In vitro synthesis of vitamin D-3 by cultured human keratinocytes and fibroblasts: action spectrum and effect of AY-9944.
With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skin's role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting.
M K
MK
Nemanic
J
J
Whitney
P M
PM
Elias
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000096:biosynthesis* / Q000378:metabolism / Q000031:analogs & derivatives; D002784:Cholesterol / Q000378:metabolism; D003510:Cyclohexanes / Q000494:pharmacology*; D003684:Dehydrocholesterols / Q000378:metabolism; D004875:Ergosterol / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D050618:Glycolaldehyde Dehydrogenase; D006801:Humans; D008854:Microscopy, Electron; D012867:Skin / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D013057:Spectrum Analysis; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride / Q000494:pharmacology*
D003510:Cyclohexanes; D003684:Dehydrocholesterols; D002762:Cholecalciferol; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride; D002784:Cholesterol; C016705:7-dehydrocholesterol; D000445:Aldehyde Oxidoreductases; D050618:Glycolaldehyde Dehydrogenase; C025168:previtamin D(3); D004875:Ergosterol