Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells

Abstract

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARγ over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARα and RARβ do not and RARγ overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-R1) having deregulated RARγ expression. Since RARγ transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRβ as playing an important role along with RARγ in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRβ, but not RXRα or RXRγ, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARγ and RXRβ resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRβ and RARγ in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARγ or RXRβ in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARγ and RXRβ co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.