Advertisement

Transient Receptor Potential Vanilloid 4 (TRPV4) Is Downregulated in Keratinocytes in Human Non-Melanoma Skin Cancer

  • Camilla Fusi
    Correspondence
    Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Largo Brambilla, 3, 50134 Florence, Italy
    Affiliations
    Department of Health Sciences, Clinical Pharmacology and Oncology Unit, University of Florence, Florence, Italy
    Search for articles by this author
  • Author Footnotes
    3 The first two authors contributed equally to this work.
    Serena Materazzi
    Footnotes
    3 The first two authors contributed equally to this work.
    Affiliations
    Department of Health Sciences, Clinical Pharmacology and Oncology Unit, University of Florence, Florence, Italy
    Search for articles by this author
  • Daiana Minocci
    Affiliations
    Department of Health Sciences, Clinical Pharmacology and Oncology Unit, University of Florence, Florence, Italy
    Search for articles by this author
  • Vincenza Maio
    Affiliations
    Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy
    Search for articles by this author
  • Teresa Oranges
    Affiliations
    Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy
    Search for articles by this author
  • Daniela Massi
    Correspondence
    Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Largo Brambilla, 3, 50134 Florence, Italy
    Affiliations
    Division of Pathological Anatomy, Department of Surgery and Translational Medicine, University of Florence, Florence, Italy
    Search for articles by this author
  • Romina Nassini
    Affiliations
    Department of Health Sciences, Clinical Pharmacology and Oncology Unit, University of Florence, Florence, Italy
    Search for articles by this author
  • Author Footnotes
    3 The first two authors contributed equally to this work.
      A subgroup of the transient receptor potential (TRP) channels, including vanilloid 1 (TRPV1), TRPV2, TRPV3, TRPV4, and TRP ankyrin 1 (TRPA1), is expressed in cutaneous peptidergic somatosensory neurons, and has been found in skin non-neuronal cells, such as keratinocytes. Different cancer cells express TRPs, where they may exert either pro- or antitumorigenic roles. Expression and function of TRPs in skin cancers have been, however, poorly investigated. Here, we have studied the distribution and expression of TRPs by immunohistochemistry and messenger RNA (mRNA) in human healthy skin and human keratinocytic tumors, including intraepidermal proliferative disorders (solar keratosis (SK) and Bowen’s disease), and non-melanoma skin cancer (NMSC; basal cell and squamous cell carcinomas). Similar TRPV1, TRPV2, and TRPV3 staining was found in keratinocytes from healthy and tumor tissues. TRPA1 staining was increased solely in SK samples. However, the marked TRPV4 staining and TRPV4 mRNA expression, observed in healthy or inflamed skin, was abrogated both in premalignant lesions and NMSC. In a human keratinocyte cell line (HaCaT), TRPV4 stimulation released IL-8, which in turn downregulated TRPV4 expression. Selective reduction in TRPV4 expression could represent an early biomarker of skin carcinogenesis. Whether the cytokine-dependent, autocrine pathway that results in TRPV4 downregulation contributes to NMSC mechanism remains to be determined.

      Abbreviations

      BCC
      basal cell carcinoma
      BD
      Bowen’s disease
      GSK
      GSK1016790A
      HaCaT
      human keratinocyte cell line
      mRNA
      messenger RNA
      NMSC
      non-melanoma skin cancer
      PGE2
      prostaglandin E2
      SCC
      squamous cell carcinoma
      SK
      solar keratosis
      TNF-α
      tumor necrosis factor-α
      TRP
      transient receptor potential
      TRPA
      TRP ankyrin
      TRPV
      TRP vanilloid
      4αPDD
      4α-phorbol-12,13-didecanoate

      Introduction

      The superfamily of transient receptor potential (TRP) channels encompasses six subfamilies, which are widely and differently expressed in various tissues and organs where they mediate pleiotropic functions in health and disease (
      • Nilius B.
      • Owsianik G.
      • Voets T.
      • et al.
      Transient receptor potential cation channels in disease.
      ). A subgroup of channels defined as thermo-TRPs because of their ability to sense changes in temperature are localized to a subpopulation of peptidergic primary sensory neurons where they transduce nociceptive signals by a heterogenous variety of physicochemical stimuli and drive neurogenic inflammatory responses (
      • Geppetti P.
      • Holzer P.
      ;
      • Nilius B.
      • Owsianik G.
      • Voets T.
      • et al.
      Transient receptor potential cation channels in disease.
      ). This subgroup of channels includes vanilloid 1 (TRPV1, the capsaicin receptor), TRPV2, TRPV3, and TRPV4, and TRP ankyrin 1 (TRPA1, the mustard oil receptor;
      • Nilius B.
      • Owsianik G.
      • Voets T.
      • et al.
      Transient receptor potential cation channels in disease.
      ). Activators of thermo-TRP may be summarized as follows: TRPV1 is activated by vanilloid compounds (capsaicin, resiniferatoxin), noxious heat (⩾43°C), low pH, and certain eicosanoids (
      • Caterina M.J.
      • Schumacher M.A.
      • Tominaga M.
      • et al.
      The capsaicin receptor: a heat-activated ion channel in the pain pathway.
      ;
      • Tominaga M.
      • Caterina M.J.
      • Malmberg A.B.
      • et al.
      The cloned capsaicin receptor integrates multiple pain-producing stimuli.
      ;
      • Ho W.S.
      • Barrett D.A.
      • Randall M.D.
      'Entourage' effects of N-palmitoylethanolamide and N-oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors.
      ); TRPV3 and TRPV2 are activated by innocuous warm temperatures (32–39°C) and noxious high temperature (>52°C), respectively; TRPV4 is activated by mild high temperature (>24°C) and by hypoosmotic stimuli (
      • Liedtke W.
      • Choe Y.
      • Marti-Renom M.A.
      • et al.
      Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.
      ;
      • Cheng X.
      • Jin J.
      • Hu L.
      • et al.
      TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.
      ); and TRPA1 is activated by moderate low temperatures and a large series of environmental irritants and reactive molecules, including reactive oxygen species and their by-products (
      • Bautista D.M.
      • Jordt S.E.
      • Nikai T.
      • et al.
      TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents.
      ;
      • Eid S.R.
      • Crown E.D.
      • Moore E.L.
      • et al.
      HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity.
      ;
      • Materazzi S.
      • Nassini R.
      • Andre E.
      • et al.
      Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.
      ;
      • Biro T.
      • Kovacs L.
      An "ice-cold" TR(i)P to skin biology: the role of TRPA1 in human epidermal keratinocytes.
      ).
      Apart from their more prominent expression in nociceptors, thermo-TRPs may be expressed in non-nociceptive neurons (
      • Chen Y.
      • Williams S.H.
      • McNulty A.L.
      • et al.
      Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.
      ). More importantly, for the present study, some thermo-TRPs are found in non-neuronal cells (
      • Earley S.
      • Gonzales A.L.
      • Crnich R.
      Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-activated K+ channels.
      ;
      • Nozawa K.
      • Kawabata-Shoda E.
      • Doihara H.
      • et al.
      TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells.
      ;
      • Nassini R.
      • Pedretti P.
      • Moretto N.
      • et al.
      Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation.
      ) including skin cells (
      • Atoyan R.
      • Shander D.
      • Botchkareva N.V.
      Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.
      ;
      • Sokabe T.
      • Fukumi-Tominaga T.
      • Yonemura S.
      • et al.
      The TRPV4 channel contributes to intercellular junction formation in keratinocytes.
      ;
      • Radtke C.
      • Sinis N.
      • Sauter M.
      • et al.
      TRPV channel expression in human skin and possible role in thermally induced cell death.
      ). TRPV1 expression has been identified in epidermal and hair follicle keratinocytes, dermal mast cells, sebaceous gland–derived sebocytes, and dendritic cells (
      • Stander S.
      • Moormann C.
      • Schumacher M.
      • et al.
      Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures.
      ;
      • Bodo E.
      • Biro T.
      • Telek A.
      • et al.
      A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.
      ). TRPV2 has been found in keratinocytes (
      • Axelsson H.E.
      • Minde J.K.
      • Sonesson A.
      • et al.
      Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain.
      ) and macrophages (
      • Link T.M.
      • Park U.
      • Vonakis B.M.
      • et al.
      TRPV2 has a pivotal role in macrophage particle binding and phagocytosis.
      ), and TRPV3 has been found in blood vessels (
      • Earley S.
      • Gonzales A.L.
      • Garcia Z.I.
      A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation.
      ) and keratinocytes (
      • Cheng X.
      • Jin J.
      • Hu L.
      • et al.
      TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.
      ). The presence of TRPV4 has been reported in basal and suprabasal keratinocytes of healthy human skin (
      • Chung M.K.
      • Lee H.
      • Caterina M.J.
      Warm temperatures activate TRPV4 in mouse 308 keratinocytes.
      ;
      • Radtke C.
      • Sinis N.
      • Sauter M.
      • et al.
      TRPV channel expression in human skin and possible role in thermally induced cell death.
      ), where its function has been related to cell survival after skin exposure to noxious heat. Additional functions have been suggested for TRPV4 localized to skin cells. TRPV4 has been defined as osmo- and mechano-sensor for its contribution in responses to mechanical and osmotic stimuli (
      • Liedtke W.
      • Kim C.
      Functionality of the TRPV subfamily of TRP ion channels: add mechano-TRP and osmo-TRP to the lexicon!.
      ). In particular, TRPV4 has been suggested to control the homeostasis of the skin permeability barrier, as a sort of osmotic pressure detector (
      • Denda M.
      • Sokabe T.
      • Fukumi-Tominaga T.
      • et al.
      Effects of skin surface temperature on epidermal permeability barrier homeostasis.
      ). More recently, a highly relevant evolutionary function of TRPV4 has been proposed in mammalian skin both at the physiological and pathophysiological levels. Indeed, TRPV4 appears to contribute to UVB-induced damage and to UVB-evoked pain behavior by increasing the expression of the proalgesic/algogenic mediator endothelin-1 (
      • Moore C.
      • Cevikbas F.
      • Pasolli H.A.
      • et al.
      UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
      ). TRPA1 has been found in keratinocytes and in melanocytes, where it is activated by UVA irradiation, and via this mechanism it may contribute to the phototransduction process (
      • Atoyan R.
      • Shander D.
      • Botchkareva N.V.
      Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.
      ;
      • Bellono N.W.
      • Kammel L.G.
      • Zimmerman A.L.
      • et al.
      UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.
      ). Thus, TRPA1 may contribute to an extraneural pathway of phototransduction, possibly contributing to a spectrum of UV-activated TRPs, whereby TRPV4 is sensing UVB and TRPA1 UVA (
      • Bellono N.W.
      • Kammel L.G.
      • Zimmerman A.L.
      • et al.
      UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.
      ;
      • Moore C.
      • Cevikbas F.
      • Pasolli H.A.
      • et al.
      UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
      ).
      Non-melanoma skin cancer (NMSC) encompasses basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), which account for ∼80 and 20%, respectively, of the total tumor burden (
      • Weinstock M.A.
      Epidemiologic investigation of nonmelanoma skin cancer mortality: the Rhode Island Follow-Back Study.
      ). Both cancer subtypes originate from the basal layer of the epidermis of the skin, but although BCC is characterized by a slow-growing rate and poor metastatic potential SCC shows opposite features, thus representing the major cause of the deaths attributable to NMSC (
      • Weinstock M.A.
      Epidemiologic investigation of nonmelanoma skin cancer mortality: the Rhode Island Follow-Back Study.
      ). BCC and SCC occur primarily on sun-exposed areas of the body and have been associated with chronic sun and UV exposure (
      • Kwa R.E.
      • Campana K.
      • Moy R.L.
      Biology of cutaneous squamous cell carcinoma.
      ;
      • Bowden G.T.
      Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling.
      ). Accumulating evidence suggests that oxidative stress and release of inflammatory mediators from epidermal cells contribute to tumor development (
      • Marnett L.J.
      Oxyradicals and DNA damage.
      ;
      • Bachelor M.A.
      • Bowden G.T.
      Ultraviolet A-induced modulation of Bcl-XL by p38 MAPK in human keratinocytes: post-transcriptional regulation through the 3'-untranslated region.
      ;
      • Perrotta R.E.
      • Giordano M.
      • Malaguarnera M.
      Non-melanoma skin cancers in elderly patients.
      ). Change in expression and identification of functions, relevant for tumor progression, have been reported for TRP channels in different types of cancer (
      • Duncan L.M.
      • Deeds J.
      • Cronin F.E.
      • et al.
      Melastatin expression and prognosis in cutaneous malignant melanoma.
      ;
      • Tsavaler L.
      • Shapero M.H.
      • Morkowski S.
      • et al.
      Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.
      ;
      • Bode A.M.
      • Cho Y.Y.
      • Zheng D.
      • et al.
      Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis.
      ;
      • Oancea E.
      • Vriens J.
      • Brauchi S.
      • et al.
      TRPM1 forms ion channels associated with melanin content in melanocytes.
      ;
      • Lehen'kyi V.
      • Raphael M.
      • Prevarskaya N.
      The role of the TRPV6 channel in cancer.
      ;
      • Santoni G.
      • Caprodossi S.
      • Farfariello V.
      • et al.
      Antioncogenic effects of transient receptor potential vanilloid 1 in the progression of transitional urothelial cancer of human bladder.
      ). However, little information exists regarding TRP channel expression and function in skin tumors, and particularly in NMSC. Here, we show that TRPV4 is markedly downregulated in both the premalignant lesions of NMSC such as solar keratosis (SK) and Bowen’s disease (BD), and in SCC and BCC. In addition, TRPV4 in cultured keratinocytes is downregulated by cell exposure to a variety of proinflammatory mediators, including IL-8, which is released from keratinocytes upon TRPV4 stimulation.

      Results

      Localization of TRP in human healthy skin

      We first investigated, by immunohistochemistry, the expression of TRP proteins in human healthy skin. Staining for TRPV1 was observed in the epidermis, basal, and suprabasal epidermal keratinocytes (Figure 1). TRPV2 and TRPV3 staining was detected in basal and suprabasal keratinocytes and endothelial cells (
      • Axelsson H.E.
      • Minde J.K.
      • Sonesson A.
      • et al.
      Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain.
      ) (Figure 2a and b). TRPV4 protein expression was diffusely observed in basal and suprabasal epidermal keratinocytes, and it was also found in adnexal structures. Intense immunostaining was evident in the epidermal and dermal part of the eccrine sweat gland ducts. The secretory portion of sweat glands showed staining in single secretory and myoepithelial cells. Endothelial cells decorating dermal blood vessels were also TRPV4 positive (Figure 3). TRPA1 immunoreactivity was detected in the basal layer of the epidermis in healthy skin (Figure 4). Specificity of staining is indicated from previous studies (
      • Nassini R.
      • Pedretti P.
      • Moretto N.
      • et al.
      Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation.
      ;
      • Sulk M.
      • Seeliger S.
      • Aubert J.
      • et al.
      Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.
      ) and from negative controls performed in the presence of an excess of the respective immunizing peptide (Supplementary Figure S1 online).
      Figure thumbnail gr1
      Figure 1Localization of transient receptor potential vanilloid 1 (TRPV1) in human healthy and cancer skin tissues. Immunohistochemical localization reveals TRPV1 protein staining in the epidermis, basal, and suprabasal epidermal keratinocytes. TRPV1 staining and semiquantitative analysis of skin samples taken from patients suffering from solar keratosis (SK), Bowen’s disease (BD), squamous cell carcinoma (SCC), and nodular (Nod), superficial (Sup), and sclerodermiform (Scl) basal cell carcinoma (BCC) do not show a significant difference in protein expression in atypical keratinocytes. Bar=100μm.
      Figure thumbnail gr2
      Figure 2Localization of transient receptor potential vanilloid 2 and 3 (TRPV2 and TRPV3) in human healthy and cancer skin tissues. Immunohistochemical localization reveals TRPV2 (a) and TRPV3 (b) protein staining in basal and suprabasal keratinocytes, endothelial cells, and neuronal structures. TRPV2 and TRPV3 staining and semiquantitative analysis of skin samples taken from patients suffering from solar keratosis (SK), Bowen’s disease (BD), squamous cell carcinoma (SCC), and nodular (Nod), superficial (Sup), and sclerodermiform (Scl) basal cell carcinoma (BCC) do not show a significant difference in protein in atypical keratinocytes. Bar=100μm.
      Figure thumbnail gr3
      Figure 3Transient receptor potential vanilloid 4 (TRPV4) protein and messenger RNA (mRNA) results downregulated in different premalignant and invasive non-melanoma skin cancers of different histotypes compared to human healthy skin. (a) Immunohistochemical localization reveals TRPV4 protein staining in basal and suprabasal epidermal keratinocytes, and in adnexal structures in samples of healthy skin. TRPV4 staining (a) and semiquantitative analysis (b) of skin samples taken from patients suffering from solar keratosis (SK), Bowen’s disease (BD), squamous cell carcinoma (SCC), and nodular (Nod), superficial (Sup), and sclerodermiform (Scl) basal cell carcinoma (BCC) show a significant downregulation of protein in atypical keratinocytes. (c) TRPV4 mRNA analysis of samples taken from SK, BD, SCC, and BCC (Nod, Sup, and Scl) paraffin-embedded tissues shows a significant downregulation of TRPV4 mRNA compared with healthy skin (HS). Samples of dermatitis (inflamed tissues, IT) show levels of TRPV4 mRNA similar to those of HS. Values are expressed as percentage compared with 18S mRNA. *P<0.05 versus HS (nonparametric, two-tailed Mann–Whitney test in b and analysis of variance (ANOVA) followed by Bonferroni’s post hoc test in c). Bar=100μm.
      Figure thumbnail gr4
      Figure 4Localization of transient receptor potential ankyrin 1 (TRPA1) in human healthy and cancer skin tissues. Immunohistochemical localization of TRPA1 reveals protein staining in the basal layer of the epidermis in healthy skin. TRPA1 semiquantitative analysis of skin samples taken from patients suffering from solar keratosis (SK), Bowen’s disease (BD), squamous cell carcinoma (SCC), and nodular (Nod), superficial (Sup), and sclerodermiform (Scl) basal cell carcinoma (BCC) show a significant increase in TRPA1 staining only in atypical keratinocytes in samples of SK, whereas in different cases of BD, SCC, and BCC, irrespective of different histotypes, TRPA1 expression does not display any significant difference in staining compared with healthy skin (HS). *P<0.05 versus HS (nonparametric, two-tailed Mann–Whitney test). Bar=100μm.

      Differential expression of TRPs in skin cancer tissues

      Immunohistochemistry was used to determine semiquantitatively the level of expression of TRPV1, TRPV2, TRPV3, TRPV4, and TRPA1 in keratinocytes of healthy skin and cancer skin samples. As the expression of all the channels was predominant in keratinocytes, the analysis was limited to this cutaneous cell type. TRPV1, TRPV2, and TRPV3 expression was similar in atypical keratinocytes of SK, BD, SCC, BCC, and in keratinocytes of healthy skin (Figure 1 and Figure 2a and b). However, analysis of TRPV4 staining showed from remarkable reduction to complete abrogation of protein expression in skin samples of BD, SCC, and BCC of different histotypes as compared with healthy skin samples (Figure 3a and b). Reduction in TRPV4 protein staining was detected in keratinocytes in all the tested conditions. In contrast, other cells, such as those of the sweat gland ducts in the various types of cancers, exhibited a TRPV4 staining intensity, unchanged as compared with that observed in control samples (as an example, see SCC in Figure 3). In SK samples, the apparently reduced expression of TRPV4 staining did not reach the significance level (Figure 3a and b). In contrast, expression of TRPA1 protein was significantly higher in SK as compared with healthy skin, whereas no significant difference was noted in any other skin cancer type (Figure 4). We further tested TRPV4 messenger RNA (mRNA) expression by real-time PCR in the same paraffin-embedded samples used for immunohistochemical studies. Remarkable downregulation of TRPV4 mRNA was observed in all the cancer types compared with the level of TRPV4 mRNA measured in healthy skin samples (Figure 3c). TRPV4 mRNA was detected in the whole paraffin-embedded slice, and its decreased level paralleled the decrease in protein staining observed in atypical or neoplastic keratinocytes. As other cells (sweat gland cells) did not show any reduction in TRPV4 protein, it is plausible that keratinocytes from normal adnexal structures contribute only marginally to the bulk of channel mRNA in the present human skin tissues.

      Inflammatory mediators downregulate TRPV4 mRNA expression

      Several cytokines have been shown to contribute to the biology of skin neoplasms, including BCC and SCC (
      • Elamin I.
      • Zecevic R.D.
      • Vojvodic D.
      • et al.
      Cytokine concentrations in basal cell carcinomas of different histological types and localization.
      ). Keratinocytes in response to different stimuli, including UVB radiation, may release additional proinflammatory mediators such as prostaglandin E2 (PGE2), which has been proposed to promote skin tumor development (
      • Pei Y.
      • Barber L.A.
      • Murphy R.C.
      • et al.
      Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.
      ;
      • Countryman N.B.
      • Pei Y.
      • Yi Q.
      • et al.
      Evidence for involvement of the epidermal platelet-activating factor receptor in ultraviolet-B-radiation-induced interleukin-8 production.
      ). The expression of TRPV4 (
      • Becker D.
      • Blase C.
      • Bereiter-Hahn J.
      • et al.
      TRPV4 exhibits a functional role in cell-volume regulation.
      ) and TRPV1 (
      • Southall M.D.
      • Li T.
      • Gharibova L.S.
      • et al.
      Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes.
      ) channels in HaCaT has been previously reported. Moreover, stimulation of HaCaT cells with the selective TRPV1 agonist, capsaicin, caused the release of IL-8 (
      • Southall M.D.
      • Li T.
      • Gharibova L.S.
      • et al.
      Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes.
      ). By using western immunoblotting and real-time PCR, we confirmed TRPV4 expression in the HaCaT cell line (Figure 5a). Human embryonic kidney 293 and human bronchial smooth muscle cells have been used as negative and positive control (
      • Jia Y.
      • Wang X.
      • Varty L.
      • et al.
      Functional TRPV4 channels are expressed in human airway smooth muscle cells.
      ), respectively (Figure 5a). Expression of TRPV4 in HaCaT cells was downregulated by incubation for 24hours with IL-8, IL-1β, tumor necrosis factor-α (TNF-α), or PGE2 (Figure 5b–d).
      Figure thumbnail gr5
      Figure 5Effect of proinflammatory mediators on transient receptor potential vanilloid 4 (TRPV4) messenger RNA (mRNA) expression and release of IL-8 by TRPV4 stimulation in human keratinocytes. The protein levels of TRPV4 have been determined by western blot in different cellular lines including the human keratinocyte cell line HaCaT (a) compared with human embryonic kidney 293 (HEK293) and human bronchial smooth muscle cells (HBSMCs) as negative and positive control, respectively. Overnight exposure to increased concentrations (0.1–10ngml−1) of IL-8 (b) and IL-1β (c) induces a significant reduction in the TRPV4 mRNA levels in HaCaT. Values are expressed as percentage compared with 18S mRNA. A statistically significant reduction in TRPV4 mRNA level has also been obtained after incubation of HaCaT cells with 10ngml−1 TNF-α and 10μgml−1 prostaglandin E2 (PGE2) (d). Each column represents the mean±SEM of at least four independent experiments. *P<0.05 versus control group (Ctl) (analysis of variance (ANOVA) followed by Bonferroni’s post hoc test). Overnight exposure to 4α-phorbol-12,13-didecanoate (4αPDD) (e) and GSK1016790A (GSK) (f) induces IL-8 release from HaCaT in a concentration-dependent manner. IL-8 release evoked by both TRPV4 agonist 4αPDD (10μM) and GSK (5μM) is reduced by pretreatment with the selective TRPV4 antagonist HC-067047 (HC, 10μM). Overnight exposure to 4αPDD did not increase the release of IL-1β (g), TNF-α (h), and PGE2 (i). Each column represents the mean±SEM of at least three independent experiments. *P<0.05 versus basal group; §P<0.05 versus 4αPDD (10μM) and versus GSK (5μM). Effect of 4αPDD and GSK exposure on cell viability evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test in HaCaT (j, k). Each column represents the mean±SEM of at least three independent experiments. *P<0.05 versus basal group (Bas) (ANOVA followed by Bonferroni’s post hoc test). (l) Real-time PCR for the measurement of IL-8 mRNA has been performed in the healthy skin (HS), solar keratosis (SK), Bowen’s disease (BD), squamous cell carcinoma (SCC), and nodular (Nod), superficial (Sup), and sclerodermiform (Scl) basal cell carcinoma (BCC), as well as in samples of dermatitis (inflamed tissues, IT). Values are expressed as percentage compared with 18S mRNA. *P<0.05 versus HS, ANOVA followed by Bonferroni’s post hoc test.

      IL-8 release and TRPV4 downregulation

      To investigate the role of TRPV4 in the regulation of proinflammatory mediators in keratinocytes, we measured the release of IL-8, IL-1β, TNF-α, and PGE2 from HaCaT cells after exposure to the selective TRPV4 agonist 4α-phorbol-12,13-didecanoate (4αPDD). 4αPDD evoked a concentration-dependent release of IL-8 (Figure 5e). The response to 4αPDD was abrogated in the presence of the TRPV4-selective antagonist HC-067047 (
      • Everaerts W.
      • Zhen X.
      • Ghosh D.
      • et al.
      Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.
      ; Figure 5e). In contrast, exposure of HaCaT to 4αPDD (1–3–10μM) did not affect the release of IL-1β, TNF-α, and PGE2 (Figure 5g–i). In addition to 4αPDD, we tested the effect of a highly selective and potent TRPV4 agonist GSK1016790A (GSK;
      • Thorneloe K.S.
      • Sulpizio A.C.
      • Lin Z.
      • et al.
      N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1 -piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.
      ). Exposure of HaCaT to GSK (0.5–1–5μM) produced a concentration-dependent increase in IL-8 release, an effect abated in the presence of HC-067047 (Figure 5f). Overnight exposure to 4αPDD or to GSK (10 or 5μM, respectively) did not affect cell viability (Figure 5j and k).
      On the basis of these results, to investigate the possible relationship between TRPV4 downregulation and IL-8, we have measured the IL-8 mRNA level in healthy skin and tumor tissues by real-time PCR. In addition, we included in this part of the study some tissues characterized by neutrophilic inflammation (pustular psoriasis, neutrophilic folliculitis, and leucocytoclastic vasculitis) where presumably IL-8 should be increased. In these samples, we measured mRNA for TRPV4 and IL-8 (Figure 5l). As expected, IL-8 mRNA expression was high in inflamed tissues, whereas it was totally absent in normal skin (Figure 5l). TRPV4 expression was similarly elevated in both inflamed and normal skin (Figure 3c). SK, BD, and malignant cancer samples exhibited variable levels of IL-8 expression, between the almost undetectable value of normal skin and the high level of inflamed tissues (Figure 5l).

      Discussion

      In line with previous reports that described the distribution of certain TRP proteins in epidermal keratinocytes in human skin (
      • Chung M.K.
      • Lee H.
      • Caterina M.J.
      Warm temperatures activate TRPV4 in mouse 308 keratinocytes.
      ;
      • Bodo E.
      • Biro T.
      • Telek A.
      • et al.
      A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.
      ;
      • Atoyan R.
      • Shander D.
      • Botchkareva N.V.
      Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.
      ;
      • Axelsson H.E.
      • Minde J.K.
      • Sonesson A.
      • et al.
      Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain.
      ;
      • Earley S.
      • Gonzales A.L.
      • Garcia Z.I.
      A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation.
      ;
      • Radtke C.
      • Sinis N.
      • Sauter M.
      • et al.
      TRPV channel expression in human skin and possible role in thermally induced cell death.
      ), we confirmed that in human healthy skin samples TRPV1, TRPV2, TRPV3, TRPV4, and TRPA1 proteins are expressed in basal and suprabasal epidermal keratinocytes. For TRPV4 protein, we also observed an intense staining in adnexal structures in the eccrine sweat gland ducts, myoepithelial cells, and endothelial cells. Previous studies established an altered expression and function of one or more TRP proteins belonging to different channel subfamilies in malignant cells of various types of cancers. For instance, expression levels of members of the TRP melastatin (
      • Duncan L.M.
      • Deeds J.
      • Cronin F.E.
      • et al.
      Melastatin expression and prognosis in cutaneous malignant melanoma.
      ;
      • Tsavaler L.
      • Shapero M.H.
      • Morkowski S.
      • et al.
      Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.
      ;
      • Oancea E.
      • Vriens J.
      • Brauchi S.
      • et al.
      TRPM1 forms ion channels associated with melanin content in melanocytes.
      ) and TRPV (
      • Fixemer T.
      • Wissenbach U.
      • Flockerzi V.
      • et al.
      Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression.
      ;
      • Lazzeri M.
      • Vannucchi M.G.
      • Spinelli M.
      • et al.
      Transient receptor potential vanilloid type 1 (TRPV1) expression changes from normal urothelium to transitional cell carcinoma of human bladder.
      ;
      • Bode A.M.
      • Cho Y.Y.
      • Zheng D.
      • et al.
      Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis.
      ;
      • Santoni G.
      • Caprodossi S.
      • Farfariello V.
      • et al.
      Antioncogenic effects of transient receptor potential vanilloid 1 in the progression of transitional urothelial cancer of human bladder.
      ) subfamilies have been proposed to be associated with the induction/progression of the different tumors. Thus, it is possible that in human skin, and particularly in keratinocytes, thermo-TRPs may undergo changes during the carcinogenesis process.
      Here, we provide evidence that in skin samples obtained from biopsies of premalignant lesions of NMSC, such as SK and BD, and in malignant BCC and SCC, among the series of TRPs analyzed, TRPA1 protein expression was increased in SK and, more importantly, TRPV4 protein displays a remarkably reduced expression in skin cancer. Immunohistochemical findings are strengthened by real-time PCR results. Remarkable downregulation of the TRPV4 mRNA was found in the same samples of skin cancer where TRPV4 protein expression was also reduced. These results indicate that, regardless of the initiating mechanism, TRPV4 downregulation occurs already at the transcriptional level. The observation that such a marked reduction, observed in cancer samples, also occurs in a precancer condition such as SK suggests that epigenetic or other factors that govern channel downregulation act similarly in both atypical and malignant skin phenotypes.
      Skin carcinogenesis has been associated with UV irradiation and the ensuing increase in oxidative stress (
      • Mukhtar H.
      • Elmets C.A.
      Photocarcinogenesis: mechanisms, models and human health implications.
      ), and TRPA1 exhibits a peculiar sensitivity for oxidative stress by-products (
      • Trevisani M.
      • Siemens J.
      • Materazzi S.
      • et al.
      4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1.
      ;
      • Andersson D.A.
      • Gentry C.
      • Moss S.
      • et al.
      Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress.
      ;
      • Materazzi S.
      • Nassini R.
      • Andre E.
      • et al.
      Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.
      ;
      • Sawada Y.
      • Hosokawa H.
      • Matsumura K.
      • et al.
      Activation of transient receptor potential ankyrin 1 by hydrogen peroxide.
      ). UV radiation represents the major causative factor for melanoma (
      • Boniol M.
      • Autier P.
      • Boyle P.
      • et al.
      Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis.
      ;
      • Mulliken J.S.
      • Russak J.E.
      • Rigel D.S.
      The effect of sunscreen on melanoma risk.
      ). Increasing information is available regarding the ability of UV to activate TRPA1. A previous study has shown that, in a TRPA1-expressing recombinant system, UVA, most probably via the generation of reactive oxygen species, induces channel activation (
      • Hill K.
      • Schaefer M.
      Ultraviolet light and photosensitising agents activate TRPA1 via generation of oxidative stress.
      ). More recently, it has been reported that UVA radiation stimulates TRPA1 in melanocytes, thus suggesting a role for the TRPA1 channel in the phototransduction process in melanocytes, and possibly in melanin synthesis (
      • Bellono N.W.
      • Kammel L.G.
      • Zimmerman A.L.
      • et al.
      UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.
      ). TRPA1 has also been detected in human keratinocytes, where it modulates the expression of an array of genes involved in cell differentiation, metabolism, signaling, and transcription (
      • Atoyan R.
      • Shander D.
      • Botchkareva N.V.
      Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.
      ). As SK is a condition that is markedly associated with sun exposure, further research may explore whether UV radiation is responsible for the upregulation of TRPA1 in SK keratinocytes. However, a very recent study reported that, at variance with TRPV4, in both human and mouse skin samples local transcription of TRPA1 is not detectable (
      • Liu B.
      • Escalera J.
      • Balakrishna S.
      • et al.
      TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.
      ).
      However, the major finding of the present study relates to TRPV4. Transformation from healthy to atypical, but not yet malignant, keratinocytes in SK was associated with a tendency (which, however, did not reach the significance level) to a reduced TRPV4 expression, which, however, was fully evident and significant in BD and in malignant forms of cancer such as SCC. This suggests a sort of progressive reduction in TRPV4 protein expression from healthy to precancerous and finally malignant skin cancer phenotypes. The implications of the present results are 2-fold. First, we confirm that in human skin TRPV4 expression is not solely confined to cutaneous nociceptors, but it is abundantly distributed in different non-neuronal cutaneous cell types, and prevalently in keratinocytes. Second, loss of TRPV4 expression in keratinocytes seems to be specifically associated with the transition from a healthy phenotype to a cancer phenotype. TRPV4 has been claimed to produce various functions in keratinocytes. Although keratinocyte TRPV4 may respond to warm temperatures (
      • Chung M.K.
      • Lee H.
      • Caterina M.J.
      Warm temperatures activate TRPV4 in mouse 308 keratinocytes.
      ), TRPV3 appears to mainly contribute to the transmission of warm temperature sensation, via ATP release in these cells (
      • Mandadi S.
      • Sokabe T.
      • Shibasaki K.
      • et al.
      TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.
      ). Additional proposed roles of TRPV4 in keratinocytes are represented by the control of the skin permeability barrier (
      • Denda M.
      • Sokabe T.
      • Fukumi-Tominaga T.
      • et al.
      Effects of skin surface temperature on epidermal permeability barrier homeostasis.
      ), and the promotion of Ca2+-dependent phenomena as the development and maturation of cell–cell junctions (
      • Sokabe T.
      • Fukumi-Tominaga T.
      • Yonemura S.
      • et al.
      The TRPV4 channel contributes to intercellular junction formation in keratinocytes.
      ). Cell proliferation and differentiation are regulated by Ca2+ ions, as high extracellular Ca2+ favors differentiation and low extracellular Ca2+ maintains an undifferentiated state. It is possible that the TRPV4 channel contributes to these phenomena in keratinocytes (
      • Lee H.
      • Caterina M.J.
      TRPV channels as thermosensory receptors in epithelial cells.
      ). A recent paper has reported that UVB exposure causes direct TRPV4 activation in keratinocytes, and that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage, thus increasing the expression of the proalgesic mediator, endothelin-1, and producing pain behavior in mice; all these effects were reduced by TRPV4 inhibition (
      • Moore C.
      • Cevikbas F.
      • Pasolli H.A.
      • et al.
      UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
      ). In addition, also in human specimens, the expression of epidermal TRPV4 and endothelin-1 is enhanced by sunburn (
      • Moore C.
      • Cevikbas F.
      • Pasolli H.A.
      • et al.
      UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
      ). Together, these data strongly highlight the role of TRPV4 expressed in keratinocytes as a therapeutic target for UVB-evoked skin tissue damage and sunburn-related pain.
      It has been widely reported that both cancer cells and surrounding cells release a wide array of cytokines in order to establish a tumor microenvironment that affects cancer cell growth differently, attenuating apoptosis and promoting tissue invasion and metastasis (
      • Elamin I.
      • Zecevic R.D.
      • Vojvodic D.
      • et al.
      Cytokine concentrations in basal cell carcinomas of different histological types and localization.
      ;
      • St John M.A.
      • Dohadwala M.
      • Luo J.
      • et al.
      Proinflammatory mediators upregulate snail in head and neck squamous cell carcinoma.
      ). Several proinflammatory and immunomodulatory cytokines are induced in epidermal cells after exposure to one of the most prominent cutaneous tumorigenic agents, UV radiation, including IL-1β, IL-6, IL-8, and TNF-α (
      • Kupper T.S.
      • Chua A.O.
      • Flood P.
      • et al.
      Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation.
      ;
      • Takashima A.
      • Bergstresser P.R.
      Impact of UVB radiation on the epidermal cytokine network.
      ;
      • Strickland I.
      • Rhodes L.E.
      • Flanagan B.F.
      • et al.
      TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.
      ). TNF-α mediates UV induction of adhesion molecule expression (
      • Krutmann J.
      • Czech W.
      • Parlow F.
      • et al.
      Ultraviolet radiation effects on human keratinocyte ICAM-1 expression: UV-induced inhibition of cytokine-induced ICAM-1 mRNA expression is transient, differentially restored for IFN gamma versus TNF alpha, and followed by ICAM-1 induction via a TNF alpha-like pathway.
      ) and Langerhans cell migration (
      • Vincek V.
      • Kurimoto I.
      • Medema J.P.
      • et al.
      Tumor necrosis factor alpha polymorphism correlates with deleterious effects of ultraviolet B light on cutaneous immunity.
      ). IL-8 is a chemokine that induces an inflammatory cell infiltrate after UV radiation (
      • Strickland I.
      • Rhodes L.E.
      • Flanagan B.F.
      • et al.
      TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.
      ); IL-1β acts as a chemoattractant, induces TNF-α (
      • Corsini E.
      • Bruccoleri A.
      • Marinovich M.
      • et al.
      In vitro mechanism(s) of ultraviolet-induced tumor necrosis factor-alpha release in a human keratinocyte cell line.
      ), and enhances keratinocyte prostaglandin synthesis (
      • Pentland A.P.
      • Mahoney M.G.
      Keratinocyte prostaglandin synthesis is enhanced by IL-1.
      ); and one prostaglandin, PGE2, increases in epidermal keratinocytes after UV irradiation (
      • Rundhaug J.E.
      • Simper M.S.
      • Surh I.
      • et al.
      The role of the EP receptors for prostaglandin E2 in skin and skin cancer.
      ). To explore the mechanism responsible for the marked reduction in TRPV4 expression in tumor keratinocytes, we measured channel mRNA expression in a keratinocyte cell line (HaCaT cells) which has been reported to express TRPV4 (
      • Becker D.
      • Blase C.
      • Bereiter-Hahn J.
      • et al.
      TRPV4 exhibits a functional role in cell-volume regulation.
      ) by real-time PCR, after exposure to IL-1β, IL-6, IL-8, and TNF-α and PGE2. All these stimuli induced a dose-dependent reduction in TRPV4 mRNA levels, thus suggesting that cytokines and prostaglandins, released within the tumor milieu, have the potential to reduce or suppress TRPV4 gene expression in keratinocytes. Next, we explored the ability of TRPV4 to release the proinflammatory mediators that downregulate channel expression. Present data demonstrate that TRPV4 stimulation by the selective agonist 4αPDD did not affect the release of IL-1β, TNF-α, and PGE2 from HaCaT cells. However, TRPV4 stimulation by 4αPDD increased IL-8 release, which should be specifically dependent on channel activation, as it was abrogated by the selective TRPV4 antagonist HC-067047. Previous studies have reported that TRP channel stimulation causes IL-8 release. TRPV1 stimulation has been shown to release IL-8 from HaCaT cells (
      • Southall M.D.
      • Li T.
      • Gharibova L.S.
      • et al.
      Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes.
      ), and it modulates IL-8 release in human corneal epithelial cells (
      • Wang Z.
      • Yang Y.
      • Yang H.
      • et al.
      NF-kappaB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells.
      ). Recently, we reported that TRPA1 activation releases IL-8 from TRPA1 expressed in non-neuronal airway cells (
      • Nassini R.
      • Pedretti P.
      • Moretto N.
      • et al.
      Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation.
      ). IL-8 release can be considered as part of a defense response orchestrated by keratinocytes, via TRPV4, to limit cancer progression. However, it is also possible that IL-8, downregulating TRPV4, may progressively reduce keratinocyte potential to release IL-8 itself. To better address this issue, we studied the expression of IL-8 mRNA in healthy skin and cancer tissues and both IL-8 and TRPV4 mRNA in skin dermatitis biopsies. The observation that, in inflamed tissues, IL-8 mRNA was, as expected, elevated, and TRPV4 expression was high and comparable to that of normal skin, indicates that increased IL-8 levels do not warrant TRPV4 downregulation. Thus, the most parsimonious explanation is that the marked reduction in TRPV4 expression documented in skin cancers is independent from, and not the consequence of, the variable and moderate increase in IL-8 observed in these conditions.
      A constraint of the present study is represented by the limited number of cases analyzed, a factor that may underestimate differences in TRPV1, TRPV2, TRPV3, and TRPA1 expression in the various types of cancer. Nevertheless, our findings, showing a remarkable channel downregulation, point to TRPV4 as an early biomarker of cutaneous cancers. The ability of IL-8 to downregulate TRPV4 and of TRPV4 to release IL-8 depict a possible autocrine circuitry carried out by keratinocytes to regulate skin homeostasis and contribute to pathophysiological events. However, the observation that TRPV4 expression is not affected by high IL-8 levels, as found in inflamed tissues, strengthens the hypothesis that TRPV4 is upstream to IL-8 and not vice versa. An additional hypothesis is that the remarkable TRPV4 downregulation in cancer, a condition that is most likely associated with cancer onset and progression, may contribute to a poor defensive inflammatory response in skin cancer.

      Materials and methods

      Tissue collection

      The study series included skin biopsies of human healthy skin (n=4), SK (n=5), BD (n=5), invasive cutaneous SCC (n=7), and BCC of different histotypes, including nodular (n=4), superficial (n=5), and sclerodermiform/morphea-like types (n=3). To examine the expression of mRNA of TRPV4 and IL-8 skin biopsies of pustular psoriasis (n=3), neutrophilic folliculitis (n=3) and leucocytoclastic vasculitis (n=3) were also included in the study. Paraffin-embedded skin specimens were retrospectively retrieved from the archive of the Division of Pathology, Department of Surgery and Translational Medicine, the University of Florence, Italy. Patients’ data, including age, sex, and anatomic tumor site, were collected. The median age of patients with SK and BD was 67 (range, 53–79) and 73.3 (range, 71–81) years, respectively. For both premalignant lesions, three patients were female and two patients were male. Tumor site distribution was the head and neck, upper extremities, and trunk. The median age of patients with SCC was 80 years (range 73–88 years). Six patients were male and one was female, and the tumor site distribution was upper extremities. The median age of patients with nodular BCC was 71 years (range, 39–89 years), eight patients were male and four were female, and the tumor site distribution was upper, lower, and trunk extremities. Ethical approval for the experiments performed on human tissue was obtained from the internal institutional review board.

      Immunohistochemistry

      Sections of 4-μm thickness were cut from tissue blocks of formalin-fixed, paraffin-embedded samples. Immunostaining was performed according to standard procedures. In brief, antigen retrieval was routinely performed by immersing the slides in a thermostat bath containing 10mM citrate buffer (pH 6.0) for 15min at 97°C followed by cooling for 20minutes at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in distilled water for 10minutes. After blocking with normal horse serum (UltraVision, Bio-Optica, Milan, Italy), sections were incubated overnight at 4°C with the following rabbit polyclonal antibodies: TRPV1 (1:100, Abcam, Cambridge, UK), TRPV2 (1:200, Acris Antibodies Herford, Germany), TRPV3 (1:100 Acris Antibodies), TRPV4 (1:50, Sigma-Aldrich, Milan, Italy), and TRPA1 (1:250, Novus Biologicals, Cambridge, UK). Bound antibodies were visualized using aminoethylcarbazol as chromogen (Bio-Optica). Nuclei were counterstained with Mayer’s hematoxylin. Negative controls were performed by preadsorption with immunizing peptide (overnight 4°C) for all the antisera. For the semiquantitative image analysis, epidermal intensity and area of immunostained cells were rated on a scale of 0–4 (0 absent, 1 weak/low, 2 moderate, 3 strong, and 4 very strong staining).

      Cell culture

      The human keratinocyte cell line HaCaT (Cell Line Service, Eppelheim, Germany), human bronchial smooth muscle cell (PromoCell, Heidelberg, Germany), and human embryonic kidney cell 293 (American Type Cell Collection, Manassas, VA) were grown in DMEM supplemented with 10% fetal bovine serum, 2mM glutamine, 100Uml−1 penicillin, and 100μgml−1 streptomycin (Sigma-Aldrich). Cells were cultured in an atmosphere of 95% air and 5% CO2 at 37°C.

      Measurement of cytokines and PGE2 production by keratinocyte cell culture

      Cells were plated at a density of 200,000 cells per well in a 24-well plate and grown for 24hours to ∼80–90% confluence, and then exposed to different concentrations (1–30μM) of the selective TRPV4 agonist, 4αPDD (Sigma-Aldrich), or its vehicle (0.3% DMSO), in a serum-free medium. In a different set of experiments, HaCaT cells were exposed to GSK (0.5, 1, 5, 10μM), another potent and selective TRPV4 agonist (
      • Thorneloe K.S.
      • Sulpizio A.C.
      • Lin Z.
      • et al.
      N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1 -piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.
      ), or its vehicle (0.5% DMSO). Selective TRPV4 antagonist HC-067047 (10μM;
      • Everaerts W.
      • Zhen X.
      • Ghosh D.
      • et al.
      Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.
      ; Tocris Bioscience, Bristol, UK), or its vehicle (0.1% DMSO), was added 30minutes before exposure to the agonists. The medium was collected 18hours after treatment, and human IL-8, IL-6, IL-1β, (TNF-α), and prostaglandin E2 (PGE2) were measured using the commercial quantitative ELISA kit (Invitrogen, Carlsbad, CA).

      Western immunoblot assay

      HaCaT, human bronchial smooth muscle cells, and human embryonic kidney 293 cells were lysed for 30minutes at 4°C in a buffer containing 50mM Tris, pH 7.5, 150mM NaCl, 2mM EGTA, 100mM NaF, 1mM Na3VO4, 1% Nonidet P40, and complete protease inhibitor cocktail (Roche, Mannheim, Germany). Cell lysates were used for the TRPV4 protein detection, as described in detail in Supplementary Materials and Methods online.

      Reverse transcriptase–PCR and real-time PCR

      Extraction of mRNA was performed in paraffin-embedded tissues as described (
      • Kalmar A.
      • Wichmann B.
      • Galamb O.
      • et al.
      Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and automated RNA isolation.
      ), with some modification. In brief, deparaffinization was performed by adding 1ml of xylene for 10minutes twice and 1ml absolute ethanol for 10minutes twice. Total RNA has been extracted from the air-dried deparaffinized sections by using the TRIZOL method (Invitrogen).
      HaCaT, human bronchial smooth muscle cells, and human embryonic kidney 293 cells were seeded in six-well plates and grown for 24hours to 90% confluence before stimulation or mRNA extraction. Then, cells were exposed for 24hours, in a serum-free medium, to different concentrations of several inflammatory mediators, including IL-8, IL-1β, IL-6, TNF-α (all 0.1–10ngml−1), and PGE2 10μgml−1, or their vehicles (cell medium without stimulus). Total cellular RNA was extracted from stimulated and unstimulated cells by using the TRIZOL method (Invitrogen). Complementary DNA was prepared from total RNA using the iScript cDNA Synthesis kit (Bio-Rad, Milan, Italy), and the real-time PCR was performed to check the TRPV4 and IL-8 mRNA levels as described in more details in Supplementary Materials and Methods online.

      Vitality assay by the tetrazolium salt method (MTT reduction assay)

      Cytotoxicity of various stimuli, including 4αPDD, GSK, or inflammatory mediators tested on HaCaT cell line, was assessed by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability test, as described in detail in Supplementary Materials and Methods online.

      Statistical analysis

      For the semiquantitative image analysis, statistical significance was determined by using a nonparametric one-way analysis of variance followed by the Kruskal–Wallis post hoc test. All other data are presented as mean±SEM. Statistical significance was determined by using one-way analysis of variance, followed by Bonferroni’s post hoc analysis for comparison of multiple groups. P<0.05 was considered significant.

      ACKNOWLEDGMENTS

      This study was supported in part by the Fondazione Ente Cassa di Risparmio di Firenze to DM and SM, and in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC MFAG, 13336) to RN.

      Supplementary Material

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      REFERENCES

        • Andersson D.A.
        • Gentry C.
        • Moss S.
        • et al.
        Transient receptor potential A1 is a sensory receptor for multiple products of oxidative stress.
        J Neurosci. 2008; 28: 2485-2494
        • Atoyan R.
        • Shander D.
        • Botchkareva N.V.
        Non-neuronal expression of transient receptor potential type A1 (TRPA1) in human skin.
        J Invest Dermatol. 2009; 129: 2312-2315
        • Axelsson H.E.
        • Minde J.K.
        • Sonesson A.
        • et al.
        Transient receptor potential vanilloid 1, vanilloid 2 and melastatin 8 immunoreactive nerve fibers in human skin from individuals with and without Norrbottnian congenital insensitivity to pain.
        Neuroscience. 2009; 162: 1322-1332
        • Bachelor M.A.
        • Bowden G.T.
        Ultraviolet A-induced modulation of Bcl-XL by p38 MAPK in human keratinocytes: post-transcriptional regulation through the 3'-untranslated region.
        J Biol Chem. 2004; 279: 42658-42668
        • Bautista D.M.
        • Jordt S.E.
        • Nikai T.
        • et al.
        TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents.
        Cell. 2006; 124: 1269-1282
        • Becker D.
        • Blase C.
        • Bereiter-Hahn J.
        • et al.
        TRPV4 exhibits a functional role in cell-volume regulation.
        J Cell Sci. 2005; 118: 2435-2440
        • Bellono N.W.
        • Kammel L.G.
        • Zimmerman A.L.
        • et al.
        UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes.
        Proc Natl Acad Sci USA. 2013; 110: 2383-2388
        • Biro T.
        • Kovacs L.
        An "ice-cold" TR(i)P to skin biology: the role of TRPA1 in human epidermal keratinocytes.
        J Invest Dermatol. 2009; 129: 2096-2099
        • Bode A.M.
        • Cho Y.Y.
        • Zheng D.
        • et al.
        Transient receptor potential type vanilloid 1 suppresses skin carcinogenesis.
        Cancer Res. 2009; 69: 905-913
        • Bodo E.
        • Biro T.
        • Telek A.
        • et al.
        A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.
        Am J Pathol. 2005; 166: 985-998
        • Boniol M.
        • Autier P.
        • Boyle P.
        • et al.
        Cutaneous melanoma attributable to sunbed use: systematic review and meta-analysis.
        BMJ. 2012; 345: e4757
        • Bowden G.T.
        Prevention of non-melanoma skin cancer by targeting ultraviolet-B-light signalling.
        Nat Rev Cancer. 2004; 4: 23-35
        • Caterina M.J.
        • Schumacher M.A.
        • Tominaga M.
        • et al.
        The capsaicin receptor: a heat-activated ion channel in the pain pathway.
        Nature. 1997; 389: 816-824
        • Chen Y.
        • Williams S.H.
        • McNulty A.L.
        • et al.
        Temporomandibular joint pain: a critical role for Trpv4 in the trigeminal ganglion.
        Pain. 2013; 154: 1295-1304
        • Cheng X.
        • Jin J.
        • Hu L.
        • et al.
        TRP channel regulates EGFR signaling in hair morphogenesis and skin barrier formation.
        Cell. 2010; 141: 331-343
        • Chung M.K.
        • Lee H.
        • Caterina M.J.
        Warm temperatures activate TRPV4 in mouse 308 keratinocytes.
        J Biol Chem. 2003; 278: 32037-32046
        • Corsini E.
        • Bruccoleri A.
        • Marinovich M.
        • et al.
        In vitro mechanism(s) of ultraviolet-induced tumor necrosis factor-alpha release in a human keratinocyte cell line.
        Photodermatol Photoimmunol Photomed. 1995; 11: 112-118
        • Countryman N.B.
        • Pei Y.
        • Yi Q.
        • et al.
        Evidence for involvement of the epidermal platelet-activating factor receptor in ultraviolet-B-radiation-induced interleukin-8 production.
        J Invest Dermatol. 2000; 115: 267-272
        • 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
        • Duncan L.M.
        • Deeds J.
        • Cronin F.E.
        • et al.
        Melastatin expression and prognosis in cutaneous malignant melanoma.
        J Clin Oncol. 2001; 19: 568-576
        • Earley S.
        • Gonzales A.L.
        • Crnich R.
        Endothelium-dependent cerebral artery dilation mediated by TRPA1 and Ca2+-activated K+ channels.
        Circ Res. 2009; 104: 987-994
        • Earley S.
        • Gonzales A.L.
        • Garcia Z.I.
        A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation.
        Mol Pharmacol. 2010; 77: 612-620
        • Eid S.R.
        • Crown E.D.
        • Moore E.L.
        • et al.
        HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity.
        Mol Pain. 2008; 4: 48-57
        • Elamin I.
        • Zecevic R.D.
        • Vojvodic D.
        • et al.
        Cytokine concentrations in basal cell carcinomas of different histological types and localization.
        Acta Dermatovenerol Alp Panonica Adriat. 2008; 17: 55-59
        • Everaerts W.
        • Zhen X.
        • Ghosh D.
        • et al.
        Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.
        Proc Natl Acad Sci USA. 2010; 107: 19084-19089
        • Fixemer T.
        • Wissenbach U.
        • Flockerzi V.
        • et al.
        Expression of the Ca2+-selective cation channel TRPV6 in human prostate cancer: a novel prognostic marker for tumor progression.
        Oncogene. 2003; 22: 7858-7861
        • Geppetti P.
        • Holzer P.
        Neurogenic Inflammation. CRC Press: Boca Raton, FL1996
        • Hill K.
        • Schaefer M.
        Ultraviolet light and photosensitising agents activate TRPA1 via generation of oxidative stress.
        Cell Calcium. 2009; 45: 155-164
        • Ho W.S.
        • Barrett D.A.
        • Randall M.D.
        'Entourage' effects of N-palmitoylethanolamide and N-oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors.
        Br J Pharmacol. 2008; 155: 837-846
        • Jia Y.
        • Wang X.
        • Varty L.
        • et al.
        Functional TRPV4 channels are expressed in human airway smooth muscle cells.
        Am J Physiol Lung Cell Mol Physiol. 2004; 287: L272-L278
        • Kalmar A.
        • Wichmann B.
        • Galamb O.
        • et al.
        Gene expression analysis of normal and colorectal cancer tissue samples from fresh frozen and matched formalin-fixed, paraffin-embedded (FFPE) specimens after manual and automated RNA isolation.
        Methods. 2013; 59: S16-S19
        • Krutmann J.
        • Czech W.
        • Parlow F.
        • et al.
        Ultraviolet radiation effects on human keratinocyte ICAM-1 expression: UV-induced inhibition of cytokine-induced ICAM-1 mRNA expression is transient, differentially restored for IFN gamma versus TNF alpha, and followed by ICAM-1 induction via a TNF alpha-like pathway.
        J Invest Dermatol. 1992; 98: 923-928
        • Kupper T.S.
        • Chua A.O.
        • Flood P.
        • et al.
        Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation.
        J Clin Invest. 1987; 80: 430-436
        • Kwa R.E.
        • Campana K.
        • Moy R.L.
        Biology of cutaneous squamous cell carcinoma.
        J Am Acad Dermatol. 1992; 26: 1-26
        • Lazzeri M.
        • Vannucchi M.G.
        • Spinelli M.
        • et al.
        Transient receptor potential vanilloid type 1 (TRPV1) expression changes from normal urothelium to transitional cell carcinoma of human bladder.
        Eur Urol. 2005; 48: 691-698
        • Lee H.
        • Caterina M.J.
        TRPV channels as thermosensory receptors in epithelial cells.
        Pflugers Arch. 2005; 451: 160-167
        • Lehen'kyi V.
        • Raphael M.
        • Prevarskaya N.
        The role of the TRPV6 channel in cancer.
        J Physiol. 2012; 590: 1369-1376
        • Liedtke W.
        • Choe Y.
        • Marti-Renom M.A.
        • et al.
        Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.
        Cell. 2000; 103: 525-535
        • Liedtke W.
        • Kim C.
        Functionality of the TRPV subfamily of TRP ion channels: add mechano-TRP and osmo-TRP to the lexicon!.
        Cell Mol Life Sci. 2005; 62: 2985-3001
        • Link T.M.
        • Park U.
        • Vonakis B.M.
        • et al.
        TRPV2 has a pivotal role in macrophage particle binding and phagocytosis.
        Nat Immunol. 2010; 11: 232-239
        • Liu B.
        • Escalera J.
        • Balakrishna S.
        • et al.
        TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.
        FASEB J. 2013; 27: 3549-3563
        • Mandadi S.
        • Sokabe T.
        • Shibasaki K.
        • et al.
        TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.
        Pflugers Arch. 2009; 458: 1093-1102
        • Marnett L.J.
        Oxyradicals and DNA damage.
        Carcinogenesis. 2000; 21: 361-370
        • Materazzi S.
        • Nassini R.
        • Andre E.
        • et al.
        Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.
        Proc Natl Acad Sci USA. 2008; 105: 12045-12050
        • Moore C.
        • Cevikbas F.
        • Pasolli H.A.
        • et al.
        UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
        Proc Natl Acad Sci USA. 2013; 110: E3225-E3234
        • Mukhtar H.
        • Elmets C.A.
        Photocarcinogenesis: mechanisms, models and human health implications.
        Photochem Photobiol. 1996; 63: 356-357
        • Mulliken J.S.
        • Russak J.E.
        • Rigel D.S.
        The effect of sunscreen on melanoma risk.
        Dermatol Clin. 2012; 30: 369-376
        • Nassini R.
        • Pedretti P.
        • Moretto N.
        • et al.
        Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation.
        PLoS One. 2012; 7: e42454
        • Nilius B.
        • Owsianik G.
        • Voets T.
        • et al.
        Transient receptor potential cation channels in disease.
        Physiol Rev. 2007; 87: 165-217
        • Nozawa K.
        • Kawabata-Shoda E.
        • Doihara H.
        • et al.
        TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells.
        Proc Natl Acad Sci USA. 2009; 106: 3408-3413
        • Oancea E.
        • Vriens J.
        • Brauchi S.
        • et al.
        TRPM1 forms ion channels associated with melanin content in melanocytes.
        Sci Signal. 2009; 2: ra21
        • Pei Y.
        • Barber L.A.
        • Murphy R.C.
        • et al.
        Activation of the epidermal platelet-activating factor receptor results in cytokine and cyclooxygenase-2 biosynthesis.
        J Immunol. 1998; 161: 1954-1961
        • Pentland A.P.
        • Mahoney M.G.
        Keratinocyte prostaglandin synthesis is enhanced by IL-1.
        J Invest Dermatol. 1990; 94: 43-46
        • Perrotta R.E.
        • Giordano M.
        • Malaguarnera M.
        Non-melanoma skin cancers in elderly patients.
        Crit Rev Oncol Hematol. 2011; 80: 474-480
        • Radtke C.
        • Sinis N.
        • Sauter M.
        • et al.
        TRPV channel expression in human skin and possible role in thermally induced cell death.
        J Burn Care Res. 2011; 32: 150-159
        • Rundhaug J.E.
        • Simper M.S.
        • Surh I.
        • et al.
        The role of the EP receptors for prostaglandin E2 in skin and skin cancer.
        Cancer Metastasis Rev. 2011; 30: 465-480
        • Santoni G.
        • Caprodossi S.
        • Farfariello V.
        • et al.
        Antioncogenic effects of transient receptor potential vanilloid 1 in the progression of transitional urothelial cancer of human bladder.
        ISRN Urol. 2012; 2012: 458238
        • Sawada Y.
        • Hosokawa H.
        • Matsumura K.
        • et al.
        Activation of transient receptor potential ankyrin 1 by hydrogen peroxide.
        Eur J Neurosci. 2008; 27: 1131-1142
        • Sokabe T.
        • Fukumi-Tominaga T.
        • Yonemura S.
        • et al.
        The TRPV4 channel contributes to intercellular junction formation in keratinocytes.
        J Biol Chem. 2010; 285: 18749-18758
        • Southall M.D.
        • Li T.
        • Gharibova L.S.
        • et al.
        Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes.
        J Pharmacol Exp Ther. 2003; 304: 217-222
        • Stander S.
        • Moormann C.
        • Schumacher M.
        • et al.
        Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures.
        Exp Dermatol. 2004; 13: 129-139
        • St John M.A.
        • Dohadwala M.
        • Luo J.
        • et al.
        Proinflammatory mediators upregulate snail in head and neck squamous cell carcinoma.
        Clin Cancer Res. 2009; 15: 6018-6027
        • Strickland I.
        • Rhodes L.E.
        • Flanagan B.F.
        • et al.
        TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.
        J Invest Dermatol. 1997; 108: 763-768
        • Sulk M.
        • Seeliger S.
        • Aubert J.
        • et al.
        Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.
        J Invest Dermatol. 2012; 132: 1253-1262
        • Takashima A.
        • Bergstresser P.R.
        Impact of UVB radiation on the epidermal cytokine network.
        Photochem Photobiol. 1996; 63: 397-400
        • Thorneloe K.S.
        • Sulpizio A.C.
        • Lin Z.
        • et al.
        N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1 -piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.
        J Pharmacol Exp Ther. 2008; 326: 432-442
        • Tominaga M.
        • Caterina M.J.
        • Malmberg A.B.
        • et al.
        The cloned capsaicin receptor integrates multiple pain-producing stimuli.
        Neuron. 1998; 21: 531-543
        • Trevisani M.
        • Siemens J.
        • Materazzi S.
        • et al.
        4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1.
        Proc Natl Acad Sci USA. 2007; 104: 13519-13524
        • Tsavaler L.
        • Shapero M.H.
        • Morkowski S.
        • et al.
        Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.
        Cancer Res. 2001; 61: 3760-3769
        • Vincek V.
        • Kurimoto I.
        • Medema J.P.
        • et al.
        Tumor necrosis factor alpha polymorphism correlates with deleterious effects of ultraviolet B light on cutaneous immunity.
        Cancer Res. 1993; 53: 728-732
        • Wang Z.
        • Yang Y.
        • Yang H.
        • et al.
        NF-kappaB feedback control of JNK1 activation modulates TRPV1-induced increases in IL-6 and IL-8 release by human corneal epithelial cells.
        Mol Vis. 2011; 17: 3137-3146
        • Weinstock M.A.
        Epidemiologic investigation of nonmelanoma skin cancer mortality: the Rhode Island Follow-Back Study.
        J Invest Dermatol. 1994; 102: 6S-9S