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

Gabriel Bidaux,Loïc Lemonnier,Stéphanie Thebault,Yuri Panchin,Yaroslav Shuba,Morad Roudbaraki,Alexis Bavencoffe,Roman Skryma,Matthieu Flourakis,Philippe Delcourt,Natalia Prevarskaya,Dimitri Gordienko

doi:10.1074/jbc.m503544200

Abstract

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.

Highlights

Nationale Contre le Cancer, l’ARC, the region Nord/Pas-de-Calais
We focused on the characterization of the endogenous TRPM8 in the model system of human LNCaP
Our results suggest that TRPM8 may be an important endoplasmic reticulum (ER) Ca2ϩ release channel potentially involved in a number of Ca2ϩ- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis

Summary

EXPERIMENTAL PROCEDURES

Cell Cultures—LNCaP cells from the American Type Culture Collection were cultured as described previously [17,18,19,20,21]. The intracellular pipette solution used to record ISOC/menthol in LNCaP cells contained (in mM): 100 NMDG, 20 NaCl, 10 HEPES, 8 EGTA, 3.1 CaCl2, 1 MgCl2 (pH adjusted to 7.3 with HCl). For all Ca2ϩ imaging and electrophysiological experiments, the temperature of the cell bath solution was adjusted to 36 °C, unless otherwise specified. For the immunofluorescence quantification of TRPM8 expression in LNCaP cells transfected with either control or anti-TRPM8 siRNAs, all confocal images were acquired under standard conditions (pinhole aperture, laser intensity, etc.) and analyzed using confocal microscope software (AIM 3.2, Zeiss) to determine the specific mean cell intensity for each experiment and to calculate the average and standard deviation for four independent areas.

Amino acid

RESULTS

DISCUSSION