Characterization of Retinoic Acid Receptor-deficient Keratinocytes

Abstract

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,...

Keratinocyte differentiation induced by calcium, phorbol ester or interferon-γ elicits distinct changes in the retinoid signalling pathways

“…Our results corroborate previous findings of an increased RARγ/RARα mRNA ratio in confluent human keratinocytes [37] and induction of RXRα in calcium-treated and post-confluent keratinocytes [38]. By extrapolating to the in vivo situation, RARα/RXRα heterodimers are likely to predominate in the basal layers of the epidermis while RARγ/RXRα should predominate in the suprabasal layers suggesting separate functions for RARα/γ and RXRα in keratinocyte differentiation [39]. In view of this, our results could have the following implication: The responsiveness of a tissue or cell to a hormonal factor such as RA is largely dependent on the abundance of the corresponding nuclear receptor.…”

“…This is supported by a report showing that RA affects different genes in keratinocytes deficient in RARα or RARγ [39]. Further studies are needed to elucidate whether a corresponding mechanism exists also in human normal keratinocytes.…”

RARγ acts as a tumor suppressor in mouse keratinocytes

“…AP-1 activity has been closely linked to epidermal tumorigenesis in vivo and cellular transformation in vitro (Jochum et al, 2001). While the basis for this effect is not fully understood, trans-repression of AP-1 has been suggested to be responsible for the growth inhibitory effects of RA in many cell types, including keratinocytes (Karin et al, 1997;Goyette et al, 2000).…”

AP-1 transrepressing retinoic acid does not deplete coactivators or AP-1 monomers but may target specific Jun or Fos containing dimers

“…Mouse JB6 cells express only RAR g and a, not RAR b, and the tumor promotion inhibiting activity has been attributed to the RAR g (Rudd et al, 1993). RAR g is functionally predominant for transrepression in keratinocytes (Goyette et al, 2000). The precise mechanism of transrepression of AP-1 by RA is not clear.…”

“…An AP-1 complex consisting of JunB and Fra-1 or another JunB-or Fra-1-containing complex appears to be one target of transrepression by RA. A recent study using RAR null keratinocytes revealed that RAR is essential for the transrepression of AP-1 activity by RA (Goyette et al, 2000). RARg plays a major role in AP-1 transrepression by RA in JB6 cells (Rudd et al, 1993).…”