bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Metabolism by Estrogen Receptor Alpha
Authors: Archer Ashley E. , Rogers Robert S. , Wheatley Joshua L. , White Kathleen , Rumi M.A. Karim , Soares Michael J. , Geiger Paige C.
Published: November 2023

Postmenopausal estrogen deficiency increases the risk for metabolic syndrome, obesity, and type 2 diabetes. Estrogen treatment improves metabolic function in postmenopausal women, and estrogen receptor α (ERα) is thought to be the primary player in these protective metabolic effects. However, the mechanisms behind ERα regulation of metabolic function have not been elucidated. The purpose of this study was to investigate the impact of ERα loss on whole body and skeletal muscle oxidative capacity in a rat model. 14 wk‐old female Wildtype (WT) and ERα knockout (ERα−/−) rats were exercised to exhaustion and one week later a glucose tolerance test was performed. At sacrifice, body weight and adipose tissue depot weights (subcutaneous, retroperitoneal and periuterine) were measured. Adipocyte cell size and mitochondrial protein content were assessed by immunohistochemistry and western blot, respectively. Intramuscular triglyceride storage was also assessed. ERα deficient rats had increased body weight and subcutaneous adipose tissue mass compared with WT rats. Although there was no increase in retroperitoneal adipose tissue mass, adipocyte size was increased in ERα deficient rats. HOMA‐IR values were also increased in ERα−/− animals indicating increased insulin resistance. ERα deficiency also decreased exercise time to exhaustion when compared with WT rats. Mitochondrial proteins Complex IV and peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC1α) were significantly decreased and intramuscular triglyceride storage was increased in skeletal muscles from ERα deficient rats. Our data demonstrate the potential regulatory role of ERα to enhance oxidative capacity and improve energy metabolism both systemically and in skeletal muscle. Pharmacological activation of ERα could be an effective strategy for the prevention and treatment of metabolic dysfunction. Selective estrogen receptor modulators currently used as treatment for various postmenopausal symptoms may have the potential to restore metabolic function in postmenopausal women and improve quality of life.

Support or Funding Information

This research was supported in part by an NIH grant (R01 AG031575) and the IRHRM Pilot grant.
Acute Expression of PGC‐1α Isoforms in Skeletal Muscle of Postmenopausal Women following Resistance Exercise
Authors: Kiwata Jacqueline L. , Tuzon Creighton , Rice Judd C. , Schroeder E. Todd , Dieli‐Conwright Christina M.
Published: November 2023

PGC‐1α4, an isoform of the transcriptional coactivator PGC‐1α, has been postulated to regulate skeletal muscle hypertrophy through modulation of anabolic and catabolic gene expression. While resting levels of PGC‐1α4 mRNA expression were found to increase in skeletal muscle of healthy adults after a resistance training program, the acute effect of resistance exercise (RE) on PGC‐1α4 expression in a population prone to progressive muscle loss, such as postmenopausal women, has not been investigated. We sought to determine whether high‐intensity eccentric RE altered mRNA expression of PGC‐1α isoforms including total PGC‐1α, PGC‐1α4 and a regulator of muscle oxidative changes, PGC‐1α1, in postmenopausal women. Further we examined whether PGC‐1α isoforms were associated with changes in anabolic gene expression of IGF‐1Ea, IGF‐1Eb, and IGF‐1Ec (MGF), and catabolic gene expression of myostatin, FOXO3A, MAFbx and MuRF1. Nine postmenopausal women (57.9 ± 3.2 yr) performed 10 sets of 10 maximal eccentric repetitions of single‐leg extension on an isokinetic dynamometer with 20 second rest periods between sets. Muscle biopsies were obtained from the vastus lateralis of the exercised leg before and 4 hours after the RE bout with mRNA expression determined by RT‐qPCR. No significant changes in the mRNA expression of PGC‐1α total, PGC‐1α1, PGC‐1α4, IGF‐1Eb or MGF were observed following acute eccentric RE (P > 0.05). However, expression of IGF‐1Ea significantly increased following RE (P < 0.05). No significant correlations were found between PGC‐1α and IGF‐1 isoforms, although PGC‐1α4 mRNA expression was associated with reduced expression of the catabolic gene myostatin (R = −0.88, P < 0.01) and increased expression of the catabolic gene MuRF1 (R = 0.78, P < 0.05). Thus, our data demonstrate an attenuated response of PGC‐1α isoforms to an acute bout of maximal eccentric exercise with short rest periods in postmenopausal women, with no association to changes in muscle growth factors involved with myogenic activity.

Support or Funding Information

The Clinical Exercise Research Center at the University of Southern California and NIH NCRR GCRC M0I RR000043 provided funds for this project.
The Correlation Between Tfeb and PGC‐1α in Skeletal Muscle
Authors: Erlich Avigail T , Vainshtein Anna , Hood David
Published: November 2023

Muscle health is strongly dependent on the maintenance of functional mitochondria for the provision of ATP through oxidative phosphorylation. The mitochondrial pool is governed by two competing processes: mitochondrial biogenesis and autophagy (mitophagy). Peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) is the master regulator of mitochondrial biogenesis, while transcription factor EB (Tfeb) regulates lysosomal biogenesis and is required for autophagy. Since the two processes are strongly linked, we sought to study their ability to regulate one another. The purpose of this study was to further elucidate the relationship between Tfeb and PGC‐1α by investigating Tfeb family members, as well as its transcription factors and downstream targets. Protein levels of Tfeb were reduced by 70% in whole body PGC‐1α KO mice compared to their WT counterparts. In contrast, muscle‐specific overexpression of PGC‐1α resulted in a 25% increase in Tfeb protein expression. Tfe3, a family member of Tfeb, was reduced by 50% in PGC‐1α KO mice. Despite this similar reduction in Tfeb and Tfe3 in KO animals, there were no significant changes observed in YY1, a transcription factor upstream of Tfeb that may regulate Tfeb expression. Since Tfeb is involved in lysosomal biogenesis, markers of chaperone‐mediated autophagy, were measured in the PGC‐1α WT and KO mice in order to see if they were affected by PGC‐1α‐mediated Tfeb downregulation. However, no changes were observed in Hsc70 or Lamp‐2A between the two genotypes. Our findings confirm the positive correlation between the two master regulators of biogenesis and autophagy, PGC‐1α and Tfeb. PGC‐1α does not seem to have an effect on markers of chaperone‐mediated autophagy, despite its regulation of Tfeb expression. Thus, it is probable that this correlation is working to promote other forms of autophagy, as well as mitochondrial biogenesis, in order regulate the turnover of the mitochondrial network.

Support or Funding Information

Supported by NSERC
Renin Overexpression Leads to Increased Titin‐Based Stiffness Contributing to Diastolic Dysfunction in Hypertensive mRen2 Rats
Authors: Csipo Tamas , Kovacs Arpad , Fulop Gabor Aron , Kovacs Andrea , Bodi Beata , Juhasz Bela , Priksz Daniel , Granzier Henk , Edes Istvan , Papp Zoltan , Barta Judit , Toth Attila
Published: November 2023

Hypertension is a major risk factor for heart failure (HF). We investigated the influence of hypertension on cardiac contraction and relaxation in transgenic renin overexpressing rats (carrying mouse Ren‐2 renin gene, mRen2, n=6). Blood pressure (BP) was measured by tail‐cuff method, echocardiography was used to characterize cardiac contractility, cellular force measurements and biochemical assays were applied to reveal molecular mechanisms. Sprague‐Dawley (SD) rats (n=6) were used as control. Systolic BP was elevated in mRen2 rats (235.11±5.32 vs. 127.03±7.56 mmHg in SD, P<0.05), resulting in increased left ventricular (LV) weight/body weight ratio (4.05±0.09 vs. 2.77±0.08 mg/g in SD, P<0.05). No effects of transgenic renin expression on systolic parameters, such as LV ejection fraction (EF, 68.50±2.09 vs. 70.17±1.45 % in SD), maximal Ca2+‐activated force (25.89±0.99 vs. 29.05±2.80 kN/m2 in SD) and Ca2+‐sensitivity of force production (pCa50, 5.83±0.01 vs. 5.87±0.05 in SD) were found. In contrast, pronounced diastolic dysfunction (DD) was observed in mRen2 rats: ratio of peak early and late LV diastolic filling (E/A) was lower (1.14±0.04 vs. 1.87±0.08, P<0.05), LV isovolumetric relaxation time was longer (43.85±0.89 vs. 28.55±1.33 ms, P<0.05) and cardiomyocyte passive stiffness was higher (1.74±0.06 vs. 1.28±0.18 kN/m2, P<0.05), compared to SD, respectively. Finally, hyperphosphorylation of titin at Ser‐12742 within the PEVK domain (P<0.05) proposed a molecular mechanism for DD. Our data suggest a link between the activation of renin‐angiotensin‐aldosterone system and increased titin‐based stiffness through phosphorylation of titin's PEVK element, contributing to DD. Accordingly, mRen2 rat might provide an in vivo model for HF with preserved EF (HFpEF).

Support or Funding Information

This work was supported by a European Union FP7 grant (MEDIA: metabolic road to diastolic heart failure) and by the Hungarian Scientific Research Fund (K116940 and K109083).
Transient Receptor Potential Melastatin 4 Channel Regulation by Inositol Trisphosphate Receptor in Human Urinary Bladder Smooth Muscle
Authors: Provence Aaron , Hristov Kiril L. , Rovner Eric S. , Petkov Georgi V.
Published: November 2023

Overactive bladder (OAB) is a highly prevalent and debilitating condition that lacks an effective therapeutic treatment. The contraction and relaxation of urinary bladder smooth muscle (UBSM) facilitates the voiding and storage of urine, the two main functions of the urinary bladder. Some forms of OAB have been directly linked to UBSM dysfunction. Thus, efforts to uncover unknown regulatory mechanisms in UBSM function are urgently needed. Among the potential novel targets for OAB, emerging evidence points to key roles for transient receptor potential melastatin 4 (TRPM4) channels in UBSM excitability and contractility. However, the cellular mechanisms regulating TRPM4 channel activity in human UBSM has been virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in UBSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R‐mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we used clinically‐characterized human UBSM tissues from 6 donor patients to investigate the molecular and functional interactions of the TRPM4 channels and IP3Rs in human UBSM. Using in situ proximity ligation assay (PLA) and amphotericin‐B perforated patch‐clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R‐mediated Ca2+ release of the SR in human UBSM. As determined by in situ PLA, we demonstrated colocalization of the TRPM4 channels and IP3Rs in human UBSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4‐IP3R regulatory mechanism in human UBSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel‐mediated transient inward cation currents (TICCs). In freshly‐isolated human UBSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+‐dependent activation of TRPM4 channels in human UBSM. In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human UBSM function. Our study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human UBSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for OAB.

Support or Funding Information

Supported by NIH grant R01‐DK106964 to Georgi V. Petkov and F31‐DK104528 to Aaron Provence.
Chronic Binge Alcohol Administration (CBA) Impairs Skeletal Muscle Mitochondrial Gene Expression in Simian Immunodeficiency Virus (SIV)‐Infected Rhesus Macaques
Authors: Duplanty Anthony A , Song Kejing , Simon Liz , Molina Patricia E
Published: November 2023

Previous studies from our laboratory have shown that CBA accentuates muscle wasting at end‐stage SIV infection and this is associated with pro‐oxidative and pro‐inflammatory skeletal muscle milieu as well as dysregulation of myoblast myogenic gene expression and myotube formation. Additionally, muscle of CBA/SIV macaques showed a greater reduction in the number and oxidative capacity (succinate dehydrogenase activity) of type 1 muscle fibers compared to that of sucrose administered SIV‐infected (SUC/SIV) and uninfected controls, Chronic alcohol consumption has been reported to result in mitochondrial DNA damage and impaired biogenesis; and this is considered an important mechanism in the pathogenesis of muscle myopathy. We hypothesized that CBA would impair the expression of genes involved in mitochondrial function in SIV‐infected macaques.

Purpose

The purpose of this study was to investigate the effects of CBA on expression of selected mitochondrial genes in the skeletal muscle of SIV‐infected rhesus macaques at end‐stage disease.

Methods

Male rhesus macaques were administered daily CBA (blood alcohol levels ~50 mM) or sucrose (SUC) intragastrically 3 months prior to intravenous SIVmac251 inoculation and continued throughout the study period. Skeletal muscle (gastrocnemius) samples were obtained at necropsy when animals met criteria for euthanasia and total RNA was extracted and mitochondrial gene expression was analyzed by qPCR.

Results

The relative expression of peroxisome proliferator‐activated receptor gamma, coactivator 1 beta (PGC‐1 beta) and mitofusin‐1 (MFN1) was significantly decreased in the skeletal muscle of CBA/SIV macaques (75% and 39% respectively; P<0.05), without altering the gene expression of MFN2, TFAM, NRF1, NRF2, or PGC‐1 alpha.

Conclusion

These results indicate that CBA administration decreases expression of genes essential for mitochondrial biogenesis in the skeletal muscle of SIV‐infected rhesus macaques and are in agreement with our previous observations of decreased oxidative capacity of type 1 muscle fibers. We speculate that impaired mitochondrial biogenesis may contribute to the underlying pathophysiology of alcoholic and HIV/AIDS associated myopathy. Developing therapeutic or life style interventions aimed at attenuating mitochondrial dysregulation in persons living with HIV/AIDS with alcohol use disorders should lead to improved muscle function and strength.

Support or Funding Information

This study was supported by grants from the National Institute on Alcohol Abuse and Alcoholism (NIAAA): T32 Postdoctoral Fellowship (AA07577) and the Comprehensive Alcohol Research Center P60 (AA09803) in the department of Physiology, LSU Heath Science Center, New Orleans, LA.
The Effects Of Anti‐Fibroblast Growth Factor 23‐Peptides Antibodies On Excreta Phosphate Of Chicks
Authors: Ren Zhouzheng , Butz Daniel E , Ebrahimi Marziyeh , Cook Mark E.
Published: November 2023

Neutralizing antibodies against fibroblast growth factor 23 (FGF23) have been previously shown to reduce the phosphate excretion of mice, rat and human. The objective of the present study was to determine the effect of passively transferred anti‐FGF23 peptides (FGF23‐3, VFFPGMNP; FGF23‐T, HRNTRSAD) antibodies on excreta phosphate content of chicks. Hens were vaccinated with a control vaccine or synthetic peptide vaccines, and eggs were collected and incubated following the peak antibody titer. Progeny chicks from each vaccine treatment were divided into 6 pens and fed a diet with either 0.13 or 0.36% available phosphate (n=3 pens per diet). After two weeks, all chicks were bled (to determine plasma phosphorus), and the total excreta from each pen were collected (for the analysis of phosphate content). Plasma phosphate and excreta phosphate were reduced in chicks fed 0.13% versus 0.36% phosphate (main effect of dietary phosphate, p<0.01). Interestingly, anti‐FGF antibodies significantly increased plasma phosphate of chicks (main effect of anti‐FGF23 antibodies, p<0.01). When compared with the control vaccine, chicks with anti‐FGF3 and anti‐FGFT antibodies showed 24.77% and 22.94% decrease, respectively in excreta phosphate in low phosphate diet; and 27.46% and 23.94% decrease, respectively in excreta phosphate in adequate phosphate diet (main effect of anti‐FGF23 antibodies, p<0.01). Since FGF23 is known to regulate the type II sodium‐phosphate co‐transporters’ expression in kidney, the current data suggest that anti‐FGF23 antibody may offer a new tool to reduce excreta phosphate in chicks.
Loss of PTPN2 in Intestinal Epithelial Cells Upregulates a Novel STAT‐Dependent Mechanism of Claudin‐2 Expression
Authors: Krishnan Moorthy , McCole Declan F.
Published: November 2023

Inflammatory bowel disease (IBD) is a multi‐factorial condition characterized by elevated levels of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Genome‐wide‐association‐studies (GWAS) revealed that individuals with single nucleotide polymorphic mutations in the locus of the protein tyrosine phosphatase non‐receptor type 2 (PTPN2) gene that encodes T‐cell protein tyrosine phosphatase (TCPTP), have an increased susceptibility to IBD. We have reported that reduced TCPTP expression increased effects of IFN‐γ on Signal Transducer and Activator of Transcription (STAT) signaling and led to a greater reduction in barrier function in intestinal epithelial cells (IEC). The protein composition of tight junctions (TJ) determines the tightness of the barrier. Claudin‐2 is a TJ protein that is upregulated in IBD and increases paracellular permeability to cations thus reducing transepithelial electrical resistance (TER). We have previously shown that transient knockdown of TCPTP permits increased expression of claudin‐2 through Janus Kinase (JAK)‐STAT signaling. The aim of the present study was to identify the molecular mechanisms underlying STAT regulation of claudin‐2 expression in PTPN2 (TCPTP)‐knockdown (PTPN2‐KD) HT‐29 IEC. Western blot and immunofluorescence studies revealed that basal STAT1 phosphorylation (Y701) and claudin‐2 expression were increased in unchallenged PTPN2‐KD cells compared to control shRNA transfected cells, and this effect was greatly enhanced by IFN‐γ treatment. Protein analysis of soluble and insoluble fractions showed increased claudin‐2 in the insoluble fraction in KD cells compared to control cells. siRNA silencing of STAT1 mRNA levels significantly decreased claudin‐2 protein and mRNA expression. To confirm STAT binding to the claudin‐2 promoter region, we used Chromatin immunoprecipitation (ChIP) assay to immunoprecipitate STAT1 from IFN‐γ‐treated PTPN2‐KD and control IEC with anti‐STAT1 antibody, and probed the associated DNA with primers against the claudin‐2 promoter. ChIP analysis showed increased STAT1 binding to the claudin‐2 promoter region in KD cells compared to control cells. Next we determined if STAT1 activates claudin‐2 promoter activity through the STAT binding site. We performed a luciferase reporter assay using a human claudin‐2 promoter‐luciferase construct expressed in PTPN2‐KD or Con‐shRNA IEC. Luciferase activity was significantly increased (214 ± 9%, p<0.0001; n=3) in PTPN2‐KD cells over control IEC. In further studies using two different constructs of the claudin‐2 promoter region with mutations of the STAT binding site, there was a significant decrease in promoter activity (43±15%, p<0.0001; n=3) when compared to full length claudin‐2 promoter activity in KD cells, but no significant difference with control cells. In summary, we have demonstrated that STAT1 regulates claudin‐2 expression through binding to its promoter region under conditions of decreased activity of the IBD candidate gene, PTPN2.
ErbB3 negatively regulates homeostatic Paneth cell numbers
Authors: Almohazey Dana , Hsieh Jonathan J , Vossler Claire V , Frey Mark R
Published: November 2023

Paneth cells (PCs) are located at the base of the intestinal crypt, where they support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by secreting antimicrobial peptides (AMPs). Loss of or functional defects in PCs are seen in Crohn's disease, linking abnormalities in these cells to inflammatory bowel disease (IBD). We recently showed that activation of the neuregulin receptor ErbB4 protects PCs against injury. However, the role of ErbB3, the other neuregulin receptor, in PCs is unknown. In this study we tested the effects of ErbB3 activation or deletion on PC numbers and AMP production.

METHODS

Ileal tissue and enteroid cultures derived from C57BL/6 (WT), ErbB4flox/flox, ErbB4flox/flox; Villin‐Cre (E4KOIE), ErbB3flox/flox, and ErbB3flox/flox; Villin‐Cre (E3KOIE) mice were used. Enteroids were given NRG1β (ErbB3/4 ligand; 10ng/ml) or NRG4 (ErbB4 ligand; 100ng/ml) for 24 h and RNA was extracted. Fixed tissue for immunofluorescence analysis, RNA for qPCR, and protein lysates for western blotting were collected from mice.

RESULTS

NRG1β treatment of WT enteroids for 24 h resulted in reduced expression of Lyz1 (by 29.47%, p=0.03) and Defa5 (by 46.60%, p= 0.001), and fewer lysozyme‐positive cells (by 18.03%, p=0.03). This effect of NRG1β was maintained in E4KOIE enteroids but was not recapitulated in NRG4‐treated WT enteroids, suggesting a specific role for ErbB3. Consistently, E3KOIE intestinal crypts displayed an increased number of lysozyme‐positive cells (by 25.36%, p=0.001) and MMP7/MUC2 double positive intermediate/precursor cells (by 16.24%, p=0.011). E3KOIE crypts also had increased Lyz1 (by 67.53%, p<0.0001) and Lgr5 (by 46.65%, p=0.012) RNA expression. This increase was not accompanied by differences in expression of other ErbB family members (Egfr, ErbB2, and ErbB4) or other secretory cell markers (Muc2 and Chga). Interestingly, E3KOIE mice had a significant increase in expression of the secretory regulator Atoh1 (by 59.20%, p=0.0009), which is required for PC development. Since MAPK signaling has been implicated in PC regulation, we performed western blot analysis for P‐Erk1/2 and P‐p38 on tissue lysates, but there was no difference between WT and E3KOIE mice. In contrast, E3KOIE mice had a lower level of P‐Akt (by 53.1%, p=0.02), suggesting potential involvement of the PI3K pathway.

CONCLUSIONS

These results suggest that ErbB3 restricts PC numbers by a mechanism that may involve PI3K/Akt and Atoh1. Understanding the role of ErbB3 receptor could identify new therapeutic targets for regulating PCs and the ISC niche.

Support or Funding Information

This work was supported by a Senior Research Award from the Crohn's and Colitis Foundation of America and by NIH award R01DK095004.
Metformin enhances intestinal epithelial health through activation of AMPK
Authors: Xue Yansong , Sun Xiaofei , Zhu Mei‐Jun
Published: November 2023

The intestinal epithelium is a vital barrier that is consistently renewed. The balance among proliferation, migration, and differentiation are tightly regulated, which is critical for intestinal epithelial homeostasis. The objective of this study was to evaluate the beneficial role of metformin, a widely used antidiabetic drug, on epithelial health using interleukin‐10 deficient (IL10KO) mice. Metformin supplementation increased the abundance of E‐cadherin, a core component of the adhesion junctions, in both WT and IL10KO mice. In addition, metformin supplementation suppressed macrophage pro‐inflammatory activity in IL10KO mice as indicated by reduced M1 macrophage abundance as well as decreased pro‐inflammatory cytokine IL‐1b, TNF‐a and INF‐g expression. As a target of metformin, AMP‐activated protein kinase (AMPK) phosphorylation was enhanced, which might explain the suppression of inflammation in metformin group. Bone morphogenetic protein (BMP) signaling is one of the key pathways regulating gastrointestinal development and homeostasis. Consistently, metformin supplementation enhanced BMP signaling in IL10KO mice. Furthermore, the expression of two key transcription factors promoting epithelial differentiation, Hes1 and Math, was promoted by metformin. To confirm the mediator role of AMPK, we further over‐expressed AMPK in Caco‐2 cells and found that the expression of BMP4 and the phosphorylation of Smad, which is a down‐stream effector of BMP signaling, was enhanced in AMPK over‐expressed Caco‐2 cells. In addition, AMPK activity was correlated with the mRNA expression and/or protein content of Notch1, Math, Hes1, which are key transcription factors regulating epithelial differentiation. In conclusion, our data demonstrated that metformin suppresses inflammation and enhanced intestinal epithelial differentiation via enhancing AMPK signaling.

Support or Funding Information

R15HD073864
Suppressive effect of nicotine on expression of MAdCAM‐1 in colitis
Authors: Maruta Koji , Kurihara Chie , Furuhashi Hirotaka , Takajo Takeshi , Yasutake Yuichi , Okada Yoshikiyo , Yoshikawa Kenichi , Higashiyama Masaaki , Watanabe Chikako , Komoto Shunsuke , Tomita Kengo , Nagao Shigeaki , Miura Soichiro , Hokari Ryota
Published: November 2023

Introduction

Adhesion molecules play important roles in inflammatory bowel disease (IBD), and modulation of gut‐specific adhesion molecules like MAdCAM‐1 and α4β7‐integrin could be a new therapeutic target. Epidemically, nicotine absorbed by smoking is reported to work protectively in some ulcerative colitis cases. We investigated the effect of nicotine on dextran sulfate sodium (DSS)‐induced murine colitis in point of adhesion molecules.

Aim

We investigated the effect of nicotine on the following things; 1) symptoms of murine colitis; 2) leukocyte adhesion to colonic microvascular endothelium; 3) expression of α4β7‐integrin on lymphocytes; and 4) expression of adhesion molecules on endothelium.

Method

1) C57BL/6J mice were treated with 3% DSS (40kDa) in drinking water for 7 days. Some mice were treated with 0.1 mg/ml nicotine in drinking water simultaneously. Disease activities of colitis were assessed by DAI score, which consists of body weight loss, stool consistency and bleeding. 2) C57BL/6J mice were treated with 0.1mg/ml nicotine in drinking water 1 day after 3% DSS treatment for 7 days. Mice were injected FITC‐labeld splenocytes (3×107 dissoleved in 0.3ml) obtained from another mouse, via cervical vein. The number of splenocytes adhering to colonic submucosal vasculature were counted with an intravital fluorescence microscope. 3) C57BL/6J mice were treated with 0.1mg/ml nicotine in drinking water 1 day or for 7 days. The expression of α4β7‐integrin on splenocytes was analyzed with a flow cytometer. 4) The bEnd.3 cells, a cell line derived from murine endothelioma, were treated with 5 ng/ml TNF‐α for 12 hours. Some groups were treated with 1 mM nicotine simultaneously. The mRNA expression of MAdCAM‐1 was measured by quantitative RT‐PCR.

Result

Nicotine treatment significantly decreased DAI score induced by DSS. In intravital microscopic study, the number of splenocytes adhering to vascular endothelium was increased by DSS treatment, and the increase was significantly attenuated by additional nicotine treatment. Expression of α4β7‐integrin on splenocytes was not affected by nicotine. In bEnd.3 cells, nicotine treatment significantly attenuated the increased expression of MAdCAM‐1 induced by TNF‐α.

Conclusion

Enhanced expression of adhesion molecules, especially MAdCAM‐1, on the microvascular endothelium in inflamed condition, was significantly inhibited by nicotine‐treatment. This study showed that nicotine might have a protective effect on inflammation of colonic mucosa of IBD via modulation of expression of MAdCAM‐1.

Support or Funding Information

None
Mechanism of copper uptake from ceruloplasmin by cells and the involvement of an additional copper uptake transporter
Authors: Dinh Lauren , Juarez Jacqueline , Vargas Rebecca , Ramos Danny , Mar David , Ishida Michael , Linder Maria C
Published: November 2023

Ceruloplasmin (Cp) is the most abundant copper‐containing protein in the blood plasma. Though well known for its ferroxidase activity, it also has other functions. We have now demonstrated it is a circulating source of copper for cells, using 64Cu‐labeled Cp purified from mouse plasma, and mouse embryonic fibroblasts (MEFs) that do and do not express the only as yet identified copper uptake transporter in mammalian cells, CTR1. During copper delivery, Cp was converted from the holo (Cu‐containing) to the apo form, as demonstrated by immunoblotting of native PAGE gels, and 64Cu‐uptake was not reduced by inhibitors of endocytosis. Uptake of Cp‐Cu was significantly greater in cells expressing vs not expressing Ctr1, indicating that Ctr1 can take up copper from Cp. However, most of the uptake was through an as yet unidentified transporter, that like Ctr1 preferred Cu(I) and was inhibited by Ag(I). Attempts to cross‐link Cp to this transporter in the Ctr1−/− MEFs did not result in the identification of plausible transporter proteins. To improve chances of cross‐linking sufficient protein for identification by MS‐MS, studies with plasma membrane fractions from brains and hearts of rats incubated at 4ºC with purified 67Cu‐labeled rat plasma Cp were initiated (a method used previously to identify Cp “receptors”; BBRC 139: 822, 1986) and Cp‐binding to the membranes was confirmed. We conclude that uptake of copper from Cp occurs at the cell surface, with formation of apoCp and delivery of the copper to Ctr1 and an additional transporter.

Support or Funding Information

R15 GM100464 from the National Institutes of Health
MicroRNA Profiling and Regulation in Hypertensive Kidney
Authors: Weber Gregory , Pushpakumar Sathnur , Mukhopadhyay Manas , Sen Utpal
Published: November 2023

Hypertension is a major risk factor for chronic kidney disease and renal inflammation is an integral part in this pathology. In recent years, hydrogen sulfide (H2S) has emerged as an important regulator of blood pressure. Studies in spontaneously hypertensive rats have shown that endogenous hydrogen sulfide (H2S) production is impaired due to decreased CBS/CSE expression, and exogenous H2S supplementation decreased blood pressure and vascular remodeling. MicroRNAs are short, single‐stranded RNA genes that regulate post‐transcriptional gene expression by targeting messenger RNAs (mRNAs), and are associated with a broad spectrum of physiological processes. While there are several studies aimed at investigating the role of non‐coding RNA in hypertension, the roles and implications of these RNAs are not well defined. In this study, we sought to identify microRNAs that are dysregulated in response to angiotensin‐induced hypertension in the kidney and whether hydrogen sulfide donor, GYY 4137, could reverse the microRNA alteration. Wild‐type mice were treated without or with Ang‐II (1000ng/Kg/Min) and without or with GYY4137 for 4 weeks. Blood pressure, renal blood flow, renal resistive index (RI) measurements were performed. microRNA microarrays were conducted and subsequent target prediction revealed genes associated with proinflammatory response. Ang‐II treated mice showed a significant increase in blood pressure accompanied by a decrease in blood flow in the renal cortex and an increase in the resistive index. These effects were attenuated in mice treated with GYY4137. Microarrays of the kidneys revealed downregulation of miR‐129 and miR‐299b in Ang‐II treated mice and upregulation following GYY4137 treatment. In contrast, miR‐369 was upregulated in Ang‐II treatment and suppressed in Ang‐II+GYY4137 mice. Quantitation of genes involved in proinflammatory response revealed upregulation of Tnfα, Il1β, Mcp1 and Mip2 mRNA. Our data suggests that downregulation of miR‐129 and ‐299b plays a significant role in Ang‐II induced renal inflammation, and GYY4137 reduces inflammation and is associated with reversal of miR expression.

Support or Funding Information

NIH: HL104103, AHA: 15SDG25840013
The Otolithic Membrane Mediates Otolith Organization
Authors: Kramer Kenneth L. , Thiessen Kevin D.
Published: November 2023

Otoconia are small biocrystals that link mechanical forces to the sensory hair cells in the mammalian utricle and saccule, a process essential for sensing linear acceleration and gravity as well as maintaining bodily balance. In fish, structurally similar otoliths mediate both balance and hearing. While research across several vertebrates has identified at least seven homologous proteins that are found in both otoliths and otoconia, little is known about how and when these proteins termed otoconins are organized. The goals of this project were to begin to characterize otoconin organization in the zebrafish otolith, determine when otoconin organization occurs during development, and identify some of the cellular and molecular mediators of otoconin organization. Using antibodies that we generated to the otoconins Otoconin‐90 and Starmaker, we found that these otoconins initial colocalize in early otoliths. Remarkably, Otoconin‐90 and Starmaker expression gradually changed after attachment of the otolith to early tether cells to become complementary by 30 hours post fertilization. When tether cells were ablated, otoconin expression fails to organize; yet otoconin organization eventually occurred in some embryos when tether cells appeared later in development. We further demonstrated that expression of Otogelin and Otogelin‐like in the otolithic membrane are necessary for otolith attachment and otoconin organization since unattached otoconins aggregate in both morphant and mutant embryos. We conclude that zebrafish otolith organization during early development is mediated by Otogelin and Otogelin‐like expression on the tether cell surface.

Support or Funding Information

This project was supported by grants from the National Center for Research Resources (5P20RR018788‐09) and the National Institute of General Medical Sciences (8 P20 GM103471‐09) from the National Institutes of Health.
Effect of ADAM17 on EMMPRIN Expression after Vesicant Injury to the Cornea
Authors: Gordon Marion K , DeSantis‐Rodrigues Andrea , Chang Yokechen , Hahn Rita A , Zhou Peihong , Heindel Ned D , Gerecke Donald R , Svoboda Kathy K. H.
Published: November 2023

In a cancer cell line expressing EMMPRIN and ADAM17, it has been shown that inhibiting EMMPRIN expression has no effect on ADAM17 levels, but inhibiting ADAM17 expression significantly inhibits EMMPRIN expression (Xu et al. Tumour Biol. 35:7575‐75‐86, 2014). To verify that this sequence of events was not a phenomenon peculiar to cancer cells, but instead represented a cell's normal response to injury, we examined whether ADAM17 was activated quickly in corneas injured by nitrogen mustard (NM), and whether inhibiting ADAM17 activation had any effect on EMMPRIN expression. Rabbit corneal organ cultures were exposed to topical NM for 0 min (i.e., immediately washed off), or 5 min, or 10 min. NM was washed off at the appointed times and the corneal proteins extracted for ADAM17 activity assays using the Innozyme TACE activity assay kit. This revealed that ADAM17 activity was turned on immediately by NM exposure. We then looked at EMMPRIN in exposed corneas, as well as in exposed corneas where ADAM17 activity was inhibited by hydroxamate NDH4417 (N‐octyl‐N‐hydroxy‐2‐[4‐hydroxy‐3‐methoxyphenyl acetamide). Western analyses showed that inhibiting ADAM17 in exposed corneas reduced EMMPRIN levels by 10 fold, as compared to NM‐exposed corneas not treated with the hydroxamate. Furthermore, reducing EMMPRIN resulted in reduced MMP‐9 activity. These data support the idea that, upon injury, ADAM17 activation impacts EMMPRIN expression, and thereby plays a key role in the downstream activation of matrix metalloproteinase, such as MMP9.

Support or Funding Information

NIAMD U54AR055073 Counter ACT award and NIEHS P30ES005022 Center for Environmental Exposures and Disease
The Non‐Receptor Tyrosine Kinase, Src, and its Regulator PTP1B are Present at Tubulobulbar Complexes
Authors: Ho Clement Dallas , Lyon Kevin RP , Vogl A Wayne
Published: November 2023

During spermiation, the intercellular junctions between Sertoli cells and the spermatids must be internalized before the spermatid can be released into the lumen of the seminiferous tubule. Prior to release from the epithelium, the spermatids are engulfed in protrusions of Sertoli cell cytoplasm which are referred to as apical processes. Unique endocytic structures termed, tubulobulbar complexes (TBCs), internalize these intercellular junctions during sperm release. TBCs have three regions: a proximal tubule, a bulbar region and a distal tubule ending in a clathrin‐coated pit. At sites of intercellular junctions between Sertoli and sperm cells, TBCs form by invaginating into the Sertoli cell and dragging the spermatid's membrane along with it through its intercellular attachments. This results in the entire structure consisting of a double membrane. The tubular regions are surrounded by a dendritic actin network, which is absent at the bulbar region. The bulbar region is a swelling in the distal third part of the TBC and is closely associated with a cisterna of endoplasmic reticulum. Eventually the bulb region of the TBC vesiculates and enters the endosomal pathway of the Sertoli cell. It has been shown by immunoprecipitation that Src, a non‐receptor tyrosine kinase, associates with components of the intercellular junctions between Sertoli cells and spermatids. One of the substrates that Src is able to phosphorylate is cortactin, a component of the dendritic actin networks at TBCs. We suspect that Src is involved in regulating the actin network at TBCs. In this study, we used immunofluorescence to evaluate whether or not Src and its phosphatase regulator, protein tyrosine phosphatase 1b (PTP1B), are associated with TBCs. Rat testes were perfusion‐fixed and cryosectioned or fragmented to get epithelial fragments. Sections and fragments were probed with antibodies against Src or PTP1B and an appropriate fluorescent secondary antibody. The Src antibody labels linear tracts in apical processes that could represent the tubular regions of TBCs. In cryosections, the PTP1B antibody has a Sertoli cell‐labeling pattern that extends from the base of the seminiferous epithelium to the apex. In fragments and cryosections, the PTP1B antibody labels regions of the Sertoli cell where TBCs would be expected to form. This evidence suggests that Src and its regulator PTP1B are present at TBCs.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants program.
Sarco/Endoplasmic Reticulum Ca2+‐ATPase 2 (SERCA2) is Present at Tubulobulbar Complexes
Authors: Lyon Kevin RP , Vogl A Wayne
Published: November 2023

Endocytic structures known as tubulobulbar complexes (TBCs) internalize intact intercellular junction proteins at sites of intercellular attachment between spermatids and Sertoli cells prior to sperm release. TBCs are highly specialized clathrin‐based endocytosis machines. They also resemble podosomes which have an elongated membrane tube and an associated dendritic actin network. However, unlike podosomes that have a membrane core of a single plasma membrane, the core of TBCs consists of two plasma membranes: the membrane of the Sertoli cell which the structure is invaginated into and the membrane of a spermatid. TBCs have a bulb region in the distal third part of the complex which does not have the cuff of actin but is closely associated with a cisterna of endoplasmic reticulum (ER). We hypothesize that the plasma membrane/ER junction is involved in Ca2+ signaling. If this is true then we would expect to find molecular machinery involved in calcium signaling mechanisms in other systems. In this study, we used immunofluorescence to probe for the calcium pump, sarco/endoplasmic reticulum Ca2+ ‐ATPase 2 (SERCA2), which pumps Ca2+ into the ER. Cryosections and epithelial fragments from perfusion‐fixed rat testes were probed with antibodies against SERCA2 and an appropriate fluorescent secondary antibody. The SERCA2 antibody has a labeling pattern that extends from the base of the epithelium to the apex which is consistent with a Sertoli cell pattern. As seen in both epithelial fragments and cryosections, the SERCA2 antibody labels regions where we would expect TBCs to be. Our results show that one possible component of a calcium signaling mechanism is present in regions containing TBCs.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants program.
Development of the Epaxial Muscles in the Human Embryo
Authors: Mekonen Hayelom Kebede , Hikspoors Jill P.J.M , Mommen Greet , Köhler S Eleonore , Lamers Wouter H
Published: November 2023

Background

The appearance of the true muscles of the back is dominated by three muscle columns, but a division into a lateral sacrospinal compartment with longissimus (medial) and iliocostal (lateral) divisions, and a medial compartment with transversospinal muscles is presently more often used. Although back muscles are defined by their embryological origin and innervation pattern, no detailed study on their development is available.

Methods

Human embryos (5–10 weeks development) were studied, using Amira® and Cinema4D® remodeling software for visualization.

Results

At Carnegie Stage (CS)15, the epaxial portions of the myotomes became identifiable laterally to the developing vertebrae. At CS16, these portions fused to form a longitudinal muscle column, which became innervated by the dorsal branch of the spinal nerve. At CS17, the longitudinal muscle mass segregated into medial and lateral columns (completed at CS18). At CS18, the medial column segregated again into intermediate and medial columns (completed at CS20). Between CS20 and CS23, the cervical portions of the three columns segregated again form lateral to medial resulting ventrolaterally in rod‐like continuations of the caudal portions of the columns and dorsomedially in spade‐like portions. The lateral and intermediate columns did not separate in the lower lumbar and sacral region. The observed topography identifies the iliocostalis and splenius as belonging to the lateral column, the longissimus to the intermediate column, and the multifidus and (semi‐)spinalis to the medial column. The medial group acquired its transverso‐spinal course during closure of the vertebral arches in the early fetal period.

Conclusion

Epaxial muscle growth is characterized by cranio‐caudal and latero‐medial gradients in development. Our ontogenetic approach shows that all back muscles can be classified as components of the erector spinae.

Support or Funding Information

NFP (Netherlands Fellowship Programmes)
Thoracic Vertebral Morphology and Locomotor Adaptation in Anthropoid Primates
Authors: Sarrazin Blake D , Middleton Emily R , Ward Carol V
Published: November 2023

Thoracic shape is related to locomotor adaptation in anthropoid primates. Pronograde quadrupeds tend to have narrow, deep rib cages, whereas those of hominoids tend to be broader mediolaterally. Further, hominoids, especially humans, have the thoracic vertebral column invaginated into the rib cage to support orthograde posture. Humans, and to a lesser extent apes, also exhibit declination of the sternal ends of the ribs. Although the ribs may be the best indicator of overall thoracic shape, they are not well preserved as fossils and are not found in articulation with the vertebral column. However, thoracic form and vertebral invagination should be reflected in orientation of the transverse processes of the thoracic vertebrae, as they articulate with the ribs.

To test this hypothesis, we examined the fifth and ninth thoracic (T5, T9) vertebrae from 168 anthropoids. Landmark data were collected using a Microscribe G2X digitizer and analyzed using Polyworks software (Innovmetric, Inc.). Using this program, planes were fit to the superior surface of the vertebral body and to the median plane. A vector was fit between costovertebral and costotransverse articular facets, and used to measure the angles of dorsal and inferior inclination of the transverse process orientation. Statistical analysis of the data was performed using the R statistical software package.

As predicted, hominoids, in particular humans, have more dorsally inclined transverse processes than monkeys. However, the semisuspensory atelines are similar to hominoids in regards to this feature, suggesting a relationship between suspension and invagination of the thoracic column into the rib cage. Differences are greater in T9 than at T5. Hominoids, including humans, were found to have more horizontally projecting T5 transverse processes than monkeys. At T9, humans are even more horizontal than apes, and all hominoids have more horizontally projecting transverse processes than do monkeys. These patterns reflect variation in rib declination among taxa and show that a relatively more superior position of the costotransverse joints contributes to declination of the sternal end of the ribs. Comparing these relationships using in silico models of thoracic shape will further refine our understanding of the geometry of all of these elements together. Overall, our results support our hypothesis that the thoracic transverse processes are able to reflect ventral invagination of the vertebral column into the rib cage and variation in rib declination, and so are a useful indicator of thoracic form in fossil anthropoids.
Ontogenetic Shape Change in the Human Cranial Base and its Relationship to Positional Behavior
Authors: McGechie Faye R
Published: November 2023

The cranial base is frequently used to make inferences about upright posture in the hominin fossil record, particularly as it relates to the earliest evidence of bipedalism. There is contention, however, regarding which aspects of cranial base morphology best reflect upright posture. The goal of this study is to evaluate how changes in cranial base morphology may relate to changes in positional behavior throughout human ontogeny. A close relationship between positional behavior and cranial base morphology would support the use of similar morphological features to reconstruct posture in fossil hominins. The study sample included an ontogenetic series of crania (n=33) ranging in age from neonate to adult. Crania were grouped based on ages at which humans undergo changes in positional behavior. Three dimensional landmark data were acquired either from computed tomography scans or digitized from an archaeological collection. Data were analyzed using Morphologika2 and MorphoJ software. Specifically, Principal Components Analysis was used to determine differences in cranial base shape among age groups. Results demonstrate age‐related changes in nuchal plane length, anterior cranial base length, and occipital condyle position congruent with the acquisition of upright posture in human infants. This analysis supports the use of features such as cranial base length and occipital condyle position as a tool to reconstruct posture in fossil hominins and identifies future avenues of study.

Support or Funding Information

Funding for analysis provided by Barrett, the Honors College at Arizona State University