Role of SF-1 and DAX-1 during differentiation of P19 cells by retinoic acid.

Retinoic acid (RA) is critical for embryonic development and cellular differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in pre-β cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents endodermal and neuronal differentiation. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor (SF-1) were found by microarray analysis to be regulated by PBX in P19 cells. To determine the roles of DAX-1 and SF-1 during RA-dependent differentiation, P19 cells that inducibly express either FLAG-DAX-1 or FLAG-SF-1 were prepared. Unexpectedly, overexpression of DAX-1 had no effect on the RA-induced differentiation of P19 cells to either endodermal or neuronal cells. However, SF-1 overexpression prevented the RA-dependent loss of OCT-4, DAX-1 and the increase in COUP-TFI, COUP-TFII, and ETS-1 mRNA levels during the commitment stages of both endodermal and neuronal differentiation. Surprisingly, continued expression of SF-1 for 7 days caused the RA-independent loss of OCT-4 protein and RA-dependent loss of SSEA-1 expression. Despite the loss of well-characterized pluripotency markers, these cells did not terminally differentiate into either endodermal or neuronal cells. Instead, the cells gained the expression of many steroidogenic enzymes with a pattern consistent with adrenal cells. Finally, we found evidence for a feedback loop in which PBX reduces SF-1 mRNA levels while continued SF-1 expression blocks the RA-dependent increase in PBX levels. Taken together, these data demonstrate that SF-1 plays a dynamic role during the differentiation of P19 cells and potentially during early embryogenesis.