bluuebunny/crossref_metadata_2025 huggingface dataset:
TARGETING TRAF6‐PAK1 SIGNALING LIMITS PROSTATE TUMOR GROWTH AND TGF&[Beta]1‐INDUCED PROSTATE CANCER CELL EPITHELIAL TO MESENCHYMAL TRANSITION
Authors: Somanath Payaningal R , Al‐Azayzih Ahmad , Gao Fei
Published: November 2023

Recent studies demonstrate the dual role of transforming growth factor beta (TGFβ) in prostate cancer. It is not clear how TGFβ1 that suppresses early prostate tumor growth induces epithelial‐to‐mesenchymal transition (EMT) in advanced stages. We studied if P21‐activated kinase1 (Pak1), a serine‐threonine kinase that mediates cytoskeletal remodeling is necessary for the TGFβ1 induced prostate cancer EMT. Effects of TGFβ1 on control prostate cancer PC3 and DU145 cells and those with Pak1 inhibitor IPA 3 and siRNA mediated Pak1 inhibition were tested for prostate tumor xenograft in vivo and EMT in vitro. TGFβ1 inhibited PC3 tumor xenograft growth via activation of P38‐MAPK and caspase‐3, 9. Long‐term stimulation with TGFβ1 induced PC3 and DU145 cell scattering and increased expression of EMT markers such as Snail and N‐cadherin through tumor necrosis factor receptor‐associated factor‐6 (TRAF6)‐mediated activation of Rac1/Pak1 pathway. Selective inhibition of Pak1 using IPA 3 or knockdown using siRNA both significantly inhibited TGFβ1‐induced prostate cancer cell EMT and expression of mesenchymal markers. Our study demonstrated that TGFβ1 induces apoptosis and EMT in prostate cancer cells via activation of P38‐MAPK and Rac1/Pak1 respectively. Our results reveal the potential therapeutic benefits of targeting TGFβ1‐TRAF6‐Pak1 pathway for advanced‐stage prostate cancer.

Support or Funding Information

Funds were provided by the UGA‐Research Foundation, Wilson Pharmacy Foundation, The American Legion, and the NIH (R01HL103952) grants to PRS. Al‐Azayzih was supported by pre‐doctoral fellowship from Jordan University of Science and Technology. This material is the result of work supported with resources and the use of facilities at the Charlie Norwood VAMC, Augusta, GA.
Transcriptional Regulation of Histone Demethylase JARID1B/KDM5B by Ikaros Tumor Suppressor in Human Lymphoblastic Leukemia
Authors: Payne Jonathon Lee , Dovat Elanora , Song Chunhua , Ding Yali , Gowda Chandrika , Payne Kimberly J , Dovat Sinisa
Published: November 2023

IKZF1 encodes the Ikaros DNA‐binding protein that is essential for normal hematopoiesis and acts as a tumor suppressor. Deletion of a single Ikaros allele is associated with the development of high‐risk B‐cell precursor acute lymphoblastic leukemia (B‐ALL) that has a poor prognosis. Despite extensive studies of Ikaros function, the molecular mechanisms by which Ikaros regulates transcription of its target genes and its role in the epigenetic control of gene expression in human leukemia remain unknown. Using chromatin immunoprecipitation followed by deep‐sequencing (ChIP‐seq), we determined that Ikaros binds to the promoter of the histone H3K4 demethylase JARID1B (KDM5B) gene in human leukemia. The objective of our study was to determine the mechanism through which Ikaros regulates transcription of JARID1B in leukemia. Overexpression of Ikaros in B‐ALL cells resulted in transcriptional repression of JARID1B as measured by qRT‐PCR and Western blot. The increase in JARID1B was associated with a global increase in the H3K4me3 epigenetic mark in leukemia cells. The use of luciferase reporter assays demonstrated that Ikaros co‐transfection directly represses transcription of JARID1B. Knockdown of Ikaros expression in B‐ALL cells with Ikaros shRNA results in increased transcription of JARID1B, as evidenced by qRT‐PCR and Western blot. Together, these experiments suggest that Ikaros regulates global levels of H3K4 tri‐methylation in leukemia by repressing JARID1B transcription. Functional assays showed that Ikaros‐mediated repression of JARID1B requires recruitment of histone deacetylase HDAC1 to the JARID1B promoter, which results in the formation of repressive chromatin. In B‐ALL, the Ikaros protein is phosphorylated by pro‐oncogenic Casein Kinase II (CK2). Functional analysis showed that CK2‐mediated phosphorylation of Ikaros severely impairs its DNA‐binding affinity toward the JARID1B promoter and its function as a transcriptional regulator in high‐risk leukemia. Treatment of leukemia cells with specific CK2 inhibitors restores Ikaros binding to the JARID1B promoter and transcriptional repression of JARID1B. In conclusion, our results demonstrated that CK2, Ikaros, and HDAC1 regulate the global level of H3K4 tri‐methylation in leukemia via transcriptional control of JARID1B expression. These data provide a novel insight into the regulatory network that controls the epigenetic signature in leukemia.
Overexpression of basonuclin 2 promotes carcinogenesis in basal cell carcinoma
Authors: Douhne Timothy , Cornett Jonathan C
Published: November 2023

Basal cell carcinoma (BCC) is the most common form of human skin cancer. To identify driver genes in BCC tumorigenesis, we carried out a piggyBac transposon mutagenesis screen in mice. Basonuclin 2 (Bnc2) was identified as a gene that, when overexpressed in the skin, likely contributes to the formation of basal cell carcinomas. However, the mechanism by which Bnc2 acts to promote tumor formation is unknown. To gain insight into its role in normal cells, we overexpressed Bnc2 in human keratinocytes. Bnc2 overexpressing cells showed increased viability compared with control cells. In addition, Bnc2 overexpression caused reduced expression of differentiation markers in human keratinocytes. Taken together, our results suggest that Bnc2 acts as a novel oncogene in BCC progression by preventing differentiation and cell death in Bnc2 overexpressing keratinocytes.

Support or Funding Information

This work was supported by a Ledford scholarship from the Appalachian College Association.
Anti‐proliferative Effect of Curcumin is Mediated by Down‐regulation of Polycomb Complex Protein, BMI1
Authors: Adeyeni Temitope Adetola , San KayKay , Ezekiel Uthayashanker
Published: November 2023

BMI1 encodes a 37‐kDa protein that is a key regulatory component of PRC1 (polycomb regulatory complex‐1). BMI1 functions as an oncogene, induces cell growth and promotes tumor growth in in vitro and in vivo animal models. BMI1 is currently recognized as a gene of interest in terms of pharmaceutical drug development. Curcumin, a major phytochemical in turmeric, inhibits the proliferation of many types of cancer cells, both in vitro and in vivo. Previously, we observed that DLD‐1 colorectal cancer cells had a suppressed BMI1 expression following curcumin treatment. In addition, there was an inhibition of cell proliferation. This statistically significant finding indicates that curcumin potentially inhibits DLD‐1 cell proliferation by targeting BMI1. Curcumin has potential to be used in colorectal cancer cell therapy, however, it has been reported to have poor bioavailability. The purpose of this study is to determine whether or not modified curcumin compounds (dimethoxycurcumin, bisdemethoxycurcumin) and curcumin metabolite (tetrahydrocurcumin), with a higher bioavailability, have the same mechanism of action as curcumin in DLD‐1 colorectal cancer cells. DLD1 cells were treated with curcumin, dimethoxycurcumin, bisdemethoxycurcumin, and tetrahydrocurcumin. After 48 hours, BMI1 gene expression and protein levels will be measured. Preliminary results suggested that dimethoxycurcumin reduced BMI1 protein expression more than curcumin.

Support or Funding Information

TAA and KS are supported by the DeNardo Education and Research Foundation.
HER2+ breast cancer metastasis mediated by E2F2 copy number changes
Authors: Andrechek Eran
Published: November 2023

The E2F family of transcription factors is classically known to regulate G1 to S‐phase transition in cell cycle. Recently, other functions have emerged including roles for E2Fs in cancer, specifically HER2+ breast cancer. We have recently shown that a loss of E2F1 or E2F2 in the MMTV‐Neu mouse model leads to a decrease in tumor metastasis. It is not known what mechanistic roles specific E2Fs are playing in this process. To address this, gene expression of tumors from MMTV‐Neu transgenic mice of wild type and E2F2 knockout backgrounds were analyzed through ACE (Analysis of CNAs by Expression data) to identify regions of chromosomal gain and loss. Conserved mouse and human copy number changes were identified using the TCGA data. These events were screened for effects on patient outcomes through the application of a predictive gene expression signature. An average of 8.8 amplification or deletion events were observed in Neu induced tumors compared to 21.8 events in similar tumors from an E2F2 knockout background (n=15, p<.01). In the E2F2 KO samples, a consistent deletion was found on chromosome 11qD (human chromosome 17q21.33). qPCR analysis of chromosome 11D indicated a significant copy number decrease in MMTV‐Neu E2F2 KO (p<.05). Importantly, further analysis revealed that in 25% of HER2+ve patients there was an amplification of this same region. Strikingly, the presence of this amplification event is associated with a worse metastasis free survival (p<.001) and high E2F2 activity (p<.05). CRISPR mediated knockout studies to determine effects on tumor cell migration in vitro and tumor metastasis in vivo for key genes, including Col1a1 and CHAD, in this amplicon reveal a role in migration. Preliminary results show marked effects on metastatic capacity with knockouts of genes within this amplicon. These results strongly suggest that the E2F family of transcription factors contributes to metastasis partially through copy number changes of regions regulating metastasis in both mouse models and human breast cancer. These findings will have an important impact in understanding the interplay between transcription factors, genomic instability, and tumor metastasis.

Support or Funding Information

NIH R01CA160514
Investigation of the Sodium‐Hydrogen Exchanger 1 and Calcineurin Homologous Protein Isoforms 1 and 2 interaction in Lung Fibroblasts
Authors: Polveroni‐Edwards Thomas , Wallert Mark , Provost Joseph John
Published: November 2023

The Sodium‐Hydrogen Exchanger 1 (NHE1) is a transmembrane protein that plays a crucial role in maintaining stable intracellular pH. Furthermore, the protein is regulated by interaction between the C‐terminus of NHE1 and several intracellular proteins, including Calcineurin Homologous Protein Isoform 1 (CHP1) and 2 (CHP2). NHE1's role and importance are easily observable in metastatic lung cancer cells as the exchanger displays higher than normal activity in order to compensate for the increased metabolism and proton production found in the metabolically active cells. Expression of CHP1 to NHE1 is ubiquitous and CHP1‐NHE1 interaction is necessary for proper function of the exchanger, whereas the expression of CHP2 to NHE1 has so far only been found in gut cells and metastatic cancer cells. To investigate the differences between CHP1 and CHP2, and their binding to NHE1, we prepared CHP1‐RFP and CHP2‐GFP and observed their expression in Chinese Hamster Lung Fibroblasts with NHE1 null expressors (PS120 cells) and in Chinese Hamster Lung Fibroblasts stably expressing human NHE1 (PSN cells). Substantial translocation of CHP1 and CHP2 to the cellular membrane was observed in the PSN cells whereas no translocation was observed in the PS120 cells. We also generated several single amino acid alterations of NHE1 to determine the importantance of these residues on binding. Furthermore expression and translocation levels were also measured in fed, serum deprived and stimulated conditions.
A Comparison of SIKE and WIT 3 Structure and Dynamics
Authors: Machek Megan , Minzer Ian , Standen Heather , Kim Seung‐Hwan , Latzer Joachim , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE shares significant homology with WIT3 (known to be involved in wound healing) including several of the phosphorylation sites. It is unknown if these sites cause differences in structure and function. SIKE and WIT3 (both his tagged) have been expressed and SIKE purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. 3Dimensional models of SIKE and WIT3 and various phosphoforms have been constructed using Phyre2 and subjected to molecular dynamics analysis to further explore structural and dynamic similarities and differences between SIKE and WIT3. In an attempt to validate the models limited proteolysis and Hydrogen Deuterium exchange studies are underway.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB
The Effects of Trifluoroethanol on the Helical Structure of SIKE
Authors: GarciaDuBar Selena , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To examine the structure of SIKE we have performed two dimensional proton NMR experiments and Circular Dichroism spectroscopy and compared the results with both secondary and tertiary structure predictions. 2D NOESY spectra obtained using several mixing times in general revealed little stable structure in the molecule as detected in the NH‐NH cross peak region, suggesting the protein may contain significant disordered regions. CD spectra suggest significant alpha helix content and the possibility of coiled coil regions of structure. Addition of Trifluoroethanol, known to stabilize alpha helical structures, led to increased CD signal at 222nm. Using a range of TFE concentrations optimal stabilization of helical structure in SIKE allowed comparison with helical secondary structure predications.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB
PTH Regulation of CEBP‐delta Expression in MAPK Phosphatase‐1 (MKP‐1) Null Osteoblasts from Male and Female Mice
Authors: Datta Nabanita S , Vengalil Nathan , Chukkapalli Sahiti
Published: November 2023

Age‐related bone loss is associated with a gradual decline in bone homeostasis characterized by increased bone resorption than formation. The mechanisms of osteoblast bone formation are incompletely understood. Parathyroid hormone (PTH) is an important regulator of bone mass and controls osteoblast (the bone forming cells) and osteoclast (the bone resorbing cells) function. MAPKs are negatively regulated by a family of dual‐specificity phosphatases known as the MAPK phosphatases (MKPs). Previous studies from this laboratory have shown that female MKP‐1 knockout (KO) mice display osteopenia, while male KO mice show osteopetrosis. Insulin‐like growth factor‐1 (IGF‐1) plays an important role in promoting osteoblastic differentiation and PTH anabolic action. CCAAT/enhancer‐binding protein delta (CEBP‐delta), a transcription factor, is an upstream regulator of IGF‐1. In order to dissect the molecular mechanism of PTH action on MKP‐1 and sexual dimorphism, this study examined the expressions of CEBP‐delta using primary osteoblasts from wild type (WT) and MKP‐1 KO mice. Calvarial (skull) osteoblasts from 13–16 week old animals were isolated and differentiated for 7‐ days with ascorbic acid. The cells were then treated with vehicle (control) or PTH (100 nM) for 1–2 hour and harvested. Total cellular RNA was isolated and quantitative real‐time PCR was performed. Results indicated sexual dimorphism on CEBP‐delta expression following PTH treatment in both sexes and genotypes. PTH increased 3 to 6‐fold CEBP‐delta expression in KO males but not in WT males. In contrast, 2 to 4‐fold increase in CEBP‐delta expression was noted in WT females compared to 1.5‐fold in KO animals. These findings suggest involvement of CEBP‐delta expression, at least in part, in increased IGF‐1 expression observed in osteoblasts in the absence of MKP‐1 in males. Taken together, our findings provide new insight into the role of MKP‐1 in CEBP‐delta/ IGF‐1 pathway in osteoblast and sexual dimorphism in bone anabolic action.

Support or Funding Information

Wayne State University Funding for Research and Education (FMRE)
Intramolecular C2‐Kinase Domain Interactions Autoinhibit Conventional Protein Kinase
Authors: Callender Julia A , Antal Corina E , Kornev Alexandr P , Yang Yuan , Igumenova Tatyana I , Taylor Susan S , Newton Alexandra C
Published: November 2023

The signaling output of protein kinase C (PKC) is exquisitely controlled, with its disruption resulting in pathophysiologies. Identifying the structural basis for autoinhibition is central to developing effective therapies for cancer, where PKC activity needs to be enhanced, or neurodegenerative diseases, where PKC activity should be inhibited. Here, we reinterpret a previously reported crystal structure of PKCβII and use docking and functional analysis to propose an alternative structure that is consistent with previous literature on PKC regulation. Mutagenesis of predicted contact residues establishes that the Ca2+‐sensing C2 domain interacts intramolecularly with the kinase domain and the carboxyl‐terminal tail, locking PKC in an inactive conformation. Applying a similar approach towards another conventional PKC isozyme, PKCα, confirmed C2 domain‐mediated autoinhibition as a shared mechanism among conventional PKC isozymes. Ca2+‐dependent bridging of the C2 domain to membranes provides the first step in activating PKC via conformational selection. Therefore, elucidation of the structural basis for autoinhibition of PKC unveils a unique direction for therapeutically targeting PKC activity.

Support or Funding Information

This work was supported by NIH grants GM43154 to A.C.N. and DK54441 to A.C.N. and S.S.T. C.E.A. was supported in part by the University of California, San Diego, Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752) and the National Science Foundation Graduate Research Fellowship (DGE1144086).
Protein kinase C participates in a cell growth network in the filamentous fungus Aspergillus nidulans
Authors: Jackson‐Hayes Loretta , Hill Terry , Myers Lance
Published: November 2023

The long‐term goal of our research is to advance understanding of the mechanisms involved in hyphal growth and the synthesis and maintenance of cell walls in filamentous fungi. Recently our work has focused on proteins that localize to sites of cell wall synthesis, namely growing cell apices (hyphal tips) and forming crosswalls (septa). PkcA, an orthologue of protein kinase C in our model organism Aspergillus nidulans, is one such protein. In previous work, we identified a mutation in the pkcA gene which results in reduced cell wall integrity. We showed that wild type PkcA localizes to growing hyphal apices and septation sites, and we identified amino acid sequences within PkcA that are required in order for PkcA to localize to these sites of cell wall synthesis. Our work, as well as work done in other labs, has also brought to light many other proteins that play roles in septation and polarized growth and that also localize to septation sites and growing tips. Here we report our work towards placing PkcA and other polarized growth related proteins in a cell growth network. Using a collection of mutant strains carrying dual labels (GFP and monomeric RFP) of PkcA and other polarized growth related proteins, we showed via fluorescence microscopy that PkcA colocalizes with the actinin‐like protein AcnA, the septin AspB, BimG phosphatase, landmark protein Bud4, Cdc14 phosphatase, septation initiation complex component MobA, Rho4 GTPase, myosin regulatory light chain (Rlc1), the formin SepA, and the IQGAP homologue SepG at hyphal tips and/or septation sites. We also observed PkcA localization in a variety of strains in which expression of other growth related proteins had been genetically disrupted. In addition, the current results have begun establishing which proteins PkcA physically binds at tips and septation sites. Using bimolecular fluorescence complementation we demonstrated that PkcA and SepA might physically interact at both tips and septation sites. Our future work will include investigating other physical interactions with PkcA and determining which complexed proteins are solely involved in localization and regulation and which are PkcA substrates.
The Role of Ste20‐like Kinase (SLK) in TGFbeta‐activated Kinase 1 (TAK1)‐mediated Pathways
Authors: Tseng Ping‐Hui , Liao Jia‐Wei , Chen I‐Ting
Published: November 2023

Mitogen‐activated Protein Kinases (MAPKs) are activated in various signaling pathways, and thus, critical for different biological functions. As the member in Ste20‐related kinases, Ste20‐like kinase, SLK, has been suggested to play an important role in apoptosis and cytokinesis. Here, we investigate the interaction between SLK and TGFbeta‐activated kinase 1 (TAK1), which is a key MAP3K in innate immunity. We found that p38 activation is declined in SLK‐silencing macrophages upon LPS engagement. On the other hands, SLK overexpression is able to specifically enhance TAK1‐mediated p38 activation, but has no effect on JNK. In conclusion, we demonstrate that a novel role of SLK in TLR pathways and the mechanism for distinct activation of MAPK signaling.

Support or Funding Information

MOST‐102‐2320‐B‐010‐027‐MY3
The Preparation and Screening of Several Novel Bacterial Homoserine Lactone Autoinducers
Authors: Sengstack Janine , Aquino Ashley , Schilling Kevin , Davies Jocelyn , Lehrer Joshua , Ng Cheryl , Armstrong Megan , Souza Lucas , Villaroman Joanne , Santos Riley , Mason Marcos , Brummitt Mary , Gruver Megan , Abadi Shayda , Meisenheimer Kristen , Fidopiastis Pat
Published: November 2023

The work described here explores the synthesis of known and potentially novel autoinducing molecules. Autoinducers, are small signaling molecules bacteria use to communicate to sense population density and control gene expression. Bacterial quorum sensing is the term used to describe this communication process. Two autoinducers have already been prepared in our lab: p‐coumaroyl homoserine lactone (p‐C‐HSL) and the novel N‐3‐oxooct‐7‐enoyl‐L‐homoserine lactone. The quorum sensing activities of both of these molecules have been tested in the Vibrio fisheri bacteria through established collaborations in the Fidopiastis lab here at Cal Poly. Luminescent/growth data suggests Vibrio fisheri responds to N‐3‐oxooct‐7‐enoyl‐L‐homoserine lactone but has diminished luminescence compared to the native autoinducer, N‐(3‐oxohexanoyl) homoserine lactone. Vibrio fischeri does not respond to p‐C‐HSL. However, p‐C‐HSL is a known autoinducer in the photosynthetic bacterium Rhodopseudomonas palustris. Reactivity profiling of both p‐C‐HSL and N‐3‐oxooct‐7‐enoyl‐L‐homoserine lactone is currently in progress to generate an extensive collection of reactivity data and novel derivatives. Lastly, the synthesis of another potential autoinducer, N‐3‐oxohept‐6‐enoyl‐L‐homoserine lactone is also being prepared in our lab; all of these molecules and derivatives will be tested for quorum sensing activity in the Vibrio fischeri bacteria.

Long Range Research Goals: Because the quorum sensing mechanism of Vibrio fischeri is similar to that in other quorum sensing bacteria, these results can be used to demonstrate that synthetic autoinducers can trigger either a similar or possibly inhibited quorum sensing response. Further research would involve extrapolating this result to other quorum sensing bacteria behaviors, such as the production of toxins or antibiotic resistance. A potential goal would be to create an autoinducer that ultimately shuts down bacterial communication thereby inhibiting the formation of toxins in the first place.

Support or Funding Information

California Polytechnic State University, College Based Fee Fund
Tumor Suppressors Klotho and 1,25‐Dihydroxyvitamin D3 Cooperate via Synergistic Inhibition of Oncogenic Wnt/β‐Catenin Signaling
Authors: Khan Zainab , Khan Sameera , Sandoval Ruby , Whitfield G. Kerr , Haussler Mark R. , Jurutka Peter W.
Published: November 2023

The human klotho gene encodes a protein that can generate two isoforms: a single‐pass transmembrane protein (mKlotho) or a secreted peptide (sKlotho) formed from the cleavage of the former. The two isoforms likely possess physiologically unique roles, as mKlotho is known to serve as a membrane‐bound coreceptor for FGF23, while sKlotho may function as an endocrine factor and/or as an enzyme that modifies glycans of cell surface glycoproteins. Due to its putative interactions with several intracellular signaling pathways, klotho has been proposed as an anti‐aging protein and tumor suppressor gene. Intracellular signaling may potentially involve the vitamin D and the Wnt/β‐catenin pathways. Vitamin D, a bioactive lipid and essential nutrient, is acquired via endogenous synthesis upon skin exposure to UVB‐radiation or through dietary intake. Activated 1,25‐dihydroxyvitamin D3 (1,25D) binds to and stimulates the nuclear vitamin D receptor (VDR), which acts as a central factor in bone and mineral homeostasis, as well as a potential chemopreventative agent through its ability to promote cellular differentiation, while inhibiting growth and division. In contrast, the Wnt/β‐catenin signaling pathway can drive enhanced cellular proliferation leading to numerous types of cancers, especially colorectal neoplasia. We hypothesized that sKlotho and vitamin D/VDR may function collectively to inhibit the tumor‐inducing properties of the β‐catenin pathway and that sKlotho may modulate VDR activity. In order to assess the influences of sKlotho and 1,25D on VDRE‐mediated transcription and Wnt/β‐catenin signaling, transcriptional assays and quantitative real time PCR (qPCR) were conducted in embryonic kidney (HEK293) and colon cancer (HCT116) cell lines. We also analyzed the effects of an NAD‐dependent deacetylase sirtuin‐1 (SIRT1), a potential VDR modulator that is activated by resveratrol, on the β‐catenin signaling pathway. Our results revealed an increase in the transactivation capacity of VDR in the presence of 1,25D, SIRT1, and sKlotho. Additionally, sKlotho, SIRT1 and 1,25D were shown to independently and synergistically inhibit β‐catenin activity. The data also indicated functional differences between the two isoforms of klotho, as mKlotho was shown to enhance β‐catenin activity, suggesting that the proposed anti‐aging effects of klotho are presumably directed by the secreted isoform. In conclusion, our study suggests a role for sKlotho as a tumor suppressor and further points to the potential for molecular crosstalk between vitamin D and klotho signaling to elicit optimal human health.

Support or Funding Information

National Institutes of Health Grants DK033351 to MRH and CA140285 to PWJ
Regulator of G Protein Signaling 6 (RGS6) Protects Against Neurodegeneration and Motor Dysfunction in the Unilateral 6‐hydroxydopamine (6‐OHDA) mouse model of Parkinson's Disease
Authors: Luo Zili , Ahlers Katelin E , Yang Jianqi , Alberico Stephanie , Narayanan Nandakumar S , Fisher Rory A
Published: November 2023

PD is a devastating, and currently untreatable, neurological disease resulting from death of a subset of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). These neurons normally project to the striatum and release DA to postsynaptic targets that control motor behavior. Loss of these SNc DA neurons leads to the hallmark motor deficits of this disease: bradykinesia, rigidity and resting tremors. The reason for the greater sensitivity of SNc neurons to degeneration is unknown. We and our collaborators recently discovered that RGS6 is required for the adult survival of SNc DA neurons whose loss causes PD. RGS6 is restrictively expressed in these neurons and knockout of RGS6 in mice profoundly reduced survival of these neurons. Specifically, RGS6−/− mice exhibited an age‐dependent degeneration of SNc DA neurons, DA loss in the SNc and striatum and motor deficits. While PD is primarily a disease of aging, environmental toxins can produce selective SNc DA neuron death in humans and vertebrates and induce PD motor symptoms. While our findings demonstrate that RGS6 suppresses development of PD in mice without a provoking stimulus we wondered if it would protect against neurotoxic insult. To test this possibility, we administered saline or a submaximal dose of the neurotoxin 6‐OHDA (1 μg) by stereotaxic injection in the SNc of 1 yr old wild type (WT) and RGS6−/− mice and assessed motor behaviors after 2 wks. 6‐OHDA injection provoked loss of RGS6 DA neurons in the SNc in WT mice as well as motor deficits in both groups of mice. Automated gait analysis revealed a decrease in stride length and increased stride frequency in 6‐OHDA vs saline treated mice though RGS6−/− mice exhibited greater changes in these parameters compared to WT mice. Similarly while 6‐OHDA injection impaired accelerating rotarod performance in both groups of mice, RGS6−/− mice had a shorter latency to fall compared to WT mice. RGS6−/− mice also showed greater amphetamine‐induced net ipsilateral rotations and less contralateral paw contact following 6‐OHDA injection compared to WT mice. We also measured forebrain dopamine and its metabolites by in both the non‐injected and 6‐OHDA injected sides of the brains of WT and RGS6−/− mice. Though 6‐OHDA injection induced a significant decrease in forebrain DA content and increases in DOPAC/DA ratio in both sets of mice, the loss of DA and the increase in DOPAC/DA was higher in RGS6−/− mice compared to WT mice. These findings provide the first evidence that RGS6 plays a critical neuroprotective role in neurotoxin‐induced neurodegeneration and motor dysfunction in the most well developed mouse model of PD. These findings identify RGS6 as a potential therapeutic target for development of neuroprotective approaches in PD.

Support or Funding Information

Supported by NIH CA161882, AHA 14GRNT20460208
STING is Involved in the Development of Aortic Aneurysm and Dissection
Authors: Guardado Christopher , Zhang Lin , Coselli Joseph S , Shen Ying H , LeMaire Scott A
Published: November 2023

Objectives

Aortic aneurysm and dissection (AAD) is associated with significant morbidity and mortality. Less than 20% of AAD cases are caused by genetic defects, whereas more than 80% are classified as sporadic. Sporadic AAD results from a progressive loss of smooth muscle cells in the tunica media. However, the molecular mechanisms that lead to the development and pathology of sporadic AAD remain poorly understood. STING (stimulator of interferon genes) is involved in inflammation in response to endoplasmic reticulum stress and DNA damage. Our preliminary data indicate that STING plays a critical role in AAD development. The objective of the current study was to elucidate the molecular pathways by which the STING‐mediated innate immune response contributes to the development of AAD.

Methods and Results

Eight‐week‐old Sting−/− mice and wild type mice were either challenged with a high‐fat diet for 8 weeks and an angiotensin II infusion during the last 4 weeks or remained unchallenged for the study period. Aortas from the Sting−/− and wild type mice were stained with markers for smooth muscle cells (SM22a), inflammatory cells (CD68 for macrophages, matrix metalloproteinase [MMP]‐2, and MMP‐9), fibroblasts (fibroblast‐specific protein 1 [FSP1]), and necroptosis (receptor‐interacting protein kinase 3 [RIP3] and mixed lineage kinase domain‐like protein [MLKL]). Qualitative analysis of the stained images showed that aortic challenge induced a significant decrease in the smooth muscle cell marker but increases in CD68, MMP‐2, FSP1, and RIP3 in the aortic smooth muscle cells in wild type mice. Importantly, these changes were significantly reduced in aortas from the challenged Sting−/− mice, as compared to those from challenged wild type mice.

Conclusions

Our study suggests that STING may contribute to a switch in smooth muscle cell phenotype and necroptosis, leading to a loss of smooth muscle cells during AAD development.

Support or Funding Information

Baylor College of Medicine and NIH grants
The Role of Nrf2 in Neuroinflammation and the Impact of Botanicals
Authors: Krepel Stacey A , Hogan Mary B , Mangum Sally , Kuhn Thomas B
Published: November 2023

Persistent inflammatory and oxidative stress is largely responsible for the progressive loss of neuronal integrity and connectivity underlying the continuous decline of cognitive function associated with most chronic CNS disorders as well as normal aging. The transcription factor Nrf2, a master redox regulator, plays a pivotal role in the antioxidant defense of non neuronal and neuronal cells. Nrf2 remains sequestered in the cytosol of cells under basal conditions tightly bound to its repressor Keap1 and upon oxidative stress dissociates from Keap1 and translocates into the nucleus to stimulate transcription of antioxidant defense mechanisms. It is not clear how duration and extent of oxidative stress impact activation and nuclear translocation of Nrf2 in neuronal cells. Exposure of SH‐SY5Y human neuroblastoma cells to peroxide (250 μM H2O2, 1 h) considerably increased the presence of Nrf2 in the nuclear fraction indicative of translocation of Nrf2 from the cytosolic to the nuclear compartment. Immunocytochemistry against Nrf2 corroborated this finding. Exposure of SH‐SY5Y neuroblastoma cells (1 h) to the proinflammatory cytokine TNFa at concentrations ranging from 50 ng/ml to 400 ng/ml triggered the translocation of Nrf2 into the nucleus where as prolonged exposure (4 h) to TNFa (200 ng/ml) revealed significantly stronger presence of Nrf2 in the nuclear compartment. However, prolonged exposure of SH‐SY5Y cells to TNFa (24 h) at concentrations of 200 ng/ml or higher also resulted in significant cell death due to oxidative stress. Together these findings suggest that, in response to TNFa stress, Nrf2‐enhanced antioxidant defenses were insufficient. One of the many health benefits of nutrition rests on the capacity of distinct botanicals to directly act on biochemical mechanisms rather than through passive antioxidant capacities. Several botanicals including some polyphenols have the potency to stimulate Nrf2 activity and thus increase antioxidant defense mechanisms. We demonstrated that supplementation of SH‐SY5Y neuroblastoma cells with crude extracts or non polar extracts (5 μg/ml each) obtained from Alaska wild bog blueberries significantly increased neuronal viability in the prolonged presence of 200 ng/ml TNFa. Incubation of SH‐SY5Y cells with distinct blueberry extracts alone stimulated Nrf2 activation, which could at least partially explain the reported neuroprotective potency of diets rich in berry fruits. Understanding the regulation of Nrf2 through botanicals could provide vital insight for developing neuroprotective strategies applicable to an array of neurodegenerative diseases.

Support or Funding Information

Supported in part by NIH grants RL56M118990, 1UL16M18991, TL4GM118992, and generous donations from the Alzheimer'r Resources of Alaska. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health
Peptide inhibition of crosstalk between autophagy and phagocytosis as a novel anti‐septic shock therapeutic strategy
Authors: Yang Chul‐Su
Published: November 2023

Sepsis is a life threatening disease characterized by inflammation of the entire body, also known as systemic inflammatory response syndrome (SIRS). Upon microbial infection, p22phox‐gp91phox NADPH oxidase complexes produce Reactive Oxygen Species (ROS) critical for the elimination of invading microbes. However, excess production of ROS represents a key element in the cascade of deleterious processes in sepsis. We have recently discovered the direct crosstalk between autophagy and phagocytosis machineries by demonstrating that the Run/cysteine‐rich‐domain‐containing‐Beclin1‐interacting autophagy protein (Rubicon) interacts with p22phox upon microbial infection, facilitating the phagosomal trafficking of the p22phox‐gp91phox NADPH oxidase complex to induce a ROS burst, inflammatory cytokine production, and thereby, potent anti‐microbial activities. Remarkably, the N‐terminal eight‐amino‐acid short peptide derived from p22phox, called the N8 peptide, was sufficient for Rubicon interaction and thus, capable of robustly blocking the Rubicon‐p22phox interaction and profoundly suppressing ROS and inflammatory cytokine production. Consequently, treatment of the N8 peptide dramatically reduced the mortality of lipopolysaccharide (LPS)‐induced endotoxic sepsis and Cecal‐Ligation‐and‐Puncture (CLP)‐induced polymicrobial sepsis in mice, and Escherichia coli infection‐induced sepsis in rhesus monkeys. This study demonstrates a new anti‐sepsis therapeutic strategy by blocking crosstalk between autophagy and phagocytosis innate immunity machineries, representing a potential paradigm shift for urgently needed therapeutic intervention against this life‐threatening SIRS.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF‐2014R1A1A1006117).
Modeling the Role of Impaired Akt Kinase Signaling in the Susceptibility to Stress
Authors: Willock Christine D , Berdux Nicholas C , Franke Thomas F
Published: November 2023

Background

Akt kinase plays a key role in neuronal cell function and survival. Biochemical studies on human postmortem brains indicate significant changes in Akt signaling in depression and suicide. AKT1 mutations also are a liability in psychiatric diseases such as schizophrenia. These noteworthy findings suggest an important role for intact Akt signaling in mood regulation.

Methods

Our studies have examined mouse models of Akt deficiency in anxiety‐and depression‐like behaviors, and their responsiveness to antidepressants and psychostimulants. In addition to genomic Akt knockout mice for all three Akt isoforms (Akt1, Akt2 and Akt3), we employed Cre‐dependent recombination to produce Akt1 deficiency in specific brain regions and neuron populations.

Results

Following the social defeat regimen, Akt1–deficient mice exhibited increased susceptibility to the detrimental effects of chronic stress. Antidepressant treatment failed to ameliorate depression‐like behaviors in the mutants when compared to wild‐type controls. Mice with conditional Akt1 deficiency exhibited a spectrum of phenotypes after completing the chronic social defeat paradigm: Akt1 ablation in the frontal cortex significantly decreased resilience, while ablation in other brain areas and specific neuron populations increased resilience. Similarly, ablation of Akt1 in different brain regions produced distinct and opposing results when measuring locomotor responsiveness to cocaine.

Discussion

Our results in Akt1‐mutant mice converge with biochemical and genetic findings in neuropsychiatric patients and confirm the critical requirement for intact Akt signaling in mood regulation. Based on our findings in conditional Akt1 mutant mice, we predict a significant role for frontal cortical Akt signaling in stress resilience and antidepressant response. Our experiments suggest conditional Akt1 mutant mice as a tractable genetic model to investigate brain region‐specific contributions of Akt signaling to mood regulation. These mutants aid in understanding how circuit‐specific Akt1 deficiencies may become liabilities Conditional Akt1‐mutant mice also illustrate specific contributions of Akt signaling to the function and output of distinct neuronal circuits.

Support or Funding Information

This work was supported in part by NIH grants DA032280, OD018339 and OD018340, and by American Foundation for Suicide Prevention (AFSP) award SRG‐1‐135‐11.
Sex‐ and Corticotropin‐releasing Factor Receptor 2‐dependent Actions of Urocortin 1 During Colitis
Authors: Hasdemir Burcu , Matloubian Mehrdad , Garnett Elizabeth A , Heyman Melvin B , Bhargava Aditi
Published: November 2023

We investigated whether corticotropin‐releasing factor receptor 2 (CRF2R) and its high affinity agonist urocortin 1 (Ucn1) mediate sex‐specific signaling and immune responses using an in vivo model of inflammatory bowel disease (IBD). Male and female wild‐type (WT) and CRF2R heterozygous (CRF2R+/−) mice, immunoassays, flow cytometry, and microscopy, were used to examine sex‐specific responses. CRF2R+/− mice of both sexes were more susceptible to colitis‐induced mortality than their WT littermates. Ucn1 rescued male CRF2R+/− mice from colitis‐induced mortality, whereas it increased mortality in female CRF2R+/− mice. Ucn1 restored colon length, spleen and adrenal mass, and decreased colonic TNF‐α and IL‐1β levels to baseline levels in male CRF2+/− mice alone. Treatment with monoclonal anti‐TNF‐a ameliorated colitis‐induced inflammation in both sexes, suggesting that Ucn1 actions are upstream of TNF‐α and gender‐specific. CRF2R+/− mice of both sexes showed decreased phosphorylation of MAPK p38 and cytoprotective heat shock protein 27 (Hsp27) levels. Ucn1 restored p‐Hsp27 levels in male CRF2R+/− mice alone. Baseline expression of the chaperone protein Hsp90 was reduced in male CRF2R+/− mice to start with and did not change during colitis or after Ucn1 treatment, suggesting that CRF2R interaction with Hsp90 is critical. Expression levels and subcellular localization of CRF2R and Ucn1 in pediatric IBD patients was dysregulated and gender‐specific. To conclude, CRF2R is a key determinant of the observed sex‐specific immune and cellular signaling responses. We established that Ucn1 mediates sex‐specific responses during IBD via CRF2R, emphasizing the need for inclusion of females in preclinical studies.

Support or Funding Information

NIH/NIDDK R01‐080787 to AB
A proinflammatary role of Mlkl dependent necroptosis in pancreatitis
Authors: Ren Junming
Published: November 2023

Rip3 was reported to play a pivotal role in various pathophysiological conditions, such as Crohn's disease, hepatitis, atherosclerosis and systemic inflammatory response syndrome. To determine whether Mlkl also plays a role in the progression of necroptosis‐associated diseases, we generated Mlkl knockout mice by gene disruption. Homozygous Mlkl‐deficient (Mlkl−/−) mice are viable, healthy, fertile, born with normal Mendelian frequency and do not display any gross physical or behavioral abnormalities. The expression pattern of Mlkl is quite different from that of Rip3. The expression level of Mlkl protein is high in thymus, colon, intestine, liver, spleen and lung, much lower in skeletal muscle, heart and kidney, and undetectable in the brain. We analyzed wild‐type and Mlkl−/− mice in an acute pancreatitis mouse model induced by cerulein stimulation. Wild‐type mice injected with cerulein every hour for 6 consecutive hours showed much more severe acinar cell necrosis than Mlkl−/− littermates under the same treatment; phosphorylation of Mlkl was detected in wild‐type but not Mlkl−/− acinar cells, and many more inflammatory cells, such as macrophages, neutrophils and other granulocytes, were found in wild‐type compared to Mlkl−/− pancreas and blood. The mRNA expression levels of 5 inflammatory cytokines, including IL‐6 and KC, were decreased significantly in the pancreas of mice lacking Mlkl. Our findings indicate that necroptosis plays a strong proinflammatory role in pancreatitis.
Biosynthesis of the FeFe hydrogenase active site
Authors: Suess Daniel Leif Migdow , Britt R. David
Published: November 2023

FeFe hydrogenases catalyze the reversible interconversion of H2 with protons and electrons, and thereby provide either an electron source or an electron sink for a variety of metabolic processes. Hydrogenase reactivity occurs at the H cluster which consists of a conventional [4Fe–4S]H subcluster bridged via a cysteine residue to an unusual organometallic [2Fe]H subcluster. The mechanism of H‐cluster bioassembly has emerged as an active topic of research that presents fresh challenges in the coordination chemistry, radical chemistry, and biochemistry of Fe‐S enzymes and is also important for developing biotechnological systems for solar fuels production. Our work in this area focuses on the radical SAM enzyme HydG, one of three maturase enzymes that build the organometallic [2Fe]H subcluster. HydG's role is particularly important because it transforms inorganic Fe2+ into an organometallic [Fe(CO)2(CN)] species that eventually comprises the [2Fe]H subcluster. Our work concerns the characterization of this and related organometallic intermediates with an emphasis on the mechanisms of their formation.
Novel Regulation of the Stability of Transcriptional Cofactors Ski and SnoN by GPCR Signals and Actin‐Cytoskeleton Dynamics
Authors: Macias‐Silva Marina , Vazquez‐Victorio Genaro , Caligaris Cassandre
Published: November 2023

TGF‐β signaling controls several cellular processes from growth arrest to apoptosis in epithelial cells. The transcriptional cofactors Ski and SnoN are main regulators of TGF‐β signaling by sequestering SMAD proteins or recruiting transcriptional repressor complexes at TGF‐β/SMADs target genes. TGF‐β signaling regulates the stability of Ski and SnoN proteins through the ubiquitin‐proteasome system. Even though the important role that Ski and SnoN proteins play in early development and homeostasis, cell cycle was the unique signal besides TGF‐β pathway that regulated their stability. Nevertheless, the mechanisms that regulate Ski and SnoN protein stability in diverse cellular processes remain unknown. In this study, we found out that the actin cytoskeleton dynamics is a novel regulator of Ski and SnoN protein stability in hepatocytes. Inhibitors of actin cytoskeleton dynamics and cell confluence status regulate Ski and SnoN protein levels. Actin polymerization is a signal for Ski protein degradation via proteasome. We also demonstrated that G protein‐coupled receptors (GPCR) signaling control Ski and SnoN protein levels through promoting and decreasing its stability by cAMP or Rho signals, respectively. In conclusion, we identified a new regulation for Ski and SnoN protein stability by changes in actin cytoskeleton rearrangements. These findings suggest that changes in Ski and SnoN protein stability could be controlled by this novel regulation in physiological and pathological events as a result of changes in actin cytoskeleton dynamics induced by cell contact, extracellular matrix remodeling and soluble ligands; thus, it will be important to evaluate TGF‐β/SMADs signaling in all these cellular contexts.

Support or Funding Information

This work is supported by grants from DGAPA/PAPIIT/UNAM (IN208115) and CONACYT (No. 240224).