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Volume 270, Number 29, Issue of July 21, pp. 17429-17436, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Evidence that Retinoid X Receptors Mediate Retinoid-dependent Transcriptional Activation of the Retinoic Acid Receptor Gene in S91 Melanoma Cells

(Received for publication, November 23, 1994; and in revised form, April 28, 1995)

From the Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute and Harvard Medical School, Boston, Massachusetts 02115

S91 melanoma cells are growth arrested and differentiate when treated with retinoids. These processes correlate with expression of the retinoic acid receptor (RAR) gene, which is induced through a retinoic acid response element (RARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RAR gene. We show that RXR and RXR are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the RARE (Complex I) in untreated cells, which can be supershifted by antibodies against RXRs but not by anti-RAR antibodies. After 48 h of treatment with retinoic acid, Complex I is replaced by a faster migrating Complex II, which can be supershifted by anti-RAR and anti-RXR antibodies. This suggests that induction of the RAR gene is largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of the RAR gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RAR into an RARRXR heterodimer complex. Thus, it appears that the RARE is sequentially occupied by RXR dimers and RARRXR heterodimers.

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