Etienne Dewailly

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.

Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78

The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca2+ homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca2+ homeostasis, and the ER Ca2+ store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca2+ leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca2+ depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca2+ leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca2+ permeability and subsequent ER stress can lead to development of a novel therapeutic approach.—Hammadi, M., Oulidi, A., Gackière, F., Katsogiannou, M., Slomianny, C., Roudbaraki, M., Dewailly, E., Delcourt, P., Lepage, G., Lotteau, S., Ducreux, S., Prevarskaya, N., Van Coppenolle, F. Modulation of ER stress and apoptosis by endoplasmic reticulum calcium leak via translocon during unfolded protein response: involvement of GRP78. FASEB J. 27, 1600–1609 (2013). www.fasebj.org

Intermediate-conductance Ca 2 -activated K channels (IK Ca1) regulate human prostate cancer cell proliferation through a close control of calcium entry

Accumulating data point to K+ channels as relevant players in controlling cell cycle progression and proliferation of human cancer cells, including prostate cancer (PCa) cells. However, the mechanism(s) by which K+ channels control PCa cell proliferation remain illusive. In this study, using the techniques of molecular biology, biochemistry, electrophysiology and calcium imaging, we studied the expression and functionality of intermediate-conductance calcium-activated potassium channels (IKCa1) in human PCa as well as their involvement in cell proliferation. We showed that IKCa1 mRNA and protein were preferentially expressed in human PCa tissues, and inhibition of the IKCa1 potassium channel suppressed PCa cell proliferation. The activation of IKCa1 hyperpolarizes membrane potential and, by promoting the driving force for calcium, induces calcium entry through TRPV6, a cation channel of the TRP (Transient Receptor Potential) family. Thus, the overexpression of the IKCa1 channel is likely to promote carcinogenesis in human prostate tissue.

Ligands of the Peripheral Benzodiazepine Receptor Are Potent Inhibitors of Plasmodium falciparum and Toxoplasma gondii In Vitro

ABSTRACT The increase in resistance of the malaria parasite Plasmodium falciparum to currently available drugs demands the development of new antimalarial agents. In this quest, we have found that ligands to the peripheral benzodiazepine receptor such as flurazepam, an agonist of the benzodiazepine family, and PK11195, an antagonist derived from isoquinoline, were active against Plasmodium falciparum. These two compounds effectively and rapidly inhibited parasite growth in vitro, irrespective of parasite resistance to chloroquine and mefloquine. Treatment with both drugs induced a sharp and consistent decline in parasitemia, a complete inhibition of parasite replication, and the destruction of parasites within the host red blood cells. Using electron microscopy, we showed that dramatic morphological changes, involving swollen endoplasmic reticulum and the reduction of hemozoin, were consistent with parasite death. The potent activities of flurazepam and PK11195 were also evaluated for antagonist or synergistic effects with currently used antimalarial drugs such as chloroquine and mefloquine. Moreover, flurazepam was found to be active against Toxoplasma gondii, another member of the phylum Apicomplexa. Taken together, our results indicated that benzodiazepines could be considered promising candidates in the treatment of both malaria and toxoplasmosis.

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.

CaV3.2 T-type Calcium Channels Are Involved in Calcium-dependent Secretion of Neuroendocrine Prostate Cancer Cells

Because prostate cancer is, in its early stages, an androgen-dependent pathology, treatments aiming at decreasing testosterone plasma concentration have been developed for many years now. However, a significant proportion of patients suffer a relapse after a few years of hormone therapy. The androgen-independent stage of prostate cancer has been shown to be associated with the development of neuroendocrine differentiation. We previously demonstrated that neuroendocrine prostate cancer cells derived from LNCaP cells overexpress CaV3.2 T-type voltage-dependent calcium channels. We demonstrate here using prostatic acid phosphatase as a marker of prostate secretion and FM1-43 fluorescence imaging of membrane trafficking that neuroendocrine differentiation is associated with an increase in calcium-dependent secretion which critically relies on CaV3.2 T-type calcium channel activity. In addition, we show that these channels are expressed by neuroendocrine cells in prostate cancer tissues obtained from patients after surgery. We propose that CaV3.2 T-type calcium channel up-regulation may account for the alteration of secretion during prostate cancer development and that these channels, by promoting the secretion of potential mitogenic factors, could participate in the progression of the disease toward an androgen-independent stage.

Differential roles of STIM1, STIM2 and Orai1 in the control of cell proliferation and SOCE amplitude in HEK293 cells

Orai1, together with STIM1 and STIM2, constitutes the molecular basis for store-operated calcium entry (SOCE) and we have investigated their role in cell proliferation and cell cycle progression in HEK293 cells. 48-h serum deprival, and a 24-h treatment with 1 mM hydroxyurea or with 10 microM RO-3306--a cyclin-dependent kinase 1 inhibitor--induced cell cycle block in G1, S and G2/M, respectively. SOCE amplitude, monitored in whole-cell voltage clamped cells, was markedly reduced (60-70%) in all conditions, with full reversibility within 4h. Silencing of Orai and STIM1 using siRNA resulted in a large inhibition of SOCE (70-80%) whereas siSTIM2 had a smaller but significant effect (30%). However, the cell population doubling time was not affected in siSTIM1 cells (18 h, the same as in control cells) but was increased in both siOrai1 cells (29 h) and in siSTIM2 (23 h) even when combined with siSTIM1. This suggests that STIM1 plays no role in cell proliferation in HEK293 cells while STIM2 is involved in both SOCE and cell proliferation in these cells. Finally, the cell cycle block induced SOCE inhibition was associated with reduced Orai1 expression with full recovery within 4h, whereas the expression of STIM1 and STIM2 remained unaltered. These observations reveal a tight relation between cell proliferation, calcium entry and Orai1 expression in HEK293 cells.

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha

Variations in calcium concentration within the endoplasmic reticulum ([Ca2+]ER) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor‐alpha (TNFalpha) as well as evaluating the possible role of [Ca2+]ER variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca2+]ER whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca2+]ER. IGF‐induced [Ca2+]ER increase is correlated to an overexpression of the sarcoendoplasmic calcium‐ATPase 2B (SERCA2b), whereas TNFalpha‐induced [Ca2+]ER decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca2+]ER reduction and SERCA2b downregulation. Reduction in [Ca2+]ER induced by thapsigargin (TG) (from 1 pM to 1 μM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 μM TG. We also show that a transient TG application (1 pM, 1 nM, 1 μM 15 min) is insufficient to induce a long lasting decrease in [Ca2+]ER, since [Ca2+]ER remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 μM, 15 min) induces apoptosis. We thus identify [Ca2+]ER and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.

Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.