Journal of Dermatology 2013; 40: 919–922
doi: 10.1111/1346-8138.12288
CONCISE COMMUNICATION
Activation of transient receptor potential melastatin 8 reduces ultraviolet B-induced prostaglandin E2 production in keratinocytes Nok-Hyun PARK,* Yong-Joo NA,* Hyang-Tae CHOI, Jun-Cheol CHO, Hae-Kwang LEE Skin Research Institute, Amorepacific R&D Center, Yongin, South Korea
ABSTRACT Transient receptor potential melastatin 8 (TRPM8) is a member of the TRP family, and is activated at temperatures below 22°C, or by cooling compounds such as menthol. In this study, it was found that a new role of TRPM8 activation on prostaglandin E2 (PGE2), an inflammatory cytokine and dendritogenesis stimulator of normal human melanocytes. Normal human keratinocytes were pretreated with menthol or incubated at 22°C for TRPM8 activation before ultraviolet (UV)-B irradiation. To examine the specificity between TRPM8 activation and PGE2 release, we inhibited TRPM8 with the antagonist (capsazepine), or introduced TRPM8 siRNA for a gene silencing experiment. UV-B irradiation significantly induced PGE2 release in normal human keratinocytes. Interestingly, activation of TRPM8 at 22°C or with menthol inhibited UV-B-induced PGE2 release. The effect of the TRPM8 agonist was completely blocked by pretreatment with the TRPM8 antagonist, capsazepine. When TRPM8 expression was suppressed by siRNA, UV-B irradiation still upregulated PGE2 in keratinocytes, but pretreatment of menthol or low temperature did not inhibit UV-B-induced PGE2. In conclusion, the activation of TRPM8 inhibits UV-B-induced PGE2 production in keratinocytes, and the activation of TRPM8 may reduce inflammatory responses in skin.
Key words:
keratinocyte, prostaglandin E2, transient receptor potential melastatin 8, ultraviolet B.
INTRODUCTION Thermo-sensing is mediated by a transient receptor potential (TRP) ion channel family.1 Recently, thermo-sensitive receptors were found in skin cells, and it has been reported that they are involved in physiological skin functions. For example, heat shock-induced matrix metalloproteinase-1 (MMP-1) was decreased by the TRP cation channel subfamily V member (TRPV)1 inhibitor.2 The TRPV4 agonist accelerated barrier recovery in normal skin after acute perturbations.3 TRPM8 is expressed in epidermal keratinocytes, and is activated by menthol and cooling temperatures. Interestingly, topical application of TRPM8 modulators affected epidermal permeability barrier homeostasis, and could reduce inflammation.4 It is well-known that acute ultraviolet (UV)-B irradiation on skin induces inflammation, including erythema, edema and immunosuppression.5 Prostaglandin E2 (PGE2) is a main prostaglandin that is induced by keratinocytes following UV exposure.6 PGE2 mediates various functions, such as immune regulation, cell growth and differentiation.7,8 Also, PGE2 stimulates melanocyte dendrite elongation and affects skin pigmentation.9 Herein, we
showed that TRPM8 activation may regulate UV-B-induced PGE2 synthesis for the first time.
METHODS Human keratinocyte culture Normal human epidermal keratinocyte (NHEK) cells were purchased from Cascade Biologics (Portland, OR, USA), and were cultured in EpiLife medium (Cascade Biologics) with human keratinocyte growth supplement (HKGS; Cascade Biologics) with 1% penicillin/streptomycin (Lonza, Walkersville, MD, USA) at 37°C in a humidified atmosphere of 5% CO2. Cells were cultured until they were 90% confluent before being passaged. For all experiments, keratinocytes were seeded on 35-mm dishes (4 9 105 cells/well) and 60-mm dishes (5 9 105 cells/ well) and were incubated for 48 h at 37°C.
PGE2 enzyme-linked immunoassay Normal human epidermal keratinocyte cells were seeded with 4 9 105 cells/well on a 35-mm dish. The next day, cells were pretreated with menthol (4 h) or cooling stimulation (22°C,
Correspondence: Hae-Kwang Lee, Ph.D., Skin Research Institute, AMOREPACIFIC R&D Center, 314-1 Bora-dong, Giheung-gu, 446-729 Yongin, South Korea. Email: [email protected] *These authors contributed to this study equally. Received 4 June 2013; accepted 14 August 2013.
© 2013 Japanese Dermatological Association
919
N.-H. Park et al.
30 min). Pretreated cells were washed twice with phosphatebuffered saline (Welgene, Daegu, Korea) and exposed to UVB irradiation (UVB lamp; Bio Sun Lamps, Vilber Lourmat, Marine, France). The total energy dose of UV-B irradiation was 20 mJ/cm2. After UV-B exposure, fresh media without drugs was added to the cells. The culture medium was harvested after 48 h. PGE2 sandwich enzyme-linked immunoassay assays (Amersham Bioscience, Piscataway, NJ, USA) was then performed according to the manufacturer’s protocols.
siRNA cell transfection For the siRNA experiment, after reaching 60–70% confluence, the NHEK cells were transfected with either ON-TARGETplus TRPM8 siRNA or non-targeting SMARTpool control siRNA (Dharmacon, Pittsburgh, PA, USA) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, NHEK cells were seeded with 5 9 105 cells/well on a 60-mm dish. The next day, a mixture of human TRPM8-siRNA (25 and 37.5 nmol/L) and Lipofectamine RNAiMAX was diluted in 500 lL of Opti-MEM (Invitrogen). After slightly agitating for 20 min at room temperature, the mixture was added to the cells. After 6 h of incubation, the mixture was removed from the cells and replaced with EpiLife medium containing HKGS. Forty-eight hours later, the transfected NHEK cells were used for the following experiments.
Sunnyvale, CA, USA) with Softmax program (Molecular Devices).
Statistical analysis Data were presented as mean standard error of the mean. The mean data were analyzed with one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference. All of the bars in the graph are standard deviations.
RESULTS As shown in the previous report,6 we also found that UV-B irradiation significantly induced PGE2 release in cultured normal keratinocytes (Fig. 1a). We confirmed that TRPM8 is expressed in normal human keratinocytes by qRT–PCR, and chemical agonist and low temperature induced TRPM8 gene expression (Table S1). Menthol, a chemical agonist, or low temperature induced intracellular Ca2+ elevation (Fig. 1b). As shown in Figure 1(a), UV-B-induced PGE2 synthesis was completely inhibited in TRPM8-activated cells by use for pretreatment with
(a)
Quantitative real-time polymerase chain reaction (qRT–PCR) analysis For the relative mRNA expression analysis of selected genes, total RNA was isolated TRIzol (Invitrogen), according to the manufacturer’s instructions. The RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). RNA (4 lg) was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen), and aliquots were kept at 20°C. qRT–PCR was performed using an ABI 7500 Fast Real-Time PCR System with commercially available TaqMan site-specific primers and probes (Applied Biosystems, Foster City, CA, USA). The cDNA samples were analyzed for TRPM8 (Hs00375481_m1). The results were normalized to the glyceraldehyde 3-phosphate dehydrogenase levels. The data were analyzed using 7500 Fast System SDS Software version 1.3.1 (Applied Biosystems). All of the data were obtained from more than two independent experiments carried out in triplicate.
Ca2+ imaging Fluo-4 NW Calcium assay kits (Molecular Probes, Eugene, OR, USA) were used for Ca2+ imaging. NHEK cells were seeded with 3 9 104 cells/well on a 96-well dark plate. The next day cells were washed Hank’s buffered salt solution buffer and cells were incubated with a dye loading solution at 37°C for 30 min, and then at room temperature for an additional 30 min. Agonist-stimulated calcium signaling in NHEK cells was measured by Flou4, with excitation at 494 nm and emission at 516 nm, using FlexStation (Molecular Devices,
920
(b)
Figure 1. Cold (22°C) or menthol reduces prostaglandin E2 (PGE2) production which is induced by ultraviolet (UV)-B irradiation. (a) Normal human epidermal keratinocyte (NHEK) cells were pre-incubated with 100 lmol/L of menthol for 4 h or incubated at 22°C for 30 min. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by kit in culture media. (b) NHEK cells were initially incubated in the absence of stimulator. Then, cold (22°C) and menthol (100 lmol/L) was applied as indicated by the arrows. Calcium signaling was measured as described in Methods. UN, untreated control. *P < 0.05 (t-test).
© 2013 Japanese Dermatological Association
Reduction of UV-B-induced PGE2 by TRPM8
cooling (22°C) or menthol. Menthol or low temperature alone did not affect PGE2 production at a basal level. To clarify whether this phenomenon is related to TRPM8dependency or not, further experiments were performed. First, capsazepine, a TRPM8 antagonist was used for pretreatment to suppress TRPM8 activation prior to either menthol or low temperature treatment. Interestingly, UV-B-induced PGE2 release was not inhibited by menthol or low temperature treatments in the capsazepine-pretreated group (Fig. 2a). Because capsazepine is also related to TRPV1,10 gene silencing (siRNA) on TRPM8 was introduced to clarify the specificity (Fig. 2b). After menthol treatment, UV-B irradiation did not induce PGE2 release in untreated and scrambled-treated groups. However, menthol treatment did not affect UV-B-induced PGE2 release in the TRPM8K/D group (Fig. 2d). TRPM8 functional silencing was confirmed by intracellular Ca2+ elevation experiment, which was almost fully suppressed in the TRPM8K/D group (Fig. 2c). These results indicated that the suppression of TRPM8 activation abolished the effect of UV-B on PGE2 production in epidermal keratinocytes.
DISCUSSION Activation of TRPM8 involves intracellular Ca2+ influx,11 and intracellular Ca2+ has been implicated in prostaglandin synthesis.12 In this study, TRPM8 activation induced intracellular calcium influx, but it did not increase PGE2 release (Fig. 1). It can be assumed that increase of calcium concentration by TRPM8 activation is not enough to induce PGE2 synthesis, or there may be a TRPM8-independent regulatory pathway that affects PGE2 synthesis. However, when TRPM8 activation was suppressed by TRPM8 antagonist or by TRPM8 gene K/D, UV-B-induced PGE2 release was maintained as untreated control level. Denda et al.11 showed that low temperature activated 60% of undifferentiated keratinocytes, and this result indicated that the epidermis is sensitive to cold stimulation. After exposure to UV light, soothing remedies for lowering the temperature of the skin are often performed, but the physiological mechanism of cooling the skin is still unclear. Our findings suggest that skin soothing with reduced temperatures activates TRPM8, and it might contribute to PGE2 reduction after UVB irradiation, and finally that could be alleviating the inflammatory response in the epidermis. There should be further
(a)
(b)
(c)
(d)
Figure 2. Transient receptor potential melastatin 8 (TRPM8) contributes to ultraviolet (UV)-B-induced prostaglandin E2 (PGE2) production. (a) Normal human epidermal keratinocyte (NHEK) cells were pre-incubated with capsazepine for 30 min before menthol or 22°C. After capsazepine treatment, cells were treated with 100 lmol/L of menthol for 4 h or incubated at 22°C for 30 min. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by enzyme-linked immunoassay (ELISA) in culture media. (b) NHEK cells were transfected with TRPM8 siRNA. The cDNA were analyzed for TRPM8 expression by quantitative reverse transcription polymerase chain reaction. TRPM8 expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase. (c) Calcium signaling was measured by stimulated with menthol (100 lmol/L) in scrambled or TRPM8 siRNA transfected NHEK cells. (d) TRPM8 siRNA transfected NHEK cells were pre-incubated with 100 lmol/L of menthol for 4 h. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by ELISA in culture media. Cont, untransfected cells; vehicle, Lipofectamine only; S.C (scrambled) siRNA, non-targeting SMARTpool control. *P < 0.05, **P < 0.01 (t-test).
© 2013 Japanese Dermatological Association
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N.-H. Park et al.
investigation to determine the exact intracellular regulation mechanism of TRPM8 activation and UV-B-induced PGE2 synthesis.
ACKNOWLEDGMENTS: This work was supported by
7
8
research grants from Amorepacific R&D Center. 9
CONFLICT OF INTEREST: The authors have no conflict of interest. 10
REFERENCES 1 Hui K, Guo Y, Feng ZP. Biophysical properties of menthol-activated cold receptor TRPM8 channels. Biochem Biophys Res Commun 2005; 333: 374–382. 2 Lee YM, Kim YK, Kim KH et al. A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells. J Cell Physiol 2009; 219: 766–775. 3 Denda M, Sokabe T, Fukumi-Tominaga T et al. Effects of skin surface temperature on epidermal permeability barrier homeostasis. J Invest Dermatol 2007; 127: 654–659. 4 Denda M, Tsutsumi M, Denda S. Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold-sensitive TRP receptors in epidermal permeability barrier homoeostasis. Exp Dermatol 2010; 19: 791–795. 5 Matsumura Y, Ananthaswamy HN. Toxic effects of ultraviolet radiation on the skin. Toxicol Appl Pharmacol 2004; 195: 298– 308. 6 Tripp CS, Blomme EA, Chinn KS et al. Epidermal COX-2 induction following ultraviolet irradiation: suggested mechanism for the role of
922
11
12
COX-2 inhibition in photoprotection. J Invest Dermatol 2003; 121: 853–861. Pentland AP, Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 1986; 77: 246–251. Kang-Rotondo CH, Miller CC, Morrison AR et al. Enhanced keratinocyte prostaglandin synthesis after UV injury is due to increased phospholipase activity. Am J Phys 1993; 264: C396–C401. Scott G, Leopardi S, Printup S et al. Proteinase-activated receptor2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity. J Invest Dermatol 2004; 122: 1214–1224. Phillips E, Reeve A, Bevan S et al. Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1. J Biol Chem 2004; 279: 17165–17172. Tsutsumi M, Denda S, Ikeyama K et al. Exposure to low temperature induces elevation of intracellular calcium in cultured human keratinocytes. J Invest Dermatol 2010; 130: 1945–1948. Miller CC, Hale P, Pentland AP. Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 1994; 269: 3529–3533.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Cold or menthol induces TRPM8 mRNA expression in keratinocytes. Fig S1. UVB-induced PGE2 production reduced WS12. Fig S2. TRPM8 protein level was reduced by siTRPM8.
© 2013 Japanese Dermatological Association
doi: 10.1111/1346-8138.12288
CONCISE COMMUNICATION
Activation of transient receptor potential melastatin 8 reduces ultraviolet B-induced prostaglandin E2 production in keratinocytes Nok-Hyun PARK,* Yong-Joo NA,* Hyang-Tae CHOI, Jun-Cheol CHO, Hae-Kwang LEE Skin Research Institute, Amorepacific R&D Center, Yongin, South Korea
ABSTRACT Transient receptor potential melastatin 8 (TRPM8) is a member of the TRP family, and is activated at temperatures below 22°C, or by cooling compounds such as menthol. In this study, it was found that a new role of TRPM8 activation on prostaglandin E2 (PGE2), an inflammatory cytokine and dendritogenesis stimulator of normal human melanocytes. Normal human keratinocytes were pretreated with menthol or incubated at 22°C for TRPM8 activation before ultraviolet (UV)-B irradiation. To examine the specificity between TRPM8 activation and PGE2 release, we inhibited TRPM8 with the antagonist (capsazepine), or introduced TRPM8 siRNA for a gene silencing experiment. UV-B irradiation significantly induced PGE2 release in normal human keratinocytes. Interestingly, activation of TRPM8 at 22°C or with menthol inhibited UV-B-induced PGE2 release. The effect of the TRPM8 agonist was completely blocked by pretreatment with the TRPM8 antagonist, capsazepine. When TRPM8 expression was suppressed by siRNA, UV-B irradiation still upregulated PGE2 in keratinocytes, but pretreatment of menthol or low temperature did not inhibit UV-B-induced PGE2. In conclusion, the activation of TRPM8 inhibits UV-B-induced PGE2 production in keratinocytes, and the activation of TRPM8 may reduce inflammatory responses in skin.
Key words:
keratinocyte, prostaglandin E2, transient receptor potential melastatin 8, ultraviolet B.
INTRODUCTION Thermo-sensing is mediated by a transient receptor potential (TRP) ion channel family.1 Recently, thermo-sensitive receptors were found in skin cells, and it has been reported that they are involved in physiological skin functions. For example, heat shock-induced matrix metalloproteinase-1 (MMP-1) was decreased by the TRP cation channel subfamily V member (TRPV)1 inhibitor.2 The TRPV4 agonist accelerated barrier recovery in normal skin after acute perturbations.3 TRPM8 is expressed in epidermal keratinocytes, and is activated by menthol and cooling temperatures. Interestingly, topical application of TRPM8 modulators affected epidermal permeability barrier homeostasis, and could reduce inflammation.4 It is well-known that acute ultraviolet (UV)-B irradiation on skin induces inflammation, including erythema, edema and immunosuppression.5 Prostaglandin E2 (PGE2) is a main prostaglandin that is induced by keratinocytes following UV exposure.6 PGE2 mediates various functions, such as immune regulation, cell growth and differentiation.7,8 Also, PGE2 stimulates melanocyte dendrite elongation and affects skin pigmentation.9 Herein, we
showed that TRPM8 activation may regulate UV-B-induced PGE2 synthesis for the first time.
METHODS Human keratinocyte culture Normal human epidermal keratinocyte (NHEK) cells were purchased from Cascade Biologics (Portland, OR, USA), and were cultured in EpiLife medium (Cascade Biologics) with human keratinocyte growth supplement (HKGS; Cascade Biologics) with 1% penicillin/streptomycin (Lonza, Walkersville, MD, USA) at 37°C in a humidified atmosphere of 5% CO2. Cells were cultured until they were 90% confluent before being passaged. For all experiments, keratinocytes were seeded on 35-mm dishes (4 9 105 cells/well) and 60-mm dishes (5 9 105 cells/ well) and were incubated for 48 h at 37°C.
PGE2 enzyme-linked immunoassay Normal human epidermal keratinocyte cells were seeded with 4 9 105 cells/well on a 35-mm dish. The next day, cells were pretreated with menthol (4 h) or cooling stimulation (22°C,
Correspondence: Hae-Kwang Lee, Ph.D., Skin Research Institute, AMOREPACIFIC R&D Center, 314-1 Bora-dong, Giheung-gu, 446-729 Yongin, South Korea. Email: [email protected] *These authors contributed to this study equally. Received 4 June 2013; accepted 14 August 2013.
© 2013 Japanese Dermatological Association
919
N.-H. Park et al.
30 min). Pretreated cells were washed twice with phosphatebuffered saline (Welgene, Daegu, Korea) and exposed to UVB irradiation (UVB lamp; Bio Sun Lamps, Vilber Lourmat, Marine, France). The total energy dose of UV-B irradiation was 20 mJ/cm2. After UV-B exposure, fresh media without drugs was added to the cells. The culture medium was harvested after 48 h. PGE2 sandwich enzyme-linked immunoassay assays (Amersham Bioscience, Piscataway, NJ, USA) was then performed according to the manufacturer’s protocols.
siRNA cell transfection For the siRNA experiment, after reaching 60–70% confluence, the NHEK cells were transfected with either ON-TARGETplus TRPM8 siRNA or non-targeting SMARTpool control siRNA (Dharmacon, Pittsburgh, PA, USA) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, San Diego, CA, USA), according to the manufacturer’s instructions. Briefly, NHEK cells were seeded with 5 9 105 cells/well on a 60-mm dish. The next day, a mixture of human TRPM8-siRNA (25 and 37.5 nmol/L) and Lipofectamine RNAiMAX was diluted in 500 lL of Opti-MEM (Invitrogen). After slightly agitating for 20 min at room temperature, the mixture was added to the cells. After 6 h of incubation, the mixture was removed from the cells and replaced with EpiLife medium containing HKGS. Forty-eight hours later, the transfected NHEK cells were used for the following experiments.
Sunnyvale, CA, USA) with Softmax program (Molecular Devices).
Statistical analysis Data were presented as mean standard error of the mean. The mean data were analyzed with one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference. All of the bars in the graph are standard deviations.
RESULTS As shown in the previous report,6 we also found that UV-B irradiation significantly induced PGE2 release in cultured normal keratinocytes (Fig. 1a). We confirmed that TRPM8 is expressed in normal human keratinocytes by qRT–PCR, and chemical agonist and low temperature induced TRPM8 gene expression (Table S1). Menthol, a chemical agonist, or low temperature induced intracellular Ca2+ elevation (Fig. 1b). As shown in Figure 1(a), UV-B-induced PGE2 synthesis was completely inhibited in TRPM8-activated cells by use for pretreatment with
(a)
Quantitative real-time polymerase chain reaction (qRT–PCR) analysis For the relative mRNA expression analysis of selected genes, total RNA was isolated TRIzol (Invitrogen), according to the manufacturer’s instructions. The RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). RNA (4 lg) was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen), and aliquots were kept at 20°C. qRT–PCR was performed using an ABI 7500 Fast Real-Time PCR System with commercially available TaqMan site-specific primers and probes (Applied Biosystems, Foster City, CA, USA). The cDNA samples were analyzed for TRPM8 (Hs00375481_m1). The results were normalized to the glyceraldehyde 3-phosphate dehydrogenase levels. The data were analyzed using 7500 Fast System SDS Software version 1.3.1 (Applied Biosystems). All of the data were obtained from more than two independent experiments carried out in triplicate.
Ca2+ imaging Fluo-4 NW Calcium assay kits (Molecular Probes, Eugene, OR, USA) were used for Ca2+ imaging. NHEK cells were seeded with 3 9 104 cells/well on a 96-well dark plate. The next day cells were washed Hank’s buffered salt solution buffer and cells were incubated with a dye loading solution at 37°C for 30 min, and then at room temperature for an additional 30 min. Agonist-stimulated calcium signaling in NHEK cells was measured by Flou4, with excitation at 494 nm and emission at 516 nm, using FlexStation (Molecular Devices,
920
(b)
Figure 1. Cold (22°C) or menthol reduces prostaglandin E2 (PGE2) production which is induced by ultraviolet (UV)-B irradiation. (a) Normal human epidermal keratinocyte (NHEK) cells were pre-incubated with 100 lmol/L of menthol for 4 h or incubated at 22°C for 30 min. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by kit in culture media. (b) NHEK cells were initially incubated in the absence of stimulator. Then, cold (22°C) and menthol (100 lmol/L) was applied as indicated by the arrows. Calcium signaling was measured as described in Methods. UN, untreated control. *P < 0.05 (t-test).
© 2013 Japanese Dermatological Association
Reduction of UV-B-induced PGE2 by TRPM8
cooling (22°C) or menthol. Menthol or low temperature alone did not affect PGE2 production at a basal level. To clarify whether this phenomenon is related to TRPM8dependency or not, further experiments were performed. First, capsazepine, a TRPM8 antagonist was used for pretreatment to suppress TRPM8 activation prior to either menthol or low temperature treatment. Interestingly, UV-B-induced PGE2 release was not inhibited by menthol or low temperature treatments in the capsazepine-pretreated group (Fig. 2a). Because capsazepine is also related to TRPV1,10 gene silencing (siRNA) on TRPM8 was introduced to clarify the specificity (Fig. 2b). After menthol treatment, UV-B irradiation did not induce PGE2 release in untreated and scrambled-treated groups. However, menthol treatment did not affect UV-B-induced PGE2 release in the TRPM8K/D group (Fig. 2d). TRPM8 functional silencing was confirmed by intracellular Ca2+ elevation experiment, which was almost fully suppressed in the TRPM8K/D group (Fig. 2c). These results indicated that the suppression of TRPM8 activation abolished the effect of UV-B on PGE2 production in epidermal keratinocytes.
DISCUSSION Activation of TRPM8 involves intracellular Ca2+ influx,11 and intracellular Ca2+ has been implicated in prostaglandin synthesis.12 In this study, TRPM8 activation induced intracellular calcium influx, but it did not increase PGE2 release (Fig. 1). It can be assumed that increase of calcium concentration by TRPM8 activation is not enough to induce PGE2 synthesis, or there may be a TRPM8-independent regulatory pathway that affects PGE2 synthesis. However, when TRPM8 activation was suppressed by TRPM8 antagonist or by TRPM8 gene K/D, UV-B-induced PGE2 release was maintained as untreated control level. Denda et al.11 showed that low temperature activated 60% of undifferentiated keratinocytes, and this result indicated that the epidermis is sensitive to cold stimulation. After exposure to UV light, soothing remedies for lowering the temperature of the skin are often performed, but the physiological mechanism of cooling the skin is still unclear. Our findings suggest that skin soothing with reduced temperatures activates TRPM8, and it might contribute to PGE2 reduction after UVB irradiation, and finally that could be alleviating the inflammatory response in the epidermis. There should be further
(a)
(b)
(c)
(d)
Figure 2. Transient receptor potential melastatin 8 (TRPM8) contributes to ultraviolet (UV)-B-induced prostaglandin E2 (PGE2) production. (a) Normal human epidermal keratinocyte (NHEK) cells were pre-incubated with capsazepine for 30 min before menthol or 22°C. After capsazepine treatment, cells were treated with 100 lmol/L of menthol for 4 h or incubated at 22°C for 30 min. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by enzyme-linked immunoassay (ELISA) in culture media. (b) NHEK cells were transfected with TRPM8 siRNA. The cDNA were analyzed for TRPM8 expression by quantitative reverse transcription polymerase chain reaction. TRPM8 expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase. (c) Calcium signaling was measured by stimulated with menthol (100 lmol/L) in scrambled or TRPM8 siRNA transfected NHEK cells. (d) TRPM8 siRNA transfected NHEK cells were pre-incubated with 100 lmol/L of menthol for 4 h. Then, cells were stimulated with 20 mJ/cm2 UV-B and further incubated with fresh media for 48 h. PGE2 protein levels were analyzed by ELISA in culture media. Cont, untransfected cells; vehicle, Lipofectamine only; S.C (scrambled) siRNA, non-targeting SMARTpool control. *P < 0.05, **P < 0.01 (t-test).
© 2013 Japanese Dermatological Association
921
N.-H. Park et al.
investigation to determine the exact intracellular regulation mechanism of TRPM8 activation and UV-B-induced PGE2 synthesis.
ACKNOWLEDGMENTS: This work was supported by
7
8
research grants from Amorepacific R&D Center. 9
CONFLICT OF INTEREST: The authors have no conflict of interest. 10
REFERENCES 1 Hui K, Guo Y, Feng ZP. Biophysical properties of menthol-activated cold receptor TRPM8 channels. Biochem Biophys Res Commun 2005; 333: 374–382. 2 Lee YM, Kim YK, Kim KH et al. A novel role for the TRPV1 channel in UV-induced matrix metalloproteinase (MMP)-1 expression in HaCaT cells. J Cell Physiol 2009; 219: 766–775. 3 Denda M, Sokabe T, Fukumi-Tominaga T et al. Effects of skin surface temperature on epidermal permeability barrier homeostasis. J Invest Dermatol 2007; 127: 654–659. 4 Denda M, Tsutsumi M, Denda S. Topical application of TRPM8 agonists accelerates skin permeability barrier recovery and reduces epidermal proliferation induced by barrier insult: role of cold-sensitive TRP receptors in epidermal permeability barrier homoeostasis. Exp Dermatol 2010; 19: 791–795. 5 Matsumura Y, Ananthaswamy HN. Toxic effects of ultraviolet radiation on the skin. Toxicol Appl Pharmacol 2004; 195: 298– 308. 6 Tripp CS, Blomme EA, Chinn KS et al. Epidermal COX-2 induction following ultraviolet irradiation: suggested mechanism for the role of
922
11
12
COX-2 inhibition in photoprotection. J Invest Dermatol 2003; 121: 853–861. Pentland AP, Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 1986; 77: 246–251. Kang-Rotondo CH, Miller CC, Morrison AR et al. Enhanced keratinocyte prostaglandin synthesis after UV injury is due to increased phospholipase activity. Am J Phys 1993; 264: C396–C401. Scott G, Leopardi S, Printup S et al. Proteinase-activated receptor2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity. J Invest Dermatol 2004; 122: 1214–1224. Phillips E, Reeve A, Bevan S et al. Identification of species-specific determinants of the action of the antagonist capsazepine and the agonist PPAHV on TRPV1. J Biol Chem 2004; 279: 17165–17172. Tsutsumi M, Denda S, Ikeyama K et al. Exposure to low temperature induces elevation of intracellular calcium in cultured human keratinocytes. J Invest Dermatol 2010; 130: 1945–1948. Miller CC, Hale P, Pentland AP. Ultraviolet B injury increases prostaglandin synthesis through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth factor receptor activation. J Biol Chem 1994; 269: 3529–3533.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Table S1. Cold or menthol induces TRPM8 mRNA expression in keratinocytes. Fig S1. UVB-induced PGE2 production reduced WS12. Fig S2. TRPM8 protein level was reduced by siTRPM8.
© 2013 Japanese Dermatological Association
