Gas chromatography/mass spectrometry characterization of corticosteroid metabolism in human immortalized keratinocytes.
To continue our studies on the cutaneous expression of a proopiomelanocortin/corticotropin-releasing hormone system, we investigated whether this is accompanied by adrenal-type enzymatic activity. Immortalized cultured human keratinocytes were incubated with radiolabeled corticosteroids. Analysis by thin-layer chromatography showed rapid transformation of both progesterone and deoxycorticosterone; one of the progesterone metabolites migrated at the same rate as deoxycorticosterone. Gas chromatography/mass spectrometry further identified as major species of deoxycorticosterone metabolites 3beta,6alpha,21-trihydroxy-5alpha-pregnan-20-one, 3alpha,6alpha,21-trihydroxy-5alpha-pregnan-20-one, and 3alpha5alpha- and 3beta5alpha-tetrahydrodeoxycorticosterone. Minor metabolites were 3alpha,21-dihydroxy-5-pregnen-20- one (3alphaDelta5-21-OHpregnenolone), 3beta,21-dihydroxy-5-pregnen-20-one (3betaDelta5-21-OHpregnenolone), 3alpha,21-dihydroxy-4-pregnen-20-one (3alphaDelta4-21-OHpregnenolone), 6-hydroxy-dihydrodeoxycorticosterone, and two 5-dihydrodeoxycorticosterone species. Thus, in addition to sex steroids keratinocytes also actively metabolize corticosteroids along similar enzymatic pathways. The surprising detection of 3alphaDelta5-21-OHpregnenolone and 3 betaDelta5-21-OHpregnenolone, indicating Delta4-ketosteroids to Delta5-hydroxysteroids conversion, provides strong evidence for the occurrence, at least in human keratinocytes, of isomerase activity that allows the reaction to proceed in reverse of its usual direction. As skin expresses 3alpha/beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerases, cutaneous reactions catalyzed by these enzymes must be reversible. In conclusion, besides elements of the corticotropin-releasing hormone/proopiomelanocortin system human keratinocytes show high levels of corticosteroid metabolizing activity. Moreover, the wide array of steroid products generated from a single substrate indicates serial progressive conversion involving 5alpha-reductase, 6alpha-hydroxylase, 3alpha/beta-hydroxysteroid dehydrogenase, and reverse Delta4minus signDelta5 isomerase enzymes. As distinct from the adrenal cortex, production of A, B, Aldo, 18OHdeoxycorticosterone, or F in keratinocytes was absent or below limits of detectability.
Slominski
Andrzej
A
Department of Pathology, Health Science Center, University of Tennessee, Memphis, Tennessee 38163, USA. aslominski@utmem.edu
Wortsman
Jacobo
J
Foecking
Mark F
MF
Shackleton
Cedric
C
Gomez-Sanchez
Celso
C
Szczesniewski
Andre
A
D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003900:Desoxycorticosterone / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism
D003900:Desoxycorticosterone; D011374:Progesterone