Spontaneous retinoic acid receptor beta 2 expression during mesoderm differentiation of P19 murine embryonal carcinoma cells

Exposure of aggregated murine P19 embryonal carcinoma cells to dimethylsulf oxide (DMSO) induces mesoderm and both embryonic cardiac and skeletal muscl e differentiation, while retinoic acid (RA) is an inducer of neuroectoderma l differentiation. P19 cells constitutively express the retinoic acid recep tor alpha (RAR alpha) and RAR gamma mRNAs while RAR beta expression is indu ced by RA through a consensus RA-response element in the RAR beta promoter. In the present study we show that the RAR beta transcript is strongly expr essed in both P19 cells and in a RA-nonresponsive derivative of P19 cells, called RAC65, during DMSO-induced mesoderm and muscle differentiation. Reve rse transcriptase-polymerase chain reaction analysis indicated that RAR bet a 2 is the predominant isoform expressed in DMSO-differentiated cells, prov iding the first evidence for RA-independent regulation of RAR beta 2 transc ript levels. Immunoblot analysis showed a 3-fold increase in the RAR beta p rotein expression over basal levels in differentiated cells, and immunohist ochemistry indicated that all cells in the culture including muscle reacted positively for the RAR beta protein. RAR beta 2 transcript expression was differentiation-dependent and occurred without transactivation of a transfe cted RARE beta 2 reporter gene. Little transcription of the RAR beta gene w as detected in nuclear run-off assays of undifferentiated P19 cells and onl y a small increase in transcription was observed in nuclei from DMSO-treate d cells. RA treatment of P19 cells stably transfected with the RA-responsiv e element from the RAR beta gene showed that RAR beta 2 mRNA expression dur ing DMSO differentiation was associated with increased sensitivity to RA. T ogether these data show that RAR beta 2 is expressed spontaneously in an ap parently RA-independent manner in differentiating mesoderm and mesoderm der ivatives, resulting in increased sensitivity to RA in these cells.