Calcium Pathways Research Articles

Pic Protein From Enteroaggregative E. coli Induces Different Mechanisms for Its Dual Activity as a Mucus Secretagogue and a Mucinase.

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other E. coli pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP3, a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.

DLDTI: a learning-based framework for drug-target interaction identification using neural networks and network representation

BackgroundDrug repositioning, the strategy of unveiling novel targets of existing drugs could reduce costs and accelerate the pace of drug development. To elucidate the novel molecular mechanism of known drugs, considering the long time and high cost of experimental determination, the efficient and feasible computational methods to predict the potential associations between drugs and targets are of great aid.MethodsA novel calculation model for drug-target interaction (DTI) prediction based on network representation learning and convolutional neural networks, called DLDTI, was generated. The proposed approach simultaneously fused the topology of complex networks and diverse information from heterogeneous data sources, and coped with the noisy, incomplete, and high-dimensional nature of large-scale biological data by learning the low-dimensional and rich depth features of drugs and proteins. The low-dimensional feature vectors were used to train DLDTI to obtain the optimal mapping space and to infer new DTIs by ranking candidates according to their proximity to the optimal mapping space. More specifically, based on the results from the DLDTI, we experimentally validated the predicted targets of tetramethylpyrazine (TMPZ) on atherosclerosis progression in vivo.ResultsThe experimental results showed that the DLDTI model achieved promising performance under fivefold cross-validations with AUC values of 0.9172, which was higher than the methods using different classifiers or different feature combination methods mentioned in this paper. For the validation study of TMPZ on atherosclerosis, a total of 288 targets were identified and 190 of them were involved in platelet activation. The pathway analysis indicated signaling pathways, namely PI3K/Akt, cAMP and calcium pathways might be the potential targets. Effects and molecular mechanism of TMPZ on atherosclerosis were experimentally confirmed in animal models.ConclusionsDLDTI model can serve as a useful tool to provide promising DTI candidates for experimental validation. Based on the predicted results of DLDTI model, we found TMPZ could attenuate atherosclerosis by inhibiting signal transductions in platelets. The source code and datasets explored in this work are available at https://github.com/CUMTzackGit/DLDTI.

Evaluation for the Genetic Association between Store-Operated Calcium Influx Pathway (STIM1 and ORAI1) and Human Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Infection.

Simple SummaryIn this study, we systematically evaluated the genetic susceptibility between the store-operated calcium (SOC) influx pathway (stromal interaction molecule 1 (STIM1) and ORAI1) and human hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB) infection. In total, 3631 patients with CHB were recruited, forty polymorphisms of STIM1 and ORAI1 were comprehensively analyzed. Three single-nucleotide polymorphisms (SNPs) of STIM1 (rs6578418, rs11030472, and rs7116520) and one SNP of ORAI1 (rs6486795) showed a trend of being significantly associated with HCC. In particular, our functional studies (images from total internal reflection fluorescence microscopy and transwell migration assay) revealed that calcium (Ca2+) signaling is essential for the migration of HCC. Based on such a comprehensively screening in 3631 patients with chronic hepatitis, our results indicate the important role of store-operated calcium pathways in HCC.Hepatocellular carcinoma (HCC) often develops from chronic hepatitis B (CHB) through replication of hepatitis B virus (HBV) infection. Calcium (Ca2+) signaling plays an essential role in HBV replication. Store-operated calcium (SOC) channels are a major pathway of Ca2+ entry into non-excitable cells such as immune cells and cancer cells. The basic components of SOC signaling include the STIM1 and ORAI1 genes. However, the roles of STIM1 and ORAI1 in HBV-mediated HCC are still unclear. Thus, long-term follow-up of HBV cohort was carried out in this study. This study recruited 3631 patients with chronic hepatitis (345 patients with HCC, 3286 patients without HCC) in a Taiwanese population. Genetic variants of the STIM1 and ORAI1 genes were detected using an Axiom CHB1 genome-wide array. Clinical associations of 40 polymorphisms were analyzed. Three of the STIM1 single-nucleotide polymorphisms (SNPs) (rs6578418, rs7116520, and rs11030472) and one SNP of ORAI1 (rs6486795) showed a trend of being associated with HCC disease (p < 0.05). However, after correction for multiple testing, none of the SNPs reached a significant level (q > 0.05); in contrast, neither STIM1 nor ORAI1 showed a significant association with HCC progression in CHB patients. Functional studies by both total internal reflection fluorescence images and transwell migration assay indicated the critical roles of SOC-mediated signaling in HCC migration. In conclusion, we reported a weak correlation between STIM1/ORAI1 polymorphisms and the risk of HCC progression in CHB patients.

Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Modulation of Calcium Homeostasis May Be Associated with Susceptibility to Renal Cell Carcinoma in Diabetic Nephropathy Rats.

IntroductionClinical studies have indicated a relationship between diabetic nephropathy (DN) and the incidence and prevalence of renal cell carcinoma (RCC). However, the mechanism linking diabetic nephropathy and renal cell carcinoma has not yet to be identified.MethodsIn this study, a total of 42 male Sprague Dawley (SD) rats were randomly assigned to a DN group (n=35) and a control group (n=7). All animals in the DN group were unilaterally nephrectomized and treated with streptozotocin with the development of blood glucose levels >16.7mmol/L and dominant proteinuria and were compared to controls without such changes. Histopathologic alterations in the kidneys were examined by HE staining and Ki-67 immunohistochemistry. Differentially expressed genes were identified and validated by RNA-seq and PCR.ResultsAs the results, except for two rats that failed to develop the DN model and were excluded from the analysis, 33 rats in the DN group with overt signs of DN demonstrated significantly higher food and water intake, urine production, and urine protein and urinary protein/creatinine ratio than controls. Overall, 15.2% (n=5/33) of DN animals developed RCC while none tumors were observed in the control group (n=0/7). RNA-seq analysis in these animals indicated different TRPV5 gene expression and calcium pathway expression in DN animals with developing tumors, when compared with animals with no obvious tumors. In addition, DN animals diagnosed with RCC showed increased expression of GLUT2 and c-met, when compared to controls and DN animals without tumors.DiscussionIn conclusion, the disordered calcium metabolism, especially disturbed TRPV5 mediated Ca2+ signal, may have been related to the development of RCC in DN rats. Further studies related to the detailed mechanism are still needed.

A transcriptional response atlas of Chrysanthemum morifolium to dodder invasion

Chrysanthemum (Chrysanthemum morifolium), as one of the most commercially important ornamental crops, is threatened by dodder (Cuscuta spp.) during cultivation. When chrysanthemum was parasitized by dodder, growth of chrysanthemum slowed down and the leaves became dry and yellow. In the study, RNA sequencing assays were used for elucidating the immune response of chrysanthemum after dodder invasion and a total of 20,789 genes were differentially expressed. KEGG enrichment analysis revealed that these differentially expressed genes were involved in multiple pathways, “plant hormone signal transduction”, “cutin, suberine and wax biosynthesis”, and “plant-pathogen interaction”, etc. Combined with weighted gene co-expression network analysis (hereafter named WGCNA), we found that when the vascular connections were formed through haustoria, chrysanthemum recognized and fended off pathogens by the two stages of plant immune systems (PTI and ETI). Membrane bound receptors RLKs and the intracellular NLR proteins perceived pathogen-derived proteins and delivered the defense signals to trigger a series of defense responses in chrysanthemum. Reactive oxygen species (ROS), calcium, and MAPK-related pathways were triggered and a series of defense genes were up‐regulated. Besides, in stems of chrysanthemum, ET and SA signal transduction pathways played vital roles and had strong regulatory relationship with each other in response to dodder invasion. Our study provides an understanding of how chrysanthemum responds to dodder’s invasion, which could lay the basis for further revealing the interaction mechanism between chrysanthemum and dodder.

Capacitation increases glucose consumption in murine sperm.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.

Abstract 3866: Investigating tumor infiltrating immune cells signature in obese triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers and TNBC patients are pathologically negative for estrogen receptor (ER−), progesterone receptor (PR−), and human epidermal growth factor receptor 2 (HER2−) amplification, which impedes the use of targeted therapies used in other breast cancer subtypes. Obesity is a chronic state of inflammation and is associated with increased secretion of pro-inflammatory cytokines, increased infiltration of immune cells and the development of a microenvironment that supports tumor growth. Several epidemiological studies support the association of obesity with TNBC; however, the underlining molecular mechanisms remain unknown. We developed an immunocompetent obese FVB (female) mouse model by feeding ‘Western diet' or control diet for four months and then injecting with syngeneic C0321 (mouse TNBC) cells to investigate the immune signatures and to study the role of obesity related factors in TNBC progression. We found significant body weight increases in ‘Western diet' fed mice group as expected. The percentage of CD8+ and CD4+ T cells, and macrophages in spleen, liver and peripheral blood in obese mice did not change significantly compared to lean mice. However, the percentages of immunosuppressive Myeloid Derived Suppressor cells (MDSCs), particularly Monocytic-MDSCs were elevated in liver and visceral fat tissue in obese mice. Importantly, tumors in ‘Western Diet' fed mice achieved significantly larger volumes. We found no noticeable difference in the percentage of CD8+ and CD4+ T cells, MDSC or macrophages in the spleen of tumor bearing obese mice. Interestingly, there was a decreasing trend in tumor infiltrating CD8+ and CD4+ T cells in tumor bearing obese mice. Further, tumor infiltrating myeloid cells, MDSCs and macrophages were significantly increased in obese mice, suggesting the formation of an immunosuppressive environment. More importantly, tumor infiltrating MDSCs from obese mice were more immunosuppressive than those from lean mice. In summary, our results reveal alterations in the number and function of tumor infiltrating immune cells in obese mice. RNA-Sequencing of lean vs obese tumors revealed significant changes in pathways related to Hypertrophic and Dilated Cardiomyopathy, Adrenergic signaling in Cardiomyocytes, as well as signaling pathways of Insulin, cGMP-PKG, Glucagon, Calcium and Adipocytokine and others. Our data warrants further investigation on the immunosuppressive tumor microenvironment in obese TNBC patients. Citation Format: Fokhrul Hossain, Deniz A Ucar, Maria Sanchez-Pino, Matthew Dean, Samarpan Majumder, Dorota Wyczechowska, Giulia Monticone, Rachel Sabol, Keli Xu, Luis D Valle, Jovanny Zabaleta, Bruce Bunnell, Lucio Miele. Investigating tumor infiltrating immune cells signature in obese triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3866.

Abstract 2575: Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas

Abstract Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here we report that normal breast epithelial cells are mechanically sensitive, responding to mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 minutes). This persistent calcium was sustained via microtubule-dependent mechano-activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on TRPM8 channels to prolong calcium signaling. In contrast, the introduction of a constitutively-active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a novel mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal new chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predicts poor clinical outcome in estrogen receptor (ER) negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal novel signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival. Citation Format: Stephen JP Pratt, Rachel M. Lee, Erick O. Hernandez-Ochoa, Eleanor C. Ory, Keyata N. Thompson, Patrick C. Bailey, Trevor J. Mathias, Julia A. Ju, Michele I. Vitolo, Martin F. Schneider, Joseph P. Stains, Christopher W. Ward, Stuart S. Martin. Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2575.

Molecular Signatures and Networks of Cardiomyocyte Differentiation in Humans and Mice

Cardiomyocyte differentiation derived from embryonic stem cells (ESCs) is a complex process involving molecular regulation of multiple levels. In this study, we first identify and compare differentially expressed gene (DEG) signatures of ESC-derived cardiomyocyte differentiation (ESCDCD) in humans and mice. Then, the multiscale embedded gene co-expression network analysis (MEGENA) of the human ESCDCD dataset is performed to identify 212 significantly co-expressed gene modules, which capture well the regulatory information of cardiomyocyte differentiation. Three modules respectively involved in the regulation of stem cell pluripotency, Wnt, and calcium pathways are enriched in the DEG signatures of the differentiation phase transition in the two species. Three human-specific cardiomyocyte differentiation phase transition modules are identified. Moreover, the potential regulation mechanisms of transcription factors during cardiomyocyte differentiation are also illustrated. Finally, several novel key drivers of ESCDCD are identified with the evidence of their expression during mouse embryonic cardiomyocyte differentiation. Using an integrative network analysis, the core molecular signatures and gene subnetworks (modules) underlying cardiomyocyte lineage commitment are identified in both humans and mice. Our findings provide a global picture of gene-gene co-regulation and identify key regulators during ESCDCD.

WNT Signaling in Melanoma.

WNT-signaling controls important cellular processes throughout embryonic development and adult life, so any deregulation of this signaling can result in a wide range of pathologies, including cancer. WNT-signaling is classified into two categories: β-catenin-dependent signaling (canonical pathway) and β-catenin-independent signaling (non-canonical pathway), the latter can be further divided into WNT/planar cell polarity (PCP) and calcium pathways. WNT ligands are considered as unique directional growth factors that contribute to both cell proliferation and polarity. Origin of cancer can be diverse and therefore tissue-specific differences can be found in WNT-signaling between cancers, including specific mutations contributing to cancer development. This review focuses on the role of the WNT-signaling pathway in melanoma. The current view on the role of WNT-signaling in cancer immunity as well as a short summary of WNT pathway-related drugs under investigation are also provided.

Postsynaptic expression of a new calcium pathway in hippocampal CA3 neurons and its influence on mossy fiber long-term potentiation.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is induced by postsynaptic Ca(2+) influx via NMDA receptors (NMDARs). However, this synaptic plasticity occurs independently of NMDARs when Ca(2+)-permeable AMPA receptors (AMPARs) are expressed at postsynaptic sites using various genetic techniques, indicating that an increase in Ca(2+) level at critical postsynaptic sites, regardless of its entry pathway, triggers the induction of LTP at CA1 synapses. In contrast, NMDARs are sparsely distributed on mossy fiber (MF) synapses in CA3 hippocampal neurons, and most evidence favors the presynaptic mechanism for LTP induction, although some reports suggested a postsynaptic mechanism. In this study, we examined whether Ca(2+) influx through the newly produced postsynaptic receptors during high-frequency stimulation affects the induction of MF LTP. For this purpose, we expressed Ca(2+)-permeable AMPARs in CA3 pyramidal neurons by Sindbis viral-mediated gene transfer of the unedited form of the glutamate receptor 2 (GluR2Q) subunit, as a new pathway for postsynaptic Ca(2+) entry, in rat hippocampal organotypic cultures. Virally expressed myc-tagged GluR2Q was detected at the complex spines known as the thorny excrescences, which serve as postsynaptic targets for MF synaptic input, on the proximal apical dendrites of CA3 pyramidal cells. Furthermore, endogenous Ca(2+)-impermeable AMPARs at MF synapses were converted into Ca(2+)-permeable receptors by GluR2Q expression. However, the postsynaptic expression of Ca(2+)-permeable AMPARs had no significant influence on the two types of MF LTP induced by different stimulus protocols. These results supported the notion that MF LTP is independent of postsynaptic Ca(2+).

Whole body deletion of Gpr68 does not change hematopoietic stem cell function.

G protein-coupled receptor 68 (GPR68) responds to extracellular protons, thus called the proton-sensing G protein-coupled receptor (GPCR), leading to activation of the phospholipase C-β (PLCβ)/calcium (Ca2+) pathway or the adenylyl cyclase (AC)/cyclic AMP (cAMP) pathway. We recently found that whole body deletion of Gpr68 (Gpr68-/- mice) reduced the number of B lymphocytes with age and during hematopoietic regeneration, such as in response to fluorouracil (5-FU) administration. This prompted us to characterize the hematopoietic stem cell (HSC) phenotype in Gpr68-/- mice. Despite high level of Gpr68 protein expression on HSC in bone marrow (BM), the pool size of HSC was unaltered in Gpr68-/- mice either under steady state or upon stress, including aging and 5-FU treatment. HSC from Gpr68-/- mice exhibited comparable cellular features, such as cell cycle quiescence and cell survival. HSC from Gpr68-/- mice also exhibited comparable competitiveness after serial transplantation. Surprisingly, cytosolic Ca2+ accumulation was increased in HSC from Gpr68-/- mice. In contrast, cAMP levels were reduced in hematopoietic stem and progenitor cells (HSPC) from Gpr68-/- mice. Intriguingly, we found high level of Gpr68 protein expression on non-hematopoietic cells in BM, especially endothelial cells that function as HSC niche. In addition, expression of other proton-sensing GPCR was upregulated in HSPC from Gpr68-/- mice. Our studies suggest that Gpr68-/- mice display insignificant phenotype on HSC biology, possibly due to the function of Gpr68 in non-hematopoietic cells and/or the compensatory effects from other proton-sensing GPCR.

Targeting Calcium Homeostasis in Myocardial Ischemia/Reperfusion Injury: An Overview of Regulatory Mechanisms and Therapeutic Reagents.

Calcium homeostasis plays an essential role in maintaining excitation–contraction coupling (ECC) in cardiomyocytes, including calcium release, recapture, and storage. Disruption of calcium homeostasis may affect heart function, leading to the development of various heart diseases. Myocardial ischemia/reperfusion (MI/R) injury may occur after revascularization, which is a treatment used in coronary heart disease. MI/R injury is a complex pathological process, and the main cause of increased mortality and disability after treatment of coronary heart disease. However, current methods and drugs for treating MI/R injury are very scarce, not ideal, and have limitations. Studies have shown that MI/R injury can cause calcium overload that can further aggravate MI/R injury. Therefore, we reviewed the effects of critical calcium pathway regulators on MI/R injury and drew an intuitive diagram of the calcium homeostasis pathway. We also summarized and analyzed calcium pathway-related or MI/R drugs under research or marketing by searching Therapeutic Target and PubMed Databases. The data analysis showed that six drugs and corresponding targets are used to treat MI/R injury and involved in calcium signaling pathways. We emphasize the relevance of further detailed investigation of MI/R injury and calcium homeostasis and the therapeutic role of calcium homeostasis in MI/R injury, which bridges basic research and clinical applications of MI/R injury.

P0421STUDY OF DIFFERENT EXPRESSION PROFILES OF “TRANSIENT RECEPTOR POTENTIAL ION CHANNEL (TRPC) GENE FAMILY” IN PATIENTS UNDERGOING RENAL BIOPSY WITH CLINICAL SUSPECTED GLOMERULONEPHRITIS

Abstract Background and Aims The transient receptor potential ion channels (TRPC) are non-selective Ca+ 2 permeable cation channels and are widely expressed in the tissues of vertebrates. They become active in response to signal transduction pathways associated with phospholipase C stimulation. TRPC6 is expressed in podocytes and is a component of the slit diaphragm. In genetic and acquired glomerular kidney diseases, TRPC6 overactivation causes podocyte damage by pathological Ca+ 2 entry. TRPC1, TRPC2, TRPC4, and TRPC5 interact with a protein called stromal-interacting molecule 1 (STIM 1), which is susceptible to intracellular calcium storage content. As a result of binding with this protein, TRPC channels bind to calcium-releasing zone in endosomes. The aim of the study was to investigate different expression profiles of TRPC family members in renal biopsy specimens in patients with clinically considered glomerulonephritis. Method This study was conducted with 108 patients admitted to Ege University Faculty of Medicine Nephrology Clinic who underwent a kidney biopsy with a preliminary diagnosis of glomerulonephritis and 37 patients who underwent a nephrectomy in urology clinic with a diagnosis of primary kidney tumour as a control case. PKD2, NPHS2, TRPC1, TRPC3, TRPC6, STIM-1 and Orai-1 mRNA levels were studied in the biopsy samples. Results When we compared the patient and the control group, the gender distribution of both groups was similar. The frequency of diabetes and hypertension was similar. The control group was statistically significantly older and glomerular filtration rates were higher than the patient group. In pathological examination, glomerulonephritis (57.5%) was diagnosed in the majority of patients. The most common etiologic factors were membranous nephropathy 23.1%, IgA nephropathy 13%, Amyloidosis 11.2%, focal segmental glomerulosclerosis 7.1%, proliferative glomerulonephritis 4.6%, minimal change disease 2.8% and membranoproliferative glomerulonephritis was 1.9%. 7.4% of the patients were diagnosed with diabetic nephropathy by renal biopsy. When we compared the TRPC expression profiles of the patient and the control group, the TRPC1, TRPC6, PKD2, NPHS2, STIM-1 and ORAi-1 mRNA levels of the patient group were statistically significantly higher than those of the control group (Figure). In contrast, TRPC3 mRNA levels were significantly lower in the patient group compared to the control group. When we performed subgroup analysis, the TRPC1, TRPC6 and STIM1 levels of the diabetic group were statistically significantly higher compared to the non-diabetic group of patients. We could not find any difference between TRPC expression profiles between the patients according to pathological diagnosis. Similarly, there was no difference between the amount of proteinuria (nephrotic level versus nephritic level). In correlation analysis, there was a negative correlation with TRPC6 and STIM1 levels with positive C4d staining of glomeruli in renal biopsy. Positive correlation was found between ORAI and glomerular sclerosis rate. Conclusion As a result, we found that other than TRPC3 mRNA level, other TRPC and related protein channels mRNA levels were statistically significantly increased in proteinuric kidney patients compared to healthy kidney tissue. We did not find a positive relationship between proteinuria severity and TRPC expression profiles. We found that TRPC6 and STIM1 expression levels were increased in diabetic patients, which supports the knowledge that intracellular calcium pathways were activated in podocyte damage. In our study, there were significantly increased STIM1 and ORAi-1 expression levels in proteinuric patients compared to the control group and their increase was closely related to TRPC6. From this we can conclude that these proteins play an important role in proteinuric kidney damage.

LncRNA BRCAT54 inhibits the tumorigenesis of non-small cell lung cancer by binding to RPS9 to transcriptionally regulate JAK-STAT and calcium pathway genes

Increasing evidence suggest that long non-coding RNAs (lncRNAs) play critical roles in cancers. However, the expression pattern and underlying mechanisms of lncRNAs in non-small cell lung cancer (NSCLC) remain incompletely understood. This study aimed to elucidate the functions and molecular mechanisms of a certain lncRNA in NSCLC. LncRNA microarray was performed to identify differential expressed lncRNAs between pre- and postoperation plasma in NSCLC patients. The expression level of candidate lncRNA in NSCLC tissues, plasma and cells was determined by quantitative real-time PCR (qRT-PCR) and in situ hybridization. The functional roles of lncRNA were assessed in vitro and in vivo. Furthermore, RNA pull-down, RNA immunoprecipitation, microarray, qRT-PCR and rescue assays were conducted to explore the mechanism action of lncRNA in NSCLC cells. We identified a novel lncRNA (BRCAT54), which was significantly upregulated in preoperative plasma, NSCLC tissues and NSCLC cells, and its higher expression was associated with better prognosis in patients with NSCLC. Overexpression of BRCAT54 inhibited proliferation, migration and activated apoptosis in NSCLC cells. Conversely, knockdown of BRCAT54 reversed the suppressive effects of BRCAT54. Moreover, overexpression of BRCAT54 repressed NSCLC cell growth in vivo. Mechanistically, BRCAT54 directly bound to RPS9. Knockdown of RPS9 substantially reversed the promoting effects of si-BRCAT54 on cell proliferation and enhanced the inhibitive effect of si-BRCAT54 on BRCAT54 expression. In addition, silencing of RPS9 activated JAK-STAT pathway and suppressed calcium signaling pathway gene expressions. This study identified BRCAT54 as a tumor suppressor in NSCLC. Targeting the BRCAT54 and RPS9 feedback loop might be a novel therapeutic strategy for NSCLC.

Targeting mitochondrial calcium pathways as a potential treatment against Parkinson’s disease

Parkinson’s disease (PD) is a major health problem worldwide affecting millions of people and is a result of neurodegeneration in a small part of the brain known as substantia nigra pars compacta. Aberration in mitochondrial Ca2+ homeostasis plays, among several other factors, an important role for the neuronal loss in PD. Mitochondria are vital for cellular physiology, e.g. for ATP generation, and mitochondrial Ca2+ is a key player in cell functioning and survival. Mitochondrial Ca2+ homeostasis is maintained by a fine balance between the activities of proteins mediating the influx and efflux of Ca2+ across mitochondrial membranes. Malfunctioning of these proteins leading to Ca2+ overload promotes ROS generation, which induces cell death by triggering the opening of mitochondrial permeability transition pore. Till now PD remains incurable and the “gold standard” drug which can only delays the disease progression is l-Dopa from the 1960s and therefore, the situation warrants the search for novel targets for the treatment of the PD patients. In this review, we summarize the current views that suggest mitochondrial Ca2+ regulatory pathways are good candidates for the treatment of PD.

Multiomics Analyses Identify Genes and Pathways Relevant to Essential Tremor.

The genetic factors and molecular mechanisms predisposing to essential tremor (ET) remains largely unknown. The objective of this study was to identify pathways and genes relevant to ET by integrating multiomics approaches. Case-control RNA sequencing of 2 cerebellar regions was done for 64 samples. A phenome-wide association study (pheWAS) of the differentially expressed genes was conducted, and a genome-wide gene association study (GWGAS) was done to identify pathways overlapping with the transcriptomic data. Finally, a transcriptome-wide association study (TWAS) was done to identify novel risk genes for ET. We identified several novel dysregulated genes, including CACNA1A and SHF. Pathways including axon guidance, olfactory loss, and calcium channel activity were significantly enriched. The ET GWGAS data found calcium ion-regulated exocytosis of neurotransmitters to be significantly enriched. The TWAS also found calcium and olfactory pathways enriched. The pheWAS identified that the underexpressed differentially expressed gene, SHF, is associated with a blood pressure medication (P = 9.3E-08), which is used to reduce tremor in ET patients. Treatment of cerebellar DAOY cells with the ET drug propranolol identified increases in SHF when treated, suggesting it may rescue the underexpression. We found that calcium-related pathways were enriched across the GWGAS, TWAS, and transcriptome. SHF was shown to have significantly decreased expression, and the pheWAS showed it was associated with blood pressure medication. The treatment of cells with propranolol showed that the drug restored levels of SHF. Overall, our findings highlight the power of integrating multiple different approaches to prioritize ET pathways and genes. © 2020 International Parkinson and Movement Disorder Society.

Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection.

Interferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

Regulator of G protein Signaling 10 (RGS10) functionally interacts with store‐operated calcium entry machinery to regulate COX‐2 and TNF‐α in Microglia

Activation of microglia is a driving factor of neuroinflammation and a hallmark of neurodegenerative diseases. The central inflammatory enzyme cyclooxygenase 2 (COX‐2) and the pro‐inflammatory cytokine tumor necrosis factor‐alpha (TNF‐α) mediate critical features of microglial activation responses, and their upregulation in the brain is a characteristic feature of many neurodegenerative diseases and brain injury. The Regulator of G protein Signaling 10 (RGS10), a small RGS protein enriched in microglia, has G protein‐independent functions in suppressing lipopolysaccharide (LPS)‐induced expression of COX‐2 and TNF‐α. Herein, we identify a biochemical and functional interaction between RGS10 and the endoplasmic reticulum (ER)‐localized calcium sensor STIM2, which is an essential component of the store‐operated calcium entry (SOCE) machinery. We determined that STIM2 and its downstream calcium pathway, particularly Orai channels and the Ca2+‐dependent phosphatase calcineurin, are essential for LPS‐induced COX‐2 and TNF‐α production in microglia, and likewise this pathway mediates the inhibitory effect of RGS10 on LPS‐signaling. We further demonstrated that ER calcium depletion amplifies LPS‐induced COX‐2 and TNF‐α expression and that RGS10 functions to suppress SOCE triggered by ER depletion. Collectively, this physical and functional interaction between RGS10 and STIM2 in microglia suggest a complex regulatory network connecting RGS10, LPS‐signaling, and SOCE, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.Support or Funding InformationNIH‐NINDS