Retinoids (vitamin A and its derivatives such as retinoic acid) profoundly influence cell differentiation and pattern formation in embryonic development. Retinoids have also been shown to inhibit the process of malignant transformation in both cell culture and animal studies. The mechanism(s) by which retinoic acid acts are not fully understood, but it is known that several high affinity RA binding proteins are involved in the actions of RA. Our overall objectives are to understand the roles of the high affinity RA binding proteins-the cellular retinoic acid binding proteins (CRABP) I and II, and the retinoic acid receptors (RARs) alpha, beta, and gamma-by employing two model systems: the murine teratocarcinoma cell differentiation system and transgenic mice. In the teratocarcinoma system we will determine the DNA regulatory elements that control the expression of the CRABP-I and II genes. We will screen for new members of the CRABP gene family. The question of whether the CRABP-I gene is involved in the intracellular catabolism of RA to inactive polar derivatives will be addressed through HPLC analysis of [3H]RA metabolites in teratocarcinoma cells that express different levels of CRABP-I protein. The question of the function of the CRABP-II protein will be addressed by stably transfecting teratocarcinoma cells with expression vectors that contain the full-length CRABP-II CDNA downstream of the metallothionein I promoter in order to overexpress the CRABP-II protein. Such cell lines will then be tested for their ability to differentiate in response to varying [RA]s. The roles of each of the RARs alpha, beta, and gamma will be defined by creating stably transfected teratocarcinoma stem cell lines that overexpress each of the RARs; conversely, gene """"""""knock-out' experiments will be performed to inactivate both alleles of RAR alpha, beta or gamma. Whether the teratocarcinoma cells that are RAR alpha -/- (or RAR gamma -/-) can still differentiate in response to RA will be tested by measuring the expression of a variety of differentiation specific markers such as ERA- 1/Hox 1.6; TAT beta; laminin B1, B2; collagen IV(alpha1);F117;J6, and J31 (Sparc). In the transgenic animal model system, we will determine which DNA regulatory elements are necessary for expression of the CRABP-I and II genes in different embryonic cell types. We will also inject an expression vector containing either the full-length CRABP-1 or II gene downstream of a heterologous strong eukaryotic promoter such as metallothionein I or beta-actin into the pronuclei of fertilized eggs to create """"""""gain-of-function"""""""" mutations in the transgenic mice. These mice will be analyzed for possible developmental defects, since CRABP-I may be involved in establishing RA gradients in some areas of in the developing embryo and its overexpression may cause developmental abnormalities. All of these lines of experimentation should provide us with important new information about the actions of RA and the binding proteins that mediate RA's effects.
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