Retinoic acid receptors as regulators of human epidermal keratinocyte differentiation

Abstract

To examine the role of nuclear retinoic acid (RA) receptors (RARs) in the regulation of squamous differentiation in normal human epidermal keratinocytes (NHEK), we analyzed binding activity, mRNA expression, and transcriptional activity of the endogenously expressed RARs. Specific RA-binding activity eluted from size-exclusion HPLC with an apparent mol wt of 50 kilodaltons and was predominantly (>95%) associated with the NHEK nuclear cell fraction. This RAR-binding activity represented in part the expression of RARα and RARγ genes, whose transcripts were expressed in similar abundance in undifferentiated NHEK. Differentiation resulted in lower mRNA expression of RARα relative to the mRNA expression of RARγ. Treatment of NHEK cells with 10-6 M RA did not induce expression of RARβ mRNA. Similarly, three squamous cell carcinoma cell lines derived from human skin and oral cavity expressed RARα and RARγ transcripts, but not RARβ transcripts. Transfection of NHEK with chloramphenicol acetyltransferase (CAT) reporter plasmids indicated that the endogenously expressed RARs could activate transcription through the RARβ response element in a concentration-dependent manner with doses of 10-9 M RA and higher. CAT expression was not activated through TRE, a palindromic thyroid hormone response element with purported RA responsiveness. The competitive binding of benzoic acid derivatives of RA to RAR correlated with the ability of each analog to suppress mRNA expression of the squamous cell markers, involucrin, type I transglutaminase, and SQ37, and to activate transcription of the RARβ response element-CAT reporter. These results demonstrate that the control of NHEK differentiation by RA is consistent with the interaction of the retinoid with RAR and the regulation of transcription by that ligand-receptor complex.