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

Statistical analysis. Results are presented as mean or mean standard deviation. Data were analyzed by one-way analysis of variance (Bonferroni method).

RESULTS

UVB-induced conversion of 7-DHC to VD3

In order to monitor UVB-induced photolysis under experimental conditions, 7-DHC in 1.2 mL culture medium was exposed to monochromatic UVB light in absence of cells. Figure 2 shows several chromatograms (mobile phase 1) obtained from extracts of DMEM containing 25 tLM 7-DHC and supplemented with 1.0% BSA at increasing incubation times after UVB irradiation at 297 2.5 nm and Deft = 30 mJ/cmz. The photolyzed product pre-VD3 was identified by cochromatography of pre-VD3 prepared by thermal isomerization of VD3 as described in "Materials and Methods" and by its UV spectrum (Xmax: 260 nm and Vim;": 230 nm). VD3 formed was identified by cochromatography of the VD3 reference substance and by its UV spectrum (Amax: 265 nm and Xmin: 229 nm). The photoisomers of pre-VD3, lumisterol and tachysterol (retention times: 9.00 and 10.96 min, respectively), were not detectable after irradiation. The time courses of the UVB-induced conversion of 7-DHC via preVD3 to VD3 in DMEM without cells and with cells after irradiation at 297 2.5 run and Deff = 30 mJ/cmz indicate that the isomerization of pre-VD3 to VD3 is completed after 16-20 h (data not shown in detail). We failed to detect endogenous 7-DHC in HaCaT cells. Figure 3 indicates that the VD3 synthesis is dependent on the wavelength of UV radiation in the presence and absence of cells. There is a shift of the wavelength corresponding to maximum synthesis rates of VD3 from 294 nm in DMEM without cells to 303 nm in presence of cells. The maximum amounts of VD3 generated in medium alone and in regular cultures were almost comparable. After irradiation at 320 nm no VD3 production was detectable in both the presence and absence of cells.

To investigate the effect of cellular transport of 7-DHC on the overall results cell cultures were incubated with 25 IiM 7-DHC; after 1 h the medium was removed, cells were washed with 1.2 mL phosphate buffered saline (PBS) twice and reconstituted with 1.2 mL PBS. The cell cultures were then irradiated at 297 nm (D,ff = 30 mJ/cmz), incubated for 16 h at 37'C in dark and extracted as described. The cellular uptake of 7-DHC was measured to be approximately 20% of the total amount of 7-DHC (30 nmol) prior added to the cell culture. The corresponding cellular VD3 level was about 37% of the total amount of VD3 (977 348 pmol, N = 3) formed in the original cell culture.

Conversion of photosynthesized VD3 to lat,25(OH)2D3 in HaCaT cells

Fractions (1-25) obtained after NP-HPLC (solvent system 2) of extracts of irradiated cell cultures (297 2.5 run, Drf = 30 mJ/cm2) and of nonirradiated controls were analyzed for calcitriol (Fig. 4). The peak in fractions 20-22 is identical with calcitriol. Minor peaks at 7 and 17 min have not been identified yet. The radioactivity peaks of 3H-25OHD3 and 3H24R(OH)2D3 appear after 7 and 12 min, respectively. However, we failed to detect 250HD3 using NP-HPLC and UV detection at 265 nm (data not shown). No calcitriol was detectable in analogous fractions of nonirradiated cultures (Fig. 4) as well as in the other controls (ii and iii, not shown). Calcitriol comigrated with synthetic 3H-la,25(OH)2D3 in both NP- and RPHPLC systems. To exclude the presence of 19-nor-, 10-keto25-hydroxyvitamin D3 (19-nor, 10-keto-25OHD3), which comigrates with la,25(OH)2D3 in NP-HPLC, 300 pg putative calcitriol was separated by RP-HPLC and the amount of calcitriol recovered was about the same as injected initially. This indicates the absence of 19-nor, 10-keto-25OHD3 which should be separable from calcitriol under these chromatographic conditions.