All-trans retinoic acid (RA), the principal biologically active form of vitamin A, is essential for many developmental process as well as homeostasis in the adult. Many lines of evidence also suggest that RA, acting through the RA receptors (RARs), can also suppress growth of tumors of diverse origin. To assess directly the role of the RARs in a model of epidermal tumorigenesis, we investigated the incidence of tumor formation using keratinocytes lacking specific RAR types. Our data suggest that loss of RARc, but not RARa, predisposed keratinocytes to v-Ha-Rasinduced squamous cell carcinoma. We also found that ablation of RARc, but not RARa, abolished RA-induced cell cycle arrest and apoptosis in these keratinocytes. Reconstitution of receptor expression into RAR-null cells restored sensitivity to RA, and reversed the tumorigenic potential of receptor-deficient keratinocytes. These data strongly support a tumor suppressor effect for the RARs, in particular endogenous RARc, in murine keratinocytes.

Vitamin A derivatives (retinoids) play central roles in embryonic development and maintenance of various tissues in the adult (1-3). Retinoids also exhibit potent antitumorigenic properties in diverse model systems and show potential for the treatment of a number of human malignancies, including diverse epithelial cancers or pre-cancerous lesions (4 -9).The retinoid signal is transduced by two families of nuclear receptors, the retinoic acid (RA) 1 receptors (RAR␣, -␤, and -␥ and their isoforms) and the retinoid x receptors (RXR␣, -␤, and -␥) (10 -13). RARs function as ligand-inducible transcription regulators by binding, together with an RXR partner, to specific cis-acting response elements (RAREs). RARs can be activated by both RA and its stereoisomer, 9-cis RA, whereas RXRs are activated only by 9-cis RA (14). RXRs are also essential heterodimeric partners for a number of other nuclear receptor signaling pathways, including thyroid hormone, vitamin D, and certain orphan receptors (15, 16). Although 9-cis RA is not obligatory for transcriptional regulation via these pathways, some results suggest that RXR-specific ligands can elicit transcriptional activation in certain settings (e.g. .RARs, like several other nuclear receptors, can function in a ligand-dependent manner to inhibit AP-1 activity, and it has been suggested that the affect of retinoids on the growth of transformed cells may occur through this trans-repression mechanism (21-23). This inhibition is believed to be due, at least in part, to competition for limiting amounts of transcriptional co-factors, such as CBP and/or its homologue p300, common to both pathways (24, 25). Other mechanisms, such as inhibition of the expression of AP-1 family members or c-Jun N-terminal kinase (JNK), may also contribute to this cross-talk (26 -29).Gene targeting of the various RARs has revealed essential and diverse roles for these receptors (2, 30, 31). However, because of perinatal or embryonic lethality inherent to many of these RAR null backgrounds, there is a void in our knowledge of RAR function in a number of contexts, such as tumorigenesis.Exogenous retinoids can attenuate the effects of tumor promoters in the two stage skin carcinogenesis protocol (9, 32). Among the retinoid receptors, normal epidermis expresses RAR␥ and RAR␣␥ as well as RXR␣ and RXR␤, with RAR␥ and RXR␣ as the predominant heterodimer (33,34). This pattern of expression prompted us to investigate the roles of RAR␣ and RAR␥ in mediating the antitumorigenic effects of retinoids in epithelial keratinocytes. To this end, we established RAR␣, RAR␥, and RAR␣␥ null keratinocyte lines by transformation with a dominant-negative p53 expression vector and compared the properties of these various lines. Our results demonstrate that RAR␣ and RAR␥ affect different aspects of retinoid response in these transformed cells, with RAR␥ being the primary mediator of RA-induced growth inhibition. However, other synthetic ligands affected proliferation independent of the RARs. RAR-dependent, but not -independent,...