UVB-induced conversion of 7-dehydrocholesterol to (1 alpha,25-dihydroxyvitamin D3) (calcitriol) in the human keratinocyte line HaCaT

Photochemistry and Photobiology, Dec 2000 by Lehmann, Bodo, Knuschke, Peter, Meurer, Michael

UVB-induced Conversion of 7-Dehydrocholesterol to lot,25-Dihydroxyvitamin D3 (Calcitriol) in the Human Keratinocyte Line HaCaT"

ABSTRACT

We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D3 (7-dehydrocholesterol, [7-DHCI) which results in the formation of vitamin D3 also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 pA,f) at 37*C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5-60 mj/ cm2), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125-1500 mJ/cm2). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin Dj in our in vitro model followed by time-dependent isomerization to vitamin D3 and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths >315 mm generated no or only trace amounts of calcitriol. The conversion of vitamin D3 generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cmz). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.

INTRODUCTION

Calcitriol (la,25-dihydroxyvitamin D3, [lu,25(OH)2D3Dt, the most potent biologically active form of vitamin D3 (VD3) is produced by a cascade of reactions including photochemical VD3 synthesis in the skin and subsequent hydroxylation at the C-25 atom in the liver and at C-lot position in the kidney (1). Calcitriol and other vitamin D analogs have antiproliferative and prodifferentiative effects on epidermal keratinocytes (2-4) and have become potent therapeutical agents for the treatment of proliferative skin disorders such as psoriasis. It has been shown that cultured keratinocytes can convert exogenous calcidiol (25-hydroxyvitamin D3 [250HD3]) to calcitriol (5-7) and we have previously shown that both keratinocytes from the HaCaT cell line (8) and from human skin equivalents can convert exogenous VD3 to calcitriol (9,10). This implicates that functionally active lotand 25-hydroxylases are present in keratinocytes. Provitamin D3 (7-dehydrocholesterol [7-DHC]) exposed to ultraviolet B (UVB) radiation, converts in vivo (11) and in vitro (12) to previtaminD3 (pre-VD3), which in turn isomerizes to VD3. Until now, the complete pathway from 7-DHC to la,25(OH)2D3 has neither been found in the skin nor in cultures of epidermal skin cells. It was the aim of this study to provide evidence that photolysis of 7-DHC could result in the formation of la,25(OH)2D3 in cultured HaCaT keratinocytes.

MATERIALS AND METHODS

Chemicals and reagents. Dulbecco modified Eagle medium (DMEM) and fetal calf serum (FCS) were provided from GIBCO (Eggenstein, Germany). Culture dishes ( 30 mm) were from Falcon (Heidelberg, Germany). la-OHD3 and la,25(OH)2D3 were kindly provided by Hoffmann-La Roche AG (Basel, Switzerland). 25-hydroxy[26,27-methyl-3H]vitamin D3 (3H-25OHD3, 177 Ci/mmol), la,25-dihydroxy[26,27-methyl-3H]vitamin DI (3H-Ils,25(OH)2D3, 173,5 Ci/mmol) and 24R,25-dihydroxy[26,27-methyl-3H]vitamin D3 (3H-24R,25(OH)2D3, 170 Ci/mmol) were purchased from Amersham (Braunschweig, Germany). The la,25(OH)2D3-radioreceptor assay kit from Nichols Institute (Bad Nauheim, Germany) was used. VD3, solvents for high performance liquid chromatography (HPLC) (nhexane, 2-propanol and methanol) were provided by Merck (Darmstadt, Germany). Lumisterol and tachysterol were donated by Dr. A. Kissmeyer (Leo Pharmaceutical Products, Ballerup, Denmark). Ketoconazole was bought from Paesel & Lorei (Frankfurt/M, Germany). Bovine serum albumin (BSA), purity -99% (product number A 0281) and 7-DHC were from Sigma (Deisenhofen, Germany). Scintillation cocktail, Ready Protein , was purchased from Beckman Instruments (Fullerton, CA). NO-bis trimethylsilyl acetamide, Ntrimethylsilylimidazole and trimethylchlorosilane were purchased from Macherey-Nagel (Dueren, Germany). Pre-VD3 was prepared by thermal treatment of an ethanolic solution of VD3 (1.0 itg/mL) at 60*C for 16 h under nitrogen according to Isler and Brubacher (13). VD3 (retention time: 10.57 min) and generated pre-VD3 (retention time: 8.22 min) were separated by normal phase (NP)-HPLC (eluent 1, see "HPLC analysis"). Pre-VD3 was fractionated from 8.00 to 9.00 min. The UV spectrum of pre-133 (X.,,: 260 nm and km;": 230 nm) was identical to that described in the literature (14).

Cell culture protocol. HaCaT cells (used with the permission of Prof. N. E. Fusenig of the German Cancer Research Center, Heidelberg) were seeded at a density of 5 x 104 cells/cmz in culture dishes (o 30 mm) and grown in DMEM supplemented with 5% (vol/ vol) FCS. Cultures were maintained at 37*C and 5% COz in air. After 2 days the medium was replaced for 18 h by serum-free DMEM in order to induce synchronization of the cell cycle. The serum-free DMEM was then replaced by FCS-supplemented medium for 8 h, followed by serum-free DMEM supplemented with 1.0% (wt/vol) of highly purified BSA (Sigma). At this time cells were preconfluent (equal to -0.7 X 106 cells/dish). Cell numbers and their viability were assessed using a CASY' 1-cell counter (Scharfe System GmbH, Reutlingen, Germany). The viability was always ?93%.

Incubation conditions. 7-DHC (30 nmol) dissolved in 6 liL ethanol, was added to the medium (1.2 mL) of the cell cultures. Control experiments were carried out (1) in the presence of 7-DHC without irradiation; (2) in the absence of 7-DHC with and without irradiation; and (3) in cell-free culture medium containing 7-DHC with irradiation. After UVB irradiation and incubation in dark the medium and detached cells were separately extracted with methanol:chloroform (1:1). The chloroform phases were used for the determination of pre-VD3, VD3 and 101,25(OH)ZDj.