmkΔIEC mice, which mediate excision of floxed alleles in the intestine. All mice were maintained on a C57BL/6J background and were 10th-generation backcrossed. Validated by MiniMUGA. Background analysis was performed by Transnetyx, Cordova, USA Mice were housed in a 12-hour light/12-hour dark cycle at 22°C and fed standard rodent chow. All research involving mice was approved by the University of Nevada, Las Vegas’s Institutional Animal Care and Use Committee (IACUC).

Proximity ligation assay and quantitation:
PLA was utilized to detect in situ protein–protein interactions as previously described. After fixation and permeabilization, cells were blocked with blocking solution supplied by PLA kit before incubation with primary antibodies as in routine immunofluorescence staining. The cells were then processed using a PLA kit (NaveniFlex Cell MR Red Cat no. NAV-NC.MR.100.RED) according to the manufacturer’s instructions. The slides after PLA were further processed for immunofluorescence staining against the DAPI (Cat.8617, Cell Signaling). The slides were then mounted with Prolong Gold antifade Reagent. A Zeiss LSM 750 confocal microscope detected PLA signals as discrete punctate foci and provided the intracellular localization of complexes. ImageJ was used to quantify and represent the number of PLA puncta per nucleus.

DSS-induced colitis model and oral InsP6 treatment.
DSS (MP Biomedicals; relative molecular mass 36,000–50,000) was added to drinking water at 3% weight/volume for 24 hr or 2% weight/volume for five or seven days. Phytic acid (InsP6) was added to 2% weight/volume (2 gm/100 ml) containing drinking water after pH neutralization with NaHCO₃ for 48 hr after that, the mouse received 2% weight/volume InsP6 and 3% DSS for 24 hr or 2% weight/volume for five or seven days..

Intestinal permeability assay.
IPMKF/F and either IPMK∆IEC mice were fasted for 4 hours with access to water before being gavaged with 4 kDa-FITC-Dextran and 70 kDa RhodamineB (Sigma-Aldrich, Cat no: #FD4-1G) at a single dose of 0.6mg/gm body weight. Untreated control group received an equal amount of PBS. Fluorescence intensity of plasma samples was measured (excitation 485 nm/emission 535 nm) after 4 hours of gavage. Value was represented as a fold change in respect without FITC Dextran and Rhodamine treated control groups after blank subtraction. All measurements were performed using GraphPad Prism (version 6.0, GraphPad Software, Inc)22,.

Generation of IPMK antibody:
Custom anti-IPMK antibody was generated in collaboration with Pro-Sci. The anti-rabbit IPMK antibody had been raised against human IPMK amino acids 295–311 (SKAYSRHRKLYAKKHQS). This sequences are also closely conserved in mice SKMYARHRKIYTKKHHS hence reacts to both mouse and human IPMK.

Western Blot analysis.
Cell lysates were prepared through syringe flush by using a salt-free (NaCl-free) Lysis buffer (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, one tablet of Roche complete protease inhibitor). We used salt-free (NaCl-free) lysis buffer for all the assays because high salt concentration dissociates higher-order inositol phosphate (HOIPs) from HDAC3, which is essential for their deacetylase activity . Samples were centrifuged at 15,000g for 10 min, and the protein concentration of the supernatant was measured. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (NuPAGE Bis-Tris Midi GEL, Life Technologies, Cat #: WG1402BX10) and transferred to Immobilion-P PVDF (Millipore-Sigma, Cat #: IPVH00010) transfer membranes. The membranes were incubated with primary antibody diluted in 3% BSA in Tris- Buffered Saline with Tween 20 (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween 20) overnight incubation at 4C. Respective antibodies for western blot anti-HDAC3 (Santa Cruz Biotechnology, Cat #: sc-376957), IPMK (Generated in our lab) anti-beta actin (ProteinTech, Cat #: 81115-1-RR), anti-lamin (ProteinTech, Cat #: 12987-1-AP), anti-NCoR1 (Bethyl Laboratories, Cat #: A301-148A), anti-Ncor1 (Cell Signaling Technology, Cat #: 5948), anti-Rcor1 (ProteinTech, Cat #: 26686-1-AP), Sin3A (ProteinTech, Cat #: 14638-1-AP), anti-DNTTIP1 (Bethyl Laboratories, Cat #: A304-048A), anti-H3 (Cell Signaling Technology, Cat #: 12648), anti-H3k8Ac (Cell Signaling Technology, Cat #: 2598), anti-H3k9Ac (Cell Signaling Technology, Cat #: 9649), anti-H3k18Ac (Cell Signaling Technology, Cat #: 13998), anti-H3k27Ac (Abcam, Cat #: ab4729) ,anti-H3k56Ac (Cell Signaling Technology, Cat #: 4243), anti-H4k8Ac (Cell Signaling Technology, Cat #: 2594), anti-H4k12Ac (Cell Signaling Technology, Cat #: 13944), anti-H4k16Ac (Cell signaling Technology, Cat #: 13534S) and anti-Myc (Invitrogen, Cat #:46-0709). Following primary antibody incubation, the PVDF membrane was washed three times with TRIS-buffered saline/Tween-20, and incubated with HRP-conjugated secondary antibody (ECL, Cat #s: NA934V and NA931V), and the bands visualized by chemiluminescence (Super Signal West Pico, Pierce, Cat #: 34579). The depicted blots are representative replicates selected from at least three experiments. Densitometric analysis was performed using Image J software. Blots for acetylated histones were stripped using Restore Western Blot stripping buffer (Thermo Scientific, Cat # 21059), then reprobed with H3 antibody (Cell Signaling Technology, Cat #: 12648S) at room temperature for 2 h followed by secondary antibody incubation and development.

HDAC3 (histone deacetylase) activity.
To analyze deacetylase activity of specific Class I HDAC members, the HDAC 3 were individually immuno-purified, followed by an activity assay using the following kit: HDAC3 Assay Kit (BPS, Cat#: 10186-628). Since high salt concentration dissociates higher-order inositol phosphates (HOIPs) from HDAC3, which are crucial for their deacetylase activity, WT and IPMK-KO HCT 116 cells were lysed in salt-free (NaCl-free) lysis buffer (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, one tablet of Roche complete protease inhibitor). Then, the lysate was centrifuged at 15,000 g for 10 min, and the protein concentration of the supernatant was measured. Equal amount, around 20 µg total protein from the lysate was incubated against anti-HDAC3 (Santa Cruz Biotechnology, Cat #: sc-81600) antibodies, respectively, at 4C for 1 hour followed by capturing antibody with EZview A/G beads (MillipoRre Sigma, Cat #: E3403). Beads were washed 3x with an ice-cold reaction buffer (supplied with the kit). After a specific substrate reaction, fluorescence was measured using a Spectra max iD5 plate reader (Molecular Devices). IgG was immunoprecipitated from Wild Type HCT 166 cells (WT) and used as a negative control. In each experiment, separate IgG immunoprecipitation was performed, and the data was expressed as a fold change relative to the respective IgG control. Although slight variations in IgG values were observed across experiments, the overall data range differed slightly in each case, but consistently showed a similar trend. All measurements were performed in triplicate, and data was analyzed using GraphPad Prism (version 6.0, GraphPad Software, Inc).

Intestinal Epithelial cell purification.
The ileum (similar length as colon moving proximal from the ileocecal junction) were dissected from mice. After five times washing with ice cold PBS, the tissues were opened longitudinally and cut into small fragments (2–3 cm in length). To prepare single cell suspension after washing three time with PBS, small intestinal fragments were incubated in 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 5 min. Isolated crypts were washed once with cold PBS and dissociated using TrypLE Express (Invitrogen Cat# 12604021) for 10 min at room temperature. Cell suspensions were passed through a 70-mm cell strainer and then labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (BioLegend, Cat# 103113), FITC-CD31 (BioLegend, Cat# 102406), PE/Cy7-TER-119 (BioLegend, Cat# 116222), and PE-EpCAM (Thermo Fisher Scientific, Cat# 12-5791-83). Dead cells were excluded using Tail Cell Cycle Solution (Invitrogen, Cat# A10798).Following doublet exclusion and single-cell population isolation through forward and side scatter analysis, the Propidium Iodide - (Ex 535nm: Em 617nm) / APC-CD45 - (Ex 651nm: Em 660nm) cell population was gated. Next, FITC-CD31 (Ex 498nm: Em 517nm) / PE-Cy7-TER119 (Ex 565nm: Em 774nm) - - cells were gated and subtracted from the PI/APC-CD45 population. Remaining cells were gated as PE-EpCAM+ (Ex 565nm: Em 576nm) and isolated as CD45/CD31/TER119 - - - /EpCAM intestinal epithelial cells. CD45/CD31/TER119/EpCAM intestinal epithelial + - - - + cells were sorted by using a SONY SH800S Cell Sorter and collected in a post-sort buffer (RPMI-1640 with 20% FBS) .
To isolate crypt from colon, the Colon (from the cecum to the rectum) were dissected out from mice as previously described33. After five times washing with ice cold PBS, the tissues were opened longitudinally and cut into small fragments (2–3 cm in length). To isolate crypts after washing three times with cold PBS, small ileum fragments were incubated in 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 5 min. Isolated crypts were washed once with cold PBS and dissociated using TrypLE Express (Invitrogen Cat# 12604021) for 10 min at room temperature. Cell suspensions were passed through a 70-µm cell strainer.

InsP6 Rescuing HDAC3 activity.
Wild Type and IPMK-Knock out (KO) HCT116 cells were lysed in salt-free (NaCl-free) lysis buffer (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, and one Roche complete protease inhibitor tablet). Samples were centrifuged at 15,000 g for 10 min, and the protein concentration of the supernatant was measured. Equal amount, 20 µg total protein from the lysate was incubated against anti-HDAC3 antibodies respectively at 4C for 1 hr, followed by capturing antibodies with EZview A/G beads (Millipore Sigma, Cat #: E3403). Beads were washed 3x with an ice-cold reaction buffer (supplied with the kit). Following washing, beads were resuspended in reaction buffer then beads were incubated with 1nM, 10nM, 100nM, 500nM, 1µM, 5µM and 10µM concentrations of InsP6 (Sigma Aldrich Cat #: P8810-100G) for 1 hr at room temperature. After incubation, beads were washed 3x with a reaction buffer. HDAC3 activity was measured by the addition of a fluorogenic substrate provided by the HDAC Assay Kit (BPS, Cat #: 10186-628) and read using a Spectramax iD5 plate reader (Molecular Devices). IgG was immunoprecipitated from IPMK Wild Type HCT 166 cells (WT) and used as a negative control. In each experiment, separate IgG immunoprecipitation was performed, and the data was expressed as a fold change relative to the respective IgG control. Although slight variations in IgG values were observed across experiments, the overall data range differed slightly in each case, but consistently showed a similar trend. All measurements were performed in triplicate data and analyzed using GraphPad Prism (version 6.0, GraphPad Software, Inc)15,13,16.

Analysis of rescuing HDAC3 activity using PIP3, InsP3, Ins (1,3,4,5) P4, Ins(1,4,5,6)P4, InsP5 and InsP6:
Wild type and IPMK-Knock out (KO) HCT116 cells were lysed in salt-free (NaCl-free) lysis buffer (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, and one Roche complete protease inhibitor tablet). Samples were centrifuged at 15,000 g for 10 min, and the protein concentration of the supernatant was measured. Equal amount, 20 µg total protein from the lysate was incubated against anti-HDAC3 antibodies respectively at 4C for 1 hr, followed by capturing antibodies with EZview A/G beads (Millipore Sigma, Cat #: E3403). Beads were washed 3x with an ice-cold reaction buffer (supplied with the kit). Following washing, beads were resuspended in reaction buffer then beads were incubated with either 10µM concentration of PIP3, InsP3, Ins(1,3,4,5)P4 or 1µM Ins(1,4,5,6)P4, InsP5 and InsP6 (Sigma Aldrich Cat #: P8810-100G) for 1 hr at room temperature. After incubation, beads were washed 3x with a reaction buffer. HDAC3 activity was measured by the addition of a fluorogenic substrate provided by the HDAC3 Assay Kit (BPS, Cat #: 10186-628) and read using a Spectramax iD5 plate reader (Molecular Devices). IgG was immunoprecipitated from Wild Type HCT 166 cells (WT) and used as a negative control. In each experiment, separate IgG immunoprecipitation was performed, and the data was expressed as a fold change relative to the respective IgG control. Although slight variations in IgG values were observed across experiments, the overall data range differed slightly in each case, but consistently showed a similar trend. All measurements were performed in triplicate data and analyzed using GraphPad Prism (version 6.0, GraphPad Software, Inc)15,13,16.

Plasmids and Recombinant Proteins.
pMX-myc, pMX-IPMK-WT-myc and pMX-IPMK-KSA-myc plasmids transiently transfected into IPMK-KO HCT 116 cells using Lipofectamine 3000 (Cat no. L3000001, Thermo Scientific) according to the manufacturer protocol. Recombinant myc-IPMK was purchased from Origene, (Cat #: TP309343) while recombinant GST-HDAC3 (Cat #: H85-30G) and GST (Cat #: G52-30U) were purchased from Signal Chem.

Cell fractionation and isolation of chromatin fraction
Approximately 7x106 cells were isolated and washed three times with chilled PBS. After that cells were lysed with ice cold cell lysis buffer (50 mM HEPES-KOH, 140 mM NaCl,1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM DTT Supplemented with 1x protease inhibitor cocktail ). After centrifugation at 1100 g for 5 min supernatant was separated as a cytoplasmic fraction. Pellets were the crude nuclei. After washing three time with cell lysis buffer, nuclei was incubated 10 min in ice with nuclear lysis buffer (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, Supplemented with 1x protease inhibitor cocktail). Gently resuspended 10 times. Then centrifuge at 1100 g for 10 min. Supernatant was separated as a nuclear fraction and pellet was the crude chromatin. After washing three times with a nuclear lysis buffer, crude unsheared chromatin was used for further experiment.

Immunoprecipitation.
To analyze endogenous binding, HCT116 cells were lysed in salt-free lysis buffer (50 mM Tris pH 7.5, 50 mM potassium acetate, 5 % v/v glycerol, 0.3 % v/v Triton X-100, and one Roche complete protease inhibitor tablet). For the endogenous immunoprecipitation (IP), respective antibodies against HDAC3 were used, followed by western blot of the binding partners. In brief, the IP was performed from 500 μug of protein lysate. Protein lysates were incubated for 2 hr at 4oC with respective antibodies, then the antibody-protein complex was captured with EZview A/G beads (Millipore Sigma, Cat #: E3403). Beads were pelleted and washed with lysis buffer 3x, followed by elution in sample buffer. Immunoprecipitated samples were resolved on a NuPAGE Bis-Tris gel, followed by western blotting 18

ChIP-qPCR:
Approximately 7x106 cells were fixed with 1% final volume from fresh 16% stock formaldehyde (Sigma-Aldrich, Cat#: F8775) at room temperature for 10 min followed by ChIP using ChIP assay Kit from iDEAL Diagenode ( Cat no.C01010051). Cells were then harvested and lysed in 500 mL of ChIP lysis Buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.5% SDS, 0.5% NP-40, 1 mM sodium butyrate) containing protease inhibitor cocktail. The lysates were subjected to sonication to shear DNA to a length of approximately between 150 and 900 bp. The lysate was then diluted in 0.75 mL of ChIP dilution buffer and incubated with control IgG (Cell Signaling Technology, Cat#: 2729S) or primary antibody H3K9 ac (Cells signalling Technology, Cat#9649T) , H3K27 ac (Abcam cat# 4729) , H4k16ac (Cells signalling Technology, Cat#13534) , HDAC3 ( Santa Cruz Biotechnology, Cat# 39717) and HDAC1 ( Santa Cruz Biotechnology, Cat# 81598X) 4C overnight. Then the lysate was incubated with Protein G magnetic bead (provided in kit) for 1 h at 4C. The beads were washed sequentially with a wash buffer provided in the kit. The immune complexes were eluted with 75 mL of elution buffer (1% SDS, 0.1 M NaHCO3) twice at 65C for 30 min. After elution, the cross-link was reversed by adding NaCl and incubated together with Proteinase K provided with the kit overnight at 65C. ChIP DNA was purified using the ChIP DNA purification kit Provided with the kit. The purified DNA was analyzed on a StepOnePlus using the power SYBR Green Master Mix. The results are presented as a fold change in respect with IgG after calculating the percentage of input. qPCR analyses were done in triplicate. We used MMP3 5′-AAAATAGAGTAGCAGAGGCAGGTA -3′ and 5′-AGAGTGGTGGCAGTGATGTGAA -3′, and MMP 13 Fwd 5′ CAACCATGGGGCTCAATCCT3′ Rev 5′ CTTACGTGGCGACTTTTTCTTTTC3′ Primer for human and MMP3 5′-TGTGGTCACTGATAGTGTGGACTGT -3′ and 5′-CCAGAAGCCCAAGCTGAATACTGT -3′, and MMP 13 Fwd 5′ CTGCCACAAACCACACTTAGG3′ Rev 5′ AGTCACCACTTTGGGGTGTG3′ Primer for mouse 18

mRNA expression by qPCR:
After extracting the total RNA using RNeasy Mini Kit (Qiagen), and checking its integrity by nanodrop, the cDNA was synthesized from 1 mg of purified total RNA using High capacity cDNA Reverse Transcription Kit (Applied Biosystem). Expression of mouse and human MMP1, MMP3 and MMP13, 18S for human and mouse GAPDH was detected using suitably designed Taqman primers (Invitrogen). The experiments were performed (real-time PCR Systems StepOne plus, Applied Biosystems) in triplicate. Data were quantified for the above genes using the comparative Ct method, as described in the Assays-on-Demand Users Manual (Applied Biosystems)18

In vitro Binding assay.
Equal amount of recombinant myc-IPMK (Origene, Cat #: TP309343) was co-incubated with either GST-HDAC3 (Signal Chem, Cat #: H85-30G) or GST (Signal Chem, Cat #: G52-30U) in lysis buffer and the complex was maintained for 30 min at 4C. After the addition of GST beads, incubation continued for an additional 45 min and washed 3x with an ice cold lysis buffer. SDS sample buffer was added, and binding was confirmed by western blotting of anti-Myc antibodies18.

Transwell assay.
Transwell filter inserts (pore size 1.0 μm, 12 mm diameter, polyester membrane, Corning, New York, USA) were coated with fibronectin (10 μg/ml; Sigma) used as insert for 24 well plate. For time dependent permeability assay CaCo2 cells were seeded (0.25 × 10 cells/well) and grown on transwell filters for 48 hours until reaching confluency. FITC-dextran 4 kDa-FITC-Dextran (Sigma-Aldrich, Cat no: #FD4-1G) was added to the top well at 25 mg/ml. at 37°C, and fluorescence was measured after 10 min by using a Spectramax iD5 plate reader (Molecular Devices, (excitation 485 nm/emission 535 nm)47. To analyze crucially of IPMK or InsP6 in cell permeability, CaCo2 cells were seeded (0.25 × 106 cells/well) and grown on transwell filters for 48 hours until reaching confluency. Soup from WT and IPMK-KO HCT116 cells and confluent monolayers of WT and IPMK-KO HCT 116 cells were treated with similar amounts of PBS (as a negative control), Ilomastat (10µM) or cell permeable InsP6 (50µM) or MMP Activator p-Aminophenylmercuric acetate (5µM) overnight. Soup from those HCT 116 cell cultures was transferred to mono layer of CaCo2 cells in the transwell plate. Incubate for four hours. FITC-dextran 4 kDa-FITC-Dextran (Sigma-Aldrich, Cat no: #FD4-1G) was added to the top well at 25 mg/ml for 10 min at 37°C, and fluorescence was measured using a Spectramax iD5 plate reader (Molecular Devices, (excitation 485 nm/emission 535 nm).

RGFP966 treatment
One million cells WT and HDAC3 or IPMK knockout HCT 116 cells were plated and let them grow overnight. Next day cells were treated with HDAC3 specific inhibitor RGFP966 at a dose of 10µM for 24 hr.

CP-InsP6 treatment
Cell permeable InsP6 was created and characterized in Prof. Dr. Henning J. Jessen Lab, University of Freiburg, Germany17. After synthesis cell permeable InsP6 was primarily dissolved in DMSO to prepare a 10 mM primary stock. One million cells IPMK KO HCT 116 cells were plated and let them grow overnight. Next day cells were treated with CP-InsP6 at a dose of 50µM for 24 hr in media.

Intracellular inositol content analysis using HPLC:
IPMK-WT and KO HCT116 cells were plated at a density of 250,000 cells per 60 mm plate, then labeled with 60 mCi (1 Ci = 37 GBq) [3H] myo-inositol (PerkinElmer) in conventional cell culture media for three days. To extract soluble inositol phosphates, cell pellets were suspended in 300 mL of ice-cold 0.6 M perchloric acid buffer (0.1 mg/mL IP6, 2 mM EDTA) and incubated on ice for 1 min. Ninety mL of 1 M potassium carbonate with 5 mM EDTA were added and incubated on ice for 1 h. Extracts were centrifuged at 12,000 rpm for 15 min. The supernatant was collected and analyzed by HPLC using a Partisphere SAX column (Whatman Inc.). The column was eluted with a gradient generated by mixing Buffer A (1 mM EDTA) and Buffer B (Buffer A plus 1.3 M (NH4)2HPO4, pH 3.8 with H3PO4). The 1 mL fractions were collected and counted using 5 mL of Ultima-Flo AP mixture (PerkinElmer).

Unlabeled Inositol phosphates purification from cells and tissue
Inositol phosphates (HOIP) purification was performed as previously described35. Briefly, for cell culture HCT116 cells total of around 30-40 million) was used and the total inositol content was normalized by the total cell count. The data is represented as picomole (pM) per 1 million cells. For the mouse intestinal crypts from the Ileum they were washed in cold PBS, and a fraction was set aside for normalization using total protein. The samples were resuspended with cold 1 M perchloric acid and incubated on ice. Titanium dioxide beads were prepared as described (4 mg/sample). Samples were centrifuged and the supernatant was transferred to Eppendorfs containing 4 mg of titanium dioxide beads and rotated at 4°C for 20 minutes. Samples were centrifuged, and the beads were washed with cold 1 M perchloric acid three times. Inositol phosphates were eluted twice by resuspending in ammonium hydroxide and rotating at 4 C for 10 minutes. Purified inositol phosphates were dried on low heat using a centrifugal evaporator.

Inositol phosphates quantification using mass spectrometry
The analysis was performed on a CE-QQQ system (Agilent 7100 CE-with Agilent 6495C Triple Quadrupole and Agilent Jet Stream electrospray ionization source, adopting an Agilent CE-ESI-MS interface). An isocratic Agilent 1200 LC pump was used to deliver the sheath liquid (50% isopropanol in water) with a final splitting flow speed of 10 µl/min via a splitter. All separation was performed with a bare fused silica capillary with a length of 100 cm (50 µm internal diameter and 365 µm outer diameter). 40 mM ammonium acetate titrated with ammonium hydroxide to pH 9.0 was used as background electrolyte (BGE). Between runs of each sample, the capillary was flushed with BGE for 400s. Samples were injected by applying 100 mbar pressure for 15s (30 nL). The MS source parameters were as follows: gas temperature was 150˚C, gas flow was 11 L/min, nebulizer pressure was 8 psi, sheath gas temperature was 175˚C and with a flow at 8 L/min, the capillary voltage was -2000V, the nozzle voltage was 2000V. Negative high-pressure RF and negative low-pressure RF were 70 V and 40 V, respectively. Set multiple reaction monitoring (MRM) as shown in the Supplementary table 1.

HOIP level quatitaion.
Internal standard (IS) stock solution of were spiked to samples for the assignment and quantification of InsPs and PP-InsPs. Quantification of 1,5-InsP8, 5-InsP7, 1-InsP7, and InsP6 was performed with known amounts of corresponding heavy isotopic references spiked into the samples. Quantification of 4/6-InsP7 was performed with 13C 5-InsP7 and Quantification of InsP3-4 of which no isotopic standards are available was performed with spiked 13C InsP6. After spiking, 20 μM [13C] 2-OH InsP6, 20 μM [13C] InsP5, 4 μM [13C] 5-InsP7, 2 μM [13C] 1-InsP7 and 1 μM [13C] 1,5-InsP8 were the final concentration inside samples. All 13C inositol references were kindly provided by Dorothea Fiedler36.

Histology and Immunohistochemistry:
10% Phosphate-buffered-paraformaldehyde fixed intestine and colon tissues was subsequently opened lengthwise and washed in cold PBS, then cut into approximately 3-4 mm fragments for sectioning and paraffinization. After that tissue was dehydrated through 70%, 90% 100% and xylene alcohol gradation and subjected to block preparation in low melting point (65°C) paraffin wax. six micron tissue sections were cut and spreaded over Mayer's albumin coated slides. Tissue slides were deparaffinized and stained with hematoxylin and eosin following standard protocol for histological evaluation under light microscope.
Mice were euthanized by CO2. Immediately following euthanasia, the intestine and colon was removed and flushed with cold PBS. The intestine was subsequently opened lengthwise and washed in cold PBS, then cut into approximately 3-4 mm fragments for sectioning and paraffinization. Samples were deparaffinized with Histo-Clear (Thermo Fisher) and rehydrated in successive washes of 100%, 90%, and 75% ethanol followed by deionized (DI) water. Sections were unmasked via heat-induced epitope retrieval (HIER) using citrate buffer, followed by blocking with 3% goat serum for 1 hour. Primary antibodies were diluted per manufacturer's recommendation in 1% Triton-X containing blocking solution, incubating overnight at 4°C in a humidified chamber. The following day, samples were washed with TBST, followed by incubation with fluorescent-tagged secondary antibodies for 60 minutes at 37°C. After five time washing with TBST, samples were stained with 4′, 6-diamidino-2-phenylindole (DAPI) diluted in PBS (1 μg/mL). Slides were imaged using an Axiovert Zeiss epifluorescence microscope and LSM800 confocal microscope. Subsequent histogram analysis in ImageJ produced signal intensities from 0-255, with 0 = darkest intensity (positive staining) and 255 representing a white, unstained signal. A threshold of 0-230 was applied to eliminate background staining.

RNA isolation and library preparation.
Total RNA form was isolated by using IRNAeasy Mini Kit (Qiagen Cat no.74104) by following manufacturer's kit protocol. For RNA-seq, libraries were prepared from total RNA by using True Seq RNA Library Prep Kit V2 (Illumina).

RNA seq analysis.
FASTQ files were retrieved from sequencing. All pre-alignment and post-alignment QA/QC and data analysis were performed in Partek Flow (v.12.0.0). Reads were aligned to the whole genome using hg19 assembly with STAR (v.2.7.8a). Aligned reads were quantified to the hg19 annotation model using HTSeq (v.0.11.0). Differential analysis was performed for IPMK knockout versus wild-type, HDAC3 knockout versus wild-type, and IPMK∆IEC versus IPMKF/F using DESeq2 (v.3.5)31.

Pathway analysis using Metascape.
Pathway analysis was performed on differentially expressed genes (FDR <= 0.05 and log2FoldChange >= 2) for HCT116 IPMK knockout versus wild-type and Vilcre versus FF using Metascape (v.3.5) with default settings. The genes were queried against GO Biological Pathways, KEGG Pathway, Reactome Gene Sets, Canonical Pathways, WikiPathways, and Panther pathways. Terms with a p-value < 0.01, a minimum count of 3, and an enrichment factor > 1.5 were considered. All genes were used as a background gene list.

GSEA pathway analysis.
Gene set enrichment analysis (GSEA) was performed using the R package cluster Profiler37 . We used all the genes after DESeq2 normalization to generate a ranked gene list sorted by Wald's test statistic, the queried relevant pathways containing MMP genes from the C2:CP:Reactome Csubcollection of the Molecular Signature Database (MSigDB)38.

ELISA analysis of ILβ and MPO:
Briefly, tissue lysates were incubated in wells precoated with the relevant antibody, and detection was carried out by measuring absorbance at 450nm. Final optical density values were blank-subtracted and normalized to total protein content.

Statistical analysis.
All plots and statistical analyses were performed with Prism 9 (GraphPad) software. Statistical significance was determined by either Student’s t-test (two-tailed) for two groups or 1-way ANOVA for multiple groups with similar samples. Error bars represent the standard deviation of the mean and indicate replicates or the number of animals employed. Results were representative of at least three independent experiments (n). Graphs were generated by using either Prism 9 (GraphPad) software or R software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Abstract: “leaky gut,” can you use more official terminology.\nIn the Introduction, there are about 4 paragraphs spent describing the results of the study. Understandably ~1 paragraph in\nthe introduction is reasonable to cover the broad findings of what is about to be presented in the Results section, but these\nparagraphs as they stand include many details including concentrations for example. I think this could be simplified for a\nmore streamlined message in the Introduction.\nAgain the use of the term “leaky gut” throughout the paper, if used, should be defined first somewhere, probably the\nIntroduction. Barrier function and permeability are mentioned but not explicitly related or to mean “leaky gut” – this is more of\na colloquial term. IN the results section there is the following sentence, which might be more useful upfront in the paper. “The\nupregulation of MMP is associated with compromised intestinal barrier function by disrupting tight junctions, leading to a\n\"leaky gut”, which is commonly observed in IBD patients”\nFigure 1, E-Cadherin misspelled\nNoteworthy Results: The study by Chetterjee, et al. identifies inositol polyphosphate multikinase (IPMK) and inositol\nhexakisphosphate (InsP6 or phytic acid),) as activators of HDAC3 leading to maintenance of intestinal barrier integrity. It\nestablishes a mechanistic link between IPMK, HDAC3 enzymatic activation, and histone deacetylation via the recruitment of\nthe DAD domain of corepressor proteins. Loss of IPMK leads to increased histone acetylation, upregulation of matrix\nmetalloproteinases (MMPs), and compromised intestinal permeability. The study demonstrates the therapeutic potential of\nInsP6 in mitigating loss of barrier integrity and implications for inflammatory bowel disease (IBD) pathogenesis (since there\nis frequent loss of IPMK in IBD patients) using cell-based assays and a DSS-induced IBD mouse model. It provides strong\nevidence for InsP6 as a dietary factor that modulates intestinal homeostasis through epigenetic regulation.\nSignificance: The work introduces an epigenetic regulatory pathway linking dietary InsP6 to HDAC3 activity, with potential\napplications in intestinal barrier function maintenance for IBD treatment. Given that pan-HDAC inhibitors cause significant\ngastrointestinal toxicity, the identification of InsP6 as a physiological HDAC3 activator offers a targeted therapeutic approach\nfor IBD in the context of intestinal permeability.\nThe use of the “the leaky pathway” in the Results section, again is very colloquial and isn’t totally clear what the pathway is,\nsince it is a complex interaction beyond only what is addressed in this manuscript.\nThe authors note that transcriptomic analysis of IBD patients showed a significant increase in MMPs (Extended Figure 4 A),\nthis analysis was done specifically in Crohn’s disease patients and then the mouse model they employ is a DSS-colitis\nmodel which mimics Ulcerative Colitis and impacts mainly the colon in terms of inflammation. The authors note this in the\nResults section (Figure 6) and also analyzed the KO mice small intestine for molecular analysis of MMP3 which were\nconsistent with their findings. But, it is hard to reconcile the relationship to IBD as an umbrella since Crohns and Ulcerative\nColitis have unique pathophysiology. Are these genes involved in Ulcerative Colitis\nThe manuscript builds on previous studies identifying HDAC3’s role in intestinal epithelial homeostasis, and IBD (Crohn’s\nanimal models). It addresses an important knowledge gap regarding the direct biochemical and physiological role of higher-\norder inositol phosphates in epigenetic regulation, and uncovers the role of InsP6 in HDAC3 activation, which had not been\npreviously appreciated in the literature based on crystal structure.\nFigure 4: More clarification on the RNA-seq analysis is needed. The authors note there is 4014 genes upregulated and 3271\ndownregulated in IPMK-KO relative to wildtype, and then focus in on MMPs, which they justify through pathway analysis.\nFigure 4B, the figures is labeled as “Pathway analysis of WT vs IPMK KO overlapped Genes” the overlapped genes wording\nis confusing, does pathway analysis here indicated the DEGs that are upregulated in the KO and the pathways that are\nenriched? Please clarify in the figure. In the methods, it seems clear that it is the upregulated genes and multiple pathway\nanalysis tools were used, but what plot is shown? Can you corroborate these findings with GSEA? And what about the\ndownregulated pathways? What pathway analysis software or program was used here (KEGG, Panther, etc). I think the\nRNA-seq analysis should be more robust and acknowledge other genes that may be involved in this process.\nThe conclusions are well-supported by a combination of biochemical, genetic, and in vivo data. The IPMK KO models\neffectively demonstrate the loss of HDAC3 activation, and InsP6 rescue experiments provide convincing evidence for its role\nin epigenetic regulation, and oral InsP6 administration rescues the IPMK-HDAC3 axis, validating its potential as a dietary\nintervention.\nAdditional clinical validation in human subjects would be beneficial to translate these findings into therapeutic applications,\nrather than leaning on RNA-seq of publicly available data which is incomplete in the context of IBD (e.g. focus on Crohn’s vs\nUC).\nFurther validate MMP gene regulation with additional ChIP-seq or transcriptomic/protein analyses could be warranted,\nespecially for additional HDAC3 target genes could further solidify the epigenetic regulatory mechanism.", "reviewer_2": "In the manuscript by Chetterjee, et al, the authors report new evidence of inositol phosphate-HDAC3 axis to maintain\nintestinal barrier integrity. While Ins(1,4,5,6)P4 has been visualized in HDAC3 crystal structure, biochemical assay showed\nthe more phosphorylated InsP5 and InsP6 have been higher affinity to HDAC3. In this manuscript, the author found that\nIPMK, the enzyme synthesizes InsP4 and InsP5, is required for HDAC3 activity in cells. IMPK physically interacts with\nHDAC3 and the locally produced InsPs are required for interaction of HDAC3 and its corepressors, and therefore HDAC3\ndeacetylation activity. HDAC3 was previously reported to regulate the expression of matrix remodeling enzymes, and indeed\nthe authors found loss of IPMK resulted in increased MMP gene transcription, compromising intestinal barrier integrity.\nOverall, the experiments were well designed, and the manuscript was clearly written.\nThe major concern of the study is which InsP is the physiological molecular regulating HDAC3 in vivo, although the authors\nsuggest InsP6 as the most potent physiological HDAC3 activator. In vitro assay demonstrated Ins(1,4,5,6)P4, InsP5 and\nInsP6 could all restore HADC3 activity in IPMK KO cells (Fig2D). If InsP6 is the dominant regulator in cells, KO of IPPK may\nresult in similar phenotype of IMPK KO, although InsP5 level may increase. For the whole manuscript, cell permeable InsP6\nwas used to deliver InsP6 into cells. However, the delivery efficiency was not examined. Also, the delivered InsP6 may got\nmetabolized in cells after 24h treatment. So, an InsP PAGE or CE-MS assay to quantify the intracellular InsPs levels will be\nneeded to help conclude to the impact of different InsPs in HDAC3 activity.\nThe author showed IPMK physically interact with HDAC3, and the kinase activity is required for the interaction of HDAC3\nwith DAD domain of its repressors. So, the direct interaction with IPMK probably provide locally synthesized InsP4, InsP5 or\nInsP6 to mediate DAD-HDAC3 interaction. Since IPPK synthesizes InsP6 from InsP5, does IPPK also interact with the\nHDAC3 complex?\nIn extended Figure 4A, IBDTaMMA analysis revealed significant transcriptional upregulation of MMP in Crohn’s patients,\nsimilar to IPMK KO HCT116 cells. Is IMPK or IPPK levels affected in patients?\nIPMKΔIEC mice have a marked increase in intestinal the leaky pathway, allowing 4KD dextran to pass through (Fig 5D).\nHowever, a previous study by Park et.al (PMID: 35988719) found IPMKΔIEC mice intestinal epithelium has no critical defect\nin its barrier function. Could you please comment?\nIn the DSS induced leaky gut model, IMPK protein level was almost abolished after DSS treatment. How did IMPK level\nchange when mice were fed with InsP6? It’s been show phytate protect gut from DSS-induced colitis (PMID: 36341915).\nTherefore, it is possible IP6 prevent DSS caused cell damage via MMP-independent pathway, especially if IPMK protein\nkept intact after InsP6 supplementation. Also, InsPs levels of colon tissues in IP6 fed mice should recorded by CE-MS since\nthe authors have the capacity.\nMinor points:\n1. Fig 1F, HDAC3 protein level should be included.\n2. Why RGFP966 (10μM) treatment needs to last for 8 or 24h?\n3. Extended Fig2C is missing.", "reviewer_3": "In the manuscript \"Phytic acid (insP6) activates HDAC3 epigenetic axis to maintain intestinal barrier function\" Chetterjee et\nal. convincing demonstrate that InsP6 activates HDAC3, independent of IPMK, through DAD binding. Activation of HDAC3\nprevented the hyperacetylation of histones, promoting the expression of various MMP family members. This work presents\nseveral interesting and novel observations, however, the authors fall short on fully convincing the reviewer that InsP6\ntreatment mitigates leaky gut or barrier function in an IBD model.\nMajor Points:\n- The authors only include observations of the impact of InsP6 after 24hrs of DSS treatment. If the DSS model were to be\nextended how does InsP6 treatment affect disease outcomes or wound healing?\n- It has been shown that HDAC3 deficient mice are sensitive to DSS colits, do IPMK deficient mice show the same\nphenotype and would treatment with InsP6 improve disease outcomes in these mice?\n- The authors should consider investigating other tight junctional proteins associated with leaky gut, such as claudin-2. In\nextended figure 5a the authors show a modest decrease in ZO-1 expression. ZO-1 is more often associated with barrier\nintegrity and not leaky gut.\n- The authors should consider extending this ZO-1 observation to their in vivo models. Does loss of IPMK impact the\nexpression of zo-1/claudin in vivo as well.\n- Given that all in vitro studies where done using cell permeable InsP6, the authors should consider showing data from the\nmurine tissue that non-cell permeable InsP6 is in fact absorbed by the intestinal cells. The same analysis performed in\nFigure 1B could be performed on tissue extracts, as well as, on fecal extracts to provide evidence that the InsP6 is being\ntaken up by the cells and also that it makes it to the colon.\n- It would add to the manuscript if the authors were to assay the expression of MMPs in the supernatants transferred to the\ncaco2 cells.\nMinor Points:\n- In the results section there were several instances of Figures being called out in the text that refered to the incorrect figure,\nspecifically Line 263, line 442\n- It is unclear from the figure legends, methods, or text on how many mice were utilized in each experiment.\n- Can you also clarify what portion of the colon that was collected for the IF images and the crypt isolation. DSS is known to\nprimarily impact the distal colon so it is unclear why the crypts from the ileum were used in figure 5 instead of crypts from the\ncolon.\n- There are grammatical errors through out the manuscript"}, "author_response": "Response to the reviewers' responses is in blue.\nReviewer 1:\n● Abstract: “leaky gut,”can you use more official terminology\n● As suggested by the reviewer, we replaced the “leaky gut” with gut permeability. Line\nnumber 37. We introduced the term 'leaky gut' (line 134) in the introduction, which\njustifies its use throughout our manuscript.\n● Understandably, ~1 paragraph in the introduction is reasonable to cover the broad\nfindings of what is about to be presented in the Results section, but these paragraphs\nas they stand include many details including concentrations for example.\n● As suggested, we concise the introduction.\n● Reviewer Comment:\nFigure 1, E-Cadherin misspelled\n● The spelling is now corrected in the revised MS.\n● Reviewer Comment:\nThe authors note that transcriptomic analysis of IBD patients showed a significant\nincrease in MMPs (Extended Figure 4A), this analysis was done specifically in\nCrohn’s disease patients, and then the mouse model they employ is a DSS-colitis\nmodel, which mimics ulcerative colitis and impacts mainly the colon in terms of\ninflammation. The authors note this in the Results section (Figure 6) and also\nanalyzed the KO mice small intestine for molecular analysis of MMP3, which were\nconsistent with their findings. But, it is hard to reconcile the relationship to IBD as an\numbrella since Crohn's and Ulcerative Colitis have unique pathophysiologies. Are\nthese genes involved in Ulcerative Colitis. Additional clinical validation in human\nsubjects would be beneficial to translate these findings into therapeutic applications.\n● We thank the reviewer for suggesting that we examine the implications of our study\nin IBD-related clinical samples. As described in the manuscript, we collaborated with\nWashington University in St. Louis and analyzed tissue biopsy samples from patients\nwith ulcerative colitis and Crohn’s disease. Notably, we observed a selective loss of\nIPMK protein levels in both patient groups, accompanied by increased histone\nacetylation and a striking elevation of MMP3/13 expression. Most notably, we\nidentified a loss of HDAC3 activity in both ulcerative colitis and Crohn’s disease\nsamples.\nFurthermore, our analysis of the Crohn’s disease and ulcerative colitis transcriptome\ndata (Extended Figur 4 II A,B) from the IBDTaMa human dataset revealed that MMP\ngenes are commonly upregulated in both conditions, mirroring the patterns observed\nin intestine-specific IPMK knockout mice and IPMK knockout cell lines.\nCollectively, these findings highlight the clinical relevance of our study in the context\nof IBD (Line 531-536, Figure 5 O-R).\n● More clarification on the RNA-seq analysis is needed. The authors note there is 4014\ngenes upregulated and 3271 downregulated in IPMK-KO relative to wildtype, and\nthen focus in on MMPs, which they justify through pathwayanalysis. Figure 4B, the\nfigures is labeled as “Pathway analysis of WT vs IPMK KO overlapped Genes”. The\noverlapped gene-swording is confusing. Does pathway analysis here indicate the\nDEGs that are upregulated in the KO and the pathways that are enriched? Please\nclarify in the figure. In the methods, it seems clear that it is the upregulated genes\nand multiple pathway analysis tools were used, but what plot is shown? Can you\ncorroborate these findings with GSEA? And what about the downregulated\npathways? What pathway analysis software or program was used here (KEGG,\nPanther, etc). I think the RNA-seq analysis should be more robust and acknowledge\nother genes that may be involved in this process.\n● As recommended by the reviewer, we conducted separate pathway analyses for\nupregulated and downregulated genes derived from RNA-seq data of IPMK knockout\ncells and IPMK-depleted murine intestinal epithelial cells (IECs). Metascape analysis\nof the upregulated genes revealed a significant enrichment of pathways related to\nextracellular matrix degradation (Fig. 4B and Extended Fig. 5 II H), primarily driven\nby Matrix Metalloproteinase (MMP) genes. This finding was further corroborated by\nGene Set Enrichment Analysis (GSEA) of differentially expressed genes from the\nmouse IECs (Extended Fig. 5 II J), which demonstrated a consistent trend. In\nparallel, we also analyzed pathways associated with transcriptionally downregulated\ngenes (Fig. 4B and Extended Fig. 5 II I).\n● We apologize for the ambiguity. To clarify the overlap between the gene sets, the\nVenn diagram (Fig. 4J) shows that 44.4% of the differentially expressed genes\n(DEGs) in HDAC3 knockout (KO) cells are also differentially expressed in IPMK KO\ncells. These represent common gene sets that are differentially expressed in both\nIPMK KO and HDAC3 KO conditions. It should be noted that this analysis includes\nboth upregulated and downregulated genes, rather than only the upregulated subset.\nReviewer 2:\nReviewer’s Comment:\n● The major concern of the study is which InsP is the physiological molecular regulating\nHDAC3 in vivo, although the authors suggest InsP6 as the most potent physiological\nHDAC3 activator. In vitro assay demonstrated Ins(1,4,5,6)P4, InsP5 and InsP6 could all\nrestore HADC3 activity in IPMK KO cells (Fig. 2D). If InsP6 is the dominant regulator in\ncells, KO of IPPK may result in a similar phenotype of IMPK KO, although InsP5 level may\nincrease. For the whole manuscript, cell-permeable InsP6 was used to deliver InsP6 into\ncells. However, the delivery efficiency was not examined. Also, the delivered InsP6 may\ngot metabolized in cells after 24h treatment. So, an InsP PAGE or CE-MS assay to quantify\nthe intracellular InsPs levels will be needed to help conclude to the impact of different InsPs\nin HDAC3 activity.\n● We thank the reviewer for this excellent question. As suggested, we performed mass\nspectrometric analysis and genetic depletion of IPPK to explore whether InsP , InsP , or\n6 5\nboth could exert epigenetic regulation of HDAC3 in an in vivo cell line. The mass\nspectrometric analysis using cell-permeable InsP6 (cp-InsP6) indicates that once inside the\ncell, cp-InsP6 markedly increases InsP levels, but it can also significantly raise InsP levels\n6 5\n(Extended Figure 2C). This suggests that cell-permeable InsP6 treatment increases both\nInsP6 and InsP5 levels inside the cells after 24 hours of treatment. However, InsP6 levels\nremain significantly high than InsP5 (Line 277-283). In this context, genetic depletion of\nIPPK is helpful, as it selectively reduces InsP6 levels while keeping InsP5 levels mostly\nunchanged. Remarkably, we found that IPPK deletion in cell lines produces the same effect\nas IPMK deletion, with loss of HDAC3 activity (Figure 7 L) and elevated MMP3/13 levels\n(Figure 7M). This suggests that in vivo, InsP6s have selectivity for HDAC3 regulation over\nInsP5 (Line 654-668).\n● The author showed IPMK physically interact with HDAC3, and the kinase activity is required\nfor the interaction of HDAC3 with DAD domain of its repressors. So, the direct interaction\nwith IPMK probably provide locally synthesized InsP4, InsP5 or InsP6 to mediate DAD-\nHDAC3 interaction. Since IPPK synthesizes InsP6 from InsP5, does IPPK also interact with\nthe HDAC3 complex?\n● We thank the reviewer for this insightful question, which prompted us to investigate and\nelucidate the complete signaling mechanism in a spatiotemporal context. We conducted\nboth immunoprecipitation (Extended Fig. 3II A) and proximity ligation-based imaging\n(Extended Fig. 3II B) studies to assess the interaction between IPPK and HDAC3. Notably,\nboth assays confirmed that IPPK interacts with HDAC3. These results indicate that both\nIPMK and IPPK bind to HDAC3, facilitating local biogenesis of InsP6, which in turn activates\nHDAC3 (Line 351-355).\n● In extended Figure 4A, IBDTaMMA analysis revealed significant transcriptional\nupregulation of MMP in Crohn’s patients, similar to IPMK KO HCT116 cells. Is IMPK or\nIPPK levels affected in patients?\n● As described in the manuscript (Fig. 5Q,R ), we collaborated with Washington University in\nSt. Louis and analyzed tissue biopsy samples from patients with ulcerative colitis and\nCrohn’s disease. Notably, we observed a selective loss of IPMK protein ( Fig5 Q,R), while\nIPPK levels remained unchanged and comparable to the control patient tissue samples.\nAdditionally, we found increased histone acetylation (Fig 5Q,R) and a significant elevation\nof MMP3/13 (Fig 5Q,R) expression in Crohn’s disease and ulcerative colitis patients. Most\nnotably, we identified a loss of HDAC3 activity (Fig 5,O,P) in both ulcerative colitis and\nCrohn’s disease samples (Line 531-536)\n● IPMKΔIEC mice have a marked increase in intestinal leaky pathway, allowing 4KD dextran\nto pass through (Fig. 5D). However, a previous study by Park et.al (PMID: 35988719) found\nthat IPMKΔIEC mice intestinal epithelium has no critical defect in its barrier function. Could\nyou please comment?\nWe thank the reviewer for raising this important point. The genetic background of mouse\nmodels is known to critically influence phenotypic outcomes1,2. To ensure a stable genetic\nbackground, our IPMK floxed mice were backcrossed for more than 10 generations to the\nC57BL/6 strain, achieving around kore than 98.7% congenic background (tested using Mini\nMouse Universal Genotyping Array by transnetyx, data enclosed [Extended Fig 7F]). This\nmeets the standard recommendation for minimizing phenotypic variability. In contrast, the\nstudy by Park et al.3 utilized mice that were backcrossed for only five generations on a\nmixed C57BL/6-129S background, as quoted “Mice were backcrossed to C57BL/6J for at\nleast 5 generations” In the Materials and Methods section under the subheading Mice, Line\nnumber 7. We attribute the discrepancy in our findings to this key difference. The mixed\ngenetic background in the Park et al. mice likely introduced modifier genes that altered the\nphenotype, a well-documented phenomenon in mouse genetics. Our use of a nearly\ncongenic strain provides a more controlled and reliable model.\n● In the DSS-induced leaky gut model, IMPK protein level was almost abolished after DSS\ntreatment. How did IMPK level change when mice were fed with InsP6? It’s been show\nphytate protect gut from DSS-induced colitis (PMID: 36341915).Therefore, it is possible IP6\nprevent DSS caused cell damage via MMP-independent pathway, especially if IPMK\nprotein kept intact after InsP6 supplementation. Also, InsPs levels of colon tissues in IP6\nfed mice should recorded by CE-MSsince the authors have the capacity.\n● Our data; (Extended Fig. 5F) show that InsP6 supplementation suppresses histone\nacetylation (Fig 5 L) and MMP expression (Fig. 5G) without restoring IPMK protein (Fig. 5L)\n(Line 497-504, 510-517). In intestine-specific IPMK knockout mice and in DSS-treated wild-\ntype C57BL/6 mice, InsP6 did not rescue IPMK protein levels (Fig 5L and Extended Figure\nMMP promoters (Fig. 5K, 6H) (Lines 517 to 521 and 571 to 576), and repressed MMPs,\nconsistent with an epigenetic mechanism of action. Physiologically, IPMK loss reduces\nInsP6, diminishing HDAC3 activity, elevating MMPs, and promoting barrier dysfunction;\nwhile this likely contributes substantially to intestinal permeability, additional mechanisms\nmay also be involved and will be explored in future studies.\n● Also, InsPs levels of colon tissues in IP6 fed mice should recorded by CE-MS since the\nauthors have the capacity\n.\nAs suggested, we performed HOIP profiling using intestinal crypt samples, and mass\nspectrometric analysis showed that oral InsP6 administration increased crypt InsP6 levels\nin IPMKΔIEC mice. In untreated IPMKΔIEC mice, InsP5 and InsP6 levels were markedly\nreduced (Fig 7K), consistent with our cell-based observations (Fig 1B) and previous reports,\nindicating a deficiency in endogenously synthesized InsP5 and 6. Because IPMK deletion\ndisrupts de novo InsP5 and InsP6 synthesis, the elevated InsP6 and InsP5 levels observed\nfollowing oral InsP6 treatment likely arise from extracellular uptake. Incrsae in InsP5 level\nmay be influenced by compensatory activity of intracellular inositol phosphatases. Overall\nsupporting the idea of exogenous InsP6 entry into IECs.\nMinor points from Reviewer’s 2:\n1. Fig 1F, HDAC3 protein level should be included.\nThank you for your kind suggestion. The HDAC3 protein level has been included in Figure\n1F.\n2. Why RGFP966 (10μM) treatment needs to last for 8 or 24h?\nAll experiments involving RGFP966 treatment were performed using a 10 μM concentration\nwith a 24-hour incubation period. This dosage was selected based on previously published\nstudies.⁴,⁵.\n3. Extended Fig. 2C is missing.\nThank you for the comment. We have now added a new Extended Figure 2C, which was\nnot included in the previous version of the manuscript.\nReviewer 3:\nMajor\nComment 1:\n● The authors only include observations of the impact of InsP6 after 24hrs of DSS\ntreatment. If the DSS model were to be extended, how would InsP6 treatment affect\ndisease outcomes or wound healing?\n● Thank you to the reviewer for this helpful question, which led us to evaluate the healing\neffects of InsP6 across a full 7-day course of 2% DSS. As shown in Figure 7 B and\nExtended Figure 7A ,B, oral InsP6 co-treatment reduced colonic injury and lowered\ninflammation (myeloperoxidase assay for neutrophil infiltration (Figure 7 C)). We also\nassessed IL-1β maturation in tissue, which drives chronic inflammation, and found that\nInsP6 co-treatment (Figure 7D) reduced tissue IL-1β level. Overall, the data indicate that\noral InsP6 attenuates DSS-induced cell death and inflammation.(Line 584-617)\n● It has been shown that HDAC3 deficient mice are sensitive to DSS colits, do IPMK-\ndeficient mice show the same phenotype, and would treatment with InsP6 improve\ndisease outcomes in these mice?\n● As the reviewer suggested, we treated intestine-specific IPMK knockout (IPMKΔIEC) mice\nwith 2% DSS for five days. This led to increased inflammatory markers such as MPO\nlevels, and tissue-level IL-1β maturation, which worsens DSS-induced colitis in intestine-\nspecific IPMKΔIEC mice compared to IPMK floxed mice (Fig 7F-J). Notably, oral\nadministration of InsP6 effectively rescued these DSS-mediated effects in the intestine-\nspecific IPMK KO mice (Fig 7F-J).\n● The authors should consider investigating other tight junctional proteins associated with\nleaky gut, such as claudin-2. In extended figure 5a the authors show a modest decrease\nin ZO-1 expression. ZO-1 is more often associated with barrier integrity and not leaky\ngut.- The authors should consider extending this ZO-1 observation to their in vivo models.\nDoes loss of IPMK impact the expression of zo-1/claudin in vivo as well.\n● As suggested by the reviewer, we performed biochemical and imaging analyses in cell-\nbased assays (Extended Fig 5IA) and intact intestinal tissue (Fig 5 G-J) and (Extended\nFig 5 II D,E,F). We found that IPMK deletion reduces ZO-1 and occludin levels, both in\nimaging and biochemical data. However, we did not find a loss of Claudin 2; instead, we\nobserved an increase in Claudin 2 (Fig 5G).\n● Given that all in vitro studies where done using cell permeable InsP6, the authors should\nconsider showing data from the murine tissue that non-cell permeable InsP6 is in fact\nabsorbed by the intestinal cells. The same analysis performed in Figure 1B could be\nperformed on tissue extracts, as well as, on fecal extracts to provide evidence that the\nInsP6 is being\ntaken up by the cells and also that it makes it to the colon.\n● As suggested, we performed HOIP profiling using intestinal crypt samples, and mass\nspectrometric analysis showed that oral InsP6 administration increased crypt InsP6\nlevels in IPMKΔIEC mice. In untreated IPMKΔIEC mice, InsP5 and InsP6 levels were markedly\nreduced (Fig 7K), consistent with our cell-based observations (Fig 1B) and previous\nreports, indicating a deficiency in endogenously synthesized InsP5 and 6. Because IPMK\ndeletion disrupts de novo InsP5 and InsP6 synthesis, the elevated InsP6 and InsP5 levels\nobserved following oral InsP6 treatment likely arise from extracellular uptake. Incrsae in\nInsP5 level may be influenced by compensatory activity of intracellular inositol\nphosphatases. Overall supporting the idea of exogenous InsP6 entry into IECs.\n● It would add to the manuscript if the authors were to assay the expression of MMPs in\nthe supernatants transferred to the caco2 cells.\n● As suggested by the reviewers, we concentrated the conditioned media from IPMK wild-\ntype and knockout cells, and subsequent Western blot analysis revealed the presence of\nsecreted MMPs exclusively in the conditioned media from IPMK knockout cells\n(Extended Fig. 2A).\nMinor Points:\n- In the results section there were several instances of Figures being called out in the text\nthat refered to the incorrect figure, specifically Line 263, line 442\nThank you for the valuable comment. As suggested, we rechecked and revised the\nmanuscript and corrected it.\n- It is unclear from the figure legends, methods, or text on how many mice were utilized in\neach experiment.\nThank you for your comment. As suggested, the total number of mice used in each\nexperiment (n) has been added to the corresponding figure legends (Lines 1661–1707,\n1721–1734, 1744–1770, and 1771–1777).\n- Can you also clarify what portion of the colon was collected for the IF images and the\ncrypt isolation?\nThank you for your valuable comment. For biochemical analyses, crypts were isolated\nfrom the mid-to-distal region of the colon. Tissue samples used for histological studies\nwere also collected from the same region to maintain consistency.\nDSS is known to primarily impact the distal colon, so it is unclear why the crypts from the\nileum were used in Figure 5 instead of crypts from the colon.\nThank you for this insightful comment. We acknowledge that DSS primarily affects the\ndistal colon. However, previous GWAS studies have identified IPMK as a risk gene for\ninflammatory bowel disease (IBD)6, including both Crohn’s disease7,9and ulcerative colitis8.\nSince Crohn’s disease primarily impacts the small intestine and ulcerative colitis targets\nthe colon, our goal was to evaluate the role of IPMK in both intestinal regions. Although\nRNA sequencing was performed on FACS-purified ileul epithelial cells, we extended and\nvalidated our RNA seq data and other biochemical and IHC analyses to include crypts\nisolated from both the ileum (Fig. 5G, H, K, and L) and the colon (Fig. 5I, J, and Extended\nFig. 5G–I). Western blot and immunohistochemistry results demonstrated that MMP3 and\nMMP13 levels were markedly increased, while ZO-1 and occludin expression were reduced\nin both ileum and colon. These findings are consistent with our observations in human\npatient samples (Fig. 5O–R), supporting a conserved role of IPMK in maintaining intestinal\nepithelial integrity across both the ileum and colon.\nReferences:\n1.Yoshiki A, Moriwaki K. Mouse phenome research: implications of genetic background.\nILAR J. 2006;47(2):94-102. doi: 10.1093/ilar.47.2.94. PMID: 16547366.\n2.Montagutelli X. Effect of the genetic background on the phenotype of mouse mutations. J\nAm Soc Nephrol. 2000 Nov;11 Suppl 16:S101-5. PMID: 11065339.\n3.Park SE, Lee D, Jeong JW, Lee SH, Park SJ, Ryu J, Oh SK, Yang H, Fang S, Kim S. Gut\nEpithelial Inositol Polyphosphate Multikinase Alleviates Experimental Colitis via Governing\nTuft Cell Homeostasis. Cell Mol Gastroenterol Hepatol. 2022;14(6):1235-1256. doi:\n10.1016/j.jcmgh.2022.08.004. Epub 2022 Aug 19. PMID: 35988719; PMCID:\nPMC9579329.\n4.Jang J, Song G, Pettit SM, Li Q, Song X, Cai CL, Kaushal S, Li D. Epicardial HDAC3\nPromotes Myocardial Growth Through a Novel MicroRNA Pathway. Circ Res. 2022 Jul\n8;131(2):151-164. doi: 10.1161/CIRCRESAHA.122.320785. Epub 2022 Jun 20. PMID:\n35722872; PMCID: PMC9308743.\n5.Stengel KR, Bhaskara S, Wang J, Liu Q, Ellis JD, Sampathi S, Hiebert SW. Histone\ndeacetylase 3 controls a transcriptional network required for B cell maturation. Nucleic Acids\nRes. 2019 Nov 18;47(20):10612-10627. doi: 10.1093/nar/gkz816. PMID: 31586401;\nPMCID: PMC6847391.\n6.McGovern DP, Kugathasan S, Cho JH. Genetics of Inflammatory Bowel Diseases.\nGastroenterology. 2015 Oct;149(5):1163-1176.e2. doi: 10.1053/j.gastro.2015.08.001. Epub\n7.Reilly L, Semenza ER, Koshkaryan G, Mishra S, Chatterjee S, Abramson E, Mishra P, Sei Y,\nWank SA, Donowitz M, Snyder SH, Guha P. Loss of PI3k activity of inositol polyphosphate\nmultikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal\nhomeostasis. iScience. 2023 Apr 11;26(5):106623. doi: 10.1016/j.isci.2023.106623. PMID:\n37216099; PMCID: PMC10197106.\n8.Park SE, Lee D, Jeong JW, Lee SH, Park SJ, Ryu J, Oh SK, Yang H, Fang S, Kim S. Gut\nEpithelial Inositol Polyphosphate Multikinase Alleviates Experimental Colitis via Governing\nTuft Cell Homeostasis. Cell Mol Gastroenterol Hepatol. 2022;14(6):1235-1256. doi:\n10.1016/j.jcmgh.2022.08.004. Epub 2022 Aug 19. PMID: 35988719; PMCID:\nPMC9579329.\n9.Yokoyama JS, Wang Y, Schork AJ, et al. Association Between Genetic Traits for Immune-\nMediated Diseases and Alzheimer Disease. JAMA Neurol. 2016;73(6):691-697.\ndoi:10.1001/jamaneurol.2016.0150"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all of my concerns completely, including the incorporation of\nadditional data and analyses that strengthen the overall findings by expanding on the\nhuman disease relevance and highlighted the strength in signal of the MMP/HDAC findings\nin their seq data.\nI will add that \"Collectively, these findings highlight the clinical relevance of our study in the\ncontext of IBD\n(Line 531-536, Figure 5 O-R)\" this line from their response to reviewers document, the lines\nof text they cite are incorrect but I found the writing. And in the Figure O,P,Q Crohn's is\nmisspelled in the figure.", "reviewer_2": "Chetterjee, et al. have addressed all my comments appropriately. The manuscript has been\nimproved significantly. I'm pleased with this revised version. Excellent work.", "reviewer_3": "The revised manuscript from Guha et al. has been greatly improved based on the reviewers\nsuggests. The authors have fully addressed all of the comments put forth. The new data is\nclear and convincing and have improved the overall manuscript. The review has no further\nconcerns."}, "author_response": "Response: We corrected as requested by the reviewer.\nResponse: We thank reviewer 2.\nResponse: We thank reviewer 3."}]

Decision letter