ABSTRACT
Retinoic acid (RA) is a positive regulator of P19 cell differentiation. Silencing of pre-B cell leukemia transcription factors (PBXs) expression in P19 cells (AS cells) results in a failure of these cells to differentiate to endodermal cells upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) is an orphan member of the steroid-thyroid hormone superfamily. RA treatment of wild type P19 cells results in a dramatic increase in the expression of COUP-TFI; however, COUP-TFI mRNA levels fail to be elevated upon RA treatment of AS cells indicating that PBX expression is required for elevation in COUP-TFI expression. To study the role of COUP-TFI during RA-dependent differentiation of P19 cells, AS cells that inducibly express various levels of COUP-TFI were prepared. Exogenous expression of COUP-TFI in AS cells, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption, and early apoptosis. Furthermore, AS cells can overcome the blockage in RA-dependent differentiation to endodermal cells when either pharmacological levels of COUP-TFI are expressed or a combination of both the expression of physiological levels of COUP-TFI and RA treatment. Additionally, the mRNA level of several pluripotency associated genes including OCT-4, DAX-1, and SF-1 in the COUP-TFI expressing AS cells are reduced. Moreover, analysis of the expression of primary RA response genes indicates that COUP-TFI is involved in the regulatory modulation of the expression of at least two genes, CYP26A1 and HoxA1. These studies demonstrate that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells.
Subject(s)
COUP Transcription Factor I/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Endoderm/cytology , Endoderm/drug effects , Tretinoin/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/genetics , COUP Transcription Factor I/metabolism , COUP Transcription Factor II/genetics , COUP Transcription Factor II/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cells, Cultured , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , DAX-1 Orphan Nuclear Receptor/genetics , DAX-1 Orphan Nuclear Receptor/metabolism , Endoderm/metabolism , Gene Expression/drug effects , Gene Expression/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Pre-B-Cell Leukemia Transcription Factor 1 , RNA, Messenger/genetics , Retinoic Acid 4-Hydroxylase , Steroidogenic Factor 1/genetics , Steroidogenic Factor 1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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-ß 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.
Subject(s)
DAX-1 Orphan Nuclear Receptor/metabolism , Steroidogenic Factor 1/metabolism , Tretinoin/pharmacology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Line, Tumor , DAX-1 Orphan Nuclear Receptor/genetics , Embryonic Development/drug effects , Embryonic Development/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Endoderm/cytology , Endoderm/drug effects , Endoderm/metabolism , Gene Expression , Mice , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Octamer Transcription Factor-3/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Steroidogenic Factor 1/geneticsABSTRACT
Retinoic acid (RA), the most potent natural form of vitamin A, plays an important role in many diverse biological processes such as embryogenesis and cellular differentiation. This chapter is a review of the mechanism of action of RA and the role of specific RA-regulated genes during the cellular differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. RA acts by binding to its nuclear receptors and inducing transcription of specific target genes. The most studied mouse EC cell lines include F9 cells, which can be induced by RA to differentiate into primitive, parietal, and visceral endodermal cells; and P19 cells, which can differentiate to endodermal and neuronal cells upon RA treatment. ES cells can be induced to differentiate into a number of different cell types; many of which require RA treatment. Over the years, many RA-regulated genes have been discovered in EC and ES cells using a diverse set of techniques. Current research focuses on the elucidation how these genes affect differentiation in EC and ES cells using a variety of molecular biology approaches. However, the exact molecule events that lead from a pluripotent stem cell to a fully differentiated cell following RA treatment are yet to be determined.
