F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-rans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10-9 or 10-1o M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered toXenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.Retinoids are derivatives of retinol (vitamin A) that are required for the appropriate functioning of a wide number of cell types involved in processes such as cell differentiation, axial pattern formation in embryogenesis, reproduction, and vision (for reviews, see refs. 1-3). Retinoids also exhibit cancer preventive actions and are used to treat some types of cancers (for review, see ref.2). The biological and transcriptional effects of retinoids are mediated by their interactions with the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (for review, see ref. 4). RARs and RXRs can heterodimerize and bind response elements in a variety of target genes (for reviews, see refs. 1 and 4). The ligands for these receptors are generally thought to be acid derivatives of retinol; all-trans-retinoic acid (at-RA) has been shown to bind to the RARs with high affinity and 9-cis-retinoic acid (9-cis-RA) binds and activates the RXRs as well the RARs (for reviews, see refs. 4 and 5). The N-terminal portion of the ligand binding domain (E domain) is essential for the recognition of at-RA by various RARs (6).Much recent research in the vitamin A field has focused on at-RA and its isomers. Retinol has been reported to be metabolized to at-RA in a large number of different cultured cells and in various tissues from developing embryos (3, 5). However, the complexity of the biological actions of vitamin A suggests that retinol derivatives other than at-RA mediate some of these actions. Consistent with this idea, two nonacidic retinol d...

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