Expression of canonical transient receptor potential (TRPC) proteins in human glomerular mesangial cells

Dept. of Integrative Physiology, Univ. of North Texas Health Science Center, Fort Worth, TX 76107, USA.
American journal of physiology. Renal physiology (Impact Factor: 3.25). 07/2006; 290(6):F1507-15. DOI: 10.1152/ajprenal.00268.2005
Source: PubMed


Mesangial cells are located within glomerular capillary loops and contribute to the physiological regulation of glomerular hemodynamics. The function of mesangial cells is controlled by a variety of ion channels in the plasma membrane, including nonselective cation channels, receptor-operated Ca2+ channels, and recently identified store-operated Ca2+ channels. Although the significance of these channels has been widely acknowledged, their molecular identities are still unknown. Recently, the members of the canonical transient receptor potential (TRPC) protein family have been demonstrated to behave as cation channels. The present study was performed to identify the isoforms of endogenous TRPC proteins in human mesangial cells (HMCs) and their interactions. Western blotting showed that TRPC1, 3, 4, and 6 were expressed in cultured HMCs. Consistently, immunofluorescent confocal microscopy revealed specific stainings for TRPC1, 3, 4, and 6 with predominant intracellular localization. However, TRPC5 and 7 were not detectable at protein level by either Western blotting or immunofluorescent staining. The expression of TRPC1, 3, 4, and 6 was also observed in rat and human glomeruli using fluorescent immunohistochemistry. Furthermore, coimmunoprecipitation experiments and immunofluorescent double staining displayed that TRPC1 had physical interaction with TRPC4 and 6, while no interactions were detected among other isoforms of TRPCs. Ca2+ fluorescent ratiometry measurement showed that store-operated Ca2+ entry in HMCs was significantly reduced by knocking down TRPC1, but enhanced by overexpressing TRPC1. These results suggest that HMCs specifically express isoforms of TRPC1, 3, 4, and 6 proteins. These isoforms of TRPCs might selectively assemble to form functional complexes.

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    • "Studies from our laboratory and other laboratories have demonstrated that human MCs express TRPC channel proteins, including isoforms of TRPC1, 3, 4, and 6 [15], [16]. In the present study, we investigated the role of TRPC channels in the CaSR activation-induced calcium influx and subsequent cell proliferation in human MCs. "
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    ABSTRACT: Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "On the right (in red) is a list of cell or whole tissue/body effects that have been suggested to be driven or potentiated by TRPC activity; in other words, if TRPC channels were to be inhibited, the opposite of the effect is predicted to occur (e.g. less pancreatitis). Example references for cell expression items: acinar gland cells (Liu et al., 2007); adipocytes (Sukumar et al., 2012); astrocytes (Shirakawa et al., 2010); cardiac myocytes (Eder and Molkentin, 2011); cochlea hair cells (Quick et al., 2012); endothelial cells (Ahmmed et al., 2004); epithelial cells (Kim et al., 2011); fibroblasts (Xu et al., 2008); hepatocytes (Rychkov and Barritt, 2011); keratinocytes (Cai et al., 2006); leukocytes (Yildirim et al., 2012); mast cells (Freichel et al., 2012); mesangial cells (Sours et al., 2006); neurones (Bollimuntha et al., 2011); osteoclasts/blasts (Abed et al., 2009); platelets (Ramanathan et al., 2012); podocytes (Dryer and Reiser, 2010); skeletal muscle (Gervasio et al., 2008); smooth muscle cells (Beech et al., 2004); and tumour cells (Thebault et al., 2006). Example references for effect items: angiogenesis (Yu et al., 2010); cancer cell drug resistance (Ma et al., 2012); cell adhesion (Smedlund et al., 2010); cell migration (Xu et al., 2006); cell proliferation (Sweeney et al., 2002); cell survival (Selvaraj et al., 2012); cell turning (Wang and Poo, 2005); efferocytosis (Tano et al., 2011); gastrointestinal motility (Tsvilovskyy et al., 2009); glomerular filtration (Dryer and Reiser, 2010); hypoadiponectinaemia (Sukumar et al., 2012); hypo-matrix metalloproteinase (Xu et al., 2008); hypertrophy (cardiac) (Eder and Molkentin, 2011); innate fear (Riccio et al., 2009); lung hyper-responsiveness (Yildirim et al., 2012); mast cell degranulation (Ma et al., 2008); motor coordination (Trebak, 2010); muscle endurance (Zanou et al., 2010); neointimal hyperplasia (Kumar et al., 2006); oedema (Weissmann et al., 2012); permeability (Tiruppathi et al., 2002); pancreatitis (Kim et al., 2011); saliva secretion (Liu et al., 2007); seizure (Phelan et al., 2013); survival after MI (myocardial infarction) (Jung et al., 2011); thrombosis (Ramanathan et al., 2012); vaso-modulation (e.g. "
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    ABSTRACT: The primary purpose of this review is to address the progress towards small molecule modulators of human Transient Receptor Potential Canonical proteins (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7). These proteins generate channels for calcium and sodium ion entry. They are relevant to many mammalian cell types including acinar gland cells, adipocytes, astrocytes, cardiac myocytes, cochlea hair cells, endothelial cells, epithelial cells, fibroblasts, hepatocytes, keratinocytes, leukocytes, mast cells, mesangial cells, neurones, osteoblasts, osteoclasts, platelets, podocytes, smooth muscle cells, skeletal muscle, and tumour cells. There are broad-ranging positive roles of the channels in cell adhesion, migration, proliferation, survival and turning, vascular permeability, hypertrophy, wound-healing, hypo-adiponectinaemia, angiogenesis, neointimal hyperplasia, oedema, thrombosis, muscle endurance, lung hyper-responsiveness, glomerular filtration, gastrointestinal motility, pancreatitis, seizure, innate fear, motor coordination, saliva secretion, mast cell degranulation, cancer cell drug resistance, survival after myocardial infarction, efferocytosis, hypo-matrix metalloproteinase, vasoconstriction and vasodilatation. Known small molecule stimulators of the channels include hyperforin, genistein and rosiglitazone, but there is more progress with inhibitors, some of which have promising potency and selectivity. The inhibitors include 2-aminoethoxydiphenyl borate, 2-aminoquinolines, 2-aminothiazoles, fatty acids, isothiourea derivatives, naphthalene sulphonamides, N-phenylanthranilic acids, phenylethylimidazoles, piperazine/piperidine analogues, polyphenols, pyrazoles, and steroids. A few of these agents are starting to be useful as tools for determining the physiological and pathophysiological functions of TRPC channels. We suggest that the pursuit of small molecule modulators for TRPC channels is important but that it requires substantial additional effort and investment before we can reap the rewards of highly potent and selective pharmacological modulators.
    Preview · Article · Jun 2013 · British Journal of Pharmacology
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    • "Store-operated calcium channels (SOCC) participate in signal transduction as major regulators of Ca2+ internal flow in kidney micro-vascular smooth muscle cells and GMCs. TRPC1/3/4/6 of GMCs are candidates for SOCC [15]. Through SOCC and other types of calcium channels, AngII can affect intracellular Ca2+, thereby regulating renal microcirculation and GMC functions [16]. "
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    ABSTRACT: This study investigated the changes in intracellular [Ca2+]i (intracellular calcium ion concentration) and TRPC6 (transient receptor potential channel 6) expression during angiotensin II (AngII)-induced glomerular mesangial cell (GMC) proliferation, as well as the inhibitory effect of losartan. GMC cultures were split into four groups treated for 24 h: Group N (blank control group), Group A (10-7 mol/L AngII), Group LT (10-7 mol/L AngII and 10-5 mol/L losartan), and Group Pred (10-7 mol/L AngII and 10-5 mol/L prednisone). GMCs proliferation was measured by the MTT and trypan blue assays. The distribution of TRPC6 was monitored by immunofluorescence, the expression of TRPC6 was detected by RT-PCR and Western blotting, and [Ca2+]i was measured by laser scanning confocal microscopy. The results showed that the maximal proliferation of GMCs was induced by treatment with 10-7 mol/L AngII for 24 h. In Group A, the distribution of TRPC6 was not uniform in the cell membrane, there was increased accumulation of this protein within the cytoplasm, and the increased expression of TRPC6 and [Ca2+]i was consistent with the proliferation of cells. In Group LT, losartan inhibited the proliferation of GMCs significantly, the levels of TRPC6 and [Ca2+]i were diminished, and the distribution of TRPC6 was improved. Prednisone also significantly inhibited the proliferation of GMCs and had no effects on the expression of TRPC6 and [Ca2+]i in Group Pred. These findings suggested that AngII could enhance the expression of TRPC6, increase [Ca2+]i, and demonstrate a time-dose-response relationship with the proliferation of GMCs, while losartan reversed the effect of AngII on GMC proliferation.
    Preview · Article · Mar 2013 · Clinical and Experimental Medicine
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