Stéphanie Thebault

Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia.

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.

Prostate cell differentiation status determines transient receptor potential melastatin member 8 channel subcellular localization and function.

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.

Novel role of cold/menthol-sensitive transient receptor potential melastatine family member 8 (TRPM8) in the activation of store-operated channels in LNCaP human prostate cancer epithelial cells.

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 (Icold/menthol) that shows inward rectification and high Ca2+ selectivity, which are dramatically different properties from “classical” TRPM8-mediated Icold/menthol. Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both Icold/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 Ca2+ 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 Ca2+ release channel, potentially involved in a number of Ca2+- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Ca2+-independent Phospholipase A2-dependent Gating of TRPM8 by Lysophospholipids

TRPM8 represents an ion channel activated by cold temperatures and cooling agents, such as menthol, that underlies the cold-induced excitation of sensory neurons. Interestingly, the only human tissue outside the peripheral nervous system, in which the expression of TRPM8 transcripts has been detected at high levels, is the prostate, a tissue not exposed to any essential temperature variations. Here we show that the TRPM8 cloned from human prostate and heterologously expressed in HEK-293 cells is regulated by the Ca2+-independent phospholipase A2 (iPLA2) signaling pathway with its end products, lysophospholipids (LPLs), acting as its endogenous ligands. LPLs induce prominent prolongation of TRPM8 channel openings that are hardly detectable with other stimuli (e.g. cold, menthol, and depolarization) and that account for more than 90% of the total channel open time. Down-regulation of iPLA2 resulted in a strong inhibition of TRPM8-mediated functional responses and abolished channel activation. The action of LPLs on TRPM8 channels involved either changes in the local lipid bilayer tension or interaction with the critical determinant(s) in the transmembrane channel core. Based on this, we propose a novel concept of TRPM8 regulation with the involvement of iPLA2 stimulation. This mechanism employs chemical rather than physical (temperature change) signaling and thus may be the main regulator of TRPM8 activation in organs not exposed to any essential temperature variations, as in the prostate gland.

Peptide Hormone Regulation of Angiogenesis

It is now apparent that regulation of blood vessel growth contributes to the classical actions of hormones on development, growth, and reproduction. Endothelial cells are ideally positioned to respond to hormones, which act in concert with locally produced chemical mediators to regulate their growth, motility, function, and survival. Hormones affect angiogenesis either directly through actions on endothelial cells or indirectly by regulating proangiogenic factors like vascular endothelial growth factor. Importantly, the local microenvironment of endothelial cells can determine the outcome of hormone action on angiogenesis. Members of the growth hormone/prolactin/placental lactogen, the renin-angiotensin, and the kallikrein-kinin systems that exert stimulatory effects on angiogenesis can acquire antiangiogenic properties after undergoing proteolytic cleavage. In view of the opposing effects of hormonal fragments and precursor molecules, the regulation of the proteases responsible for specific protein cleavage represents an efficient mechanism for balancing angiogenesis. This review presents an overview of the actions on angiogenesis of the above-mentioned peptide hormonal families and addresses how specific proteolysis alters the final outcome of these actions in the context of health and disease.

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line.

Prostate smooth muscle cells predominantly express α1‐adrenoceptors (α1‐AR). α1‐AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca2+ entry pathways associated with the activation of α1‐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 α1A‐ARs, α‐agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca2+ influx which depended on the extracellular Ca2+ 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 Ca2+ influx whose properties were similar to those induced by the α‐agonists. Sensitivity to 2‐aminoethyl diphenylborate (2‐APB), SKF‐96365 and flufenamate implies that Ca2+‐permeable channels mediated both α‐agonist‐ and OAG‐evoked Ca2+ influx. Following the sarcoplasmic reticulum (SR) Ca2+ store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca2+ influx. However, OAG failed to enhance Ca2+ influx when added in the presence of an α‐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 α‐agonist‐ and OAG‐induced Ca2+ influx required TRPC3 and TRPC6, whereas the Tg‐activated (“capacitative”) Ca2+ entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor‐ and store‐operated Ca2+ entry pathways.

Vasoinhibins prevent retinal vasopermeability associated with diabetic retinopathy in rats via protein phosphatase 2A–dependent eNOS inactivation

Increased retinal vasopermeability contributes to diabetic retinopathy, the leading cause of blindness in working-age adults. Despite clinical progress, effective therapy remains a major need. Vasoinhibins, a family of peptides derived from the protein hormone prolactin (and inclusive of the 16-kDa fragment of prolactin), antagonize the proangiogenic effects of VEGF, a primary mediator of retinal vasopermeability. Here, we demonstrate what we believe to be a novel function of vasoinhibins as inhibitors of the increased retinal vasopermeability associated with diabetic retinopathy. Vasoinhibins inhibited VEGF-induced vasopermeability in bovine aortic and rat retinal capillary endothelial cells in vitro. In vivo, vasoinhibins blocked retinal vasopermeability in diabetic rats and in response to intravitreous injection of VEGF or of vitreous from patients with diabetic retinopathy. Inhibition by vasoinhibins was similar to that achieved following immunodepletion of VEGF from human diabetic retinopathy vitreous or blockage of NO synthesis, suggesting that vasoinhibins inhibit VEGF-induced NOS activation. We further showed that vasoinhibins activate protein phosphatase 2A (PP2A), leading to eNOS dephosphorylation at Ser1179 and, thereby, eNOS inactivation. Moreover, intravitreous injection of okadaic acid, a PP2A inhibitor, blocked the vasoinhibin effect on endothelial cell permeability and retinal vasopermeability. These results suggest that vasoinhibins have the potential to be developed as new therapeutic agents to control the excessive retinal vasopermeability observed in diabetic retinopathy and other vasoproliferative retinopathies.

α1-adrenergic receptors activate Ca2+-permeable cationic channels in prostate cancer epithelial cells

The prostate gland is a rich source of alpha1-adrenergic receptors (alpha1-ARs). alpha1-AR antagonists are commonly used in the treatment of benign prostatic hyperplasia symptoms, due to their action on smooth muscle cells. However, virtually nothing is known about the role of alpha1-ARs in epithelial cells. Here, by using two human prostate cancer epithelial (hPCE) cell models - primary cells from resection specimens (primary hPCE cells) and an LNCaP (lymph node carcinoma of the prostate) cell line - we identify an alpha1A subtype of adrenergic receptor (alpha1A-AR) and show its functional coupling to plasmalemmal cationic channels via direct diacylglycerol (DAG) gating. In both cell types, agonist-mediated stimulation of alpha1A-ARs and DAG analogues activated similar cationic membrane currents and Ca(2+) influx. These currents were sensitive to the alpha1A-AR antagonists, prazosin and WB4101, and to transient receptor potential (TRP) channel blockers, 2-aminophenyl borate and SK&F 96365. Chronic activation of alpha1A-ARs enhanced LNCaP cell proliferation, which could be antagonized by alpha1A-AR and TRP inhibitors. Collectively, our results suggest that alpha1-ARs play a role in promoting hPCE cell proliferation via TRP channels.

HIGH LEVELS OF SERUM PROLACTIN PROTECT AGAINST DIABETIC RETINOPATHY BY INCREASING OCULAR VASOINHIBINS

OBJECTIVE Increased retinal vasopermeability (RVP) occurs early in diabetes and is crucial for the development of sight-threatening proliferative diabetic retinopathy (DR). The hormone prolactin (PRL) is proteolytically processed to vasoinhibins, a family of peptides that inhibit the excessive RVP related to DR. Here, we investigate the circulating levels of PRL in association with DR in men and test whether increased circulating PRL, by serving as a source of ocular vasoinhibins, can reduce the pathological RVP in diabetes. RESEARCH DESIGN AND METHODS Serum PRL was evaluated in 40 nondiabetic and 181 diabetic men at various stages of DR. Retinal vasoinhibins were measured in rats rendered hyperprolactinemic by placing two anterior pituitary grafts under the kidney capsule and in PRL receptor–null mice. RVP was determined in hyperprolactinemic rats subjected to the intraocular injection of vascular endothelial growth factor (VEGF) or made diabetic with streptozotocin. RESULTS The circulating levels of PRL increased in diabetes and were higher in diabetic patients without retinopathy than in those with proliferative DR. In rodents, hyperprolactinemia led to vasoinhibin accumulation within the retina; genetic deletion of the PRL receptor prevented this effect, indicating receptor-mediated incorporation of systemic PRL into the eye. Hyperprolactinemia reduced both VEGF-induced and diabetes-induced increase of RVP. This reduction was blocked by bromocriptine, an inhibitor of pituitary PRL secretion, which lowers the levels of circulating PRL and retinal vasoinhibins. CONCLUSIONS Circulating PRL influences the progression of DR after its intraocular conversion to vasoinhibins. Inducing hyperprolactinemia may represent a novel therapy against DR.

Prolactin promotes oxytocin and vasopressin release by activating neuronal nitric oxide synthase in the supraoptic and paraventricular nuclei

Prolactin (PRL) stimulates the secretion of oxytocin (OXT) and arginine AVP as part of the maternal adaptations facilitating parturition and lactation. Both neurohormones are under the regulation of nitric oxide. Here, we investigate whether the activation of neuronal nitric oxide synthase (nNOS) in the hypothalamo-neurohypophyseal system mediates the effect of PRL on OXT and AVP release and whether these effects operate in males. Plasma levels of OXT and AVP were measured in male rats after the intracerebroventricular injection of PRL or after inducing hyperprolactinemia by placing two anterior pituitary glands under the kidney capsule. NOS activity was evaluated in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei by NADPH-diaphorase histochemistry and in hypothalamic extracts by the phosphorylation/inactivation of nNOS at Ser(847). Elevated central and systemic PRL correlated with increased NOS activity in the PVN and SON and with higher OXT and AVP circulating levels. Notably, treatment with 7-nitroindazole, a selective inhibitor of nNOS, prevented PRL-induced stimulation of the release of both neurohormones. Also, phosphorylation of nNOS was reduced in hyperprolactinemic rats, and treatment with bromocriptine, an inhibitor of anterior pituitary PRL secretion, suppressed this effect. These findings suggest that PRL enhances nNOS activity in the PVN and SON, thereby contributing to the regulation of OXT and AVP release. This mechanism likely contributes to the regulation of processes beyond those of female reproduction.