SF-1, BUT NOT DAX-1, PREVENTS P19 CELLS FROM DIFFERENTIATING TO EITHER TROMA-1 OR TUJ1 POSITIVE CELLS UPON RA-TREATMENT

Abstract

Retinoic acid (RA) is critical for embryonic development and cell differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in Pre-â cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents them from differentiating to either endodermal or neuronal cells. This suggests that PBX is an important regulator of RA-induced differentiation of P19 cells. A microarray analysis was performed to identify PBX regulated genes, utilizing the empty vector P19 (TO3) and antisense to PBX (AS2) cell lines, during RA-induced differentiation of P19 cells into endodermal or neuronal cells. Among the genes identified by the microarray, Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor 1 (SF-1) were identified to be directly or indirectly regulated by PBX. Both DAX-1 and SF-1 proteins have only recently been reported to be present in preimplantation mouse embryos prior to the expression of steroidogenic enzymes, suggesting they may play a role in early mouse embryogenesis. 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 upon removal of doxicyclin were prepared. We found that overexpression of FLAG-DAX-1 had no effect on the RA-induced differentiation of P19 cells. However, FLAG-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 seven days caused a RA-independent loss of Oct-4 protein. However, cells which continued to express SF-1 for seven days did not terminally differentiate into endodermal or neuronal cells in response to RA treatment. In addition, we found evidence for a feedback loop, where PBX reduces SF-1 mRNA expression and continued SF-1 expression blocks the RA-dependent increase in PBX protein levels. Our findings suggest that SF-1 plays a novel role in P19 cells where its level of expression is critical for the differentiation state of the cells. At basal levels SF-1 maintains the pluripotent state of the cells, while SF-1 levels must be dramatically reduced for cells to differentiate into both endodermal and neuronal cells upon RA treatment. However, at elevated levels above basal, SF-1 inhibits Oct-4 expression and leads to the induction of the expression of steroidogenic enzymes with a pattern consistent with adrenal cells in a RA-independent fashion. Taken together these data suggest that SF-1 plays a much more dynamic role in P19 cells than previously reported.