A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ

Abstract

Cannabinoid receptors (CB) are expressed throughout human skin epithelium. CB1 activation inhibits human hair growth and decreases proliferation of epidermal keratinocytes. Since psoriasis is a chronic hyperproliferative, inflammatory skin disease, it is conceivable that the therapeutic modulation of CB signaling, which can inhibit both proliferation and inflammation, could win a place in future psoriasis management. Given that psoriasis is characterized by up-regulation of keratins K6 and K16, we have investigated whether CB1 stimulation modulates their expression in human epidermis. Treatment of organ-cultured human skin with the CB1-specific agonist, arachidonoyl-chloro-ethanolamide (ACEA), decreased K6 and K16 staining intensity in situ. At the gene and protein levels, ACEA also decreased K6 expression of cultured HaCaT keratinocytes, which show some similarities to psoriatic keratinocytes. These effects were partly antagonized by the CB1-specific antagonist, AM251. While CB1-mediated signaling also significantly inhibited human epidermal keratinocyte proliferation in situ, as shown by K6/Ki-67-double immunofluorescence, the inhibitory effect of ACEA on K6 expression in situ was independent of its anti-proliferative effect. Given recent appreciation of the role of K6 as a functionally important protein that regulates epithelial wound healing in mice, it is conceivable that the novel CB1-mediated regulation of keratin 6/16 revealed here also is relevant to wound healing. Taken together, our results suggest that cannabinoids and their receptors constitute a novel, clinically relevant control element of human K6 and K16 expression.

Keywords: Cannabinoid; Keratin; Psoriasis; Wound healing.

Figure 1. The CB1 specific agonist, ACEA significantly inhibits K6 and K16 expression in situ.

(A) Representative images of K6 immunofluorescence with organ cultured human skin treated with ACEA/AM251 (1-day). (B) Statistical analysis of K6 immunofluorescence intensity in organ cultured human skin (quantitative immunohistomorphemtry, ImageJ); stimulation with ACEA (30 µM), AM251 (1 µM) or both for 1-day. n = 9–22 skin sections/group. (C) Representative images of K16 immunohistochemistry with organ cultured human skin samples with ACEA (1-day). (D) Quantitative K16 immunohistomorphometry within the epidermis of organ-cultured human skin samples after 1-day of stimulation with ACEA (30 µM). n = 4 skin sections/group. Data are expressed as mean + SEM. *p < 0.05, **p < 0.01.

Figure 2. The CB1 specific agonist, ACEA significantly inhibits K6 expression in cultured HaCaT cells.

(A) Representative images of K6 immunofluorescence of cultured HaCaT KCs with ACEA (1 µM), AM251 (100 nM) or both for 1-day. (B) Statistical analysis of K6 immunofluorescence intensity of cultured HaCaT cells. n = 6 colonies/group (C) Statistical analysis of K6 gene expression in HaCaT cells treated with vehicle control or ACEA (1 µM) for 8 h. Data are expressed as mean + SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3. The CB1 specific agonist, ACEA significantly decreases human epidermal keratinocyte proliferation in situ.

(A) Representative images of K6 (green) and Ki67 (red) double-immunofluorescence with organ cultured human skin treated with ACEA/AM251 (1-day). (B) Quantitative analysis of the percentage of Ki67 + KCs within organ cultured human epidermis. *p < 0.05; ***p < 0.001. n = 5–12 skin sections/group.

Figure 4. The CB1 specific agonist, ACEA, significantly decreases K6 expression in suprabasal cells in a proliferation-independent manner.

(A) Representative images of K6 (green) and Ki-67 (red) double immunofluorescence. Dotted rectangles indicate the reference area for quantitative immunohistomorphometry of K6 fluorescence intensity. (B) Quantitative analysis of K6 fluorescence intensity in non-proliferating (i.e. Ki67-negative) cells within human epidermis in situ. Data are expressed as mean + SEM. *p < 0.05. n = 5–7 skin sections/group. (C) K6 (green) and CB1 (red) double-immunofluorescence study. Yellow arrows denote double-positive KCs.