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

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 differentiation. Through gain of function experiments, this report highlight 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 phenotype. 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 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.