All‐trans retinoic acid inhibits binding of 1,25‐dihydroxy‐vitamin D₃ to the vitamin D receptor in cultured human keratinocytes

Abstract

Psoriasis is characterized by hyperproliferation and impared differentiation of epidermal keratinocytes (KCs). Psoriasis can be treated with derivatives of retinoic acid (RA) and vitamin D3. Analogues of vitamin D3 are able to inhibit proliferation and stimulate differentiation of KCs. In contrast, RA inhibits terminal differentiation of KCs. Interactions are known to occur between RA and vitamin D3 signalling pathways. The purpose of the present study was to determine the effect of all-trans RA on the binding of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) to the vitamin D3 receptor (VDR) of cultured human KCs. Cultured KCs from normal adults were incubated with or without RA (10-9-10-7M) for 4-24 h. Cells were then harvested, homogenized and ultrasonicated. The extracted protein was incubated with 3H-1,25 (OH)2D3 (0.015-1.0 nM) with or without 250-fold excess nonradioactive 1,25 (OH)2D3 for 24 h and specific binding was determined by use of the dextran coated charcoal binding assay. Western blot analysis utilizing the monoclonal antibody 9A7 gamma to VDR was performed on protein extracted from the KCs. The bands resulting from Western blot analysis were visualized by enhanced chemiluminescence. From Scatchard analysis it was found that KCs bind 1,25 (OH)2D3 with high affinity (Kd = 0.175 nM). This binding was dose and time dependently inhibited by RA (60% inhibition at 10-7 M after 24 h of incubation). By Western blot analysis, RA had no effect on the amount of protein extracted from KCs at any of the RA concentrations tested. In conclusion, these results show that binding of vitamin D3 to its receptor of human KCs can be inhibited markedly by RA without effecting the amount of protein. These results are in contrast to results with other cell types in which RA upregulates binding of 1,25 (OH)2D3 to the VDR. Because interaction between retinoids and vitamin D3 may occur at different levels during signal transduction, it is not possible to predict from our results whether RA will inhibit the effects of vitamin D3 in vivo.

Effects of drugs and anticytokine antibodies on expression of ATP2A2 and ATP2C1 in cultured normal human keratinocytes

“…The cultured keratinocytes were grown in standard medium (Epilife‐KGM ® ) until 80–85% subconfluence and then washed in phosphate‐buffered saline (PBS). Medium was replaced with fresh standard medium without hydrocortisone to which several drugs were added at various concentrations as previously described: 10,16–23 100 nmol L −1 prednisolone (Wako Pure Chemical; Osaka, Japan), 10 µmol L −1 FK‐506, 10 µmol L −1 ciclosporin A, 10 nmol L −1 vitamin D 3 (Calbiochem, San Diego, CA, U.S.A.) or 10 µmol L −1 retinoic acid (Biomol, Plymouth Meeting, PA, U.S.A.). Twenty‐four hours after the addition of prednisolone, retinoic acid or vitamin D 3 , or 4 h after addition of ciclosporin A or FK‐506, the keratinocytes were washed with PBS and exposed to 20 mJ cm −2 UVB from two fluorescent bulbs with an emission peak at 312 nm (AB‐1500/HP‐15M; ATTO Co., Tokyo, Japan).…”

“…In previous reports, DD and HHD are improved by therapy with corticosteroids, retinoids, ciclosporin, vitamin D 3 and tacrolimus 11–15 . These drugs have various effects for human keratinocytes, 10,16–23 but it is uncertain whether drugs have influences for the regulation of DD and HHD genes.…”

Effects of Diisopropylamine Dichloroacetate on Proliferation and Differentiation of Normal Human Keratinocytes in vitro

“…As a biomaterial improving skin disease, retinoids are well known and are applied for the therapy and the care of skin [31]. But differently from DADA, retinoids are reported to suppress differentiation [32] and proliferation [33] of keratinocytes in vitro.…”

Towards a renaissance of dermatoendocrinology: Selected current frontiers

“…[1][2][3][4][5][6][7][8] Ever since its inception, Experimental Dermatology has accompanied, mirrored and promoted the systematic exploration of dermatoendocrinology as a distinct, clinically highly relevant field of skin research. Beyond a conventional focus on steroid hormones, [9][10][11] early on, the journal has championed skin research into neuroendocrine signalling and non-steroid hormones. [12][13][14][15] Since the foundation of a working group within the Arbeitsgemeinschaft Dermatologische Forschung (ADF) devoted to dermatoendocrinology, its founders, Christos Zouboulis, Ralf Paus and Markus Böhm have been eager to promote research, stimulate scientific exchange and increase the overall visibility of this emerging field, for example by editing special supplemental issues of Experimental Dermatology.…”