bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone Deacetylase 3 Supports Cystathionine β‐synthase Mediated Bone formation by Controlling Cytokine Signaling and Matrix Remodeling
Authors: Behera Jyotirmaya , Zhai Yuankun , Bala Jyoti , Tyagi Suresh C , Tyagi Neetu
Published: November 2023

Cystathionine beta‐synthase (CBS) is a crucial enzyme in homocysteine (Hcy) metabolism. CBS deficiency cause hyperhomocysteinemia (HHcy) which confers diverse clinical manifestations, notably characteristic skeletal abnormalities. However, several clinical trials demonstrated that despite reduction in Hcy levels, bone disease outcome remained unaffected, thus the mechanism of bone formation is poorly defined. Therefore, we hypothesize that CBS as a novel molecular regulator in osteoblasts which promotes bone development. To test this hypothesis, we used 8–12 weeks old mice in our study; wild type mice (C57BL/6, WT) and CBS+/− mice. Silencing CBS function in osteoblast by either inhibitor hydroxylamine (HA) or CRISPR/CAS‐9 mediated gene knockout in vitro and bone marrow derived CBS‐deficient (CBS+/−) osteoblast causes reduced osteoblast differentiation and mineralization and increased osteoclast activity. Bone marrow derived CBS‐deficient (CBS+/−) osteoblasts exhibited exaggerated oxidative stress mechanism and further increased the expression of inflammatory cytokine and matrix‐degrading genes (IL‐6, MMP‐9, MMP‐13) and reduced expression of genes related to extracellular matrix production, bone development (ALP, Col1A, RUNX2, OSTERIX, OCN, OPN). ELISA assay identified among others IL‐6 to be present in plasma of CBS(+/−) mice but not in plasma of wild type mice (C57BL/6, WT). Furthermore, histone deacetylase activity 3 also decreased under oxidative stress mechanism via phosphorylation dependent manner. This leads to activation and acetylation of nuclear factor Kb (NF‐kB). This increased inflammatory cytokine production further suppresses the osteoblast differentiation and paracrine activation of osteoclasts and bone resorption via IL‐6 signaling. This increased resorptive activity is further confirmed with immunostaining of tibia cross section by TRAP‐activity and increased osteoclasts number in bone tissue of CBS+/− mice. Blockade of HDAC3 activation in CBS‐deficient (CBS+/−) osteoblast by RGFP566 (HDAC3 Inhibitor) leads to the remodelling of histone landscapes in genome and thereby accelerates histone acetylation via recognising the acetylated lysine residue in histone (H3K27ac) of chromatin. This eventually resulted in transcriptional activation of MMP9,‐13 and IL‐6 gene expression. Thus, CBS controls the temporal and spatial expression of tissue‐remodeling genes and inflammatory responses in osteoblast to ensure proper bone development. In conclusion, we demonstrate that CBS control the osteoblasts balances, osteoblast differentiation/mineralization and osteoclastgenesis via HDAC3 dependent manner during bone development.

Support or Funding Information

This work is financial supported from National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledged.
Inflammatory Mediators during the Pre‐implantation Period Play a Key Role in the Pathogenesis of Preeclampsia in the Spontaneous Preeclamptic‐like BPH/5 Mouse
Authors: Sones Jenny Liford , Merriam Audrey A , Grenier Jen , Douglas Nataki A , Davisson Robin L
Published: November 2023

Preeclampsia (PE) is a hypertensive disease of pregnancy that affects up to 10% of pregnant women in the United States and is a leading cause of maternal and fetal morbidity/mortality. PE is responsible for 42% of maternal deaths worldwide annually. Despite the prevalence and severity of PE, the cause is unknown. The BPH/5 mouse model spontaneously phenocopies the maternal syndrome of PE, including late‐gestational hypertension and placental pathologies seen in preeclamptic women. Defective implantation is thought to cause ripple effects leading to abnormal placentation and poor pregnancy outcomes, including PE. The exact mechanism by which implantation defects contribute to the development of PE is not fully understood. Recent published data supports decidual cyclooxygenase 2 as a principle dysregulated factor during the periimplantation period in BPH/5 mice playing a causal role in maternal and fetal adverse pregnancy outcomes. Anti‐inflammatory therapy early in BPH/5 pregnancy at embryonic day (e) 6.5, ameliorated late‐gestational hypertension and fetal growth restriction. To further characterize the decidual inflammatory transcriptome in BPH/5 pregnant mice, molecular analyses were carried out in e7.5 implantation sites, as maximal decidual inflammation was observed at this time point, and e10.5 placenta. We hypothesized that inflammatory genes would be upregulated in BPH/5 at these critical pregnancy time points. RNA was isolated from BPH/5 and control C57 implantation sites at e7.5, and then subjected to RNA Sequencing and validation by qRT‐PCR. 484 differentially expressed genes were identified (≥2‐fold; n=4/group, p<0.05), of which 21 were inflammatory response genes. Interestingly, several of these genes were from the complement pathway. C3 gene expression was upregulated (20‐fold v C57, p<0.05) and C3 deposition at the decidual mesometrial pole, the site of future placenta development, was increased in BPH/5 mice at e7.5 (1.690e+008 ± 3.387e+007 v C57: 4.867e+007 ± 4.048e+006 corrected total cell fluorescence; n=6, p<0.05). Interestingly, C3 gene expression was persistently upregulated in BPH/5 placenta tissue at e10.5 (20‐fold v C57, p<0.05). These findings are of significance as BPH/5 and preeclamptic pregnancies are characterized by over activation of the innate immune system with an unknown etiology. Dysregulations in the early pregnancy decidual milieu may lead to aberrant trophoblast invasion and placenta formation in preeclamptic pregnancies before presentation of the maternal syndrome. Therefore, future studies involving determination of the local signals leading to complement activation and decidual inflammation in BPH/5 mice will greatly contribute to our understanding of the PE maternal syndrome and fetal growth restriction.
Bone Marrow Transfer from Rats Fed Low or High Salt Diets Does Not Influence Salt‐Sensitive Blood Pressure and Kidney Disease in Dahl SS Recipient Rats
Authors: Fehrenbach Daniel J , Petrova Galina , Lund Hayley , Abais‐Battad Justine M , Dasinger John Henry , Mattson David L
Published: November 2023

Increasing evidence indicates a role for the immune system in the development of hypertension and renal disease. Studies in our laboratory have demonstrated that pharmacological or genetic inhibition of adaptive immunity attenuates salt‐sensitive hypertension in the Dahl SS rat. Specifically, Dahl SS rats with a mutation in CD247 (SSCD247−/−), a gene encoding the CD3 zeta chain, lack CD3+ T‐cells and have a marked attenuation of salt‐sensitive hypertension and renal damage. The present study was designed to investigate the potential role of memory cells in the bone marrow in the development of salt‐sensitive disease. Age‐matched Dahl SS rats were kept on either a low salt (0.4% NaCl) or a high salt (4.0% NaCl) diet for 3 weeks starting at 3 weeks of age. Bone marrow from these SS rats was then transferred into age‐matched, SSCD247−/− rats (n=4 low salt bone marrow; n=5 high salt bone marrow) that underwent total body irradiation (TBI) at a dose of 11 Gy and a rate of 1.83 Gy/min. The recipient SSCD247−/− rats that underwent TBI had been maintained throughout life on the low salt diet and received bone marrow from SS rats fed the low (LSBM) or high salt (HSBM) diet. Any T cells reconstituted in the SSCD247−/− rats which underwent TBI therefore arose from the donor SS. After two weeks of recovery, the LSBM and HSBM rats were instrumented for blood pressure measurement by telemetry. While maintained on a low salt diet, there was no difference in the MAP or urine albumin excretion (Ualb) rate in the LSBM (108±2 mmHg and 0.8±0.3 mg/day) or HSBM rats (111±4mmHg and 2.4±1.6 mg/day). The rats were then switched to a high salt diet and followed for 7 days. When the rats were switched to a high salt diet, MAP and Ualb tended to increase compared to baseline in the LSBM group (127±5 mmHg and 41.4±13.5 mg/day) and MAP but not Ualb significantly increased in the HSBM group (129±6 mmHg and 60.9±27.7 mg/day), but there were no significant differences between groups after 7 days. A subsequent flow cytometry analysis demonstrated reconstitution of T‐lymphocyte (CD45+/CD3+), helper T cell (CD4+), cytotoxic T cell (CD8+), and T‐memory cell (CD44h) populations, but there were no significant differences between the two groups at this time in the any of the tissues collected (PBMCs, spleen, bone marrow, and kidneys). Though significant differences were not detected between groups in the time frame of this experiment, we plan to repeat this experiment for an extended high salt period as previous work has demonstrated the effects of immune cells on hypertension and renal damage in Dahl SS rats typically begins at approximately Day 14 of high salt.

Support or Funding Information

Supported by DK96859, HL116264, and 15SFRN2391002
Effects of Whole Milk and Full‐Fat Dairy Products on Blood Pressure in Patients with Elevated Blood Pressure
Authors: Roy Stephen , Baker Brett , LaPierre Stephanie , Geary Wesley , Delfausse Laura , Pasha Evan , Tanaka Hirofumi
Published: November 2023

We have previously demonstrated that the solitary addition of nonfat dairy products to the normal routine diet was capable of lowering blood pressure. Currently, it is not known if whole milk and full‐fat dairy products could exert similar effects as most of the available cohort studies reported no association between full‐fat dairy products and blood pressure. Therefore, the purpose of the study was to determine the efficacy of conventional full‐fat dairy products as part of the normal routine diet for reducing blood pressure in otherwise healthy adults with elevated blood pressure. After a 2‐week run‐in period, 35 subjects (13 men and 22 women) with pre‐ or stage I hypertension underwent a randomized controlled cross‐over dietary intervention trial encompassing 4 servings of dairy (whole milk, cheese, or yogurt) per day and 4 weeks of an isocaloric non‐dairy control (coconut milk, orange juice, peanuts, and applesauce) per day. All the dairy products as well as isocaloric non‐dairy products were provided in daily packages by the investigators. Two weeks of washout period was inserted between the dietary conditions. Body weight did not change throughout the experimental period. Casual (office) systolic blood pressure decreased slightly but significantly in both the dairy (135±1 vs. 131±2 mmHg) and the control (136±2 vs.133±2 mmHg) conditions. There were no significant changes in casual diastolic or mean blood pressure. Ambulatory (24 hour) systolic blood pressure did not change significantly in both dairy (131±2 vs. 130±2 mmHg) and control (131±3 vs. 129±2 mmHg) conditions. Similarly, daytime and nighttime blood pressure did not change significantly in both conditions. These data suggest that unlike non‐fat milk and non‐fat dairy products, whole milk and full‐fat dairy products added to the normal routine diet did not exert hypotensive effects in patients with elevated blood pressure.

Support or Funding Information

Supported by the Dairy Research Institute
Exercise and Dairy Protein Interactions in the Treatment of Obesity
Authors: Trottier Sarah
Published: November 2023

The purpose of this investigation was to compare the individual and combined effects of dairy protein and endurance exercise training in reducing weight gain and adiposity in a rodent model of diet‐induced obesity. A 6‐week feeding intervention of high fat, high sucrose (HFHS) diet was used to rapidly induce obesity and to impair glucose homeostasis in male Sprague Dawley rats (6‐weeks of age). Rats were then randomized to one of four weight matched, isocaloric groups (4.6 kcal/gram) for 6 weeks, where the dairy protein source was supplemented in the form of non‐fat skim milk powder: 1) HFHS‐casein, no treatment, 2) HFHS‐casein, exercise 3) HFHS, dairy protein and 4) HFHS, dairy protein, exercise. The exercise training intervention took place 5 days/week followed by a 2‐day recovery period (60 minutes of treadmill running: 20m/min, 10% incline). The effects of the exercise training along with the dairy protein were greater than either intervention alone in attenuating increases in body weight, along with epididymal and inguinal adipose tissue mass, while no significant differences were observed in food intake, total energy expenditure (TEE) and respiratory exchange ratio (RER) amongst all four treatment groups. The total weight of feces excreted over a 36‐hour period was higher in the dairy group, and this was associated with an increase in the total amount of lipid excreted in the HFHS dairy when compared to the casein group. This study provides new evidence that dairy protein is as effective as exercise training in preventing further weight gain in obese rats. While exercise training reduces weight gain through increases in energy expenditure with each bout of exercise, dairy protein would appear to reduce the amount of lipid that is absorbed.
Mesenchymal Stem Cells Derived Exosomes Promotes Bone Regeneration through Cystathionine‐β‐synthase Enhanced Angiogenesis and Osteogenesis during Bone Development
Authors: Behera Jyotirmaya , Zhai Yuankun , Bala Jyoti , Tyagi Suresh C , Tyagi Neetu
Published: November 2023

It is critical that bone formation and angiogenesis are tightly coordinated during bone development and fracture healing, but the molecular regulator of such intercellular communication in bone microenvironment are not well studied. Cystathionine‐β‐synthase (CBS), an enzyme in homocysteine (Hcy) metabolic pathway play crucial role in osteoblast differentiation. Therefore, we hypothesize that CBS as a novel molecular regulator in osteoblasts, which regulates vascularization during bone development in osteoblast derived exosome. To test this hypothesis, we have used 7–10 weeks old male mice in our study; wild type mice (C57BL/6, WT) and CBS+/− mice. Blocking the CBS function was done by either inhibitor hydroxylamine (HA) or SiRNA prevented osteoblast differentiation and mineralization. This was supported by studies using osteoblasts cultured from bone marrow of CBS deficient (CBS+/−) mice. Exosomes of mesenchymal stem cell derived osteoblasts cultured stimulated endothelial migration and angiogenesis, which was prevented by blocking CBS in osteoblasts using HA or SiRNA. Mass spectrometry analysis and ELISA assay identified among others vascular endothelial growth factor (VEGF)‐A and VEGF homolog placental growth factor (PIGF) to be present in control osteoblasts culture medium and CBS(+/+) mice osteoblasts but not in osteoblasts treated with HA and CBS+/− osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of CBS+/− mice. However, osteopenia phenotype was confirmed in CBS+/− mice by microCT analysis. CBS+/− mice showed significantly reduced bone mineral density (BMD) and bone volume/tissue volume (BV/TV) and showed increased plasma Hcy levels compared to CBC+/+ mice (WT). However, CBS+/− mice exhibited a more significant increase in the number of TRAP‐positive osteoclasts and higher levels of RANKL than CBS+/+ mice. This suggests that Hcy levels may be associated with osteoclast activity. Furthermore, real‐time PCR and western blot analyses revealed significant decreases Alkaline phosphatase (ALP) and Runt‐related transcription factor 2 (RUNX2) expression in CBS‐deficient (CBS+/−) mouse bone marrow cells. In conclusion, we demonstrate that CBS in osteoblasts is at the crossroad of osteoblast differentiation/mineralization and angiogenesis. These findings uncover the previously undefined molecular understanding of CBS that promote angiogenesis and osteogenesis in bone development and fracture repair.

Support or Funding Information

This work is financial supported from National Institute of health grant AR‐067667 and HL‐107640‐NT are greatly acknowledged.
Serum Adipokines and Metabolic Syndrome Risk Factors in Hispanic Children
Authors: Peterson Jonathan M , Clark William Andrew , Marrs Jo‐Ann , Alamian Arsham
Published: November 2023

Childhood obesity is a relatively new but growing epidemic in the Unites States, with 1 in 5 children currently classified as obese. Obese children are at a significantly higher risk for developing many types of cancers, cardiovascular disease, diabetes and Metabolic Syndrome (MetS). This project is focused on understanding the associations between adipose tissue health and adipose tissue‐derived secreted factors/hormones/cytokines (hereafter referred to as adipokines) and obesity and MetS in young children. Adiponectin, Leptin, and C1q TNF Related Protein 3 (CTRP3) are all adipokines which have been linked to human health, but have not been explored in children with MetS.

METHODS

Anthropomorphic measurements and blood collections were performed at a well child visit for Hispanic children aged 2–10 years. Lipid panel was completed at a clinical laboratory facility. Multiplex analysis (Bio‐Plex Multiplex System) was used to measure adiponectin, insulin, and leptin level. Circulating CTRP3 levels were determined by ELISA (enzyme‐linked immunosorbent assay). Risk factors for MetS were established as follows: abdominal obesity, elevated blood pressure, elevated triglycerides, low HDL and elevated plasma glucose concentrations. Pearson correlation coefficients were calculated for continuous variables and Spearman rank‐order correlation was performed for categorical variables to evaluate associations between adipokines and risk factors for MetS.

RESULTS

Briefly, 44 of 114 Hispanic children had 2 or more risk factors for the development of MetS. The association between Adiponectin levels, glucose, insulin, total triglyceride and Body Mass Index (BMI) trended towards significance (P>0.08), whereas leptin was significantly positively associated with BMI. CTRP3 levels were not associated with any individual measure variable, but were negatively associated with the presence of 2 or more risks factors for MetS.

CONCLUSION

Adipose tissue secretes many bioactive molecules that circulate in blood, and dysregulation of adipose tissue endocrine function may promote the occurrence of MetS. The data presented in this project demonstrate the association between adipokines and MetS.

Support or Funding Information

This research was supported in part National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612, Tennessee Board of Regents Diversity Research Grant (2014–2015), and by the East Tennessee State University Research Development Committee (E82262).
TNFα exposure induces neuroinflammation and insulin resistance in a rat‐derived hypothalamic cell model, rHypoE‐7
Authors: Clemenzi Matthew N , Aljghami Makram E , Wellhauser Leigh , Belsham Denise D
Published: November 2023

Obesity is approaching epidemic levels. One of the main characteristics of obesity is the development of insulin resistance, leading to other comorbidities, such as type 2 diabetes mellitus (T2DM) and cardiovascular disease. Insulin resistance has been shown to be induced with obesity in peripheral tissues, such as liver, muscle, and adipocytes, by the pro‐inflammatory cytokine tumor necrosis factor alpha (TNFα). In the hypothalamus, the main brain region for energy regulation, neuroinflammation‐induced insulin resistance has been demonstrated to occur upon prolonged treatment with high levels of insulin and palmitate, and we are exploring whether TNFα, a downstream surrogate for palmitate, has similar effects. We hypothesized that TNFα‐treated rat hypothalamic neurons would exhibit cellular neuroinflammation and insulin resistance as evidenced by increased expression of pro‐inflammatory genes and attenuated phosphorylation of Akt (pAkt) in the insulin signaling pathway. Using an immortalized rat hypothalamic cell line (rHypoE‐7), a well‐characterized NPY‐expressing neuronal line derived from male embryonic hypothalamii, we studied changes in pAkt and inflammatory gene marker mRNA expression after TNFα treatment by Western blot and quantitative real‐time PCR, respectively. A qPCR array of 84 inflammatory markers (receptors, chemokines, and cytokines) demonstrated an increase in the expression of a number of genes encoding inflammatory markers, including Tollip and Caspase‐1, upon exposure to 100 ng/mL TNFα for 4 h. Neurons pre‐exposed to TNFα (50 ng/mL) for 6 or 16 h exhibited a significant reduction in pAkt compared to control after insulin treatment. TNFα also significantly increased mRNA expression of TNF, IK‐κB, Tollip, Tnfrsf1a, and IL6 at 4 h. In conclusion, pre‐treatment with the inflammatory cytokine TNFα causes cellular insulin resistance in the rHypoE‐7 neurons, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin‐ and palmitate‐induced insulin resistance in hypothalamic neurons. Further, these results demonstrate that components of the IKK‐β/NF‐κB pathway appear to be activated upon exposure to TNFα, providing a putative target to reverse neuroinflammation and insulin resistance with anti‐inflammatory agents.

Support or Funding Information

This research was supported by grants from the CIHR, CRC, CFI, NSERC, APS and BBDC.
PREFERENTIAL RENAL SYMPATHETIC HYPERACTIVITY IN EXPERIMENTAL DIABETES MELLITUS
Authors: Oliveira Tales L , Shimoura Caroline G , Lincevicius Gisele S , Garcia Michelle L , Boim Mirian A , Gomes Guiomar N , Bergamaschi Cassia T , Campos Ruy R
Published: November 2023

Autonomic, cardiovascular and renal dysfunction has been observed in diabetic patients and animal models of diabetes mellitus (DM), including the streptozotocin‐induced type 1 diabetes model. The aim of this study was to evaluate the effects of the experimental DM on efferent post‐ganglionic sympathetic nerve activity, arterial baroreceptor reflex sensitivity to heart rate (HR), renal (rSNA) and lumbar (lSNA) sympathetic nerves in DM rats. The DM was induced by Streptozotocin (60 mg/kg, ip) 4 weeks before experimental analysis. Male Wistar rats (n=6–8 per group), 8 weeks of age, 230–250 g of body weight were used. The animals were separated into control (CTR) and diabetic (DM) groups. Protocol CEUA/UNIFESP 1787250714. The results were expressed as mean ± SEM and compared with t‐Student test (p<0.05). DM was confirmed by the reduction in body weight (CTR: 320.1±6.3; DM: 242.1±6.7 g, p<0.05) and significantly increased in blood glucose (CTR: 100.3±3.7; DM: 509.1±14.4 mg/dL, p<0.05) in the DM group. A significant increase in feed intake (CTR: 75.8±3; DM: 158.3±6.4 g/kg), water intake (CTR: 98±4.3; DM: 608±31,3 ml/kg), and urinary flow in the DM rats evaluated for 24h (CTR: 40±3.1; DM: 562±25,1 ml/kg) was found. Sodium urinary excretion (CTR: 5.1±0.5; DM: 17.4±1.2 mmol/kg), potassium (CTR: 16±0.9; DM: 36.4±1 mEq/kg), glucose (CTR: 12±1.4; DM: 2719±192 mg/kg), and albumin urinary excretion (CTR: 82.3±8; DM: 394.3±42.7 mg/kg) were significantly increased in DM group. In addition, sodium (CTR: 123±1.7; DM: 141.4±1.6 mmol/L) and potassium plasma concentration (CTR: 4.4±0.1; DM: 5.2±0.1 mEq/L) were significantly increased in DM rats compared to control group. The creatinine clearance was reduced in the DM rats (CTR: 1.4±0.1; DM: 1±0.1 mL/min). DM reduced heart rate (HR) (CTR: 352.6±013.5; DM: 292.2±11 bpm, p<0.05), but did not change basal values of blood pressure (BP) compared to control group. rSNA was significantly increased (CTR: 85.5±3.1; DM: 133.5±2.5 pps) but lSNA did not change in DM rats. The arterial baroceptor function was significantly decreased to rSNA, (CTR: 1.3±0.1; DM: 0.9±0.1 pps/mmHg), lSNA (CTR: 1.4±0.1, DM: 0.8±0.1 pps/mmHg) and HR control (tachycardic response = CTR: −1.4 ± 0.1; DM: 0.8 ± 0.05 and bradycardic response = CTR: 1.3 ± 0.05; DM: 0.7 ± 0.03 bpm/mmHg, respectively) in the DM group compared to control rats. Taken altogether, the results suggest that autonomic dysfunction, characterized by impaired baroreceptor function, preferential increasein renal sympathetic nerve activity associated with impaired renal function are involved in the development and/or maintenance of experimental DM. Whether the renal sympathoexcitation in DM is a cause or a consequence of renal dysfunction remains to be determined.

Support or Funding Information

CAPES and FAPESP.
Leptin Activates Transient Receptor Potential Melastatin 7 (TRPM7) Channels in Leptin‐Receptor Expressing Pheochromocytoma (PC12) Cells
Authors: Liu Xiao‐Ru , Paudel Omkar , Shin Mi‐Kyung , Tang Wan‐Yee , Shirahata Machiko , Polotsky Vsevolod Y. , Sham James S.K.
Published: November 2023

Obesity is a leading cause of high cardiovascular morbidity and mortality. It is characterized by an increased leptin level, which has been implicated in increased sympathetic activity and the pathogenesis of hypertension. However, the mechanism by which leptin enhances sympathetic activity is unclear. Leptin receptors (LEPRs) are expressed in the carotid bodies, the most important arterial chemoreceptors for the cardiovascular‐respiratory chemoreflex. We have previously demonstrated that leptin enhances carotid sinus nerve activity in response to hypoxia; this effect is abolished by non‐selective blockers of transient receptor potential (TRP) channels; and the TRPM7 channel is the most abundant TRP subtype expressed in CB (Shirahata et al., Adv Exp Med Biol. 2015;860:153–9). Since leptin is known to regulate many different ion channels, we hypothesize that leptin may exert its effects through modulation of TRPM7 channel activity. To test this hypothesis, we used LEPR (LEPRb) expressing pheochromocytoma (PC12LEPRb) cells, which express a high level of TRPM7 channels, as the cell model. Non‐selective cation channels were recorded under amphotericin‐B perforated‐patch techniques with K+ replaced by Cs+ in the presence of voltage‐gated Ca2+ channel and Cl− channel blockers. Voltage‐ramp from −100 to 100 mV activated an outward‐rectifying current. Removal of extracellular divalent ions enhanced the inward current, consistent with the inward divalent ion selectivity of TRPM7. The specific TRPM7 agonist naltriben caused significant increase of the current, which was abolished by the TRPM7 antagonist NS8593 and FTY720, indicating that functional TRPM7 channels are present in PC12LEPRb cells. More importantly, leptin at concentration of 10–100 ng/ml caused concentration‐dependent increase in the outward rectifying current. The enhanced current was completely blocked by NS8593 and FTY720. These results for the first time demonstrated that stimulation of leptin receptor is capable of activating TRPM7 channels, and suggest that leptin may exert its physiological effects through modulation of TRPM7 activity.

Support or Funding Information

This work is supported by the NIH grant HL133100
Regulation and role of ER‐Golgi contact sites
Authors: De Matteis Antonella , Venditti Rosella , Masone Maria Chiara , Rega Laura , Polishchuk Elena , Santoro Michele , Di Tullio Giuseppe , La Montagna Raffaele
Published: November 2023

Despite their early identification, there are still many unknowns regarding the functions and composition of ER‐TGN membrane contact sites (MCSs) due to the lack of efficient visualization methods at the optical microscopy level. We developed a FRET‐based strategy to follow the ER‐TGN MCSs and used it to run a High Content Screen to identify components and regulators of these structures. We identified, among other components, the VAPA and VAPB proteins as structural determinants for this class of contact sites. Intriguingly, the VAP proteins are not only scaffolding components of ER‐Golgi contact sites but also play an active role in the regulation of Golgi PI4P levels by interacting directly with the Sac1 phosphatase and positioning it at the ER‐TGN MCSs. By contrast, we found that proteins previously described to localize at other MCSs, such as the oxysterol binding proteins OSBP1 and ORP9, are dispensable for the maintenance of the ER‐TGN MCSs. Using a proteomics approach we identified the PH‐domain– containing protein FAPP1 as a novel partner for Sac1 and VAPs at the ER‐Golgi MCSs. We show that FAPP1 depletion results in an increase in PI4P at the Golgi complex, particularly at the trans Golgi network (TGN). Thus FAPP1 acts as a PI4P sensor at the TGN activating the Sac1 phosphatase at the ER‐TGN MCSs.
Molecular probes to study the subcellular localization and dynamics of phospholipids and cholesterol
Authors: Fairn Gregory D.
Published: November 2023

Cellular lipids are critical components of the membrane bilayers that delineate individual organelles and separate the cell from its environment. Additionally, many classes of lipids serve important roles in signal transduction and vesicular transport. To complement traditional biochemical techniques used to quantify individual classes and species of lipids, researchers have developed a number of genetically encoded probes and antibodies to investigate the subcellular localization and dynamics of lipids of interest. This presentation will highlight the use of specific probes to study the inhomogeneous distribution and dynamics of phospholipids and cholesterol within the cell and during the process of phagocytosis in macrophage.

Support or Funding Information

Supported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Government of Ontario Early Researcher Award, and the Canadian Consortium on Neurodegeneration in Aging.
Cryptosporidium parvum infection disrupts intestinal epithelial barrier function via decreasing expression of specific tight junction and adherens junction proteins
Authors: KUMAR ANOOP , Chaterjee Ishita , Anbazhagan Arivarasu Natarajan , Priyamvada Shubha , Jayawardena Dulari , Alrefai Waddah , Borthakur Alip , Dudeja Pradeep Kumar
Published: November 2023

Background

Enteric infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrheal disease. CP sporozoites released in the lumen invade epithelial cells of the small intestine and complete the infectious cycle therein. However, mechanisms of CP infection‐induced diarrhea are not fully understood. Decreased barrier function and increased epithelial permeability are known to contribute to the pathophysiology of diarrheal diseases caused by enteric infections. Intestinal epithelial barrier integrity is maintained by tight junctions (TJ) and adherens junctions (AJ). However, there are virtually no studies on CP effects on the expression of TJ and AJ proteins and their relevance to cryptosporidiosis.

Aim

The current studies were performed to examine the effects of CP infection on paracellular permeability and on the expression of occludin and claudins (key proteins of TJ assembly), and E‐cadherin (a major AJ protein) utilizing Caco‐2 cell monolayers, ex vivo mouse enteroids and in vivo mouse model.

Methods

Transepithelial resistance (TER) was measured by a Volt Ohm Meter, paracellular permeability was measured by apical to basolateral flux of FITC‐dextran, mRNA and protein levels of the TJ/AJ components were measured by real‐time RT‐PCR and immunoblotting/immunofluorescence, respectively.

Results

CP infection (0.5 × 106 oocysts/well) for 24 h decreased TER and increased paracellular permeability in Caco‐2 cell monolayers. CP infection significantly decreased protein levels of occludin, claudin‐4 and E‐cadherin with no effects on other claudins in Caco‐2 monolayers, mouse‐enteroid‐derived monolayers, and in the mucosa of ileum and jejunum in C57BL/6 mice (infected with 1.0 × 107 oocysts/mouse for 24 hr). mRNA levels of these three proteins were also significantly decreased in CP infected mouse ileum and jejunum, however, CP infection of Caco‐2 cells did not alter occludin and claudin‐4 mRNA levels. Further, bafilomycin, an inhibitor of lysosomal proton pump, partially abrogated CP‐induced downregulation of occludin expression, suggesting a potential role of post‐translational mechanisms such as protein degradation in CP‐induced downregulation of occludin in Caco‐2 cells.

Conclusion

Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could contribute to CP infection‐induced diarrhea.

Support or Funding Information

Supported by NIDDK and Department of Veterans Affairs
An Inherent Defect in Tight Junction Structure and Permeability is Apparent in Colonoids from Crohn's Disease Patients
Authors: Gadeock Safina , Schultz Michael , Butt Grant
Published: November 2023

An increase in intestinal permeability is associated with inflammatory bowel disease (IBD). However, whether the increased permeability is a cause or consequence of the inflammation is unknown. Here we have used epithelial derived colonoids from healthy individuals and Crohn's (CD) patients to determine whether there is an inherent defect in intestinal epithelial permeability associated with CD. Colonoids were grown for 15 days from biopsies from the transverse colon of healthy patients with no known history of IBD and non‐inflamed regions of the transverse colon of CD patients. The epithelial structure was characterised with both conventional and electron microscopy, and the expression of the tight junction proteins occludin, ZO‐1 and claudin 2, quantified with qPCR and western blot. The distribution of the proteins was determined with immunohistochemistry, while the relative permeability of the colonoids was measured by adding 0.1 mg ml−1 of FITC labelled dextran 4000 (FD4) to the growth media for 24 h and then measuring the fluorescence intensity in the organoid lumen by confocal microscopy. Surface area and volume of the organoids was calculated from the measured radius to allow calculation of the FD4 flux (μg cm−2 24 h−1). Macroscopically there were no obvious differences between the control and CD colonoids but, although the epithelium in the CD colonoids consisted of columnar epithelial cells with basal nuclei, the cells in the CD colonoids had extensive apical protrusions and ≈20% of the CD colonoids were inside out. Comparison of the control and CD colonoids indicated there was no difference in the levels of expression of either transcript or protein of occludin, ZO1 and claudin 2. There was, however, a marked redistribution of the tight junction proteins in the CD colonoids. In the control colonoids, ZO‐1 and occludin were concentrated at the apical pole of the epithelial cells, whereas in the CD colonoids they were located in the basolateral membrane and cytoplasm. In contrast, in control colonoids claudin 2 was located in the basolateral membrane, whereas in the CD colonoids it was concentrated at the apical pole. Associated with this redistribution of the tight junction proteins was a marked increase (P<0.001) in the FD4 flux from 0.071±0.07 to 0.425±0.081 μg cm−2 24 h−1 in control (80 colonoids from 5 patients) and CD (98 colonoids from 5 patients) colonoids, respectively. Collectively, these data suggest that an inherent defect develops in the CD organoids independent of inflammatory or bacterial effects. This supports the proposal that in vivo an increased permeability contributes to the development of Crohn's disease.

Support or Funding Information

Supported by a University of Otago Research Grant and Grants from the Department of Physiology and the Dean's Funds, Otago School of Biomedical Sciences and Dunedin School of Medicine
Claudin‐4 Disrupts Claudin‐2 Anchoring and Pore Function via Cis, but not Trans, Interactions at the Tight Junction
Authors: France Marion , Turner Jerrold
Published: November 2023

Tight junction strands are composed of claudin (cldn) proteins. The consensus view is that cldns either form pores that mediate paracellular flux or form seals that prevent such flux. An alternative view is that cldns that reduce paracellular flux do so by competitively disrupting pore‐forming cldn function. Both trans‐ and cis‐interactions between similar (homotypic) and dissimilar (heterotypic) cldns have been implicated in strand assembly and anchoring at the tight junction. We sought to test the cldn competition model and define the topology of cldn‐cldn interactions involved in cldn2 anchoring. mEGFP‐cldn‐2 expression in monolayers of high resistance MDCK I cells, which lack endogenous cldn2, reduced transepithelial electrical resistance (TER) by 38%±3%. Conversely, mCh‐cldn4 expression increased TER of mEGFP‐cldn‐2 expressing monolayers. These data indicate that cldn4 can oppose cldn2‐dependent paracellular pore function, but do not distinguish between pore‐seal and competition models of cldn function. The latter is, however, favored by the observation that cldn4 knockout does not significantly affect TER of MDCK I monolayers. Fluorescence recovery after photobleaching (FRAP) analyses demonstrated an mEGFP‐cldn2 mobile fraction (Mf) of 18%±3%, while mCherry‐cldn4 displayed a much higher Mf of 44%±4%. When mCherry‐cldn4 was expressed with mEGFP‐cldn2 at symmetric tight junctions, mEGFP‐cldn2 FRAP increased markedly, with an overall Mf of 35%±3%. This suggests that cldn4 increases TER by destabilizing cldn2 at the tight junction. Cldn2 stabilization at the tight junction did not require cldn2‐cldn2 trans‐interactions, as the mEGFP‐cldn2 Mf was similar at asymmetric junctions between cells expressing mEGFP‐cldn2 and non‐transfected MDCK I cells (23%±4%). Further, cldn4‐mediated cldn2 destabilization was not dependent on cldn4‐cldn2 trans‐interactions as demonstrated by similar mEGFP‐cldn2 Mf at asymmetric junctions between cells expressing mEGFP‐cldn2 and those expressing mCherry‐cldn4 (25%±3%). Cldn4 therefore modifies cldn2 anchoring at the tight junction via cis‐interactions within individual cells. These data support the cldn competition model and suggest that cldn4 increases TER by disrupting cldn2‐cldn2 cis‐interactions that are required for anchoring and pore function.

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DK061931
TFF2, CXCR4 and EGF‐R mediated gastric wound repair in vitro in gastric organoids
Authors: Engevik Kristen , Aihara Eitaro , Matthis Andrea , Montrose Marshall
Published: November 2023

Background

Trefoil factor 2 (TFF2), a secreted peptide found in the stomach mucosa, is known to play an important role in promoting gastric epithelial repair following damage. We previously demonstrated in vivo that TFF2 acts via the chemokine C‐X‐C receptor 4 (CXCR4). Due to the complexity of dissecting molecular pathways in vivo, the mechanisms of TFF2 action remain unclear. Epidermal growth factor receptor (EGF‐R) is implicated in repair and has been shown to be involved in the TFF2 pathway outside of the gastrointestinal tract. Utilizing the 3D primary culture of gastric epithelial cells known as gastric organoids, which mimic in vivo gastric epithelium, we tested the role of EGF‐R and TFF2‐driven restitution. We tested the hypothesis that TFF2 activates CXCR4 and transactivates EGF‐R to promote gastric restitution.

Methods

Gastric organoids were generated from isolated corpus tissue of wild‐type (WT) and TFF2 knockout (TFF2−/−) mice. Gastric organoids were cultured for 4–5 days prior to experiments. Damage was induced in gastric organoids by 5s exposure of single epithelial cells to high intensity two‐photon 720 nm light (photodamage), resulting in damage to the targeted cell and subsequent cell death. Progression of damage and repair was evaluated using confocal/2‐photon microscopy, imaging cell nuclei (10 μg/ml Hoechst33342 vital DNA stain) and measuring the intensity of Lucifer yellow ([LY] 20 μM added to culture medium) in the luminal side and extracellular space outside of the organoid.

Results

Unperturbed organoids maintain the integrity of the epithelial barrier and show no sign of basal cell migration. Photodamage injury in WT organoids resulted in the simultaneous events of dead cell exfoliation and migration of undamaged neighboring cells to restore a continuous epithelium in the damaged site. To assess repair of damage, we measured the rate of cell exfoliation (μm/min) as determined by nuclear (Hoechst) and fluorescence (LY) confocal images. Photodamaged TFF2−/− gastric organoids showed slowed repair of damage (0.09 ± 0.02 μm/min, n=5) compared to WT organoids (0.5 ± 0.045 μm/min, n=5). Addition of 1 μg/mL AMD3100 (CXCR4 inhibitor) to WT organoids similarly slowed repair (0.08 ± 0.01 μm/min, n=6), suggesting the presence of CXCR4 receptor in the gastric organoid model. Moreover, photodamage in WT organoids exposed to 200 nM AG1478 (EGF‐R inhibitor) resulted in a slower repair at 0.14 ± 0.03 μm/min (n=3).

Conclusion

Results demonstrate that TFF2, CXCR4 and EGF‐R are required for efficient epithelial restitution in gastric organoids. Outcomes suggest that gastric organoids can be a useful tool to elucidate cellular signaling mechanisms involved in epithelial repair of gastric damage.

Support or Funding Information

Supported by NIH R01 DK102551
Alteration of colonic motility by protease activated receptors in DSS‐induced colitis
Authors: Sung Tae Sik , Sanders Kenton , Koh Sang Don
Published: November 2023

The fact that 4.5% of all human genes encode proteases in the human genome reflects the importance of proteases in the human body under physiological and pathophysiological conditions. Proteases can trigger cell signaling pathways via activation of protease activated receptors (PARs). Activation PARs appear to play a significant role in GI inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, and GI motor dysfunction (leading to diarrhea or constipation). Although the role of PARs in GI mucosa and lamina propria has been reported, information of PARs expression and their pathological significance of PAR modulation in GI muscles layer are limited. Inflammation was induced by exposing to DSS for 7 days in C57BL/6 mice. Inflammatory colon showed bloody stool, erosion and inflammatory cell infiltration. PAR1 agonist‐induced initial relaxation was reduced in DSS‐treated colons. PAR2 agonist‐induced responses were not affected on DSS‐treated colitis. DSS‐treated colonic smooth muscle revealed the depolarized the resting membrane potentials and EFS‐induced purinergic inhibitory junction potentials which are mediated by activation of small conductance Ca2+‐activated K+ channels (SK) were also reduced in DSS‐colitis. Quantitative transcriptional analysis showed that expressions of Pdgra and Kcnn3 were reduced in DSS‐treated colons. Since PAR activation can be coupled to Ca2+ sensitization pathways, we examined the changes in protein expression of MYPT1 and CPI‐17. MYPT1 and CPI‐17 phosphorylation were increased in DSS‐treated colons. In conclusion, the alteration of motility by PAR1 is due to changes in Pdgfra and Kcnn3 with Ca2+‐sensitization pathways. These findings suggest a role for PARs in contributing to IBD associated functional dysmotility.

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P01‐DK41315
Transcriptional Regulation of SLC26A3 (DRA) by CDX2
Authors: Chatterjee Ishita , Kumar Anoop , Madrigal Rosa Mª Castilla , Pellon‐Cardenas Oscar , Borthakur Alip , Gill Ravinder , Alrefai Waddah , Verzi Michael , Dudeja Pradeep Kumar
Published: November 2023

Background

SLC26A3 (Down Regulated in Adenoma, DRA) plays a key role in mammalian intestinal NaCl absorption via mediating apical membrane Cl−/HCO3− exchange. DRA function and expression is significantly decreased in diarrheal diseases associated with infections and inflammatory bowel diseases (IBD). Since, DRA is now being touted as an important therapeutic target for infectious and IBD associated diarrhea, it is critical to understand the detailed mechanisms of regulation of DRA expression at the molecular and cellular level in health and disease. Earlier studies showed that, the intestinal epithelial caudal‐related homeobox transcription factor 2 (CDX2) is an essential regulator of intestinal epithelial homeostasis and is also involved in differentiation. Interestingly, DRA expression is higher in differentiated cells and in silico analysis of DRA promoter showed 2 consensus sites for CDX2 binding.

Aim

Current studies were, therefore, undertaken to test the hypothesis that CDX2 regulates DRA transcription.

Methods

Cdx2Fl/Fl;Villin‐CreERT2 mice, [where tamoxifen (TAM) deletes Cdx2 in intestine], were used for these studies and Caco2 cells were used as an in vitro model to study the overexpression and silencing effects of CDX2 by transfecting the cDNA and siRNA, respectively. mRNA and protein levels were assessed by real time RT‐PCR and immunoblotting/immuno‐staining, respectively. DRA promoter activity was determined by luciferase assay. Direct binding of CDX2 to DRA promoter region was assessed by chromatin immuno‐precipitation (CHIP) and electrophoretic mobility shift assay (EMSA).

Results

CDX2− /− mice showed marked reduction in DRA mRNA in ileum (Arbitrary units: WT=2.73 ± 1.04, KO=0.029 ± 0.02 (P<0.05), proximal colon (PC) WT=1.87 ± 0.53, KO=0.51 ± 0.08; P <0.05) and distal colon (DC) (WT=1.75 ± 0.46, KO=0.02 ±0.02, P<0.05) as well as in protein levels as compared to wild type mice. Similarly, silencing of CDX2 in Caco2 cells resulted in a marked (~ 50%) decrease in DRA mRNA and protein levels, whereas, ectopic over‐expression of CDX2 significantly up‐regulated DRA expression. CDX2 overexpression also stimulated DRA promoter activity by ~10‐fold, indicating its role in transcriptional regulation. We next utilized EMSA and ChIP assays to demonstrate direct binding of CDX2 to one putative cis elements identified in the 645 to 663 (+) region of the DRA promoter.

Conclusion

Our studies, for the first time, demonstrate a direct transcriptional regulation of DRA by CDX2. Given that CDX2 has been shown to be downregulated in UC (ulcerative colitis) and CD (crohn's disease) patients, CDX2 may at least partly be responsible for the observed decrease in DRA expression in IBD.

Support or Funding Information

Supported by NIDDK/Department of Veteran Affairs