Characterization of a human keratinocyte HaCaT cell line model to study the regulation of CYP2S1.
CYP2S1 is an extrahepatic cytochrome P450 (P450) that shows marked individuality in constitutive and inducible expression. CYP2S1 mRNA expression is increased in psoriasis and by treatments for psoriasis, including retinoids and UV radiation, although endogenous substrates remain poorly characterized. Because previous model systems have overexpressed modified CYP2S1 in bacteria, human HaCaT keratinocyte cells were screened for constitutive and regulatable CYP2S1 expression and CYP2S1 activity in HaCaT cells compared with a novel Chinese hamster ovary (CHO)-based cell line engineered to stably coexpress CYP2S1 and NADPH cytochrome P450 reductase. Constitutive mRNA expression for CYP2S1 and additional P450s, retinoid acid receptors (RARα, RARβ, RARγ), and retinoid X receptors (RXRα, RXRβ and RXRγ) was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in HaCaT cells. Cells were then exposed to retinoids or to UV radiation (UVR), and changes in CYP2S1 mRNA abundance were further examined by qRT-PCR analysis. P450 expression in HaCaT cells was similar to human skin, with abundant CYP2S1 expression. RARα and RARγ (but not RARβ) and all RXR isoforms were also detectable. All-trans retinoic acid (atRA) induced CYPS1 mRNA expression more potently than 9-cis RA or 13-cis RA. P450-dependent atRA metabolism was demonstrated in HaCaT cells, with a very similar metabolite profile to that produced by our CYP2S1-expressing CHO cells. CYP2S1 mRNA expression was also induced by UVR, more potently than CYP1B1, a known UVR-inducible P450. Our results demonstrate regulatable and functional CYP2S1 expression in HaCaT cells, thus identifying a human cell line model with utility for further analysis of CYP2S1 regulation and substrate specificity.
Centre for Neuroscience, Division of Medical Sciences, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. a.d.mcneilly@dundee.ac.uk
Alison D
AD
McNeilly
Julie A
JA
Woods
Sally H
SH
Ibbotson
C Roland
CR
Wolf
Gillian
G
Smith
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003879:Dermatologic Agents / Q000378:metabolism / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004790:Enzyme Induction / Q000187:drug effects* / Q000528:radiation effects*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology; D014466:Ultraviolet Rays
D003879:Dermatologic Agents; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; C422564:CYP2S1 protein, human; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D009251:NADPH-Ferrihemoprotein Reductase