bluuebunny/crossref_metadata_2025 huggingface dataset:
GPCRs are regulators of MRTF‐A and YAP mediated CCN1 expression (1066.4)
Authors: Yu Olivia , Miyamoto Shigeki , Heller Brown Joan
Published: June 2021

Cysteine‐rich 61 (Cyr61/CCN1) is a multifunctional matricellular, secreted protein involved in myriad cellular processes including tumor cell growth, inflammation, and wound healing. It is part of a family of three immediate early genes all largely subserving regulatory rather than structural roles in signaling. CCN1 functions in the extracellular matrix through binding to integrins on the cell surface. The role of G‐protein coupled receptors (GPCRs) in CCN1 regulation has remained largely unexplored. However, recent evidence from our laboratory indicates that CCN1 is highly and rapidly induced via a subset of GPCRs, in particular the protease activated receptor (PAR1) thrombin receptor and the receptors for lysophospholipids, LPA and S1P through Rho activation. Myocardin‐related transcription factor A (MRTF‐A), a transcription coactivator has been shown to regulate CCN1 expression, as has Yes‐associated protein (YAP). Whether these transcriptional co‐activators work in parallel or in synergy to induce CCN1 expression is not known, nor are the distinct signals that activate these pathways defined. Here we report that stimulation of GPCRs that activate Rho leads to YAP dephosphorylation and MRTF‐A dissociation from the actin cytoskeleton, both of which result in nuclear translocation and gene activation. Whereas both responses are Rho‐dependent and inhibited with C3 exoenzyme, inhibiton of Rho kinase blocks MRTF‐A but not YAP activation. Studies using reporter genes confirm these findings. Knockdown of either MRTF‐A or YAP significantly reduces agonist induced CCN1 expression and we are currently examining other genes to determine whether regulation of multiple genes requires activation by these two signals. Our observations establish that MRTF‐A and YAP are activated through different signals downstream of RhoA, but CCN1 serves as a downstream effector where these two important transcriptional hubs converge.
Structural basis of membrane deformation by the ESCRT‐III family of proteins (107.4)
Authors: Frost Adam , Sundquist Wes , McCullough John , Saunders Marissa
Published: June 2021

I will report high resolution electron cryo‐microscopy (cryoEM) reconstructions of ESCRT‐III filaments in lipid‐free and membrane‐bound states. These structures reveal that homo‐ and hetero‐dimers are the basic protomers of ESCRT‐III filaments. This unexpected finding illuminates why ESCRT‐III proteins have preferred “pairings” in different biological contexts. For example, mammalian genomes contain ~12 ESCRT‐III proteins and, for example, the protein CHMP2 pairs with CHMP3 during MVB formation whereas IST1 pairs with CHMP1 during cell division. Incorporation of different subunits may alter the architecture of the ESCRT‐III lattice to change its curvature, rigidity or propensity to bundle for different processes. These and related models will be discussed in the context of new structural models.
CGRP release from perivascular capsaicin‐sensitive sensory nerves regulates vascular function through pannexin‐1 channel opening (1075.4)
Authors: Gaete Pablo , Lillo Mauricio , Poblete Inés , Figueroa Xavier
Published: June 2021

Resistance arteries are inervated by capsaicin‐sensitive sensory nerves (CSSNs) that release calcitonin gene‐related peptide (CGRP). CGRP is a vasodilator, but its participation in the regulation of vascular signaling has not been explored. We assessed if CSSNs regulate the expression of key proteins involved in the control of vascular function. The isolated mesenteric arterial bed of rat was perfused with 1 µM capsaicin for 20 min. Protein expression was evaluated by Western blot and cell permeability through hemichannels such as pannexin (Panx) channels was assessed by ethidium uptake. Expression of eNOS and Panx‐1, but not connexin (Cx) 32, Cx43 or Panx‐2, was reduced 3 h after capsaicin treatment. CGRP8‐37, a CGRP‐1 receptor blocker, or probenecid, a Panx‐1 channel inhibitor, prevented the reduction of eNOS and Panx‐1 levels. Capsaicin and CGRP (100 nM) increased ethidium uptake in mesenteric arteries, but only CGRP in cultured endothelial cells. Capsaicin‐ or CGRP‐induced ethidium uptake was prevented by CGRP8‐37, probenecid, KT‐5720 (PKA inhibitor) or NG‐nitro‐L‐arginine (NOS blocker). These data suggest that transient activation of CSSNs leads to down‐regulation of eNOS and Panx‐1 expression by a Panx‐1 channel‐dependent pathway. Panx‐1 channel opening depends on CGRP‐1 receptor activation, which initiates a PKA‐mediated signaling and NO production.

Grant Funding Source: Supported by FONDECYT 1100850 and AT 24121388.
LRRC26 is a functional auxiliary γ subunit for BKCa channels in arterial smooth muscle cells (1077.1)
Authors: Evanson Kirk , Bannister John , Leo M , Jaggar Jonathan
Published: June 2021

Smooth muscle cell (myocyte) large‐conductance calcium (Ca2+)‐activated potassium (BKCa) channels are functionally significant modulators of arterial contractility. Leucine‐rich repeat containing (LRRC) proteins are novel auxiliary γ subunits that were recently identified to elevate the voltage‐sensitivity of recombinant or LNCaP cell BKCa channels. Here, we investigated the expression and physiological functions of LRRC26 in arterial myocytes. RT‐PCR and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence and immunofluorescence resonance energy transfer microscopy indicated that LRRC26 was primarily plasma membrane‐localized and in close spatial proximity to BKCa α subunits. LRRC26 knockdown using RNAi reduced arterial LRRC26, but did not alter BKCa α or β1 protein. Patch‐clamp electrophysiology demonstrated that LRRC26 knockdown reduced BKCa channel voltage‐ and apparent Ca2+‐sensitivity and transient BKCa current frequency and amplitude in arterial myocytes. LRRC26 knockdown increased myogenic tone over a range (40 ‐ 100 mmHg) of intravascular pressures and reduced functional responses to iberiotoxin, a BKCa channel inhibitor. In summary, we show that LRRC26 is expressed, associates with BKCa α subunits, and elevates channel voltage‐ and Ca2+‐sensitivity in arterial myocytes to induce vasodilation.

Grant Funding Source: Supported by NHLBI/NIH (HL67061 and HL110347 to J.H.J and HL116175 to K.W.E)
CaV3.2 knockout mice display enhanced myogenic tone due to reduced BKCa‐mediated feedback (1077.3)
Authors: Harraz Osama , Brett Suzanne , Wilson Sean , Welsh Donald
Published: June 2021

In resistance arteries, there is an emerging view that CaV3.2 channel restrains vascular constriction through a feedback response that involves ryanodine receptors (RyR), Ca2+ spark generation and the modulation of large‐conductance Ca2+ activated K+ channel (BKCa). In the present study, we tested this hypothesis by examining the vasomotor, electrophysiological and Ca2+ responses of mesenteric arterial smooth muscle isolated from wild type (WT) and CaV3.2 knockout (KO) mice. Using vessel myography, we first observed enhanced myogenic tone in KO arteries, a finding consistent with the loss of feedback response. Control myography and electrophysiological experiments subsequently confirmed the inability of Ni2+ (CaV3.2 blocker) to either constrict arteries or suppress T‐type currents in KO tissues. Line scan analysis, performed on opened arteries loaded with fluo‐4, illustrated the presence of Ca2+ sparks in all arteries with the subsequent application of Ni2+ only affecting WT arteries. Consistent with Ca2+ imaging, the frequency of BKCa‐induced spontaneous transient outward currents (STOC) dropped in WT but not KO cells upon the pharmacological suppression of CaV3.2 channel. Overall, our findings support the view that CaV3.2 channels drive a local Ca2+‐induced Ca2+ release‐like process that restrains arterial constriction through the induction of Ca2+ sparks and the activation of BKCa channels.
Reperfusion but not ischemia initiates ERS and inhibition of ERS protects the heart from reperfusion injury (1080.5)
Authors: Xu Zhelong , Xi Jinkun
Published: June 2021

Reperfusion but Not Ischemia Initiates ERS and Inhibition of ERS Protects the Heart from Reperfusion Injury Jinkun Xi, Zhelong Xu Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, China The exact role of endoplasmic reticulum stress (ERS) in myocardial ischemia/reperfusion injury remains unknown. We aimed to test if and how ERS contributes to the pathogenesis of ischemia/reperfusion injury in the heart. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion or sham operation. Myocardial biopsies were taken from ischemic area. Ischemia did not increase the levels of GRP‐78, an established marker of ERS, compared to the sham group. In contrast, GRP‐78 was markedly increased upon reperfusion (1.3, 1.5, 1.9, and 1.6‐fold increases at 10, 30, 60, and 120 min of reperfusion). These observations suggest that reperfusion but not ischemia triggers ERS. In support of this finding, electron microscopic analysis revealed a significant increase in the number of endoplasmic reticulum swelling in myocardium upon reperfusion but not during ischemia. To confirm that the above observations were of ERS origin, we tested if TUDCA, a classic inhibitor of ERS, prevents reperfusion‐induced GRP‐78 increase. Our data showed that TUDCA given at reperfusion prevented GRP‐78 increases, assuring that reperfusion indeed induces ERS. Interestingly, TUDCA also reduced infarct size in rat hearts subjected to 30 min ischemia plus 2 h of reperfusion (28.45± 2.9 % vs. 37.9 ± 3.1 % in control), suggesting that ERS during reperfusion is detrimental and prevention ERS leads to cardioprotection. These data suggest that reperfusion but not ischemia initiates ERS in isolated rat hearts and inhibition of ERS may serve as a unique strategy for prevention of reperfusion injury.
Effects of epicatechin on glucose transporters in cardiac tissue and peripheral nerves in type 2 diabetes (1086.5)
Authors: Gonzalez Nelson
Published: June 2021

An abnormally high level of blood glucose is the key characteristic of type 2 diabetes (T2D). Studies have established that this results from decreased translocation of glucose transporters. Due to the consequences of T2D, i.e. cardiovascular disease and diabetic neuropathy, two glucose transporters of interest are the type 4 (GLUT4) and the glucose transporter type 3 (GLUT3). T2D has been linked to energy deficiency leading to cardiovascular disease. A key protein in an insulin independent pathway is 5' adenosine monophosphate‐activated protein kinase (AMPK), which is directly involved with the translocation of GLUT4. Thus, one of the main objectives of this study is to investigate the translocation of GLUT4 and relative AMPK expression in T2D. Moreover, studies have shown that hyperglycemia can cause peripheral nerve damage. GLUT3 translocation is independent of insulin signaling and can lead to an accumulation of glucose resulting in damage to the nerves. Peripheral nerve damage triggers the innate immune response which exacerbates the degeneration of peripheral nerves. In particular, the pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) has been observed to play a role. Therefore, another objective of this study is to investigate the expression of GLUT3 and TNF‐α in T2D to evaluate the progression of diabetic neuropathy. Using an in vivo high fat, streptozotocin (STZ) induced T2D model, we will inspect whether (‐)‐epicatechin (EPI), a naturally occurring flavonoid with demonstrated anti‐diabetic properties, alters the relative expression of the aforementioned proteins. Using samples collected from the heart and sciatic nerves of these animals, biochemical and immunohistological analysis will be performed to evaluate the expression and location of the proteins of interest. Ultimately, these results will further validate the anti‐diabetic properties of EPI and provide crucial data on its relatively elusive mechanism.

Grant Funding Source: Supporter by Dr. Katrina Yamazaki and IMPACT LA
Fatal pulmonary arterial hypertension in female Fischer 344 rats (1089.20)
Authors: Crockett Edward , McLendon Jared , Alzoubi Abdallah , McMurtry Ivan , Stevens Troy
Published: June 2021

Background: Male Fischer 344 (F344) rats exposed to Sugen‐5416, hypoxia, and then normoxia (Su/Hx/Nx) develop severe pulmonary arterial hypertension that is accompanied by death within 8 weeks. The mechanism responsible for death in these animals is incompletely understood, although acute heart failure was suspected. Here, we test the hypothesis that development of pulmonary arterial hypertension is accompanied by decreased right ventricular perfusion, which corresponds with decreased right ventricular function and decreased cardiac output. Methods: Six female F344 rats were injected with Sugen‐5416 (20 mg/kg) followed by three weeks of hypoxia (10% normobaric oxygen) and then five weeks of normoxia (21% oxygen at sea level). Survival was plotted as a Kaplan‐Meier curve. Echocardiography was utilized to monitor the development and severity of pulmonary arterial hypertension. Microsphere analysis was used to assess coronary and microvascular perfusion in the right ventricle. Histology was used to determine right ventricular matrix deposition, and right ventricular hypertrophy was estimated using the Fulton index (RV/[LV+S]). Preliminary Results: Five of six female F344 PAH rats died within 5‐weeks following SU/Hx/Nx exposure. Echocardiography showed right ventricular dilation and septal encroachment on the left ventricle, as well as decreased cardiac output. RV/[LV+S] confirmed significant hypertrophy of the right ventricular wall. Microsphere analysis indicated a substantial decrease in right ventricular coronary and microvascular perfusion. Histological trichrome staining displayed increased extracellular matrix deposition surrounding coronary arteries and cardiac myocytes. Conclusion: Decreased coronary and microvascular perfusion, increased fibrosis of the hypertrophied right ventricle and decreased cardiac output contribute to maladaptive cardiac remodeling and death in female F344 rats with severe pulmonary arterial hypertension.

Grant Funding Source: Sponsred by NIH
Ribosome structural dynamics during translation (109.2)
Authors: Noller Harry , Zhou Jie , Lancaster Laura , Donohue John , Guo Zhuojun , Li Xun
Published: June 2021

During each step of elongation of the polypeptide chain, coupled translocation of mRNA and tRNAs through the ribosome is catalyzed by the GTPase elongation factor EF‐G. This complex mechanical process is accompanied by large‐scale structural changes in the ribosome, including intersubunit rotation and rotational movements of the head of the small subunit. The nature of these molecular movements can be pieced together from crystal structures of trapped translocation intermediates. The timing of the different dynamic events can be determined by FRET and other biophysical methods. A detailed description of the molecular mechanism of translocation is emerging from a combination of these different approaches.
Utilizing transgenic rats with eGFP‐tagged serotonergic (5‐HT) neurons to assess differential gene expression associated with age‐dependent changes in cellular CO2/H+ chemosensitivity (1092.7)
Authors: Puissant Madeleine M. , Mouradian Gary C. , Geurts Aron M. , Hodges Matthew R.
Published: June 2021

The ventilatory response to hypercapnia in rodents increases throughout postnatal development, which parallels the increases in cellular CO2 sensitivity of serotonergic (5‐HT) neurons in primary cultures. Herein we begin to test the hypothesis that specific changes in gene expression in 5‐HT neurons during development contribute to the changes in cellular and/or organismal CO2/pH sensitivity. To begin to test this, acutely dissociated medullary raphe neurons obtained from adult (P56) transgenic rats (n=2) that express enhanced green fluorescent protein (eGFP) in cells expressing Pet‐1 (ePet‐1:eGFP rats) were sorted into pools (n= 5000 cells) using Fluorescent‐Assisted Cell Sorting (FACS). Total RNA was then extracted, and RNA quality and abundance measured before quantifying transcript abundance (Δ ct) with qRT‐PCR of 5‐HT neuron‐specific (slc6a4 (SERT); slc18a2 ( VMAT2); Tph2, and Trh) or non‐5‐HT specific raphe (Gad2 and Hprt) genes. We found high expression of 5‐HT neuron‐specific genes in the eGFP+ cell pools, including TRH (∆ct 6.94), TPH (∆ct 6.95), SERT (∆ct 3.18), and VMAT2 (∆ct 2.27), with little or no detectable expression of these genes in eGFP‐ cell pools. Expression levels of Gad2 and the housekeeping gene Hprt were comparable in both eGFP+ and eGFP‐ cells. These data indicate effective sorting of 5‐HT and non‐5‐HT raphe neurons in adult transgenic rats, and allow for further studies utilizing RNA sequencing to identify, measure and compare age‐related differences in 5‐HT neuron and non‐5‐HT neuron raphe transcriptomes.

Grant Funding Source: NIH HL097033
The role of O‐linked‐N‐acetylglucosamine transferase in regulating autophagy (1094.7)
Authors: McLarty Jennifer , Paterson Andrew , Chatham John
Published: June 2021

OGT catalyzes the addition of β‐N‐acetylglucosamine (O‐GlcNAc) to the hydroxyl group to serine/threonine residues of nuclear and cytoplasmic proteins, which regulates many biological processes critical for normal cell function. Autophagy is a highly conserved cellular self repair and survival mechanism also critical for normal cell function. We have previously reported that increases in cellular O‐GlcNAc levels interfere with autophagic signaling; therefore, we hypothesized that protein O‐GlcNAcylation may also regulate autophagy. O‐GlcNAc turnover was modulated in cultured human embryonic kidney 293 (HEK293) cells by overexpressing either OGT or a dominant negative OGT (dnOGT). Autophagy was induced either by nutrient deprivation (i.e., ND, serum and glucose free media) or rapamycin (RAP, 100 nM) for 3 hours, which was associated with an increase in overall O‐GlcNAc levels as well as O‐GlcNAcylation of Beclin‐1. Overexpressing OGT increased basal O‐GlcNAc levels and was associated with a decrease in the autophagic response in both ND and RAP treatment. Overexpression of dnOGT decreased overall basal O‐GlcNAcylation as well as attenuated the autophagic response in both ND and RAP groups. This study demonstrates that normal O‐GlcNAc cycling may play a critical role in the regulation of autophagy and provides novel mechanism by which changes in cellular O‐GlcNAcylation mediate cell survival responses.

Grant Funding Source: Supported by NIH‐NHLBI grant RO1‐HL101192
Redox‐sensitive activation of PI3K/Akt pathway in RVLM on endoplasmic reticulum stress‐associated neurogenic hypertension (1095.10)
Authors: Chao Yung‐Mei , Chan Julie
Published: June 2021

We reported previously that endoplasmic reticulum (ER) stress in the rostral ventrolateral medulla (RVLM), where the sympathetic premotor neurons for maintenance of basal vasomotor tone reside, is engaged in neural mechanism of hypertension. We investigated in the present study the role of the PI3K/Akt signaling in RVLM in the redox‐sensitive and ER stress‐associated neurogenic hypertension. In comparison to normotensive WKY rats, expressions of PI3K and phosphorylated Akt were greater in RVLM of the spontaneously hypertensive rats (SHR), accompanied with increased expressions of ER stress markers, GRP78 and phosphorylated elF2α. Inhibition of PI3K expression in the RVLM of SHR resulted in a significant decrease in mean arterial pressure (MAP), alongside suppressions of the augmented ER stress. However, protect the RVLM from the ER stress with salubrinal had no effect on the augmented PI3K expression or AKT activity, although the MAP was decreased. Treatment of the superoxide dismutase mimetic, tempol, not only abrogated oxidative stress in RVLM, but also blunted the increased expression of PI3K/Akt signaling and ER stress markers, resulting in an decrease in MAP of the SHR. These results suggest that the redox‐sensitive activation of PI3K/Akt pathway in the RVLM may account for the ER stress‐associated neurogenic hypertension in the SHR. Supported by NMRPG8C0261 from National science council in Taiwan.
cAMP/PKA‐mediated regulation of amino acid metabolism in murine T‐lymphoma cells (1095.16)
Authors: Wilderman Andrea , Guo Yurong , Taylor Susan , Insel Paul
Published: June 2021

The second messenger cyclic AMP (cAMP), acting through PKA, influences gene and protein expression through CREB and other transcription factors. In order to define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we conducted quantitative proteomic studies of wild‐type (WT) and kin‐ (PKA‐null) S49 murine T lymphoma cells and compared the impact on protein expression of increases in cAMP by a cell‐permeable analog (8‐CPT‐cAMP, CPT). Numerous proteins involved in branched chain amino acid metabolism had markedly increased expression in WT S49 cells, but not kin‐ cells, treated with CPT for 16h. Expression of mRNA levels of these genes also increased in WT, but not kin‐, S49 cells in response to treatment with CPT. Consistent with this finding, incubation of WT S49 cells with CPT enhanced cell survival in growth media lacking glutamine, whereas kin‐ S49 cells show decreased viability in such media even in the presence of 8‐CPT‐cAMP. We conclude that cAMP acts via PKA to regulate expression of genes and proteins for branched chain amino acids, an action that may help protect cells from a decrease in viability in glutamine‐deficient media. Since not all of the cAMP/PKA‐regulated branched chain amino acid metabolism genes with increased expression have strong predicted binding of CREB, regulation of those genes by cAMP/PKA appears to occurs through as‐yet to be defined transcriptional pathways.

Grant Funding Source: Supported by HHMI (S. S.T.), and DK54441 (S.S.T.), GM066232 (P.A.I.) and HL107200 (P.A.I.) from NIH
Anti‐aging gene klotho regulates cellular antioxidant capacity via binding to S‐fromylglutathione hydrolase through the N285 glycosylation site (1095.3)
Authors: Xu Yuechi , Dutcher Lincoln , Sun Zhongjie
Published: June 2021

Background. Klotho is a recently‐identified aging‐suppressor gene. Mutation of klotho causes extensive premature aging phenotypes and shortens lifespan while overexpression of this gene extends lifespan by 20%. The secreted Klotho (Skl) circulates in the blood. However, the receptor of Skl is unknown. The purpose of this study is to identify the binding sites of Skl.

Methods & Results. We apply proteomics method to identify the associated proteins of Skl. Mass spectrometry anal[[Unsupported Character ‐ Codename ­]]ysis and coimmunoprecipitation identified a direct interaction between Skl and S‐formylglutathione hydrolase (FGH), which was confirmed by western blotting and immune fluorescent staining. Overexpression of Skl upregulated FGH protein levels in cells. In KL‐/‐ mice, FGH was decreased in kidneys and red blood cells. Skl decreased cellular O2‐ levels (DHE staining) and increased FGH activity (ratio of Glutathione/Glutathione disulfide). Knocked down FGH by siRNA abolished the Skl‐induced increase in anti‐oxidant activity, suggesting that the anti‐oxidant function of Skl is mediated by FGH. The point mutation experiment confirmed 3 N‐glycosylation sites Asp 161, 285 and 346 on Skl. The Asp 285 is the key site that determines Skl anti‐oxidant activity.

Conclusion. Skl exerts anti‐oxidant activity by binding to FGH. The Asp 285 is the key site to the anti‐oxidant activity of Skl.

Grant Funding Source: Supported by HL105302 and HL 102074.
Endothelial cell surface expressed chemotaxis and apoptosis regulator controls white adipose tissue lipolysis through modulation of PI3/AKT pathway (1095.8)
Authors: Kilari Sreenivasulu , Remadevi Indulekha , Sahoo Daisy , Ramchandran Ramani , Wilkinson George
Published: June 2021

Endothelial cell specific chemotaxis receptor (ECSCR), a cell surface glycoprotein is thought be specifically expressed in Endothelial cells (EC). ECSCR is involved in EC migration, apoptosis and proliferation (Verma et al., 2010). Our previous results indicate that VEGF stimulation induces ECSCR and VEGFR2 complex formation, while loss of ECSCR reduced VEGF induced VEGFR2 activation and impaired AKT and ERK phosphorylation (Kilari et al., 2013). Here, we demonstrate that ECSCR protein is highly expressed in white adipose tissues (WAT) and lungs of mice. Immunohistochemistry of mouse tissue sections reveal ECSCR protein in blood vessels of lung and skeletal muscles, and in precursors and mature white adipocytes. Furthermore, ECSCR protein increases with progression of adipocyte differentiation in 3T3L1 cell pre‐adipocyte model, suggesting that ECSCR contributes to adipocyte maturation. Therefore, we investigated the function of ECSCR in WAT and mice physiology using our ECSCR knockout model. Our results indicate that homozygous ECSCR null mouse are viable and fertile with elevated circulatory triglycerides and increased triglyceride partition into VLDL and free fatty acids without changes in total cholesterol. These results suggest increased lipolysis in ECSCR null mice. Interestingly, histology of WAT of ECSCR null mice shows fewer fully inflated “signet” adipocyte profiles, but no overt adipose pathology. In line with these results, WAT of ECSCR null mice show reduced AKT phosphorylation without changes in total AKT levels. Moreover, we demonstrate direct association of ECSCR and PTEN, a lipid phosphatase suppressor of AKT activation, which dephosphorylates PIP3, suggesting that loss of ECSCR directly modulates the AKT pathway. Taken together, these results indicate that ECSCR regulates adipose tissue lipolysis, via modulating PI3K/AKT pathway. Future studies will examine control of lipolysis in ECSCR deficient cells following adrenergic or insulin stimulation.
c‐Jun N‐terminal kinase inhibits TNFα‐induced NF‐κB activation through endosomal Nox1 in vascular smooth muscle cells (1096.4)
Authors: Choi Hyehun , Lamb Fred
Published: June 2021

Tumor necrosis factor‐α (TNFα), a pro‐inflammatory cytokine, can cause either vascular smooth muscle cell (VSMC) apoptosis (JNK‐dependent) or proliferation (NF‐κB‐dependent). We hypothesized that JNK modifies NADPH oxidase (Nox1)‐dependent NF‐κB stimulation by TNFα in VSMC. We quantified the impact of siRNA targeting JNK1 or 2 on TNFα dependent NF‐κB activation using an adenoviral reporter construct in cultured aortic VSMC from wildtype (WT) or Nox1 knockout (Nox1KO) mice. NF‐κB activation was potentiated 1.5 + 0.5 fold (n=6) by siRNA targeting JNK1 and 2 in WT cells, however, there was no effect on NF‐κB activation in Nox1KO cells (0.9 + 0.1 fold, n=4). NF‐κB activation was endocytosis‐dependent as it was blocked by dominant‐negative dynamin (Ad‐DynK44A, 61% reduction, n=4). JNK inhibition with SP600125 did not alter endocytosis of dextran‐conjugated Texas Red. Endosomal ROS production is required for NF‐κB activation by TNFα in VSMC. JNK regulates NF‐κB activation either at the level of Nox1 activation or at a step downstream of ROS. Reduction of NF‐κB activation by JNK may tip the balance of signaling to favor cell death over proliferation.

Grant Funding Source: Supported by American Heart Association
Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying ouabain‐triggered Ca2+ oscillations (1097.10)
Authors: Fontana Jacopo , Kruusmägi Markus , Brismar Hjalmar , Aperia Anita
Published: June 2021

Our group has studied the origin of Ca2+ oscillations triggered by the cardiotonic steroid ouabain, a highly specific ligand of Na+‐K+‐ATPase. It has been shown that these low frequency oscillations protect the cells from apoptosis by activating the NF κB/p65 survival subunit and inhibiting the intrinsic mitochondrial apoptotic pathway by reversing an imbalance between the apoptotic factor Bax and the antiapoptotic factor Bcl‐xL.

The aim of this study is to reveal the identity and role of the intracellular compartments that are involved in ouabain‐triggered Ca2+ oscillations.

Studies were first performed on COS7 cells which expressed genetic Ca2+ indicators targeted to mitochondria and cytosol. Following application of low doses of ouabain, we found that each cytosolic calcium wave was followed by a delayed mitochondrial calcium transient. The recovery of Ca2+ to normal levels was slower in mitochondria compared to cytosol, while the initial rate of increase was found to be similar in both compartments. Next, using an endoplasmic reticulum (ER)‐targeted genetic Ca2+ indicator, we observed rhythmic and simultaneous Ca2+ transients in both mitochondria and ER after ouabain application.

We propose that Ca2+ shuttling between the ER and mitochondria may have a pivotal role in the propagation of ouabain triggered slow Ca2+ oscillations.

Grant Funding Source: Swedish Research Council, Family Erling‐Persson Foundation, Märta and Gunnar V Philipson
MED28 regulates the development of epithelial‐mesenchymal transition and invasion in human lung cancer cells (1097.14)
Authors: Hsieh Nien‐Tsu , Li Chun‐I , Huang Chun‐Yin , Lee Ming‐Fen
Published: June 2021

The development of the epithelial tumors from epithelial‐mesenchymal transition (EMT) to metastasis includes degradation and remodeling of the extracellular matrix and acquisition of a motile and invasive phenotype. MED28, facilitating transcriptional activation as a Mediator subunit, is also found highly expressed in epithelial tumors. Recently, we have reported that MED28 enhanced migration and invasion in human MCF7 and MDA‐MB‐231 breast cancer cells. The aim of this current study is to explore the role of MED28 in EMT and invasion in human non‐small cell lung cancer (NSCLC). Suppression of MED28 reduced the expression of vimentin, a mesenchymal marker, in human A549 NSCLC cells. In addition, TNFα induced EMT, whereas MED28 knockdown alleviated the morphological alterations. MED28 modulated TNFα‐induced Matrigel invasion and NFκB functional activity as assessed by heterologous promoter/reporter assay. The increased expression of several TNFα‐enhanced proteins, including MMP9, vimentin, p‐p65, and pIκB, was suppressed upon MED28 knockdown. Out data indicate a role of MED28 in the development of EMT and invasion in human NSCLC cells.

Grant Funding Source: Supported by NSC101‐2320‐B‐309‐001 and NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee
Serotonin and its influence on feeding behavior of larval yellow fever mosquitoes (1100.6)
Authors: Onken Horst , Yuzon Pierre , Citozi Enri , Medina Edward , Moffett David
Published: June 2021

It has been demonstrated that serotonin (5HT) stimulates anterior midgut alkalinization, posterior midgut re‐acidification, midgut nutrient absorption, and midgut peristalsis in larval yellow fever mosquitoes (Aedes aegypti). The present study investigates, if serotonin also plays an essential role in the regulation of feeding behavior of larval mosquitoes. To this purpose, the feeding dives of fed or unfed larval yellow fever mosquitoes were observed and quantified at different water depth. Observations were repeated in the presence of 10 μM Methiothepin (antagonist of different metabotropic 5HT and dopamine receptors that had an influence on larval mosquito midgut acid/base transport), Dolasetron (antagonist of ionotropic 5HT3 receptors), or Dapoxetine (selective serotonin reuptake inhibitor). The results clearly show the expected dependence of feeding dive frequency on the depth of the water. The drugs that influence the action of serotonin modify the frequency of feeding dives in fed and unfed larvae in different ways, suggesting a rather complex involvement of serotonin in the regulation of feeding behavior.
Myocardial infarction rapidly induces diaphragm muscle weakness (1102.11)
Authors: Bowen T Scott , Mangner Norman , Werner Sarah , Glaser Stephanie , Schrepper Andrea , Doenst Torsten , Linke Axel , Schuler Gerhard , Adams Volker
Published: June 2021

Chronic heart failure (CHF) induced by myocardial infarction (MI) results in diaphragm muscle weakness, with increased inflammation and oxidative stress directly implicated. It remains unknown, however, if diaphragm muscle function is impaired immediately post‐MI and whether this is associated with increased markers of inflammation and oxidative stress.

Methods: Ligation of the left coronary artery to induce MI (n=21; confirmed by echocardiography) or sham operation (n=22) was performed on 8 wk old C57BL/6 mice. Three days later, in vitro isometric force of diaphragm muscle fibre bundles was assessed.

Results: Specific force was depressed (p<0.05) between frequencies of 80‐300 Hz post‐MI, as was maximal tetanic force (25±1 vs. 21±1 N/cm²). MI had no influence on local mRNA expression of TNF‐α and IL‐6, but both were increased (p<0.05) at the systemic level. Compared to sham, MI increased (p<0.05) mRNA expression and enzyme activity of xanthine oxidase (65% and 19%, respectively) and NADPH oxidase (140% and 44%, respectively), however antioxidant enzymes remained unchanged.

Conclusion: Diaphragmatic contractile dysfunction is rapidly induced following MI, and this is associated with increased systemic inflammation and markers of local oxidant production. These findings suggest diaphragm muscle weakness is early‐onset in CHF, potentially mediated by inflammation and oxidative stress.

Grant Funding Source: Supported by the Alexander von Humboldt Foundation
Mechanisms of skeletal muscle deformity in hyperhomocysteinemia (1102.24)
Authors: Veeranki Sudhakar , Givvimani Srikanth , Sathnur Pushpakumar , Metreveli Naira , Tyagi Suresh
Published: June 2021

Hyperhomocysteinemia (HHcy) is associated with elderly frailty, skeletal muscle malfunction and injury, reduced vascular integrity and function and mortality. Although HHcy was implicated in impairment of vasculogenesis after hind limb ischemia in murine models, the underlying mechanisms are unclear. We hypothesize that HHcy compromises perfusion, collateral formation and vasculogenesis through diminishing post‐ischemic vasculogenic responses in the muscle fibers. To test this hypothesis, we created femoral artery ligation in wild type (WT) and CBS+/‐ mice (a model for HHcy) and assessed tissue perfusion, collateral formation and skeletal muscle function using Laser Doppler perfusion imaging, barium angiography and fatigue tests. In addition, we evaluated post‐ischemic levels of VEGF, and levels of its muscle specific regulators: HIF‐1α and PGC‐1α in the skeletal muscle fibers. The observations indicated that dysregulation of VEGF, HIF‐1 alpha and PGC‐1 alpha levels in the CBS+/‐ mice ischemic skeletal muscles. Moreover, there was enhanced FOXO dependent atrogene transcription in the muscles and reduced muscle function after ischemia in the CBS+/‐ mice. These results suggest that HHcy adversely affects muscle specific ischemic responses and contribute to muscle frailty.

Grant Funding Source: 1RO1HL108621
Dysregulation of dysferlin during 7 days mechanical unloading is mediated by ROS in rat soleus muscle (1102.27)
Authors: Lee Yang , Lawler Matthew , Botchlett Rachel , Hord Jeff , Lawler John
Published: June 2021

Dysferlin plays an important role in membrane repair of damaged sarcolemma, is bound to neuronal nitric oxide synthase‐mu (nNOSµ) during disuse, and can be regulated by reactive oxygen species (ROS). Previously, we showed that oxidative stress increased caveolin‐3 which contributed to nNOSµ dislocation from the sarcolemma and thus atrophy. However, the underlying mechanisms that contribute to nNOS translocation and muscle atrophy remain unknown. We hypothesized that oxidative stress would affect dysferlin localization during mechanical unloading, and thus nNOSµ translocation. F344 rats were divided (n=9/group) into control (CON), hindlimb unloaded for 7days (7DHU), and 7D HU+ EUK (EUK) groups. The superoxide dismutase/catalase mimetic EUK‐134 was injected daily (3 mg/kg/d) beginning 24 hours prior to HU. Upregulated ROS sources included sarcolemma (Nox2) and mitochondria (Nox4) during 7DHU were downregulated by EUK‐134. Dystrophin and beta‐sarcoglycan are not altered during 7 days mechanical unloading period. However, nNOS was translocated from sarcolemma to sarcoplasm in 7DHU group. Dysferlin was also downregulated and disrupted from sarcolemma in 7DHU groups, which might exacerbate perturbation of the membrane environment. EUK‐134 partially prevented translocation or nNOS and dysregulation of dysferlin, suggesting changes in these proteins were redox dependent. Our data indicated that oxidative stress is involved in unloading‐induced nNOSµ translocation that is coupled with dysferlin in the rat soleus.

Grant Funding Source: Supported by NASA Space Biology grant (NNX12AR62G)
Quantitative phosphoproteomics studies reveal different signaling cascades in cultured distal convoluted tubular cells upon vasopressin or angiotensin II stimulation (1109.10)
Authors: Cheng Lei , Pistikun Trairak , Fenton Robert
Published: June 2021

The thiazide‐sensitive NaCl cotransporter (NCC) plays an important role in sodium reabsorption in the kidney distal convoluted tubules (DCT). Both angiotensin II (ANGII) and vasopressin (AVP) are hormones known to exert effects in this segment and induce phosphorylation of NCC, which regulates NCC function. However, the signaling pathway from AVP to NCC and possible interplay between AVP and ANGII signaling cascades remain unknown. Here we performed large scale quantitative phosphoproteomics of cultured mpkDCT cells to map global changes in protein phosphorylation events upon treatment with ANG II or the V2‐selective analog dDAVP (30min). Experiments were performed using the stable isotope labeling by amino acids in cell culture (SILAC) quantification approach. LC‐ESI‐MS/MS identified 21616 quantifiable phosphopeptides and 12848 different phosphorylation sites, of which 7149 were confidently assigned . Kinase prediction of the regulated phosphorylation sites using NetworKIN indicated that ANGII stimulation increased activity of the kinase families CDK, MAPKs or PAK, whereas dDAVP stimulation increased the activity of the kinases PKA, DMPK, PIM2, PAK, or CLK. Motif analysis indicated that RRXSXXX (PKA substrate motif) was a preferential target for upregulated phosphorylation sites upon dDAVP stimulation, whereas for downregulated phosphorylation sites XXXSPXX (CDK substrate motif) was more prominent. cAMP assays showed that dDAVP induced an increase in cAMP levels in mpkDCT cells, supporting the PKA signaling cascades identified from proteomic analysis. Our results suggest that highly distinct signaling cascades are activated in cultured mpkDCT cells upon acute treatment by either dDAVP or ANG II, analysis of which will likely identify novel mechanisms for NCC regulation.

Grant Funding Source: Supported by Lundbeck Foundation and the Danish Medical Research Foudation
Curcumin relaxes cholecystokinin‐ or KCl‐induced tension in male guinea pig gallbladder strips through multiple signaling pathways (1110.1)
Authors: Kline Loren , Karpinski Edward
Published: June 2021

Curcumin (diferuloymethane) is the active ingredient of the spice turmeric. In this laboratory, curcumin relaxed both cholecystokinin octapeptide‐ (CCK) and KCl‐induced tension in a concentration dependent manner. An in vitro technique was used to determine which second messenger system(s) mediated the relaxation. Paired t‐tests were used for statistical analysis. To determine if the PKA/cAMP second messenger system mediated the curcumin‐induced relaxation, PKA inhibitor 14‐22 amide myristolated (PKA‐IM; 180 nM) was used. PKA‐IM had no significant effect on the amount of curcumin‐induced relaxation. The PKC inhibitors bisindolymaleimide IV (0.5 μM) and chelerythrine Cl‐ (5 mM) were used together; a significant (p<0.01) reduction in the curcumin‐induced relaxation (46±6.1 vs. 32.8±3.9%) was observed. TEA (5 μM) significantly decreased (p<0.01) the amount of relaxation (31.2±2.4 vs. 23.8±1.5%). Adding curcumin prior to KCl significantly decreased (p<0.001) the KCl‐induced tension (0.84±0.07 vs. 0.63±0.04 g). Curcumin‐induced relaxation is mediated by the PKC second messenger system, blocking extracellular Ca2+ entry, and inhibition of K+ channels. . In conclusion, the curcumin‐induced relaxation is mediated by multiple signaling pathways.
Endogenous hydrogen sulfide decreases Rho kinase and cGMP‐specific phosphodiesterase (PDE5) activity and induces relaxation of colonic smooth muscle (1110.3)
Authors: Nalli Ancy , Kendig Derek , Mahavadi Sunila , Grider John , Murthy Karnam
Published: June 2021

H2S, synthesized endogenously from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS), play a role in the regulation of gastrointestinal (GI) motility and secretion. Neither the expression of enzymes nor the mechanism of action of H2S in GI smooth muscle is fully known. Methods: Expression of CSE and CBS was analyzed by RT‐PCR and western blot. The effect of H2S on carbachol (CCh)‐induced Rho kinase activity and sustained contraction, and nitric oxide‐induced PDE5 activity, cGMP formation and muscle relaxation was examined using L‐cysteine (activator of CSE) or NaHS (H2S donor). Results: CSE, but not CBS was expressed in colonic smooth muscle cells of mouse, rabbit and human. CCh‐induced contraction in muscle strips and cells, and increased Rho kinase activity were inhibited by L‐cysteine and NaHS in a concentration‐dependent manner. Inhibition of Rho kinase activity and muscle contraction by L‐cysteine (but not NaHS) was blocked by CSE siRNA and CSE inhibitor, propargylglycine, respectively. L‐cysteine and NaHS also inhibited PDE5 activity, and augmented cGMP formation and muscle relaxation leading to increased (35%) colonic pellet propulsion. Conclusion: Activation of H2S‐producing CSE causes muscle relaxation via a dual mechanism involving inhibition of Rho kinase and PDE5 activity leading to downregulation of RhoA/Rho kinase pathway and upregulation of cGMP/PKG pathway.

Grant Funding Source: DK‐28300
High resolution microscopy reveals claudin‐18 as a basolateral and not a tight junction‐associated protein in stomach (1111.6)
Authors: Hagen Susan , Ang Lay‐Hong , Zheng Yi
Published: June 2021

Background: Claudin‐18 (Cldn18) is the most highly expressed tight junction protein in the stomach. Cldn18, however, also localizes to the basolateral membrane of gastric epithelial cells. We aimed to determine whether cldn18 is actually a tight junction protein using high resolution microscopy techniques. Methods: Immunostaining for cldn18 and for ZO‐1, a well‐established protein that is limited to tight junctions, was done on sections from mouse stomach. Structured illumination microscopy (SIM) was used to examine the high resolution 3‐D pattern of cldn18 and ZO‐1 localization in parietal and chief cells. Results: 3‐D SIM data revealed that cldn18 is solely a basolateral membrane‐associated cldn that runs in discontinuous anastomosing bundles along the basolateral membrane (in a basket‐like configuration) to completely enclose parietal cells. Although still present, the discontinuous bundles of cldn18 are spaced further apart in chief cells. ZO‐1 in parietal cells localizes to a shallow cleft bounded by cldn18 while in chief cells, cldn18 spirals around ZO‐1 with wide spaces between each spiral. Conclusions: Despite current thinking, cldn18 is spatially distinct from the tight junction. Our data suggests that the regulation of cation permeation by cldn18 is due to specific structural constraints on ion movement through the paracellular pathway rather than by distinct properties of the tight junction.
Effect of the bile acids unconjugated and conjugated, chenodeoxycholic acid and deoxycholic acid, on epithelial tight junction barrier function in a human colon carcinoma cell line, T84 (1113.3)
Authors: French Sydney , Framarin Nicole , Badar Ayesha , Akhtar Imran , Ao Mei , Domingue Jada , Rao Mrinalini , Sarathy Jayashree
Published: June 2021

The human colon is exposed to cBA, and ucBA and excess BAs can increase fluid secretion and perturb the epithelial barrier. We showed in T84 cells (AJP 305: C447, ’13) that: a) BAs preferentially stimulate Cl‐ secretion from the basolateral surface (BLM); b) Overnight exposure (O/N) to proinflammatory cytokine (PiC), decreases transepithelial electrical resistance (TER) and then, luminal (AM) BA can stimulate Cl‐ secretion. We hypothesize that high AM [BAs] directly decrease TER, and this is enhanced by PiC. Confluent T84 cells (TER >1000 Ohm.cm2) were treated with 5‐500µM BAs for 0.5‐24h + PiCs (TNFα+IL‐1ß+IFNγ). Cell viability and toxicity were assessed by Accuri flow cytometry and lactate dehydrogenase respectively and barrier function by TER and dextran fluxes. Viability and cytotoxicity were dose and time dependent. UcBAs (500μM, O/N) had higher cytotoxicity (CDCA:28.6%; DCA:27.8%) than cBAs (TCDC:15.5%; TDC:13.3%). Physiological doses (5‐50μM) were less toxic (8‐10%). O/N PiC decreased TER by ~50% and 0.5mM CDCA (20’) decreased this further by 20%. AM, not BLM CDCA (0.5mM), increased (~2‐fold) 10‐kDa blue‐dextran AM to BLM flux over time (8h); PiC increased this further (+2‐fold). In summary, AM BAs alter epithelial barrier integrity allowing luminal BAs to access the BLM and this is enhanced by PiC. This will provide the basis for future studies on the role of BA‐induced diarrhea in inflammatory diseases.

Grant Funding Source: Supported by COS, Benedictine U.
The role of protein synthesis in saturated fatty acid‐induced ER stress in H4IIE liver cells (1116.7)
Authors: Estrada Andrea , Peelor Frederick , Wang Dong , Wei Yuren , Hamilton Karyn , Miller Benjamin , Pagliassotti Michael
Published: June 2021

Elevated fatty acids play a role in non‐alcoholic fatty liver disease (NAFLD). Disruption of endoplasmic reticulum (ER) homeostasis, or ER stress, is a characteristic feature of NAFLD. Elevated fatty acids, in particular saturated fatty acids, induce ER stress in numerous cell types including hepatocytes. However, the specific mechanisms that link fatty acids to ER stress in the liver are poorly understood. ER stress is the result of an imbalance between the protein load delivered to the ER from cellular protein synthesis, and the capacity of the ER to process, transport and/or degrade the client protein load. In this study, we examined the hypothesis that saturated fatty acid‐mediated ER stress resulted from increased cellular protein synthesis. H4IIE liver cells (n=3) were treated with palmitate (250µM), a saturated fatty acid that induces ER stress, oleate (250µM), an unsaturated fatty acid that does not induce ER stress, or a combination of both palmitate and oleate that does not induce ER stress. Protein synthesis was measured utilizing mass spectrometry based analysis of deuterated water incorporation into total cellular proteins and polysome profiling. Protein synthesis as determined by both techniques was not significantly different among groups. These data suggest that increased cellular protein synthesis does not mediate palmitate‐induced ER stress in H4IIE liver cells.

Grant Funding Source: Supported by National Institutes of Health, DK‐072017, AG031829‐01