Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.

Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.     

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.     

Characterization of a retinoic acid responsive element isolated by whole genome PCR.

We have used whole PCR in an attempt to isolate novel retinoic acid (RA) responsive genes. We cloned several small genomic fragments from total human DNA containing putative retinoic acid responsive elements (RAREs) selected by direct binding to the retinoic acid receptor alpha (RAR alpha). We report here that an oligonucleotide containing a sequence from one of the cloned human DNA fragments, and referred to as alpha 1, functions as an authentic RARE. It is shown that both RAR alpha and RAR beta produced in Cos cells as well as in vitro translated RAR alpha bind directly and sequence-specifically to the alpha 1RARE. By mutational analysis it is demonstrated that the alpha 1RARE consists of an imperfect direct repeat of the estrogen- and thyroid hormone-related AGGTCA half-site motif separated by a 5 bp spacer. The orientation and spacing of the half-site repeats are shown to play a critical role in RAR recognition. When cloned upstream of a TK-Luc reporter, the alpha 1RARE is shown to confer responsiveness to RA in an orientation-independent fashion in F9 and CV-1 cells. The magnitude of the RA response mediated by the alpha 1RARE differed in these cell lines.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Cloning and characterization of the human retinoid X receptor alpha gene: conservation of structure with the mouse homolog

Retinoid X receptors (RXRs) are members of the steroid/thyroid hormone receptor superfamily which, along with retinoic acid receptors (RARs), mediate the biological effects of retinoids. These effects include the regulation of many aspects of embryonic development, reproductive and visual function, and the maintenance of epithelial homeostasis throughout life. The genes for three distinct retinoid X receptors, RXRalpha, beta, and gamma, have been localized to separate chromosomes. In order to determine the organization of the human RXRalpha gene, we have isolated a clone containing the majority of the gene from a human genomic bacterial artificial chromosome (BAC) library and generated a physical map. The gene spans over 40 kilobases in size and contains at least 10 exons. Comparison with mapped portions of the mouse RXRalpha gene indicates highly conserved intron-exon positioning. These results provide information necessary to generate constructs for targeting the RXRalpha gene in human cell lines, which may eventually lead to an understanding of the function of RXRalpha in human cancer.     

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.