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Transient Receptor Potential Vanilloid 4 (TRPV4) Is Downregulated in Keratinocytes in Human Non-Melanoma Skin Cancer  Camilla Fusi, Serena Materazzi,

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Presentation on theme: "Transient Receptor Potential Vanilloid 4 (TRPV4) Is Downregulated in Keratinocytes in Human Non-Melanoma Skin Cancer  Camilla Fusi, Serena Materazzi,"— Presentation transcript:

1 Transient Receptor Potential Vanilloid 4 (TRPV4) Is Downregulated in Keratinocytes in Human Non-Melanoma Skin Cancer  Camilla Fusi, Serena Materazzi, Daiana Minocci, Vincenza Maio, Teresa Oranges, Daniela Massi, Romina Nassini  Journal of Investigative Dermatology  Volume 134, Issue 9, Pages (September 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Localization 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Localization 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Transient 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Localization 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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effect 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 GSK A (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 (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. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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