The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.

Bilirubin clearance is one of the numerous important functions of the liver. Defects in this process result in jaundice, which is particularly common in neonates. Elevated bilirubin levels can be decreased by treatment with phenobarbital. Because the nuclear hormone receptor constitutive androstane receptor (CAR) mediates hepatic effects of this xenobiotic inducer, we hypothesized that CAR could be a regulator of bilirubin clearance. Activation of the nuclear hormone receptor CAR increases hepatic expression of each of five components of the bilirubin-clearance pathway. This induction is absent in homozygous CAR null mice but is observed in mice expressing human CAR instead of mouse CAR. Pretreatment with xenobiotic inducers markedly increases the rate of clearance of an exogenous bilirubin load in wild-type but not CAR knockout animals. Bilirubin itself can also activate CAR, and mice lacking CAR are defective in clearing chronically elevated bilirubin levels. Unexpectedly, CAR expression is very low in livers of neonatal mice and humans. We conclude that CAR directs a protective response to elevated bilirubin levels and suggest that a functional deficit of CAR activity may contribute to neonatal jaundice

We have identified the xenobiotic receptor CAR (constitutive androstane receptor) as a key regulator of acetaminophen metabolism and hepatotoxicity. Known CAR activators as well as high doses of acetaminophen induced expression of three acetaminophen-metabolizing enzymes in wild-type but not in CAR null mice, and the CAR null mice were resistant to acetaminophen toxicity. Inhibition of CAR activity by administration of the inverse agonist ligand androstanol 1 hour after acetaminophen treatment blocked hepatotoxicity in wild type but not in CAR null mice. These results suggest an innovative therapeutic approach for treating the adverse effects of acetaminophen and potentially other hepatotoxic agents.

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Steroid receptor RNA activator (SRA) is an RNA that coactivates steroid hormone receptor-mediated transcription in vitro. Its expression is strongly up-regulated in many human tumors of the breast, uterus, and ovary, suggesting a potential role in pathogenesis. To assess SRA function in vivo, a transgenic-mouse model was generated to enable robust human SRA expression by using the transcriptional activity of the mouse mammary tumor virus long terminal repeat. Transgenic SRA was expressed in the nuclei of luminal epithelial cells of the mammary gland and tissues of the male accessory sex glands. Distinctive evidence for SRA function in vivo was obtained from the elevated levels of estrogen-controlled expression of progesterone receptor in transgenic mammary glands. Although overexpression of SRA showed strong promoting activities on cellular proliferation and differentiation, no alterations progressed to malignancy. Epithelial hyperplasia was accompanied by increased apoptosis, and preneoplastic lesions were cleared by focal degenerative transformations. In bitransgenic mice, SRA also antagonized ras-induced tumor formation. This work indicates that although coactivation of steroid-dependent transcription by SRA is accompanied by a proliferative response, overexpression is not in itself sufficient to induce turmorigenesis. Our results underline an intricate relationship between the different physiological roles of steroid receptors in conjunction with the RNA activator in the regulation of development, tissue homeostasis, and reproduction.Cancer is still a major cause of morbidity in humans due to the limited arsenal of therapies available. This lack of treatment options reflects a dearth in both general and specific scientific knowledge of tumorigenesis that exists largely because we still have not identified all the key molecular players responsible for controlled gene expression.We have previously reported the isolation and functional characterization of a novel transcriptional coactivator, steroid receptor RNA activator (SRA) (18). SRA acts as an RNA transcript by regulating eukaryotic gene expression mediated by the steroid receptors (SRs), which play critical roles in eukaryotic development, metabolism, reproduction, and disease (23, 45). In mammalian tissue culture cells, recombinant SRA showed potent coactivation activity with the receptors for androgens (AR), estrogens (ER), glucocorticoids, and progestins (PR). However, no evidence has yet been obtained for a direct binding of SRA to SRs. We found SRA in a protein complex together with SRC coactivators and therefore proposed that SRA-containing ribonucleoprotein complexes accentuate transcriptional specificity by integrating coregulator activities upon selective binding to SRs (18). Endorsing this model, SRA was shown to associate with the DEAD box proteins p72/p68 to act as an ER␣-specific ribonucleoprotein coactivator complex, which stimulates the amino-terminal activation domain of the receptor while concurrently integrating SRC/p160-mediated AF2 coa...

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.The nuclear hormone receptor LRH-1 (NR5A2) is the mammalian homolog of the Drosophila transcription factor Fushi Tarazu-Factor1, which regulates Drosophila metamorphosis (1, 2). A mouse protein termed LRH-1 3 (liver receptor homolog-1) was the first isolated mammalian orthologue 4 and further analysis of rat and human homologs revealed several isoforms (4 -7). Unlike the majority of nuclear receptors that function as dimers, LRH-1 binds as a monomer to sites found in a number of target genes that consist of a consensus nuclear receptor half-site with an upstream extension: 5Ј-(T/C)(C/A)AAGGX(C/T)X-3Ј (5-14). LRH-1 is closely related to the orphan receptor SF-1, which also binds DNA as a monomer and has important functions in the development and differentiation of steroidogenic tissues and in steroidogenesis and sexual determination (15). LRH-1 and SF-1 have more than 95% amino acid sequence identity in their DNA binding domains and recognize the same DNA sequences. These and other NR5A subfamily members contain a distinctive 26-amino acid motif, called the Fushi Tarazu-Factor1 domain, which is located just C-terminal to the conserved zinc finger DNA binding domain and contacts the upstream motif in the binding site (16).Nuclear hormone receptors are typically ligand-dependent transcription factors. The ligand binding domain adopts a three-layer ␣-helical structure and upon ligand binding the C-terminal helix 12 is held in close proximity to the ligand binding pocket, forming a surface recognized by transcriptional coactivators (17,18). An initial crystal structure of the mouse LRH-1 ligand binding domain revealed that, unlike most other superfamily members, the LBD can form a stable active monomeric structure in the absence of ligand, coa...

Cdc25 A and B are dual-speci®city phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely unde®ned. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia.

The orphan nuclear hormone receptor small heterodimer partner (SHP) regulates the expression of several genes involved in bile acid homeostasis in the liver. Because bile acid toxicity is a major source of liver injury in cholestatic disease, we explored the role of SHP in liver damage induced by common bile duct ligation (BDL). Shp ؊/؊ mice show increased sensitivity in this model of acute obstructive cholestasis, with greater numbers of bile infarcts and higher mortality than wild-type C57BL/6 mice. This increased sensitivity could not be accounted for by differences in expression of bile acid homeostatic genes 2 or 5 days after BDL. Instead, higher basal expression of such genes, including the key biosynthetic enzyme cholesterol 7␣ hydroxylase (Cyp7A1) and the bile salt export pump, is associated with both an increase in bile flow prior to BDL and an increase in acute liver damage at only 1.5 hours after BDL in Shp ؊/؊ mice, as shown by bile infarcts. At 3 hours, Cyp7A1 expression still remained elevated in Shp ؊/؊ with respect to wild-type mice, and the hepatic and serum bile acid levels and total hepatobiliary bile acid pool were significantly increased. The increased sensitivity of mice lacking SHP contrasts with the decreased sensitivity of mice lacking the farnesoid X receptor (FXR; nuclear receptor subfamily 1, group H, member 4) to BDL, which has been associated with decreased intraductal pressure and fewer bile infarcts. Conclusion: We propose that differences in acute responses to BDL, particularly the early formation of bile infarcts, are a primary determinant of the differences in longer term sensitivity of the Fxr ؊/؊ and Shp ؊/؊ mice to acute obstructive cholestasis. (HEPATOLOGY 2008;