SXR, a novel steroid and xenobiotic-sensing nuclear receptor.

An important requirement for physiologic homeostasis is the detoxification and removal of endogenous hormones and xenobiotic compounds with biological activity. Much of the detoxification is performed by cytochrome P-450 enzymes, many of which have broad substrate specificity and are inducible by hundreds of different compounds, including steroids. The ingestion of dietary steroids and lipids induces the same enzymes; therefore, they would appear to be integrated into a coordinated metabolic pathway. Instead of possessing hundreds of receptors, one for each inducing compound, we propose the existence of a few broad specificity, low-affinity sensing receptors that would monitor aggregate levels of inducers to trigger production of metabolizing enzymes. In support of this model, we have isolated a novel nuclear receptor, termed the steroid and xenobiotic receptor (SXR), which activates transcription in response to a diversity of natural and synthetic compounds. SXR forms a heterodimer with RXR that can bind to and induce transcription from response elements present in steroid-inducible cytochrome P-450 genes and is expressed in tissues in which these catabolic enzymes are expressed. These results strongly support the steroid sensor hypothesis and suggest that broad specificity sensing receptors may represent a novel branch of the nuclear receptor superfamily.

BXR, an embryonic orphan nuclear receptor activated by a novel class of endogenous benzoate metabolites.

Nuclear receptors are ligand-modulated transcription factors that respond to steroids, retinoids, and thyroid hormones to control development and body physiology. Orphan nuclear receptors, which lack identified ligands, provide a unique, and largely untapped, resource to discover new principles of physiologic homeostasis. We describe the isolation and characterization of the vertebrate orphan receptor, BXR, which heterodimerizes with RXR and binds high-affinity DNA sites composed of a variant thyroid hormone response element. A bioactivity-guided screen of embryonic extracts revealed that BXR is activatable by low-molecular-weight molecules with spectral patterns distinct from known nuclear receptor ligands. Mass spectrometry and 1H NMR analysis identified alkyl esters of amino and hydroxy benzoic acids as potent, stereoselective activators. In vitro cofactor association studies, along with competable binding of radiolabeled compounds, establish these molecules as bona fide ligands. Benzoates comprise a new molecular class of nuclear receptor ligand and their activity suggests that BXR may control a previously unsuspected vertebrate signaling pathway.

An essential role for retinoid signaling in anteroposterior neural patterning.

The vertebrate central nervous system (CNS) is induced by signals emanating from the dorsal mesoderm, or organizer, that divert the ectoderm away from an epidermal and towards a neural fate. Additional signals from the organizer pattern the neural ectoderm along the anteroposterior axis. We devised highly specific methods utilizing constitutively active or dominant negative receptors to evaluate the role of retinoids in neural patterning. Microinjection of these reagents either augments or reduces retinoid signaling in specific regions of the embryo. We show that increased receptor activity suppresses anterior neural structures while dominant negative receptors lead to anterior enhancement. Similarly, microinjection of the dominant negative receptor leads to the loss of posterior marker genes. We demonstrate that retinoid receptors comprise a critical component in neural posteriorization and are required for proper neuronal differentiation. These results support a quantitative role for retinoid signaling in regionalization of the CNS.

Novel retinoic acid receptor ligands in Xenopus embryos.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.

4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.

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-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 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 to Xenopus 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.

A mouse cdc25 homolog is differentially and developmentally expressed.

The timing and activation of the p34cdc2 kinase in mammals is associated with dephosphorylation of phosphotyrosine and phosphothreonine residues on the p34cdc2 kinase. For fission yeast, the timing of mitosis is regulated by cyclic accumulation of cdc25, which promotes dephosphorylation of p34cdc2 and concomitant protein kinase activation. We report the identification and characterization of a structural and functional mouse homolog, Cdc25M2, of the cdc25 phosphatase. Cdc25M2 shows high sequence identity to the previously reported human homolog cdc25Hu2. Cdc25M2 can functionally complement for a Schizosaccharomyces pombe cdc25ts mutation, and when expressed in Escherichia coli and purified, Cdc25M2 is an active phosphatase. cdc25M2 mRNA shows variation in expression in different tissues in the mouse embryo and is expressed in a developmental and cell-cycle-dependent fashion. We suggest that the expression and accumulation of the cdc25 mitotic inducer may play a critical role in the regulation of mouse development.

Retinoic acid is a negative regulator of AP-1-responsive genes.

We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.

Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor.

We present evidence that the glucocorticoid receptor (GR) and transcription factor Jun/AP-1 can reciprocally repress one another's transcriptional activation by a novel mechanism that is independent of DNA binding. Overexpression of c-Jun prevents the glucocorticoid-induced activation of genes carrying a functional glucocorticoid response element (GRE). Conversely, GR is able to repress AP-1-mediated transcriptional activation. Mutant analysis reveals that the ligand binding and DNA binding domains of GR and the region including the leucine zipper of c-Jun are required for repression. Gel retardation analysis demonstrates that bacterially expressed c-Jun disrupts GR-GRE complexes. These data indicate that members of two distinct classes of transcription factors can oppose one another's activity through a mechanism likely involving protein-protein interactions.

Jun-Fos and receptors for vitamins A and D recognize a common response element in the human osteocalcin gene.

We present evidence that the vitamin D response element in the human osteocalcin gene confers responsiveness to the vitamin A metabolite, retinoic acid. Retinoic acid receptor (RAR) expressed in E. coli binds to this sequence in vitro. Transfection of RAR expression vectors in cultured cells activates heterologous promoters containing this sequence in vivo. This response element contains a consensus AP-1 site TGACTCA and in vitro is bound by the Jun-Fos complex. Unexpectedly, cotransfection of Jun and Fos expression vectors suppresses basal level transcription of the osteocalcin gene and suppresses induction by both retinoic acid and vitamin D3. Additional studies delimit an 11 nucleotide segment as a minimal hormone response element containing the AP-1 site as its core. These results indicate that two distinct classes of transcription factors can recognize common regulatory sequences, a phenomenon we refer to as cross-coupling.