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 (_PMTRPM8) channels. Moreover, PCa epithelial cells obtained from in situ PCa were characterized by a significantly stronger _PMTRPM8-mediated current than that in normal cells. This _PMTRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. However, we found that in contrast to _PMTRPM8, endoplasmic reticulum TRPM8 (_ERTRPM8) retained its function as an ER Ca²⁺ release channel, independent of cell differentiation. We hypothesize that the constitutive activity of _ERTRPM8 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 _ERTRPM8 or _PMTRPM8 may be useful, depending on the stage and androgen sensitivity of the targeted PCa.

Aberrant cell differentiation is considered to be a key mechanism in the onset of prostate cancer (PCa) and benign prostatic hyperplasia (BPH) (1). In normal prostatic epithelium, cells coexist in many stages in a continuum of differentiated phenotypes, progressing from stem cells to secretory mature luminal cells via a transient amplifying population (2). Deregulated differentiation and proliferation modify prostate epithelial homeostasis and are thus major causes of tumorigenesis (1, 3). One standard therapy for PCa is androgen ablation, which causes tumor regression by inhibiting both proliferation and apical differentiation. However, under antiandrogen therapy, PCa and metastases progress into an androgen-independent stage, causing cancer relapse with a more aggressive phenotype. Therefore, is it critical to understand the mechanisms involved in PCa progression in order to develop reliable prognostic markers and define new therapeutic treatment strategies.

The role of Ca²⁺ in global cancer-related cell signaling pathways is uncontested. Alterations in Ca²⁺ homeostasis increase proliferation (4, 5) and induce differentiation (6) and apoptosis (7–9). According to a growing number of studies, cationic channels of the transient receptor potential (TRP) family represent key players in calcium homeostasis and cell physiopathology (10). In recent years, the TRP melastatin member 8 (TRPM8) channel has emerged as a promising prognosis marker and putative therapeutic target in PCa (11). Indeed, (a) high levels of TRPM8 mRNA have been found in both BPH and PCa compared with normal prostate (NP) epithelial cells (12, 13); (b) significant differences in expression were found for TRPM8, but not for PCa markers prostate-specific antigen (PSA), hK2, and PSCA, between malignant and nonmalignant tissue samples in identified groups of low- and high-grade PCa, suggesting that TRPM8 is a more specific indicator of PCa (14); (c) TRPM8 loss was observed in prostate tissues from patients treated preoperatively with antiandrogen therapy (15); and (d) TRPM8 expression requires functional androgen receptors (ARs) (16). However, despite all the existing evidence on TRPM8 involvement in PCa, the mechanisms of prostate-specific regulation and functional evolution of TRPM8 during PCa progression remain unclear.

Interestingly, although TRPM8 was originally cloned from the prostate (13), recent studies have firmly established its function as a cold receptor in sensory neurons (17), where it has been functionally characterized as a plasma membrane (PM) cationic channel involved in cold-evoked excitation. However, the features of classical plasma membrane TRPM8 (_PMTRPM8) have not yet been firmly established in prostate cells. While many hypotheses have been put forward, the prostate-specific function of TRPM8 and the role of Ca²⁺/Na⁺ inflow that it carries in prostate physiology and carcinogenesis remain unknown. Indeed, 2 recent studies (18, 19) functionally characterized the TRPM8 channel in the human PCa lymph node carcinoma of the prostate (LNCaP) cell line. In these cells TRPM8 is highly expressed, but is most exclusively localized in the ER membrane, where it acts as an ER Ca²⁺ release channel that supports the androgen-dependent component of store-operated Ca²⁺ entry (SOCE). However, _PMTRPM8 is not functional in LNCaP cells. These findings raise a number of intriguing questions. Does prostatic TRPM8 act exclusively on the ER? What mechanisms determine PM versus ER localization? How is TRPM8 function regulated during prostate cell differentiation and carcinogenesis? In light of our previous findings concerning the differential involvement of ER Ca²⁺ release and PM Ca²⁺ entry in the proliferation and apoptosis of PCa cells (5), modifications in TRPM8 localization and activity during carcinogenesis could explain the aberrant cancer cell growth phenotype. Therefore, understanding TRPM8 function in PCa is crucial for making justified conclusions regarding use of this channel as a diagnostic and therapeutic target.

The present study was designed to investigate how TRPM8 localization and activity are regulated depending on the differentiation and oncogenic status of prostate primary epithelial (PrPE) cells. We applied a combination of electrophysiology, Ca²⁺ imaging, and molecular and cell biology approaches to primary cultures of human NP, BPH, and PCa epithelial cells. Our findings show, for the first time to our knowledge, that only highly differentiated PrPE luminal cells expressed functional _PMTRPM8 channels. Importantly, prostate primary cancer (PrPCa) cells obtained from in situ PCa biopsies were characterized by _PMTRPM8-mediated current density that was significantly stronger than that of NP or BPH cells. This _PMTRPM8 activity was abolished in dedifferentiated PrPE cells that had lost their luminal secretory phenotype. In contrast, endoplasmic reticulum TRPM8 (_ERTRPM8) remained functional regardless of the differentiation status of prostate cells. This differential regulation of TRPM8 activity can be explained by the complex regulation of _ERTRPM8 and _PMTRPM8 isoforms by ARs. Considering that ER Ca²⁺ content ([Ca²⁺]_ER) is known to regulate cancer cell growth, our finding that _ERTRPM8 was functional in dedifferentiated PCa cells with downregulated AR provides insight into the role of this channel in PCa progression and may be important for developing therapeutic strategies for metastasized PCa. Finally, we propose a model for the differential regulation of _PMTRPM8 and _ERTRPM8 during PCa progression.

Expression of functional TRPM8 in human prostate apical epithelial cells. We initially studied TRPM8 protein expression in several human prostate resection samples by immunoblotting (Figure 1A). As expected, NP, BPH, and PCa extracts expressed 128-kDa TRPM8 protein and its associated glycosylated form (20). Positive control was performed using recombinant TRPM8 protein produced from HEK293 cells with inducible TRPM8 expression (HEK-TRPM8 cells). An androgen-insensitive PCa cell line, PC-3, was also assessed. Although it has previously been reported that TRPM8 is functional in PC-3 cells (19), we did not detect marked TRPM8 protein expression. Immunohistochemical analysis of several NP, BPH, and PCa samples confirmed that TRPM8 colocalized with the apical epithelial phenotype marker cytokeratin 18 (CK18) in acini luminal cells (Figure 1B). PrPE cell cultures were prepared from 3, 6, and 2 NP, BPH, and PCa samples, respectively. As no difference was noted in TRPM8 functional properties between cells from NP and BPH samples, the cells from these 2 sources were unified under the common designation of PrPE cells, while primary epithelial cells from PCa were defined as PrPCa.

Figure 1

TRPM8 channel expression and activity in human prostate cells. (A) Immunoblot showing detection of 128-kDa protein in representative human NP, BPH, and PCa samples. PC-3 cells were used for negative control; detection of recombinant (rec) TRPM8-His fusion protein was used for positive control. Calnexin was used to control the amount of proteins. (B) Confocal examination of immunohistochemical sections reporting specific expression of the TRPM8 protein (green) in CK18-positive (red) cells in PCa. Arrows denote TRPM8 expression on the luminal side membrane of apical epithelial cells. Boxed area in top left panel is shown at a higher magnification in the other panels. (C) Representative confocal image of a PrPE cell from human BPH showing colocalization of TRPM8 (green) with CK18 (red) 6 days after tissue dissociation. Note that a thin green signal was localized on PM. Top right panel shows the cell viewed with transmitted light. (D) PM localization of TRPM8 (green) in PrPE cells was confirmed by its colocalization with membrane marker CD10 (red). (E) Representative time courses of menthol-activated i_TRPM8 in PrPE cells transfected with 50 nM of either siTRPM8 or scramble siRNA (siCon). Inset shows the representative current/voltage relationships of the menthol-activated membrane currents. Scale bars: 10 μm.

TRPM8 protein was expressed in CK18-positive cells, with intense intracellular labeling largely coinciding with expression of this apical phenotype marker (Figure 1C). However, in contrast to our previous findings in LNCaP cells (18), TRPM8 labeling also provided a thin but intense signal on the cell’s perimeter, which we attributed to its plasmalemmal localization in PrPE cells. This result was further confirmed by the colocalization of TRPM8 with the CD10 PM marker (Figure 1D).

In view of earlier findings on TRPM8 expression in prostate and prostate-derived epithelial cell lines (18), we investigated whether native prostate epithelial cells exhibit functional responses and analyzed their biophysical properties. Figure 1E shows that PrPE cells responded to the application of menthol (100 μM) at physiological temperatures (36°C) by developing a strong membrane menthol current (i_menthol). Similar to the i_TRPM8 in HEK-TRPM8 cells, i_menthol was characterized by a current/voltage relationship with sharp outward rectification, close to 0 mV reversal potential (Figure 1E, inset), which suggests that it was carried through the endogenous _PMTRPM8 in PrPE cells. In order to demonstrate the association of i_menthol with the endogenous TRPM8 in PrPE cells, we used siRNA against TRPM8 (siTRPM8) to selectively knock down TRPM8 mRNA, as was reported in our previous work (18). Figure 1E shows that 48 hours after transfection of PrPE cells with 50 nM siTRPM8-1, i_menthol density was almost completely suppressed (siTRPM8-1, 1.83 ± 0.54 pA/pF, n = 11; control, 79.1 ± 5.64 pA/pF, n = 12), indicating a causal link between TRPM8 levels and current magnitude. The siTRPM8 efficiency in TRPM8 knockdown was confirmed by real-time PCR, Western blot, and electrophysiological analysis performed on HEK-TRPM8 cells (Supplemental Figure 2; supplemental material available online with this article; doi:10.1172/JCI30168DS1). Collectively, these data demonstrate that _PMTRPM8 is functional in PrPE cells and that i_menthol can be attributed to TRPM8 activity and thus be defined as i_TRPM8.

Transition to PCa increases both _ERTRPM8 and _PMTRPM8 activity. Because TRPM8 expression is enhanced in PCa (13–15), but TRPM8 activity in LNCaP cells (derived from metastatic human prostate adenocarcinoma) is found only in the ER membrane (18, 19), we investigated whether PCa development was associated with the shift in TRPM8 localization and function from the PM to the ER by comparing i_TRPM8 in PrPE and PrPCa cells. Figure 2, A and B, shows membrane currents in representative PrPE and PrPCa cells (the latter derived from a nonmetastatic high-grade PCa, Gleason score 7) before and after exposure to icilin (10 μM) at 36°C. As quantified in Figure 2D, exposure to icilin evoked a 4-fold higher current density in PrPCa cells than in PrPE cells at 100 mV (PrPCa, 74.81 ± 15.31 pA/pF, n = 8; PrPE, 16.62 ± 5.11 pA/pF, n = 6). Enhanced i_TRPM8 density correlated with higher TRPM8 mRNA levels in PrPCa cells compared with PrPE cells (Figure 2A, inset). We found no current development in response to icilin in the androgen-insensitive PC-3 cell line (Figure 2, C and D).

Figure 2

i_TRPM8 is functional in PrPE cells and presents increased activity in PCa. (A–C) Representative time courses of icilin-activated i_TRPM8 in PrPE (A), PrPCa (B), and PC-3 cells (C). Insets show the representative current/voltage relationships of the icilin-activated i_TRPM8. Inset in A shows an agarose gel indicating the enhancement of TRPM8 expression in PCa. M, protein ladder. (D) Cumulative data (mean ± SEM) of maximum currents measured at 100 mV. (E–G) Typical traces of the estimated passive Ca²⁺ leak induced by 10 μM icilin in digitotin-permeabilized PrPE (E), PrPCa (F), and PC-3 cells (G). IM, ionomycin. (H) Cumulative data (mean ± SEM) of icilin-evoked ER Ca²⁺ release. *P < 0.05; **P < 0.01; ***P < 0.001.

Next, we sought to determine whether these cell types also reveal TRPM8 activity characteristic of its ER membrane localization. We used compartmentalized fluorescent Ca²⁺ Mag–Fluo-4 dye on permeabilized cells to monitor the effect of TRPM8 activation on [Ca²⁺]_ER. In this series of experiments, we used icilin to activate TRPM8, whereas ionomycin and EGTA were used to induce full ER Ca²⁺ store depletion. As shown in Figure 2, E, F, and H, exposure to icilin evoked ER Ca²⁺ release in PrPCa that was 2-fold that of PrPE cells (PrPCa, 73.5% ± 9.37% of total [Ca²⁺]_ER, n = 23; PrPE, 39.4% ± 6.68% of total [Ca²⁺]_ER, n = 62). Interestingly, icilin in PC-3 cells also induced a measurable decrease in [Ca²⁺]_ER (17.8% ± 4.66% of total [Ca²⁺]_ER, n = 29; Figure 2, G and H), which suggests preferred TRPM8 expression and function on the ER membrane, as previously reported (19). These results demonstrated that transition to the PCa did not eliminate _PMTRPM8 function, as we suggested in our previous report based on LNCaP cell line studies (18), but instead upregulated TRPM8 mRNA expression and enhanced TRPM8 activity in both PM and ER.

TRPM8-mediated PM current disappears during dedifferentiation of prostate apical epithelial cells to the transit amplifying/intermediate phenotype. Because PCa is composed of both transit amplifying/intermediate (TA/I) and apical mature epithelial cells (2, 21, 22), we have investigated TRPM8 regulation during the transition of human prostate epithelial cells from the apical to the intermediate phenotype.

Primary cultures of prostate cells are characterized by the gradual reversion of cell differentiation from the apical phenotype to the TA/I phenotype, which is accompanied by the loss of both AR expression and activity (22). We have previously shown that at least 80% of PrPE cells after 6 days in culture (PrPE-6d) express TRPM8 and apical phenotype markers CK8 and CK18 (16). After 20 days in culture (PrPE-20d), a marked increase in the expression of basal phenotype markers CK5 and CK14 is usually observed, suggesting a reversion of the apical terminally differentiated phenotype to the TA/I phenotype (23, 24). High-magnification confocal images clearly showed the loss of _PMTRPM8 expression in PrPE-20d cells, as well as deep modifications of intracellular TRPM8 distribution — from a fibrillary pattern to a dotted one — compared with PrPE-6d cells (Figure 3, A and B). Thus, reversion of the differentiation characteristic of PrPE-20d cells correlated with the loss of _PMTRPM8 expression.

Figure 3

TRPM8 localization and activity in PrPE cells depends on the phenotype of the differentiated epithelial cells. (A and B) Confocal images showing immunolocalization of TRPM8 in either PrPE-6d (A) or PrPE-20d cells (B). Higher magnifications of boxed areas are presented in the insets (original magnification, ×600). Scale bars: 10 μm. (C) Representative time courses of menthol-activated (100 μM) i_TRPM8 in PrPE-6d and PrPE-20d cells at 36°C. Currents were recorded from voltage ramps at 100 mV. Inset shows the representative current/voltage relationships of i_TRPM8. (D) Typical [Ca²⁺]_c in response to menthol (100 μM) in PrPE-6d and PrPE-20d cells. CCE, capacitative Ca²⁺ entry.

Given the change in preferred TRPM8 localization depending on the differentiation status of human prostate cells, we next examined possible differences in the biophysical properties of PrPE-6d and PrPE-20d cell responses to menthol. Figure 3C shows that exposure to 100 μM menthol elicited the development of a strong outward i_TRPM8 (91.09 ± 9.72 pA/pF at 100 mV, n = 14) only in PrPE-6d cells. Similar results were obtained with icilin (Supplemental Figure 1A), suggesting that _PMTRPM8 is functional only in mature, fully differentiated human prostate apical epithelial cells.

Imaging experiments on Fluo-2AM–loaded cells aimed at testing for ER membrane TRPM8 activity showed that in the absence of extracellular Ca²⁺, exposure of both PrPE-6d and PrPE-20d cells to menthol caused a transient increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), obviously the result of ER Ca²⁺ store depletion (Figure 3D). However, the [Ca²⁺]_c increase appeared much more pronounced in PrPE-6d than in PrPE-20d cells (PrPE-6d, 22.3% ± 5.36%, n = 28; PrPE-20d, 10.4% ± 3.72%, n = 32). This transient [Ca²⁺]_c increase was followed by sustained [Ca²⁺]_c elevation upon reintroduction of extracellular Ca²⁺, a result of store-operated channel (SOC) activity and associated capacitative Ca²⁺ entry, which was also higher in PrPE-6d cells (Figure 3D and Supplemental Figure 1B). These results indicate that TRPM8 is also present in the ER membrane of PrPE cells, where it functions as a Ca²⁺ release channel involved in SOC activation. However, the fact that _ERTRPM8, unlike _PMTRPM8, remained functional in cells that lost their apical phenotype suggests that these represent different forms of TRPM8: _PMTRPM8 is specific to the fully differentiated, apical epithelial cell phenotype, and _ERTRPM8 does not show preferred expression depending on the differentiation status of prostate epithelial cells. To verify this hypothesis, we used a well-characterized model of apical epithelial cell differentiation consisting of artificial overexpression of AR in the inherently androgen-independent PC-3 PCa cell line.

ARs differentially regulate _ERTRPM8 and _PMTRPM8 activities. Given that (a) _ERTRPM8 activity may take 2 forms depending on AR activity of epithelial prostate cells, (b) classic TRPM8 expression is dependent on the AR (15, 16, 19), and (c) AR controls both the differentiation and proliferation of prostate epithelial cells (25, 26), we next sought to determine the relationship among AR activity, _PMTRPM8 activity, and _ERTRPM8 activity.

We investigated whether heterologous AR overexpression in PC-3 cells affects TRPM8 expression and function. First, we compared TRPM8 and AR mRNA expression in wild-type control and AR-transfected PC-3 cells by PCR (Figure 4A). The expression of AR-dependent PSA mRNA served as a control for AR activity. As shown in Figure 4, A and B, control PC-3 cells were found to express very low levels of AR and TRPM8 mRNAs. However, 5 days after AR transfection, quantification of PCR products revealed strong upregulation of TRPM8 (from 107% ± 17% to 191% ± 42%) as well as PSA (from 98% ± 3% to 180% ± 61%) mRNAs (Figure 4B). Western blot experiments showed no specific AR protein expression in the control PC-3 cells and its strong elevation in AR-transfected PC-3 cells (Figure 4C). Classic 128-kDa TRPM8 protein and its posttranslationally modified (glycosylated) 145-kDa form became clearly detectable on the fifth day after AR transfection (Figure 4C). Detection of TRPM8 proteins by Western blot was confirmed on PC-3 cells directly transfected with recombinant human TRPM8 (Figure 4C).

Figure 4

TRPM8 expression is stimulated by AR in PCa cells. (A) Agarose gel showing amplification of the 2 TRPM8 amplicons (TRPM8F12/R15), AR and PSA in PC-3 transfected with either empty vector (Con) or AR for 3 or 5 days (d3 and d5, respectively). GAPDH was used as an internal reporter. (B) Quantification of PCR experiment in A. (C) Immunoblotting showing detection of TRPM8 proteins and AR in PC-3 cells transfected with empty vector, with AR for 3 or 5 days, or with TRPM8 encoding vector. Actin was used to control protein loading. **P < 0.01.

Ca²⁺ imaging and electrophysiological experiments showed that enhancement of endogenous TRPM8 expression in PC-3 cells following AR transfection was paralleled by increased functional responses. Menthol induced a modest decrease in [Ca²⁺]_ER 3 days after AR transfection (18% ± 7.2%, n = 31), which suggests the presence of very low basal TRPM8 activity in the ER membrane in control PC-3 cells, whereas ER Ca²⁺ store depletion by menthol was approximately 1.5-fold that of control 3 days after transfection (i.e., 33% ± 6.77% increase, n = 33), and approximately 3-fold that of control 5 days after transfection (i.e., 53% ± 6.2% increase, n = 28) (Figure 5, A and B). Interestingly, in TRPM8-overexpressing PC-3 cells, menthol induced almost the same ER Ca²⁺ store depletion (61% ± 5.2%, n = 45) as in the PC-3 cells 5 days after AR transfection (Figure 5, A and B).

Figure 5

TRPM8 activity correlated with AR expression levels in PC3 cells. (A) Typical traces of the estimated ER Ca²⁺ release induced by 250 μM menthol in digitotin-permeabilized control PC-3 cells, PC3 cells transfected with AR for 3 or 5 days, and TRPM8-transfected PC3 cells. (B) Cumulative data (mean ± SEM) for percent release from internal Ca²⁺ stores measured at 375 s. (C and D) Representative time courses of menthol-activated (250 μM) i_TRPM8 in control PC3 cells and in PC3 cells transfected with AR for 3 or 5 days (C) as well as in TRPM8-transfected PC3 cells (D). Insets show the representative current/voltage relationships of the baseline and menthol-activated membrane currents. (E) Cumulative data (mean ± SEM) of maximum currents measured at 100 mV. **P < 0.01.

Figure 5, C–E, demonstrates that PC-3 cells 5 days after AR transfection also acquired the ability to generate classic outwardly rectifying menthol-activated i_TRPM8 (3.34 ± 0.55 pA/pF, n = 12), which suggests that _PMTRPM8 expression and function is strictly regulated by the AR. In comparison, the response to menthol in TRPM8-overexpressing PC-3 cells’ outwardly rectifying i_TRPM8 reached a density as high as 117 ± 22 pA/pF (n = 14; Figure 5, D and E). These data suggest that TRPM8 in PC-3 cells is expressed at some low, baseline level in an AR-independent manner and is exclusively functional in the ER membrane. Furthermore, upon reaching full functionality, the AR promotes the expression of classic AR-dependent TRPM8, which targets both PM and ER membranes. Thus, functional AR seems to be imperative for _PMTRPM8 activity, while the activity of _ERTRPM8 may take place even in AR-deficient cells.

To resolve the specific AR dependence of the _ERTRPM8 and _PMTRPM8 we observed that (a) although classic TRPM8 mRNA was undetectable in wild-type PC-3 and PrPE-20d cells, menthol and icilin evoked Ca²⁺ release in those cells, (b) TRPM8-overexpressing PC-3 cells presented significantly higher menthol- and icilin-evoked Ca²⁺ release than did HEK-TRPM8 cells (Figure 5, B and E), and (c) TRPM8-overexpressing LNCaP cells did not develop i_TRPM8 because of ER retention of TRPM8 (18), we hypothesized the existence of 2 TRPM8 isoforms whose function and subcellular targeting are differentially regulated by the AR.

A new TRPM8 isoform may be partially responsible for _ERTRPM8 activity. It is important to note that in the pioneering paper by Tsavaler et al. (13), the expression of 2 different transcripts (6.2 kb and 5.2 kb) of TRPM8 was demonstrated in the prostate, although only the longer one has been cloned to date. Thus, we hypothesized that the expression of a truncated TRPM8 splice variant could be responsible for basal, AR-independent _ERTRPM8 activity. Figure 6A shows the alignment of known TRPM8 mRNAs and the putative genomic structure of the trpm8 gene. Although trpm8 is composed of 27 exons, only 24 of them are involved in the formation of cold/menthol receptor (13). Using differential real-time PCR, we measured the expression intensity of the amplification products using the primer pairs F8/R9 (flanking the region between exons 8 and 9) and F21/R22 (flanking exons 21 and 22) in PrPE-6d and PrPE-20d cells (Figure 6B). Standardization of amplifications had previously been validated on TRPM8 DNA and cDNA from HEK-TRPM8 cells (18). The proportion of each amplicon was used to determine the ratio of F8/R9 (representing the classical TRPM8) to F21/R22 (representing the classical TRPM8 and TRPM8 isoform) (Figure 6C). We found that differentiated PrPE-6d cells were characterized by a F8/R9-to-F21/R22 ratio close to 1 (i.e., 0.86 ± 0.2), which was comparable to that of HEK-TRPM8 cells (1.04 ± 0.11), while dedifferentiated PrPE-20d cells and PC-3 cells showed ratios of 0.10 ± 0.07 and 0.16 ± 0.05, respectively (Figure 6C). These results indicated that the level of classic TRPM8 mRNA in PC-3 and PrPE cells constitutes no more than 16% of the total TRPM8 mRNA. Thus, we hypothesized that the expression of the truncated splice variant may explain our functional result.

Figure 6

The trpm8 gene encodes for classical TRPM8 channel and a putative truncated TRPM8 splice variant. (A) The trpm8 gene localized on chromosome 2 in position 37.1. Alignments of several TRPM8 mRNAs and proposed structures of TRPM8 genomic DNA and mRNA to scale. (B) Genomic map of TRPM8 (not to scale; numbered boxes denote exons) with its associated protein structure (boxes 1–6 represent putative transmembrane domains). The 3 pairs of PCR primers and siTRPM8-1 and -2 are aligned with their matching exons. (C) Real-time analysis of exons 8 to 9 amplicon (F8/R9) and exons 21 to 22 amplicon (F21/R22). Data are presented as a ratio of F8/R9 to F21/R22. HEK-TRPM8 cells represent the control condition with only 1 TRPM8 mRNA variant. (D) Ratio of TRPM8 F8/R9 to F21/R22 expression normalized to hypoxanthine-guanine phosphoribosyl transferase (HPRT) expression after quantification of PCR products obtained from single cells. Each population is represented by 10 prostate apical epithelial cells. ***P < 0.001.

In order to determine to what degree these 2 mRNAs were present in individual cells, we performed single-cell real-time PCR on apical prostate epithelial cells. These experiments enabled us to identify 2 populations of cells, for which the calculated ratios of F8/R9 to F21/R22 were 1.06 ± 0.33 and 0.22 ± 0.17 (Figure 6D). In the framework of our hypothesis, we posited that the former population was likely to express predominately the classic TRPM8, while the latter would probably express both classic TRPM8 and truncated TRPM8 mRNAs (Figure 6D).

We also developed specific siRNA directed against exon 8 (i.e., siTRPM8-1) or exon 21 (i.e., siTRPM8-2) in order to selectively ablate either the classic TRPM8 mRNA or both the classic and the truncated TRPM8 mRNAs. We performed experiments on PC-3 cells and quantified TRPM8 mRNA levels by real-time PCR. Both siTRPM8-1 and siTRPM8-2 specifically suppressed the classic TRPM8 mRNA (7-fold and 3-fold decrease, respectively), as assayed by RT-PCR with the F8/R9 pair of primers (Figure 7A). We performed [Ca²⁺]_ER measurements to study the effect of siRNAs on _ERTRPM8 activity. Figure 7B shows that only PC-3 cells transfected with siTRPM8-2 were characterized by decreased menthol-evoked Ca²⁺ release, which constituted 30.19% ± 16.42% that of control cells. The same experiments conducted on PrPE-20d cells transfected with siTRPM8-2 provided a similar reduction (22.58% ± 21.48% of control; Figure 7C). Control experiments conducted in HEK-TRPM8 cells under the same conditions revealed similar potency in the 2 siRNAs. However, siTRPM8-1 induced weak silencing (2-fold decrease) compared with the strong (10-fold) siTRPM8-2–mediated decrease of TRPM8 mRNA detected with the F21/R22 primer pair, although both of them had the same efficiency (10-fold decrease) in HEK-TRPM8 cells (Figure 7D and Supplemental Figure 2A). Control experiments on HEK-TRPM8 cells showed that siTRPM8-1 and siTRPM8-2 inhibited both _PMTRPM8 and _ERTRPM8 activity with the same efficiency (Figure 7, E and F, and Supplemental Figure 2, B and C).

Figure 7

Specific siRNA-mediated ablation of either classical TRPM8 or total TRPM8 mRNAs has different effects on menthol-evoked Ca²⁺ release. (A and D) Real-time quantification of TRPM8 F8/R9 and F21/R22 amplicon normalized to hypoxanthine-guanine phosphoribosyl transferase expression in PC-3 cells (A) or HEK-TRPM8 cells (D) transfected with 100 nM of control siRNA, siTRPM8-1, or siTRPM8-2. Each experiment was performed 6 times. (B, C, and F) Typical traces of the estimated ER Ca²⁺ release induced by 250 μM menthol in digitotin-permeabilized PC-3 (B), PrPE-20d (C), and HEK-TRPM8 cells (F). Insets show cumulative data (mean ± SEM) for percentage of menthol-evoked Ca²⁺ release from internal Ca²⁺ stores. Each condition included at least 40 cells from 3 independent experiments. (E) Cumulative data (mean ± SEM) of cold- and menthol-activated i_TRPM8 in HEK-TRPM8 cells with control siRNA, siTRPM8-1, or siTRPM8-2 (currents recorded from voltage ramps at 100 mV). *P < 0.05; **P < 0.01; ***P < 0.001.

Taken together, these results suggest that the truncated splice variant coding for the shorter, but still functional, TRPM8 isoform is expressed in AR-independent prostate epithelial cells. This truncated TRPM8 isoform is likely to represent ER-localized protein, which, in addition to functioning as an ER release channel, may also be responsible for the retention of the classic TRPM8 form in the ER, as was proposed for LNCaP cells (18).

In this study, we report 4 major findings. First, primary epithelial cells from human NP, BPH, and PCa specimens exhibited classical cold/menthol receptor–like responses (i.e., characteristic of neuronal TRPM8) that were mediated by endogenous _PMTRPM8 activation. Second, the density of _PMTRPM8 membrane current increased in PCa in situ. Third, TRPM8 in PrPE cells also functioned as a Ca²⁺ release channel, _ERTRPM8. Finally, expression and activity of _ERTRPM8 and _PMTRPM8 depend on the differentiation and oncogenic status of prostate epithelial cells and are likely mediated by 2 TRPM8 isoforms that are differentially regulated by androgens.

Cold/menthol responsiveness of the human TRPM8 channel in prostate cells. This study is the first demonstration to our knowledge of functional _PMTRPM8 activity in human prostate cells. The biophysical properties of membrane current (strong outward rectification and close to 0 mV reversal potential) matched those of neuronal TRPM8 cold/menthol receptor–mediated current in PrPE and PrPCa cells (17, 27). Our siRNA-mediated TRPM8 knockout experiments proved the association of these responses with the expression of classical TRPM8 activity. In Ca²⁺ imaging experiments, menthol and icilin also induced [Ca²⁺]_c increases in PrPE cells. As was previously reported for LNCaP cells (18, 19), it should be noted that the menthol-evoked [Ca²⁺]_c increase in PrPE cells may be indicative of Ca²⁺ entry and SOCE via _PMTRPM8 and _ERTRPM8 activity, respectively. The distinct functions of _PMTRPM8 and _ERTRPM8 may be of great importance in understanding the physiology of prostate cells as well as other cells with dual TRPM8 localization.

Differential expression of _PMTRPM8 and _ERTRPM8 based on epithelial cell differentiation status and possible physiological roles of TRPM8 in NP cells. Prostate epithelium contains 3 types of epithelial cells: basal, TA/I, and apical/luminal (28, 29). The basal cells express CK14 and CK5 but not TRPM8 (16, 30); TA/I cells show a concomitant expression of basal marker CK5 and apical markers CK8/CK18 as well as low levels of AR and TRPM8 (16, 30). Apical cells (terminally differentiated secretory mature cells) express CK8/CK18 and PSA as well as high levels of AR and TRPM8 (16, 30). Moreover, our results showed that TRPM8 colocalized with CK18 (Figure 1C). It has previously been shown in kidney cells that TRP family member TRPP1 interacted directly with intermediated filament proteins, including CK18, which suggests that TRPP1 helps to stabilize epithelial cell sheets mechanically (31). It is also plausible that TRPM8 functions as an “epithelial phenotype stabilizer” in prostate epithelium.

Our results showed that PrPE-6d cells, which express the apical epithelial phenotype markers (16), exhibited both _PMTRPM8 and _ERTRPM8 activity, while the dedifferentiated PrPE-20d cells, which are characterized by the TA/I phenotype (23, 24), only conserved _ERTRPM8. Interestingly, only _ERTRPM8 activity has been observed in PC-3 cells, previously described to have common features with early TA/I cells (absence of AR, CK5, and CK18 expression) (32). Forced AR expression in PC-3 cells induced _PMTRPM8 function with the same kinetics as the expression of the PSA apical phenotype marker, suggesting that _PMTRPM8 is tightly regulated by the AR and appears during apical epithelial differentiation. Therefore, the loss of _PMTRPM8 function in TA/I cells was likely due to the loss of functional AR induced by dedifferentiation. AR regulation of _ERTRPM8 seems more complex. In PC-3 cells 3 days after AR transfection, Ca²⁺ release via _ERTRPM8 was apparently greater than that in control PC-3 cells, although the classical TRPM8 isoform was virtually undetectable and _PMTRPM8 was not functional. Furthermore, expression of the _ERTRPM8 isoform, mediating _ERTRPM8 activity, was considerably less sensitive to AR than was classical TRPM8 (Figure 5A). It will be necessary to clone this ER-specific TRPM8 isoform to understand its preferential targeting of the ER membrane and regulation by AR. The subject of the mechanism underlying androgen regulation of TRPM8 in the prostate has previously been addressed (13, 15, 16, 19), and several putative androgen response elements have been detected. However, an alternative promoter of trpm8 may make the _ERTRPM8 isoform less sensitive to androgens than _PMTRPM8.

The observation that the _ERTRPM8 isoform is retained in the prostate cell ER membrane is also important for future research. Two scenarios have emerged on the basis of studies reporting ER retention of TRP channels: variations in TRP primary sequence (33) or partnership-evoked retention. For instance, subcellular localization and function of TRPP2, shown to act as a Ca²⁺ release ER channel, are controlled by phosphofurin acidic cluster sorting protein–1 (PACS-1) and PACS-2 (34). A reciprocal regulation between _ERTRPM8 and _PMTRPM8 is relatively likely and could also explain the retention of classical TRPM8 on the ER in LNCaP cells (18).

The physiological role of TRPM8 in the NP remains unknown. TA/I cells have been reported to be the most proliferative phenotype in NP epithelium (22), whereas apical cells are basically involved in the secretory activity of prostate acini. _ERTRPM8 activity may be involved in apical differentiation, while our data demonstrating specific _PMTRPM8 activity in human apical prostate cells suggest that the classical TRPM8 channel plays a role in the secretory function of prostate cells and is thereby indirectly involved in sperm motility and fertilization. Moreover, a cold receptor function of TRPM8 in the prostate was suggested by Stein et al. (35). This last hypothesis remains to be demonstrated since prostate is not physiologically exposed to low temperature. Finally, the results of recent studies demonstrating that phosphatidylinositol 4,5-bisphosphate tightly controls TRPM8 activity (36) emphasize the possibility that a membrane metabotropic receptor pathway may be involved in TRPM8 opening.

TRPM8 involvement in PCa. Early observations that TRPM8 upregulation correlated with the PCa stage led to the hypothesis that the trpm8 gene was an oncogene (13–15), so TRPM8 was suggested as a putative target for anticancer therapies. Because AR has been reported to play an essential role in prostate carcinogenesis and TRPM8 is tightly regulated by AR, AR deregulation during PCa progression was expected to influence the physiological function of TRPM8. A convincing model of AR pathway evolution has recently been proposed (3), in which (a) AR regulates genes involved in the differentiation of secretory epithelial cells in normal prostate; (b) enhanced AR activity in the early stages of well-differentiated PCa activates growth-promoting genes and maintains apical epithelial phenotype; and (c) progression from high-grade PCa to metastasis is mediated by selective downregulation of the AR pathway genes. This downregulation results in a higher proliferation rate and enhances the potential to metastasize. Interestingly, in this study shows that the upregulation of the CK8 apical phenotype marker in PCa and its downregulation in metastasis coincide with our results on _PMTRPM8. Indeed, because intracapsular PCas are mainly composed of secretory apical cells (2) and metastatic cells are known to be close to the TA/I phenotype, we suggest that ARs play a key role in the development of _ERTRPM8 and _PMTRPM8 expression and activity during carcinogenesis of prostate cells (Figure 8).

Figure 8

Schematic diagram summarizing the principal findings of this study and showing a simplified representation of differential TRPM8 localization and function depending on the AR activity, differentiation, and oncogenic status of human prostate epithelial cells. (A) General pattern of nondifferentiated TA/I cells and dedifferentiated metastatic cells. In these cells AR level was low, and only the _ERTRPM8 isoform was expressed in the ER membrane. _ERTRPM8 functioned as a Ca²⁺-release ER channel, which, by depleting ER stores, activated SOC localized on the PM. Under these conditions, TRPM8 agonists did not induce classical _PMTRPM8-mediated current. (B) Normal, fully differentiated prostate cell with an apical secretory phenotype. In these cells, with high AR levels, the AR-dependent classical TRPM8 channel was expressed on both ER and PM. Under these conditions, TRPM8 agonists may induce not only SOCE, but also substantial _PMTRPM8-mediated current. (C) Differentiated apical secretory in situ cancer cells. In these cells, with enhanced AR activity, the AR-dependent classical TRPM8 channel was overexpressed and TRPM8 agonists induced high levels of _PMTRPM8-mediated current. i_TRPM8 traces are schematic.

Our data suggest that _PMTRPM8 and _ERTRPM8 may determine specific, oncogenic status–dependent Ca²⁺ signatures required for the progression of Ca²⁺-dependent processes that are critical for carcinogenesis, such as proliferation (5, 37) and apoptosis (6, 9). Proliferation mostly relies on cytosolic Ca²⁺ signaling involving short, repeated Ca²⁺ entry mechanisms (37, 38). We therefore hypothesize that _PMTRPM8 is important for the Ca²⁺ signaling involved in proliferation. Moreover, store depletion and SOCE were previously shown to be critical in promoting growth arrest and apoptosis of PCa epithelial cells (7, 39). Indeed, reduced basal filling of intracellular Ca²⁺ stores is also the hallmark of the apoptosis-resistant cell phenotypes characteristic of advanced PCa (6, 9, 39). Thus, as _ERTRPM8 is a molecular entity capable of influencing the filling of ER stores, its activity may be considered a substantial factor in controlling the growth of advanced PCa metastatic cells. In light of the recent demonstration of the role of TRPM8 in LNCaP cell survival (19), we speculate that any shift in the balance between classical TRPM8 and _ERTRPM8 isoform expression may modify the Ca²⁺ signature, thus increasing the potential for either proliferation or apoptosis. Thus, TRPM8 may be an attractive target for therapeutic interventions: specific inhibition of either _ERTRPM8 or _PMTRPM8 activity should be considered, depending on the stage and androgen sensitivity of the targeted PCa. We believe these results provide novel insight and expect them to influence future research into the selective targeting of TRPM8 during PCa progression.

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This work was supported by grants from INSERM; Ministère de l’Education Nationale; and La Ligue Nationale Contre le Cancer, la region Nord/Pas-de-Calais. Y. Shuba was supported by a grant from INTAS (011-8223). D. Gkikia was supported by a long-term fellowship from the European Molecular Biology Organization (ALTF-161-2006).

Address correspondence to: Natalia Prevarskaya, Laboratoire de Physiologie Cellulaire, INSERM U800, Bâtiment SN3, USTL, Villeneuve d’Ascq 59655, France. Phone: 33-3-20-43-40-77; Fax: 33-3-20-43-40-66; E-mail: natacha.prevarskaya@univ-lille1.fr.

Nonstandard abbreviations used: -6d, 6 days in culture; AR, androgen receptor; BPH, benign prostatic hyperplasia; [Ca²⁺]_c, cytosolic Ca²⁺ concentration; [Ca²⁺]_ER, ER Ca²⁺ content; CK, cytokeratin; _ERTRPM8, endoplasmic reticulum TRPM8; HEK-TRPM8 cell, HEK293 cell with inducible TRPM8 expression; LNCaP, lymph node carcinoma of the prostate; NP, normal prostate; PCa, prostate cancer; PM, plasma membrane; _PMTRPM8, plasma membrane TRPM8; PrPCa, prostate primary cancer (cell); PrPE, prostate primary epithelial (cell); PSA, prostate-specific antigen; siTRPM8, siRNA against TRPM8; SOC, store-operated channel; SOCE, store-operated Ca²⁺ entry; TA/I, transit amplifying/intermediate (apical epithelial cells); TRP, transient receptor potential; TRPM8, TRP melastatin member 8.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:1647–1657 (2007). doi:10.1172/JCI30168

Gabriel Bidaux and Matthieu Flourakis contributed equally to this work.

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