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

Figure 8 demonstrates, that HaCaT cells preincubated with 25 pM 7-DHC and irradiated with (1) monochromatic light at 297 2.5 nm and 310 2.5 nm (D,ff = 30 mJ/ cm2); and (2) UVB from the narrow-band TL-01 lamp (dominated by a strong and narrow peak around 312 nm) generated very different amounts of calcitriol. Notably, the monochromatic light at 310 nm initiated the synthesis of only small amounts of calcitriol (--50.1 pmol/106 cells) as compared to the TL-01 lamp (-0.7 pmol/106 cells).

DISCUSSION

UVB light causes photolysis of 7-DHC to pre-VD3, which undergoes a rearrangement of its double bond structure to form the thermodynamically more stable VD3 (11,12). Previously we have shown that cultured keratinocytes can hydroxylate exogenous VD3 to la,25(OH)2D3 (9,10). In this study we investigated the generation of la,25(OH)2D3 from VD3 produced after UVB-induced photolysis of the precursor 7-DHC by HaCaT keratinocytes.

We first established the UVB-induced nonenzymatic isomerization of 7-DHC via pre-VD3 to VD3 in the absence and presence of HaCaT cells. The identities of generated pre-13 and VD3 were confirmed by NP-HPLC as well as by their UV spectra. Our results obtained under cell-free conditions are in accordance with earlier findings which show that UVB light (optimum wavelengths between 295 and 300 nm) causes photolysis of 7-DHC to pre-13 in human skin (11,15) and in cultures of human keratinocytes (12). The isomerization of pre-VD3 to VD3 is a time- and temperaturedependent process (11,15,16). Our results indicate that the conversion of pre-VD3 to VD3 at 370C in the presence of HaCaT cells is almost completed after 16 h. This is comparable to human skin where this isomerization needs approximately 20 h (16). Notably, 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 the presence of HaCaT cells. This observation is in contrast to earlier findings (11,12,15) which demonstrated that wavelengths between 295 and 300 nm optimally photolyze 7DHC to pre-133 in human skin (11,15) and in cultures of human keratinocytes (12). We have no explanation for this discrepancy at present. On the other hand, our results are very similar to observations made in rat skin (17), where the optimum wavelength for the synthesis of VD3 (305 nm) was longer than in organic solvents (295 run). It is a fact that both kinetics and thermodynamics of the reaction pre-VD3 VD3 may change significantly in many anisotropic microenvironments (16,18). Based on a liposomal model that mimics the cutaneous generation of VD3 it has been hypothesized by Tian and Holick (19) that cellular membrane phosholipids which interact with pre-VD3 stabilize pre-VD3 in its cZc-conformation, the only conformer that can convert to VD3. The stronger these interactions are, the more pre-13 is in its cZc-conformation and the faster is the rate of its conversion to VD3. It is conceivable that these phospholipidpre VD3 interactions are influenced by wavelength-dependent photodegradation of membrane phospholipids resulting in different rates of VD3 formation. Such wavelength-dependent processes also might explain the changeability of VD3 production between 285 and 295 nm. In agreement with literature (15), we found no synthesis of VD3 at wavelengths >315 nm.

These results show for the first time that VD3 generated by UVB-induced photolysis of 7-DHC is further metabolized to la,25(OH)2D3 in cultured HaCaT cells. Interestingly, two other metabolites were found which possessed crossreactivity with 1(x,25(OH)2D3 in the radioreceptor assay. The identity of the two metabolites is not known at the present time. The isolated putative calcitriol was clearly identified as 1t,25(OH)2D3 by both NP- and RP-HPLC as well as by GCMS. The mass spectrum was identical with that of the reference substance and followed a fragmentation pattern as previously described (20-22). In control experiments without cells using medium alone or in unirradiated controls with cells no calcitriol was detected in the culture extracts. Thus, the generation of calcitriol requires the presence of HaCaT cells as well as a UVB-induced VD3 synthesis.

Studies using ketoconazole, a cytochrome P450 enzyme inhibitor (23), clearly implicated the involvement of cytochrome P450 mixed-function oxidases in the formation of la,25(OH)2D3. 1,2-Dianilinoethane (10 VJP, a known radical scavenger and antioxidant (24) in contrast, exerted only a slight quench effect on the hydroxylations of VD3. These and our previous results argue in favor of the presence of both la-and 25-hydroxylase activity in HaCaT cells.

The time course of the UVB-induced generation of calcitriol in HaCaT cells shows a maximum at 16 h incubation time, followed by a gradual decrease. Calcitriol probably induces its own catabolism to calcitrioic acid (25) during this time period. Inhibition of lot-hydroxylase by lcL,25(OH)2D3 (5,7), may also contribute to a reduction in generation of calcitriol.

We found that monochromatic UVB dose-dependently (Dew = 7.50.0 mJ/cm2) induced generation of 10x,25(OH)2D3 from VD3, which was formed after photoconversion of 7DHC at 297 2.5 run in our in vitro system. For comparison, an effective dose of 30 mJ/cm2 at 297 - 2.5 nm corresponds to -1.5 minimal erythemal dose (MED) for hypopigmented Caucasian skin. The concentration of 7-DHC used in our experiments (30 nmol/1.2 mL medium equivalent to 4.25 nmol/cm2 cell monolayer) was comparable to the level of 7-DHC (=2.7 nmol/cm2) found in hypopigmented Caucasian human leg skin (11) and in human neonatal foreskin (-6.1 nmol/cmz) (26). The production rate of calcitriol showed a positive correlation to the amount of VD3 generated after UVB irradiation at 297 run up to De = 30 mJ/cm2. Apparently, UVB inactivates intracellular hydroxylases at Deft - 30 mJ/cm2, while nonenzymatic synthesis of VD3 remains unaffected. It should be noted that the conversion rate of VD3 obtained by photolysis of 7-DHC to calcitriol was approximately two to five-fold higher than that of comparable concentrations of added VD3 (9). We assume that the UV induced extra- and intracellular generation of VD3 contributes to a very efficient metabolism to calcitriol more than in cells which depend on the diffusion of extracellular VD3 through the cell membrane.