Characterization of a Human Keratinocyte HaCaT Cell Line Model to Study the Regulation of CYP2S1

• DOI: 10.1124/dmd.111.042085
• Publication Date: 2011-10-29T02:42:47Z
• Authors: Alison D. McNeilly; Julie A. Woods; Sally H. Ibbotson; C. Roland Wolf; Gillian Smith
• Keywords: EXPRESSION; Keratinocytes; 0301 basic medicine; XENOBIOTIC-METABOLIZING ENZYMES; Ultraviolet Rays; Drug Evaluation, Preclinical; 610; Tretinoin; CHO Cells; Cell Line; 03 medical and health sciences; Cytochrome P-450 Enzyme System; Cricetinae; Microsomes; Animals; Humans; TRANSCRIPTION; RNA, Messenger; NADPH-Ferrihemoprotein Reductase; P450CYP2S1; PSORIASIS; IDENTIFICATION; RECEPTOR; Osmolar Concentration; RETINOIC ACID METABOLISM; Recombinant Proteins; 3. Good health; Enzyme Induction; Cytochrome P-450 CYP1B1; Aryl Hydrocarbon Hydroxylases; Dermatologic Agents; HUMAN CYTOCHROME P4502S1; B EXPOSURE

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.