Transient Receptor Potential Melastatin 8, a sensor of cold temperatures mediates expression of cyclin-dependent kinase inhibitor, p21/Cip1, a regulator of epidermal cell proliferation

Abstract

Transient Receptor Potential Melastatin 8 (TRPM8) is a calcium-permeable, non-selective cation channel of the transient receptor potential superfamily, required for the transduction of moderate cold temperatures. TRPM8 is also known to regulate proliferation of prostate, pancreatic, breast, and melanoma carcinoma cells. Here, we examined a key factor in the regulation of TRPM8-mediated proliferation of epidermal cells, which are directly affected by cold temperatures. Experiments involving knockdown and ectopic expression of TRPM8 in normal keratinocyte HaCaT and squamous carcinoma SAS cells suggest that TRPM8 inhibits cell proliferation by upregulating the expression of cyclin-dependent inhibitor p21/Cip1. Whereas these findings were observed in the absence of an endogenous agonists, additions of the synthetic TRPM8 agonist icilin reduced DNA synthesis in HaCaT cells but stimulated that in SAS cells by altering p21/Cip1 levels in a TRPM8-independent manner, indicating that icilin poses a risk of stimulating carcinoma cell proliferation. Unexpectedly, the TRPM8 blocker, used for the treatment of overactive bladder and bladder pain, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl] oxy}-N-(2-thienylmethyl) benzamide hydrochloride salt (AMTB) reduced DNA synthesis by upregulating p21/Cip1 expression. However, another TRPM8 blocker, N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin- 2-yl) tetrahydropyrazine-1 (2H)-carbox-amide (BCTC), stimulated DNA synthesis by downregulating p21/Cip1 expression, indicating that it may pose a risk of carcinogenesis associated with dysregulated cell cycles when used to treat overactive bladder and bladder pain.

INTRODUCTION

The transient receptor potential (TRP) superfamily of ion channels now consists of more than 30 cationic channels, most of which are permeable to Ca²⁺, and some also to Mg²⁺. This superfamily of TRP channels can be divided into seven main families based on sequence homology, namely the TRPC or canonical family, the TRPV or vanilloid family, the TRPM or melastatin family, the TRPP or polycystin family, the TRPML or mucolipin family, the TRPA or ankyrin family, and the TRPN or NOMPC family. Interestingly, several TRPM members are involved in carcinoma cell proliferation (Hantute-Ghesquier et al., 2018).

TRPM8 is a calcium-permeable, non-selective cation channel of the transient receptor potential superfamily, required for the transduction of moderate cold temperatures (Peier et al., 2002). TRPM8 mRNA has been detected in malignant cells, and has been extensively studied in prostate cancer (Valero et al., 2012). TRPM8 expression is also markedly up-regulated in human pancreatic adenocarcinoma cell lines and tissues, and is important for cell proliferation. A deficiency in TRPM8 in pancreatic cancer cells leads to impaired proliferation and cell cycle progression with elevated levels of cyclin-dependent kinase (CDK) inhibitors (Hantute-Ghesquier et al., 2018; Yee et al., 2010). TRPM8 is highly expressed at both the mRNA and protein levels in the MCF-7 breast cancer cell line, and breast adenocarcinomas, and is especially correlated with estrogen receptor positive (ER+) tumors (Hantute-Ghesquier et al., 2018; Chen et al., 2014). Interestingly, while TRPM8 stimulates the proliferation of breast and pancreatic cell lines, it negatively regulates melanoma proliferation (Hantute-Ghesquier et al., 2018; Guo et al., 2012).

Here, we examined a key factor involved in the regulation of TRPM8-mediated proliferation of epidermal cells, which are directly affected by cold temperatures. We focused on the CDK inhibitor p21/Cip1 which causes G1 arrest resulting in decreased DNA synthesis while downregulation of p21/Cip1 is associated with carcinogenesis (Paramio et al., 2001; Jackson et al., 2002). In terms of regulation of p21/Cip1 expression, there is obvious difference between certain normal and carcinoma cells. Indeed, p21/Cip1 expression in renal adenocarcinoma cells is regulated by nuclear receptor farnesoid X receptor (FXR) but that in normal renal cells is not affected by FXR (Fujino et al., 2017). We therefore performed experiments at HaCaT cells derived from normal human keratinocytes (CLS Cell Lines Service, 300493) (Boukamp et al., 1988) and squamous carcinoma SAS (JCRB cell bank: JCRB0260) (Takahashi et al., 1989) cells.

MATERIALS AND METHODS

Materials

Antibody (Ab) specific to β-actin and siRNAs against p21/Cip1 and TRPM8 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Icilin was obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Ab specific to TRPM8 (ab3243) was purchased from Abcam (Cambridge, UK). ECL^TM anti-mouse IgG, horseradish peroxidase-linked whole antibody (from sheep) and ECL^TM anti-rabbit IgG, horseradish peroxidase-linked whole antibody (from donkey) were purchased from GE Healthcare (Buckinghamshire, UK). AMTB hydrochloride was purchased from Tocris Bioscience (Tokyo, Japan) and BCTC from MedChemExpress (Tokyo, Japan). Human TRPM8 expression vector (pRP[Exp]-EGFP/Puro-CAG>hTRPM8[NM_024080.4]) was purchased from Vector Builder Japan (Kanagawa, Japan).

Cell culture

HaCaT cells derived from normal human keratinocytes, squamous carcinoma SAS cells, and breast cancer cell line MCF-7 were maintained in Dulbecco’s modified eagle medium containing 10% fetal calf serum, 50 units/mL penicillin G sodium salt, and 50 μg/mL streptomycin sulfate and cultured in a humidified atmosphere of 8.5% CO₂ at 37°C.

RNA interference experiments

To knock down endogenous p21/Cip1 and TRPM8, cells were seeded on 60-mm dishes at a density of 2.0 × 10⁵ cells per dish and transfected with siRNA against p21/Cip1 and TRPM8 (10 nM each) using HiPerfect Transfection Reagent (Qiagen, Venlo, Netherland) according to the manufacturer’s instructions. After incubating for 24 hr, total RNA was extracted for real-time polymerase chain reaction (PCR). In RNA interference experiments, “Nonsilencing Control”siRNA (#1022076) from Qiagen was used as a control.

Quantification of mRNA

Quantification of mRNA was performed using real-time PCR. Briefly, 4 μg of total RNA was reverse-transcribed using a ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The resultant cDNA was subjected to real-time PCR analysis using a TaqMan Gene Expression Assay kit (Applied Biosystems, Tokyo, Japan). mRNA levels were determined using TaqMan assay mixtures for TRPM8 (Hs00368574), p16/INK4a (Hs99999189), p21/Cip1 (Hs01121172), p27/Kip1 (Hs00153277), and β-actin (4310881E). Amplification and quantification were performed using the StepOne Real-Time PCR System (Applied Biosystems). mRNA levels were normalized to those of β-actin as an internal control.

Ectopic expression of TRPM8

HaCaT and SAS cells were seeded at 5.0 × 10⁵ cells and cultured for 24 hr. The cells were then transfected with pRP[Exp]-EGFP/Puro-CAG as a control and pRP[Exp]-EGFP/Puro-CAG>hTRPM8[NM_024080.4] using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA).

Immunoblotting

Cells were washed with phosphate buffered saline, and cell extracts were prepared using sodium dodecyl sulfate (SDS) sample buffer without loading dye. Dye was added after normalizing the protein content via the protein assay, and samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting analyses. To detect TRPM8 and β-actin, polyvinylidene fluoride (PVDF) membranes were incubated with primary antibody (1:200) for 2 hr and then with secondary antibody (ECL^TM anti-mouse IgG, horseradish peroxidase-linked whole antibody or ECL^TM anti-rabbit IgG, horseradish peroxidase-linked whole antibody) for 1 hr. Immunocomplexes on the PVDF membranes were visualized on X-ray film using enhanced chemiluminescence western blotting detection reagents (GE Healthcare). Quantification of bands was conducted using densitometric analysis (Image Gauge 4.0).

Measurement of DNA synthesis

DNA synthesis was measured using the CytoSelect™ BrdU Cell Proliferation ELISA Kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions.

Statistical analysis

Data are presented as the mean ± S.E.M. of three experiments performed in triplicate and were analyzed using Student’s t-test.

RESULTS

TRPM8 regulates expression of CDK inhibitor p21/Cip1 in epidermal cells

Based on our previous study, which showed that expression levels of CDK inhibitor p21/Cip1 are associated with cell proliferation (Fujino et al., 2015a, 2015b, 2017), we examined whether p21/Cip1 is a key factor in the proliferation of epidermal cell line HaCaT derived from normal human keratinocytes and squamous carcinoma SAS cells. As shown in Fig. 1A-1, DNA synthesis in epidermal HaCaT and SAS cells increased following knockdown of p21/Cip1 (Fig. 1B), leading to focus on this CDK inhibitor. To examine whether TRPM8 regulates the expression of p21/Cip1, TRPM8 knockdown was performed using siRNA against human TRPM8 in HaCaT and SAS cells. Figure 1C-1, 2 show that p21/Cip1 levels reduces following TRPM8 decrease caused by TRPM8 knockdown. Consistent with this, ectopic expression of TRPM8 (Fig. 1D-1) upregulated p21/Cip1 expression in HaCaT and SAS cells (Fig. 1D-2, 3). These results suggest that TRPM8 positively regulates p21/Cip1 expression in epidermal cells. The changes of p21/Cip1 levels following knockdown and ectopic expression of TRPM8 reflected the changes observed in DNA synthesis (Fig. 1A-2, Fig. 1A-3) and cell numbers (Fig. 1A-4). In contrast to HaCaT and SAS cells, TRPM8 knockdown did not affect p21/Cip1 levels in MCF-7 breast cancer cell line (Fig. 1E), indicating that TRPM8 does not regulate expression of p21/Cip1 in MCF cells. Additionally, TRPM8 knockdown led to decrease in the levels of other CDK inhibitors, namely p16/INK4A and p27/Kip1, in HaCaT cells (Fig. 1F), indicating that TRPM8 widely affects the expression of CDK inhibitors in epidermal cells.

Fig. 1

Changes of TRPM8 levels affect the expression of CDK inhibitor p21/Cip1, DNA synthesis, and cell numbers in epidermal cells. A-1, A-2, A-4: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were transfected with siRNA against p21/Cip1 and TRPM8, or control siRNA. After 24 hr (A-1) or 12 and 24 hr (A-2, 4), DNA synthesis (A-1, 2) was measured as described in the Materials and Methods and cell number was counted (A-4). A-3: HaCaT and SAS cells seeded at 5.0 × 10⁵ cells/60-mm dish were transfected with TRPM8 expression vector or control vector. After 24 hr, DNA synthesis was measured. B, C-1, 2: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were transfected with siRNA against p21/Cip1 and TRPM8, or control siRNA. After 12 and 24 hr, total RNA was quantified to determine p21/Cip1 and TRPM8 mRNA levels as described in the Materials and Methods (C-1) or cell extracts were subjected to immunoblotting to detect p21/Cip1 and β-actin protein as described in the Materials and Methods (C-2). D-1, 2, 3: HaCaT and SAS cells seeded at 5.0 × 10⁵ cells/60-mm dish were transfected with TRPM8 expression vector or control vector. After 24 hr, cell extracts were subjected to immunoblotting to detect TRPM8 (D-1), p21/Cip1 (D-3), and β-actin protein or total RNA was extracted to determine of p21/Cip1 mRNA levels (D-2). E: MCF-7 cells cells seeded at 2.5 × 10⁵ cells/60-mm dish were transfected with siRNA against p21/Cip1 or control siRNA. After 24 hr, total RNA was extracted to quantify p21/Cip1 and TRPM8 mRNA levels. F: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were transfected with siRNA against TRPM8, or control siRNA. After 24 hr, total RNA was extracted to quantify p16/INK4A and p27/Kip1 mRNA levels. Data were analyzed using Student’s t-test and are presented as the mean ± S.E.M. of three experiments performed in triplicate. a, d: significant compared to “control siRNA”; a: P < 0.05, d: P < 0.01. b: significant compared to “control siRNA24h”; P < 0.01. c: significant compared to “control vector”; P < 0.01. e: significant compared to “control siRNA12h”; P < 0.05.

TRPM8 agonist icilin downregulates p21/Cip1 expression in a TRPM8-independent manner

Given that TRPM8 knockdown decreases CDK inhibitor level and TRPM8 overexpression in the absence of an exogenous agonist increases it (Fig. 1), we hypothesized that TRPM8 may regulate the expression of CDK inhibitors following activation by an endogenous agonist. Besides cold temperatures, TRPM8 is activated by several substances, including icilin (Chuang et al., 2004) and menthol (Peier et al., 2002). To examine whether these agonists affect the TRPM8-mediated regulation of CDK inhibitors, we treated epidermal cells with icilin and determined p21/Cip1 levels. As shown in Fig. 2A-1, treatment with icilin increased p21/Cip1 mRNA levels in HaCaT cells but decreased them in SAS cells. The p21/Cip1 level was similarly increased in HaCaT cells and decreased in SAS cells by icilin even when TRPM8 was knocked down (Fig. 2B-1), suggesting that icilin regulates p21/Cip1 expression, adversely in normal HaCaT and carcinoma SAS cells, through a TRPM8-independent mechanism. Given that icilin-induced changes of p21/Cip1 levels reflected the changes observed in DNA synthesis and icilin did not further altered p21/Cip1 levels and DNA synthesis in p21/Cip1-knockdown cells (Fig. 2B-2), icilin affects DNA synthesis by regulating p21/Cip1 expression.

Fig. 2

TRPM8 agonist and blockers affect the expression of p21/Cip1 and DNA synthesis. A-1, 2: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were treated with 2 μg/mL icilin, 10 μΜ AMTB, 10 μΜ BCTC, or DMSO as a control. After 24 hr, total RNA was extracted to quantify p21/Cip1 and TRPM8 mRNA levels. B-1, 2: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were transfected with siRNA against TRPM8, p21/Cip1, or control siRNA, and treated with 2 μg/mL icilin or DMSO as a control. After 24 hr, total RNA was extracted to quantify p21/Cip1 and TRPM8 mRNA levels (B-1) or DNA synthesis was measured (B-2). C-1, 2, 3: HaCaT and SAS cells seeded at 2.0 × 10⁵ cells/60-mm dish were transfected with siRNA against TRPM8, p21/Cip1, or control siRNA, and treated with 10 μΜ AMTB, 10μΜ BCTC, or DMSO as a control. After 24 hr, total RNA was extracted to quantify p21/Cip1 and TRPM8 mRNA levels (C-1, 2) or DNA synthesis was measured (C-3). Data were analyzed using Student’s t-test and are presented as the mean ± S.E.M. of three experiments performed in triplicate. a, b: significant compared to “DMSO”; a: P < 0.05, b < 0.01. c, e: significant compared to “control siRNA/DMSO”; c: P < 0.05, e: P < 0.01. d, f: significant compared to “TRPM8 siRNA/DMSO”; d: P < 0.05, f: P < 0.01.

TRPM8 blockers affect the expression of p21/Cip1

Given that TRPM8 was originally identified as a channel required for the transduction of moderate cold temperatures (Peier et al., 2002), blockers of the TRPM8 channel, such as AMTB (Lashinger et al., 2008) and BCTC (Weil et al., 2005) were developed for the treatment of overactive bladder and bladder pain. As described above, TRPM8 downregulation decreased p21/Cip1 levels (Fig. 1C), thereby increasing the risk of carcinogenesis. We therefore examined whether TRPM8 blockers affect p21/Cip1 expression in HaCaT and SAS cells. Surprisingly, while AMTB increased p21/Cip1 levels, BCTC decreased them (Fig. 2A-1). The unexpected AMTB-mediated increase in p21/Cip1 probably arises from an increase in TRPM8, whereas BCTC does not affect TRPM8 levels (Fig. 2A-2). The BCTC-mediated decrease in p21/Cip1 levels may be associated with TRPM8 blocker activity, since p21/Cip1 levels in TRPM8 knockdown cells did not further decrease following treatment with BCTC (left panel of Fig. 2C-1, 2). In contrast, AMTB increased p21/Cip1 levels even in TRPM8 knockdown cells (left panel of Fig. 2C-1, 2), suggesting that AMTB-mediated p21/Cip1 increase is not associated with blocker activity. Given that AMTB and BCTC-induced changes of p21/Cip1 levels reflected the changes observed in DNA synthesis and AMTB and BCTC did not further altered p21/Cip1 levels and DNA synthesis in p21/Cip1-knockdown cells (Fig. 2C-3), AMTB and BCTC affect DNA synthesis by regulating p21/Cip1 expression.

DISCUSSION

We showed that TRPM8, a sensor of cold temperatures, mediates the expression of cyclin-dependent kinase inhibitor, p21/Cip1, which regulates the proliferation of epidermal cells. This is the first report to show that p21/Cip1 is a key factor in TRPM8-mediated regulation of epidermal cell proliferation.

As shown in Fig. 1C-1, 2, we found that p21/Cip1 levels were reduced in TRPM8 knockdown cells, suggesting that TRPM8 downregulation may increase the risk of carcinogenesis attributed to a decrease in p21/Cip1 (Paramio et al 2001; Jackson et al 2002). Moreover, the decrease in p21/Cip1 following TRPM8 downregulation probably stimulates proliferation of carcinoma cells. In fact, TRPM8 knockdown increased DNA synthesis and cell numbers (Fig. 1A-2, 4). Several reports have suggested that steroid hormones and lipids may regulate TRPM8 expression or activity (Mohandass et al., 2020; Liu et al., 2019). Given that p21/Cip1 expression is regulated by nuclear receptors, peroxisome proliferator-activated receptor-gamma (Han et al., 2004) and FXR (Fujino et al., 2017), further studies should examine whether these receptors are involved in TRPM8 regulation.

Besides cold temperatures, TRPM8 is also activated by agonists such as icilin (Chuang et al., 2004) and menthol (Peier et al., 2002). These agents produce a “cooling” sensation without actually lowering the body temperature, thus avoiding pathogenesis associated with low body temperatures. However, as shown in Fig. 2B, icilin stimulates DNA synthesis by downregulating p21/Cip1 expression in carcinoma SAS cells in a TRPM8-independent manner, indicating that it poses a risk for stimulating proliferation of carcinoma cells by reducing levels of p21/Cip1. Thus, it is important to consider such side-effects when developing future “cooling” agents.

There is obvious difference between certain normal and carcinoma cells in terms of p21/Cip1 expression. We previously showed that p21/Cip1 expression in renal adenocarcinoma cells is regulated by FXR but that in normal renal cells is not affected by FXR (Fujino et al., 2017). Whether FXR regulates p21/Cip1 expression and icilin affects FXR activity in epidermal cells is of next interest, in order to reveal why icilin increases p21/Cip1 expression of HaCaT cells derived from normal human keratinocytes but reduces that of squamous carcinoma SAS cells (Fig. 2A-1).

We also showed that while the TRPM8 blocker AMTB upregulated p21/Cip1 expression, BCTC downregulated it (Fig. 2A-1). AMTB-mediated upregulation of p21/Cip1 expression may be attributed to the increase in TRPM8 induced by AMTB, which is not associated with TRPM8 blocker activity. In contrast, BCTC-mediated downregulation of p21/Cip1 may be associated with BCTC’s blocker activity. AMTB is known as the selective TRPM8 blocker, however, it may act as a weak blocker for other TRP family, TRPV1 and TRPV4 (Lashinger et al., 2008). Moreover, BCTC exhibits blocker activity for TRPV1 and TRPM8 (Weil et al., 2005). Thus, AMTB-induced increase in TRPM8 is possibly attributed to activity of TRP families other than TRPM8 and absence of TRPM8 increase by BCTC may be attributed to the difference between AMTB and BCTC in terms of blocker activity for TRP families other than TRPM8. Caution is needed when using TRPM8 blockers for clinical purposes, such as the treatment of overactive bladder and bladder pain, based on their effect on p21/Cip1 expression.

ACKNOWLEDGMENTS

We thank Atsushi Shoji, Kazuhiro Morioka, Yoshinori Inoue, Makio Hayakawa, and Toshiyuki Oshima for their encouragement. This work was supported in part by a grant from the Japan Private School Promotion Foundation.

Conflict of interest

The authors declare that there is no conflict of interest.

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