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Contains fulltext : 80878.pdf (publisher's version ) (Open Access), Recent identification of a mutation in the EGF gene that causes isolated recessive hypomagnesemia led to the finding that EGF increases the activity of the epithelial magnesium (Mg2+) channel transient receptor potential M6 (TRPM6). To investigate the molecular mechanism mediating this effect, we performed whole-cell patch-clamp recordings of TRPM6 expressed in human embryonic kidney 293 (HEK293) cells. Stimulation of the EGF receptor increased current through TRPM6 but not TRPM7. The carboxy-terminal alpha-kinase domain of TRPM6 did not participate in the EGF receptor-mediated increase in channel activity. This activation relied on both the Src family of tyrosine kinases and the downstream effector Rac1. Activation of Rac1 increased the mobility of TRPM6, assessed by fluorescence recovery after photobleaching, and a constitutively active mutant of Rac1 mimicked the stimulatory effect of EGF on TRPM6 mobility and activity. Ultimately, TRPM6 activation resulted from increased cell surface abundance. In contrast, dominant negative Rac1 decreased TRPM6 mobility, abrogated current development, and prevented the EGF-mediated increase in channel activity. In summary, EGF-mediated stimulation of TRPM6 occurs via signaling through Src kinases and Rac1, thereby redistributing endomembrane TRPM6 to the plasma membrane. These results describe a regulatory mechanism for transepithelial Mg2+ transport and consequently whole-body Mg2+ homeostasis.

Contains fulltext : 71094.pdf (Publisher’s version ) (Open Access), Transient receptor potential melastatin 6 (TRPM6) plays an essential role in epithelial Mg(2+) transport. TRPM6 and its closest homologue, TRPM7, both combine a cation channel with an alpha-kinase domain. However, the role of this alpha-kinase domain in TRPM6 channel activity remains elusive. The aim of this study was to investigate the regulation of TRPM6 channel activity by intracellular ATP and the involvement of its alpha-kinase domain. We demonstrated that intracellular Na- and Mg-ATP decreased the TRPM6 current in HEK293 cells heterogeneously expressing the channel, whereas Na-CTP or Na-GTP had no effect on channel activity. Whole cell recordings in TRPM6-expressing HEK293 cells showed that deletion of the alpha-kinase domain prevented the inhibitory effect of intracellular ATP without abrogating channel activity. Mutation of the conserved putative ATP-binding motif GXG(A)XXG (G1955D) in the alpha-kinase domain of TRPM6 inhibited the ATP action, whereas this effect remained preserved in the TRPM6 phosphotransferase-deficient mutant K1804R. Mutation of the TRPM6 autophosphorylation site, Thr(1851), into either an alanine or an aspartate, resulted in functional channels that could still be inhibited by ATP. In conclusion, intracellular ATP regulates TRPM6 channel activity via its alpha-kinase domain independently of alpha-kinase activity.

Contains fulltext : 71290.pdf (publisher's version ) (Closed access), BACKGROUND: The maintenance of the body's Mg(2+) balance is of great importance because of its involvement in numerous enzymatic systems and its intervention in neuromuscular excitability, protein synthesis, and nucleic acid stability. Recently, the transient receptor potential melastatin 6 (TRPM6) was identified as the gatekeeper of active Mg(2+) transport and therefore plays a crucial role in the regulation of Mg(2+) homeostasis. Remarkably, TRPM6 combines a Mg(2+) channel with an alpha-kinase domain whose function remains elusive. RESULTS: Here, we identify the receptor for activated C-kinase 1 (RACK1) as the first regulatory protein of TRPM6 that associates with the alpha-kinase domain. RACK1 and TRPM6 are both present in renal Mg(2+)-transporting distal convoluted tubules. We demonstrate that RACK1 inhibits channel activity in an alpha-kinase activity-dependent manner, whereas small interference (si) RNA-mediated knockdown of RACK1 increases the current. Moreover, threonine(1851) in the alpha-kinase domain was identified as an autophosphorylation site of which the phosphorylation state is essential for the inhibitory effect of RACK1. Importantly, threonine(1851) was crucial for the Mg(2+) sensitivity of TRPM6 autophosphorylation and channel activity. TRPM6 channel activity was less sensitive to Mg(2+) when RACK1 was knocked down by siRNA. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity. CONCLUSIONS: We propose a unique mode of TRPM6 regulation in which the Mg(2+) influx is controlled by RACK1 through its interaction with the alpha-kinase and the phosphorylation state of the threonine(1851) residue.

Contains fulltext : 52958.pdf (publisher's version ) (Open Access), Primary hypomagnesemia constitutes a rare heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg(2+)) wasting resulting in generally shared symptoms of Mg(2+) depletion, such as tetany and generalized convulsions, and often including associated disturbances in calcium excretion. However, most of the genes involved in the physiology of Mg(2+) handling are unknown. Through the discovery of a mutation in the EGF gene in isolated autosomal recessive renal hypomagnesemia, we have, for what we believe is the first time, identified a magnesiotropic hormone crucial for total body Mg(2+) balance. The mutation leads to impaired basolateral sorting of pro-EGF. As a consequence, the renal EGFR is inadequately stimulated, resulting in insufficient activation of the epithelial Mg(2+) channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6) and thereby Mg(2+) loss. Furthermore, we show that colorectal cancer patients treated with cetuximab, an antagonist of the EGFR, develop hypomagnesemia, emphasizing the significance of EGF in maintaining Mg(2+) balance.

Contains fulltext : 52400.pdf (publisher's version ) (Closed access), TRPM6 and its closest relative TRPM7 are members of the Transient Receptor Potential Melastatin (TRPM) subfamily of cation channels and are known to be Mg2+ permeable. By aligning the sequence of the putative TRPM6 pore with the pore sequences of the other subfamily members, we located in the loop between the fifth and the sixth transmembrane domain, a stretch of amino acids residues, 1028GEIDVC1033, as the potential selectivity filter. Two negatively charged residues, E1024 (conserved in TRPM6, TRPM7, TRPM1 and TRPM3) and D1031 (conserved along the entire TRPM subfamily), were identified as important determinants of cation permeation through TRPM6, because neutralization of both residues into an alanine resulted in non-functional channels. Neutralization of E1029 (conserved in TRPM6, TRPM7, TRPM4 and TRPM5) resulted in channels with increased conductance for Ba2+ and Zn2+, decreased ruthenium red sensitivity and larger pore diameter compared to wild-type TRPM6. Changing the residue I1030 into methionine, resulted in channels with lower conductance for Ni2+, decreased sensitivity to ruthenium red block and reduced pore diameter. Thus, these data demonstrate that amino acid residues E1024, I1030 and D1031 are important for channel function and that subtle amino acid variation in the pore region accounts for TRPM6 permeation properties.

Contains fulltext : 53165thebault.pdf (publisher's version ) (Open Access), In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer (PCa). However, the mechanisms of prostate-specific regulation and functional evolution of TRPM8 during PCa progression remain unclear. Here we show, for the first time to our knowledge, that only secretory mature differentiated human prostate primary epithelial (PrPE) luminal cells expressed functional plasma membrane TRPM8 ((PM)TRPM8) channels. Moreover, PCa epithelial cells obtained from in situ PCa were characterized by a significantly stronger (PM)TRPM8-mediated current than that in normal cells. This (PM)TRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. However, we found that in contrast to (PM)TRPM8, endoplasmic reticulum TRPM8 ((ER)TRPM8) retained its function as an ER Ca(2+) release channel, independent of cell differentiation. We hypothesize that the constitutive activity of (ER)TRPM8 may result from the expression of a truncated TRPM8 splice variant. Our study provides insight into the role of TRPM8 in PCa progression and suggests that TRPM8 is a potentially attractive target for therapeutic intervention: specific inhibition of either (ER)TRPM8 or (PM)TRPM8 may be useful, depending on the stage and androgen sensitivity of the targeted PCa.

Contains fulltext : 53160thebault.pdf (publisher's version ) (Closed access), One of the best-studied temperature-gated channels is transient receptor potential melastatin 8 (TRPM8), which is activated by cold and cooling agents, such as menthol. Besides inducing a cooling sensation in sensory neurons, TRPM8 channel activation also plays a major role in physiopathology. Indeed, TRPMP8 expression increases in early stages of prostate cancer and its involvement in prostate cell apoptosis has recently been demonstrated. Thus, as TRPM8 is a tumor marker with significant potential use in diagnosis, as well as a target for cancer therapy, there is a need for new TRPM8-specific ligands. In this study, we investigated the action of "WS" compounds on TRPM8 channels. We compared the affinity of these molecules to that of menthol and icilin. This enabled us to identify new TRPM8 agonists. The menthol analog with the highest affinity, WS-12, had an EC(50) value about 2000 times lower than that of menthol and is, therefore, the highest-affinity TRPM8 ligand known to date. Finally, incorporating a fluorine atom in the WS-12 retained 75% of the activity of the parent compound. The high affinity of this new TRPM8 ligand and the possibility of incorporating a radiohalogen could thus be useful for diagnosis, monitoring and, perhaps, even therapy of prostate cancer.

Contains fulltext : 52666thebault.pdf (publisher's version ) (Closed access), Prolactin, a hormone fundamental for lactation, was recently shown to mediate postpartum cardiomyopathy, a life-threatening disease in late-term and lactating mothers. The detrimental effect of prolactin results from myocardial upregulation of cathepsin-D, which in turn cleaves prolactin to a 16 kDa fragment (vasoinhibin) with anti-angiogenic and pro-apoptotic properties that impair developing cardiac vasculature.

Contains fulltext : 51766thebault.pdf (publisher's version ) (Closed access), A major clinical problem with PC (prostate cancer) is the cell's ability to survive and proliferate upon androgen withdrawal. Indeed, deregulated cell differentiation and proliferation, together with the suppression of apoptosis, provides the condition for abnormal tissue growth. Here, we examine the differential role of TRP (transient receptor potential) channels in the control of Ca(2+) homoeostasis and growth of PC cells.

Contains fulltext : 49423.pdf (publisher's version ) (Closed access), Many physiological functions rely on the precise maintenance of body calcium (Ca2+) and magnesium (Mg2+) balance, which is tightly regulated by the concerted actions of intestinal absorption, renal reabsorption, and exchange with bone. The kidney plays an important role in the homeostasis of divalent ions. Most Ca2+ and Mg2+ reabsorption occurs in the proximal tubules and the thick ascending limb of Henle's loop via a passive paracellular pathway. At the level of the distal convoluted tubule (DCT) and the connecting tubule (CNT), Ca2+ and Mg2+ are reabsorbed via an active transcellular route. Reabsorption of divalents in these latter segments is regulated in a Ca2+ and Mg2+-specific manner and determines the final excretion in the urine. Importantly, genetic studies, as well as molecular cloning strategies, recently identified epithelial ion channels as the gatekeepers of active Ca2+ and Mg2+ reabsorption. These channels are members of the transient receptor potential (TRP) superfamily. TRP vanilloid 5 (TRPV5) is responsible for the rate-limiting Ca2+ entry, and TRP melastatin 6 (TRPM6) constitutes the apical entry step in Mg2+ reabsorption. Dysregulation or malfunction of these influx pathways has been associated with renal Ca2+ and Mg2+ wasting. This review updates the current knowledge and the recent advances of Ca2+ and Mg2+ reabsorption and related disorders.

Contains fulltext : 51069.pdf (publisher's version ) (Closed access), The transient receptor potential vanilloid 5 (TRPV5) channel determines urinary Ca(2+) excretion, and is therefore critical for Ca(2+) homeostasis. Interestingly, mice lacking the serine protease tissue kallikrein (TK) exhibit robust hypercalciuria comparable to the Ca(2+) leak in TRPV5 knockout mice. Here, we delineated the molecular mechanism through which TK stimulates Ca(2+) reabsorption. Using TRPV5-expressing primary cultures of renal Ca(2+)-transporting epithelial cells, we showed that TK activates Ca(2+) reabsorption. The stimulatory effect of TK was mimicked by bradykinin (BK) and could be reversed by application of JE049, a BK receptor type 2 antagonist. A cell permeable analog of DAG increased TRPV5 activity within 30 min via protein kinase C activation of the channel since mutation of TRPV5 at the putative PKC phosphorylation sites S299 and S654 prevented the stimulatory effect of TK. Cell surface labeling revealed that TK enhances the amount of wild-type TRPV5 channels, but not of the TRPV5 S299A and S654A mutants, at the plasma membrane by delaying its retrieval. In conclusion, TK stimulates Ca(2+) reabsorption via the BK-activated PLC/DAG/PKC pathway and the subsequent stabilization of the TRPV5 channel at the plasma membrane.

Contains fulltext : 50745thebault.pdf (publisher's version ) (Closed access), One major clinical problem with prostate cancer is the cells' ability to survive and proliferate upon androgen withdrawal. Because Ca2+ is central to growth control, understanding the mechanisms of Ca2+ homeostasis involved in prostate cancer cell proliferation is imperative for new therapeutic strategies. Here, we show that agonist-mediated stimulation of alpha1-adrenergic receptors (alpha1-AR) promotes proliferation of the primary human prostate cancer epithelial (hPCE) cells by inducing store-independent Ca2+ entry and subsequent activation of nuclear factor of activated T cells (NFAT) transcription factor. Such an agonist-induced Ca2+ entry (ACE) relied mostly on transient receptor potential canonical 6 (TRPC6) channels, whose silencing by antisense hybrid depletion decreased both hPCE cell proliferation and ACE. In contrast, ACE and related growth arrest associated with purinergic receptors (P2Y-R) stimulation involved neither TRPC6 nor NFAT. Our findings show that alpha1-AR signaling requires the coupled activation of TRPC6 channels and NFAT to promote proliferation of hPCE cells and thereby suggest TRPC6 as a novel potential therapeutic target.

Contains fulltext : 48641thebault.pdf (publisher's version ) (Closed access), Neuroendocrine (NE) differentiation of prostate epithelial/basal cells is a hallmark of advanced, androgen-independent prostate cancer, for which there is no successful therapy. Here we report for the first time on alterations in regulatory volume decrease (RVD) and its key determinant, swelling-activated Cl- current (I(Cl,swell)), associated with NE differentiation of androgen-dependent LNCaP prostate cancer epithelial cells. NE-differentiating regimens, namely, chronic cAMP elevation or androgen deprivation, resulted in generally augmented I(Cl,swell) and enhanced RVD. This occurred as a result of both the increased endogenous expression of ClC-3, which is a volume-sensitive Cl- channel involved, as we show, in I(Cl,swell) in LNCaP (lymph-node carcinoma of the prostate) cells and the weaker negative I(Cl,swell) control from Ca2+ entering via store-dependent pathways. The changes in the RVD of NE-differentiated cells generally mimicked those reported for Bcl-2-conferred apoptotic resistance. Our results suggest that strengthening the mechanism that helps to maintain volume constancy may contribute to better survival rates of apoptosis-resistant NE cells.

Contains fulltext : 48999thebault.pdf (publisher's version ) (Open Access), The genome of the human T-cell leukemia virus type I (HTLV-I) codes for a basic leucine zipper protein, HBZ, capable of repressing JUN activity and viral transcription. Transient expression in mammalian cells showed that HBZ was targeted to the nucleus, where it accumulated in nuclear speckles. By using a complementary set of deletion mutants, we report here that the nuclear targeting of HBZ is mediated by three distinct nuclear localization signals and that at least two are necessary for the translocation of HBZ to the nucleus. Moreover, the resulting mutant proteins distribute throughout the nucleoplasm and/or into the nucleoli, whereas the wild-type HBZ exclusively accumulates in nuclear speckles, suggesting that the integrity of the protein is required for its speckle localization. We also demonstrate that the HBZ-containing speckles do not correspond to Cajal bodies, splicing factor compartments, or promyelocytic leukemia oncoprotein bodies. Unexpectedly, by using immunogold electron microscopy, we found HBZ localized to heterochromatin. Until now, such characteristics had never been described for a transcription factor and could explain the inhibitory activity of HBZ.

Contains fulltext : 47464thebault.pdf (publisher's version ) (Closed access), TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

Contains fulltext : 47422thebault.pdf (publisher's version ) (Closed access), Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) inf

Contains fulltext : 48991.pdf (Publisher’s version ) (Open Access), Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.