Inhibition Of Calcium Release Research Articles

Dantrolene corrects cellular disease features of Darier disease and may be a novel treatment

Darier disease (DD) is a rare severe acantholytic skin disease caused by mutations in the ATP2A2 gene that encodes for the sarco/endoplasmic reticulum calcium ATPase isoform 2 (SERCA2). SERCA2 maintains endoplasmic reticulum calcium homeostasis by pumping calcium into the ER, critical for regulating cellular calcium dynamics and cellular function. To date, there is no treatment that specifically targets the disease mechanisms in DD. Dantrolene sodium (Dl) is a ryanodine receptor antagonist that inhibits calcium release from ER to increase ER calcium levels and is currently used for non-dermatological indications. In this study, we first identified dysregulated genes and molecular pathways in DD patient skin, demonstrating downregulation of cell adhesion and calcium homeostasis pathways, as well as upregulation of ER stress and apoptosis. We then show in various in vitro models of DD and SERCA2 inhibition that Dl aided in the retention of ER calcium and promoted cell adhesion. In addition, Dl treatment reduced ER stress and suppressed apoptosis. Our findings suggest that Dl specifically targets pathogenic mechanisms of DD and may be a potential treatment.

Ryanodine receptor 2 promotes colorectal cancer metastasis by the ROS/BACH1 axis.

There is no targeted therapy for KRAS proto-oncogene, GTPase (KRAS)-mutant metastatic colorectal cancer (mCRC) because the underlying mechanism remains obscure. Based on bioinformatic analysis, this study aims to elucidate a potential gene target for which an approved drug is available, and to reveal the function as well as the underlying mechanism of the candidate gene. Here, we identified that ryanodine receptor 2 (RyR2) expression was upregulated in KRAS-mutant mCRC, and that this promoted cancer cell metastasis. S107, an approved drug to inhibit calcium release from RyR2 in the clinic, inhibited cancer cell metastasis both in vitro and in vivo. High expression of RyR2 predicts poor survival in our patient cohort. CRC patients with serosa invasion and vascular tumor thrombus are characterized by high RyR2 expression. Analysis of expression profiles upon RyR2 knockdown and inhibition, revealed a set of metastasis-related molecules, and identified BTB domain and CNC homolog 1 (BACH1) as the main transcription factor regulated by RyR2. RyR2 regulates cellular reactive oxygen species (ROS) levels, which activates nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2) and HMOX1 expression, and thus BACH1 accumulation. Collectively, this study provides evidence that the RyR2/ROS/BACH1 axis may be a potential intervention target for CRC metastasis.

Human-induced pluripotent stem cell-derived keratinocyte-like cells for research on protease-activated receptor 2 in non-histaminergic cascades of atopic dermatitis.

Keratinocytes are the most abundant cells in the epidermis, and as part of the frontline immunological defense system, keratinocytes function as a barrier to exogenous attacks. Protease-activated receptor 2 (PAR2) is expressed in human keratinocytes and activated in several inflammatory conditions, such as atopic dermatitis (AD). In this study, we demonstrated the differentiation of human induced pluripotent stem cell (iPSC) into keratinocytes by the improved robust procedure, and confirmed that human-induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express PAR2, which is activated by external addition of the ligand peptide and trypsin. The activation of PAR2 led to the release of calcium from intracellular calcium storage, followed by the release of the pro-inflammatory cytokine, tumor necrosis factor-alpha. Moreover, PAR2 antagonist, I-191, inhibited calcium release in a dose-dependent manner. This is the first study to demonstrate that iKera expresses a functional PAR2 protein. Furthermore, our results indicate cross-talk between the PAR2- and IL-4-mediated inflammatory axes in iKera, suggesting that iKera can be used as a platform for a broad range of mechanism-targeted drug screening in AD. Significance Statement This is the first study to provide evidence that human-induced pluripotent stem cell-derived keratinocyte-like cells (iKera) express functional protease-activated receptor 2 (PAR2). Furthermore, we demonstrated in iKera that the IL-4 inflammatory axis can cross-talk with the PAR2 mediated inflammatory axis in keratinocytes. To the best of our knowledge, this is the first report to indicate that iKera can be used for research and as a drug screening platform for atopic dermatitis (AD).

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

In This Issue

HomeCirculation ResearchVol. 130, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published3 Mar 2022https://doi.org/10.1161/RES.0000000000000536Circulation Research. 2022;130:691is related toEro1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac ArrhythmiaGLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body InhibitionInhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan SyndromeGLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition (p 694)Gut hormone receptors in the carotid body may be treatment targets for metabolic syndrome, say Pauza et al.Download figureDownload PowerPointHypertension and diabetes are risk factors for cardiovascular disease (CVD) and yet, for many patients with these two conditions, lowering blood pressure and blood sugar is insufficient for eliminating the risk. Because both hypertension and diabetes are linked to increased sympathetic nerve activation, and because the carotid body (CB)—a clusters of sensory cells in the carotid artery—regulates sympathetic nerve activity, Pauza and colleagues investigated the CB’s possible involvement. They performed a transcriptome analysis of CB tissue from rats with and without spontaneous hypertension and found, among many differently expressed genes, that the transcript encoding glucagon-like peptide-1 receptor (GLP1R) was considerably less abundant in the hypertensive animals. This was of particular interest because the gut hormone GLP1 promotes insulin secretion and tends to be suppressed in type 2 diabetes. Moreover, GLP1R agonists are already used as diabetes treatments. The team now show that treating rat CBs with such an agonist suppresses sympathetic nerve activation and arterial blood pressure, suggesting such drugs may provide benefits in more ways than one, and that the CB could be a novel target for lowering CVD risk in metabolic syndrome.Ero1α-Dependent ERp44 Dissociation from RyR2 Contributes to Cardiac Arrhythmia (p 711)Blocking oxidoreductase Ero1α could reduce arrhythmia risk in cardiovascular disease, say Hamilton et al.Download figureDownload PowerPointThe oxidative stress associated with cardiovascular disease (CVD) can lead to heart arrhythmia and sudden death. At the cellular level, the increased oxidation in the sarcoplasmic reticulum (SR) triggers aberrant release of calcium—via the ryanodine receptor (RyR2) channel—and this in turn ramps up muscle contractions. However, the precise molecular details of the pathology are lacking. Cellular stress is known to raise the level of SR oxidoreductase Ero1α, which interacts with an inhibitor of calcium release called ERp44. Now, Hamilton and colleagues complete the molecular puzzle, showing that ERp44 normally binds and inhibits RyR2 in rat cardiomyocytes. The stress-induced increase in Ero1α expression (in hypertrophic rat cardiomyocytes) causes dissociation of ERp44 from RyR2, they report, thus boosting calcium release. Importantly, the team also showed that inhibiting Ero1α reduced oxidation in the SR, prevented ERp44-RyR2 disassociation and reduced RyR2-mediated calcium release. It also curbed arrhythmia in isolated hypertrophic hearts. Because myocardium from heart failure patients also exhibited increased Ero1α expression compared with controls, the authors say, Ero1α inhibition could be a clinical strategy for preventing CVD-associated arrhythmia.Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan Syndrome (p 728)Lim et al suggest cytokine IL-11 as a new target for the treatment of Marfan syndrome.Download figureDownload PowerPointPeople with the genetic connective tissue disorder Marfan syndrome (MFS) are typically tall and thin with long limbs, and are prone to skeletal, eye and cardiovascular problems including a life-threatening weakening of the aorta. While MFS patients commonly take blood-pressure lowering treatments to minimize the risk of aortic aneurysm and dissection, there is no cure for MFS nor even a targeted therapy. With the aim of finding novel MFS treatments, Lim and colleagues focused their attention on IL-11. This cytokine is strongly induced in vascular smooth muscle cells (VSMCs) upon treatment with the growth factor TGFβ—itself overactivated in MFS patients and considered a key feature of the syndrome’s molecular pathology. The team now show that IL-11 is indeed strongly upregulated in the aortas of MFS model mice and that genetically eliminating IL-11 in these animals can protect them against the aortic pathology they normally exhibit—including dilation, fibrosis, inflammation, elastin degradation and loss of VSMCs. Furthermore, treating MFS mice with an anti-IL-11 neutralizing antibody had the same beneficial effects. The results therefore suggest that inhibiting IL-11 activity could be a novel approach for protecting the aortas of MFS patients. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesEro1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac ArrhythmiaShanna Hamilton, et al. Circulation Research. 2022;130:711-724GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body InhibitionAudrys G. Pauza, et al. Circulation Research. 2022;130:694-707Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan SyndromeWei-Wen Lim, et al. Circulation Research. 2022;130:728-740 March 4, 2022Vol 130, Issue 5Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000536 Originally publishedMarch 3, 2022 PDF download Advertisement

Drug library screen identifies inhibitors of toxic astrogliosis

BackgroundMultiple sclerosis (MS) is a chronic neuroinflammatory disorder, in which activated immune cells directly or indirectly induce demyelination and axonal degradation. Inflammatory stimuli also change the phenotype of astrocytes, making them neurotoxic. The resulting ‘toxic astrocyte’ phenotype has been observed in animal models of neuroinflammation and in MS lesions. Proteins secreted by toxic astrocytes are elevated in the cerebrospinal fluid (CSF) of MS patients and reproducibly correlate with the rates of accumulation of neurological disability and brain atrophy. This suggests a pathogenic role for neurotoxic astrocytes in MS. MethodsHere, we applied a commercially available library of small molecules that are either Food and Drug Administration-approved or in clinical development to an in vitro model of toxic astrogliosis to identify drugs and signaling pathways that inhibit inflammatory transformation of astrocytes to a neurotoxic phenotype. ResultsInhibitors of three pathways related to the endoplasmic reticulum stress: (1) proteasome, (2) heat shock protein 90 and (3) mammalian target of rapamycin reproducibly decreased inflammation-induced conversion of astrocytes to toxic phenotype. Dantrolene, an anti-spasticity drug that inhibits calcium release through ryanodine receptors expressed in the endoplasmic reticulum of central nervous system cells, also exerted inhibitory effect at in vivo achievable concentrations. Finally, we established CSF SERPINA3 as a relevant pharmacodynamic marker for inhibiting toxic astrocytes in clinical trials. ConclusionDrug library screening provides mechanistic insight into the generation of toxic astrocytes and identifies candidates for immediate proof-of-principle clinical trial(s).

Inhibiting Calcium Release from Ryanodine Receptors Protects Axons after Spinal Cord Injury.

Ryanodine receptors (RyRs) mediate calcium release from calcium stores and have been implicated in axonal degeneration. Here, we use an intravital imaging approach to determine axonal fate after spinal cord injury (SCI) in real-time and assess the efficacy of ryanodine receptor inhibition as a potential therapeutic approach to prevent intra-axonal calcium-mediated axonal degeneration. Adult 6-8 week old Thy1YFP transgenic mice that express YFP in axons, as well as triple transgenic Avil-Cre:Ai9:Ai95 mice that express the genetically-encoded calcium indicator GCaMP6f in tdTomato positive axons, were used to visualize axons and calcium changes in axons, respectively. Mice received a mild SCI at the T12 level of the spinal cord. Ryanodine, a RyR antagonist, was given at a concentration of 50 μM intrathecally within 15 min of SCI or delayed 3 h after injury and compared with vehicle-treated mice. RyR inhibition within 15 min of SCI significantly reduced axonal spheroid formation from 1 h to 24 h after SCI and increased axonal survival compared with vehicle controls. Delayed ryanodine treatment increased axonal survival and reduced intra-axonal calcium levels at 24 h after SCI but had no effect on axonal spheroid formation. Together, our results support a role for RyR in secondary axonal degeneration.

Structural and Chemical Alterations to the Intestinal Mucus Barrier during Giardia duodenalis Infection

Background During the course of infection with the protozoan parasite Giardia duodenalis, both mucus production and structure of the mucus layers have been shown to be altered throughout the large and small intestines. This potentially has significant impacts on mucosal barrier function, predisposing a host to mucosal inflammation, which contributes both to Giardia pathogenesis and to development of post-infectious complications. We have attempted to characterize the mechanisms by which Giardia modulates mucus production and secretion by intestinal goblet cells, as well as chemical alterations to mucin proteins that occur in response to Giardia infection. Several effects have been found to be dependent on Giardia cysteine protease activity, suggesting a potential mechanism involving protease activated receptors. Methods The human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR). Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using WGA and qPCR was performed for Muc2 and Muc5ac in the small and large intestines. Expression of glycosyltransferase enzymes was assessed using qPCR. Results Giardia NF, S2, and WB, but not GSM, upregulate MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or ERK1/2 signaling in LS174T cells. Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non-infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice. Expression of the fucosyltransferase Fut2 was increased in the jejunums of WT mice, while the sulfotransferase Chst4 was decreased. Expression of the glycan core synthase C2GnT1 was increased in the WT jejunum, while C1GalT1 expression was slightly decreased in the jejunum and colon. Conclusions Giardia-induced alterations to mucin gene expression in vitro and in vivo are dependent on PAR2 signaling. Canonical PAR2 signaling, characterized by calcium release and ERK1/2 pathway activation, contribute to altered expression of the MUC2 mucin in colonic goblet cell cultures. Expression of several glycosyltransferases, responsible for the addition of glycan groups to intestinal mucins, are altered during Giardia infection in vivo, suggesting mucosal alterations may involve chemical modifications of mucin proteins.

A50 MODULATION OF GOBLET CELL ACTIVITY DURING GIARDIA DUODENALIS INFECTION: A ROLE FOR PAR2

Abstract Background Giardia duodenalis has been shown to alter the structure of the intestinal mucus layers during infection via obscure mechanisms. We hypothesize that goblet cell activity may be disrupted in part due to proteolytic activation of protease-activated receptor 2 (PAR2) by Giardia proteases, resulting in disruption of mucus production and secretion by intestinal goblet cells. Aims Characterize alterations in goblet cell activity during Giardia infection, focusing on the roles of Giardia protease activity and PAR2. Methods Chinese hamster ovary cells transfected with nano-luciferase tagged PAR2 were incubated with Giardia NF or GSM trophozoites. Cleavage within the activation domain results in release of enzymes into the supernatant. Luminescence in the supernatant was measured as an indication of PAR cleavage by Giardia. LS174T, a human colonic mucus-producing cell line, was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad-spectrum cysteine protease inhibitor, and LS174T were treated with a PAR2 antagonist, a calcium chelator, or an ERK1/2 inhibitor. Quantitative PCR (qPCR) was performed for the MUC2 mucin gene. Wild-type (WT) and PAR2 knockout (KO) mice were infected with Giardia. Colonic mucus was stained using fluorescein-coupled wheat-germ agglutinin (WGA), and qPCR was performed for Muc2 and Muc5ac. Results Giardia trophozoites cleaved PAR2 within the N-terminal activation domain in a cysteine protease-dependent manner. Cleavage was isolate dependent, with isolates that show higher protease activity cleaving at a higher rate. High protease activity Giardia isolates increased MUC2 gene expression in LS714T. This increase was attenuated by inhibition of Giardia cysteine protease activity, and by antagonism of PAR2, inhibition of calcium release, or inhibition of ERK1/2 activity in LS174T cells. Both Muc2 and Muc5ac expression were upregulated in the colons of WT mice in response to Giardia infection, while in the jejunum Muc2 expression decreased and Muc5ac expression increased. In KO, no changes in gene expression were seen in the colon in response to Giardia infection, while in the jejunum, Muc2 expression was unchanged and Muc5ac expression decreased. Both WT infected and KO noninfected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in KO infected mice. Conclusions PAR2 plays a significant role in the regulation of mucin gene expression in mice and in a human colonic cell line. Results suggest that Giardia cysteine proteases cleave and activate PAR2, leading to calcium release and activation of the MAPK pathway in goblet cells, ultimately leading to altered mucin gene expression. Findings identify a novel regulatory pathway for mucus production by intestinal goblet cells. Funding Agencies CAG, CCC

Alterations to Mucus Production and Secretion during Giardia Infection Involve Giardia Protease Activity and PAR2 Activation

BackgroundThe protozoan parasite Giardia duodenalis has been shown to disrupt the intestinal mucus barrier via unknown mechanisms, potentially disrupting host mucosal immunity and contributing to development of post‐infectious disorders. Protease‐activated receptor 2 (PAR2) has been identified as a potential target for secreted Giardia cysteine proteases, and may play a role in disruption of mucus production and secretion in the intestines.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR), and intracellular mucin content was quantified by staining with fluorescein‐coupled wheat germ agglutinin (WGA) and imaging using confocal microscopy.Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using WGA and qPCR was performed for Muc2 and Muc5ac in the small and large intestines.ResultsGiardia NF, S2, and WB, but not GSM, upregulate MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or ERK1/2 signaling in LS174T cells. Preliminary results suggest that WGA staining is decreased upon exposure of LS174T to Giardia NF trophozoites.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsGiardia‐induced alterations to mucin gene expression in vitro and in vivo are dependent on PAR2 signaling. Giardia cysteine proteases cleave and activate PAR2, leading to both calcium release and MAPK pathway activation, which in turn contributes to altered gene transcription. Further, Giardia induces altered mucus secretion in vitro, which may contribute to thinning of the colonic mucus gel in vivo. Further study is required to determine the role of PAR2 in mucus secretion during Giardia infection.Support or Funding InformationCrohn’s Colitis Canada

Transient Receptor Potential V Channels Are Essential for Glucose Sensing by Aldolase and AMPK

SummaryFructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD+ levels in aged muscles, rejuvenating the animals’ running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.

Palmitate is not an effective fuel for pancreatic islets and amplifies insulin secretion independent of calcium release from endoplasmic reticulum

ABSTRACTThe aim of the study was to determine the acute contribution of fuel oxidation in mediating the increase in insulin secretion rate (ISR) in response to fatty acids. Measures of mitochondrial metabolism, as reflected by oxygen consumption rate (OCR) and cytochrome c reduction, calcium signaling, and ISR by rat islets were used to evaluate processes stimulated by acute exposure to palmitic acid (PA). The contribution of mitochondrial oxidation of PA was determined in the presence and absence of a blocker of mitochondrial transport of fatty acids (etomoxir) at different glucose concentrations. Subsequent to increasing glucose from 3 to 20 mM, PA caused small increases in OCR and cytosolic calcium (about 20% of the effect of glucose). In contrast, the effect of PA on ISR was almost 3 times that by glucose, suggesting that the metabolism of PA is not the dominant mechanism mediating PA’s effect on ISR. This was further supported by lack of inhibition of PA-stimulated OCR and ISR when blocking entry of PA into mitochondria (with etomoxir), and PA’s lack of stimulation of reduced cytochrome c in the presence of high glucose. Consistent with the lack of metabolic stimulation by PA, an inhibitor of calcium release from the endoplasmic reticulum, but not a blocker of L-type calcium channels, abolished the PA-induced elevation of cytosolic calcium. Notably, ISR was unaffected by thapsigargin showing the dissociation of endoplasmic reticulum calcium release and second phase insulin secretion. In conclusion, stimulation of ISR by PA was mediated by mechanisms largely independent of the oxidation of the fuel.

Giardia‐Induced Alterations to Intestinal Mucus Production Involve Protease‐Activated Receptor‐2‐Mediated Activation of MAPK and Calcium Release

IntroductionDisruption of the intestinal mucus layers is associated with increased rates of infection and inflammation and has been implicated in a wide variety of intestinal disorders. Giardia duodenalis has been shown to disrupt mucus production and secretion [1] and may therefore be used as a model system to study regulation of these processes in health and disease.AimsDetermine the mechanisms involved in Giardia mediated disruptions to mucus production by intestinal goblet cells. The roles of protease‐activated receptor‐2 (PAR2) and Giardia cysteine proteases were investigated.MethodsThe human colonic epithelial cell line LS174T was infected with Giardia trophozoites (isolates NF, WB, S2, and GSM). Prior to infection, trophozoites were treated with E64, a broad‐spectrum cysteine protease inhibitor, and LS174T were treated with a pepducin PAR2 antagonist, BAPTA, a calcium chelator, or U0126, an ERK1/2 inhibitor. MUC2 mucin gene expression was assessed using quantitative PCR (qPCR).Chinese hamster ovary cells transfected with nano‐luciferase tagged PAR2 were incubated with Giardia trophozoites. Release of enzymes due to cleavage at the receptor N‐terminus by Giardia resulted in a luminescent signal proportional to the number of cleaved receptors.Wild type (WT) and PAR2 deficient (PAR2 −/−) mice were infected with Giardia trophozoites. The colonic mucus layer was stained using fluorescein‐coupled wheat germ agglutinin, and qPCR was performed for Muc2 and Muc5ac.ResultsGiardia isolates NF, S2, and WB, but not GSM, increased MUC2 expression in LS174T cells. This increase was attenuated by inhibition of Giardia cysteine proteases and by antagonism of PAR2 or inhibition of calcium release or MAPK signaling in LS174T cells.Giardia trophozoites cleaved PAR2 at the N‐terminus, suggesting they are capable of activating the receptor. The amount of cleavage was isolate‐dependent, reflecting differences in protease activity, and cleavage was significantly reduced by E64 treatment for all isolates.Giardia infected WT mice show increased Muc2 and Muc5ac expression in the colon, and increased Muc5ac but decreased Muc2 expression in the jejunum. These changes were not seen in PAR2−/− mice. Both WT infected and PAR2−/− non‐infected mice showed thinning of the colonic mucus layer compared to WT controls. There was some recovery in thickness in PAR2−/− infected mice.ConclusionsResults demonstrate that PAR2 plays a significant role in the regulation of mucin gene expression in mice and a human colonic cell line, and indicate a potential pathway in which Giardia cysteine proteases cleave PAR2 at the N‐terminus, activating canonical PAR2 signaling which involves both calcium release and MAPK activation. These pathways in turn contribute to altered mucin gene expression.Support or Funding InformationCrohn's Colitis Canada (CCC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bile Canaliculi Contractility Is Regulated by Canalicular Pressure Sensing via PIEZO1

Hepatocytes have the ability to form tubular compartment known as bile canaliculi (BC). These BC are secretory lumens which expand and contract to propel bile, failure to which can lead to cholestasis and jaundice. However, the mechanism of BC expansion and contraction is unknown. Previously, we have reported that the dynamic nature of BC is an interplay of the canalicular pressure and the pericanalicular actomyosin. Here we examined the role of BC calcium in regulating the BC contractions using various calcium probes and live imaging of BC contractions. We find that the BC contractions are associated with the release of Ca2+ from BC and transient increase of Ca2+ in the cytoplasm near canaliculi. Furthermore, we also found that the release of Ca2+ from BC is dependent on the membrane tension sensing via mechanosensitive cation channel: PIEZO-1, as the presence of 10μM GsMTx-4 (PIEZO-1 inhibitor) inhibit calcium release from the BC and BC contractility is enhanced in presence of YODA1 (PIEZO-1 activator). BC expand due to secretion via transporter proteins. However, when a threshold volume is reached, increased membrane tension is sensed by PIEZO-1 and it open up to release Ca2+ into cell. This Ca2+ causes actomyosin to contract BC and this cycle repeats itself to periodically expand and contract BC. This novel mechanism present in BC to regulate of its own contractility by releasing Ca2+ in the cell above a certain membrane tension provides a mechanism to make this system autonomous. This study also opens up new avenues to study similar autonomous contractility in other lumenal structures present in salivary glands, mammary glands and pancreas.

A calcium sensor calcineurin B-like 9 negatively regulates cold tolerance via calcium signaling in Arabidopsis thaliana

ABSTRACTCalcineurin B-like protein 9 (CBL9) plays important roles in response to ABA, K+ deprivation in plants. However, whether CBL9 modulates plant adaptation to low-temperature stress is elusive. In this study, we demonstrated that the cbl9 mutants increased freezing tolerance under both cold-acclimating and nonacclimating conditions in Arabidopsis. Cold-induced changes of cytosolic free calcium concentration ([Ca2+]cyt) were then monitored by aequorin-expressed Arabidopsis plants. The results showed that the cold-triggered increases in [Ca2+]cyt levels in cbl9 mutants were clearly higher than those in wild type (WT) plants, while cold-affected changes in free calcium concentration within cytosolic microdomains adjacent to the vacuolar membrane ([Ca2+]md) in cbl9 mutants were similar to those in WT plants. In addition, treatments of seedlings with Ca2+ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and Ca2+ channel blocker lanthanum chloride markedly inhibit changes of [Ca2+]cyt in cbl9 mutants, while the inhibition of calcium release by lithium chloride from intracellular pools demonstrated consistent suppression of [Ca2+]cyt in cbl9 mutants and WT plants. Together, these results indicate that CBL9 negatively modulates cold tolerance through decreasing [Ca2+]cyt in Arabidopsis.

Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model.

The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate. Hearts were arrested and stored in Celsior preservation solution supplemented with 0.2–40 μM dantrolene for 6 hours at 4°C, then reperfused (15 min Langendorff, 30 min working mode). In 3-month hearts, supplementation with 1 μM dantrolene significantly improved aortic flow and cardiac output compared to unsupplemented controls however lactate dehydrogenase (LDH) release and contraction bands were comparable. In contrast, 40 μM dantrolene-supplementation yielded poor cardiac recovery, increased post-reperfusion LDH but reduced contraction bands. All 3-month hearts stored in dantrolene displayed significantly reduced cleaved-caspase 3 intensities compared to controls. Analysis of cardioprotective signalling pathways showed no changes in AMPKα however dantrolene increased STAT3 and ERK1/2 signaling in a manner unrelated to functional recovery and AKT activity was reduced in 1 μM dantrolene-stored hearts. In contrast to 3-month hearts, no significant improvements were observed in the functional recovery of 12-month hearts following prolonged storage in 1 μM dantrolene. Conclusions: Dantrolene supplementation at 1 μM during hypothermic heart preservation improved functional recovery of young, but not older (12 month) hearts. Although the molecular mechanisms responsible for dantrolene-mediated cardioprotection are unclear, our studies show no correlation between improved functional recovery and SAFE and RISK pathway activation.

Slow calcium waves mediate furrow microtubule reorganization and germ plasm compaction in the early zebrafish embryo.

Zebrafish germ plasm ribonucleoparticles (RNPs) become recruited to furrows of early zebrafish embryos through their association with astral microtubules ends. During the initiation of cytokinesis, microtubules are remodeled into a furrow microtubule array (FMA), which is thought to be analogous to the mammalian midbody involved in membrane abscission. During furrow maturation, RNPs and FMA tubules transition from their original distribution along the furrow to enrichments at the furrow distal ends, which facilitates germ plasm mass compaction. We show that nebel mutants exhibit reduced furrow-associated slow calcium waves (SCWs), caused at least in part by defective enrichment of calcium stores. RNP and FMA distal enrichment mirrors the medial-to-distal polarity of SCWs, and inhibition of calcium release or downstream mediators such as Calmodulin affects RNP and FMA distal enrichment. Blastomeres with reduced or lacking SCWs, such as early blastomeres in nebel mutants and wild-type blastomeres at later stages, exhibit medially bundling microtubules similar to midbodies in other cell types. Our data indicate that SCWs provide medial-to-distal directionality along the furrow to facilitate germ plasm RNP enrichment at the furrow ends.

Calcium release from intracellular stores is involved in mitochondria depolarization after lowering extracellular pH in rat brain synaptosomes

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.

AIP limits neurotransmitter release by inhibiting calcium bursts from the ryanodine receptor

Pituitary tumors are frequently associated with mutations in the AIP gene and are sometimes associated with hypersecretion of growth hormone. It is unclear whether other factors besides an enlarged pituitary contribute to the hypersecretion. In a genetic screen for suppressors of reduced neurotransmitter release, we identified a mutation in Caenorhabditis elegans AIPR-1 (AIP-related-1), which causes profound increases in evoked and spontaneous neurotransmitter release, a high frequency of spontaneous calcium transients in motor neurons and an enlarged readily releasable pool of vesicles. Calcium bursts and hypersecretion are reversed by mutations in the ryanodine receptor but not in the voltage-gated calcium channel, indicating that these phenotypes are caused by a leaky ryanodine receptor. AIPR-1 is physically associated with the ryanodine receptor at synapses. Finally, the phenotypes in aipr-1 mutants can be rescued by presynaptic expression of mouse AIP, demonstrating that a conserved function of AIP proteins is to inhibit calcium release from ryanodine receptors.

Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets.

Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway i.e., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times vs. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.