SUMMARY Liver fibrosis is a reversible wound-healing response involving TGFβ1 activation of hepatic stellate cells (HSCs). Here we show that vitamin D receptor (VDR) ligands inhibit HSC activation and abrogate liver fibrosis, while Vdr knockout mice spontaneously developed hepatic fibrosis. Mechanistically, we describe a pronounced redistribution of genome wide VDR binding sites (VDR cistrome) in HSCs elicited by a TGFβ1 pro-fibrotic insult. This TGFβ1-induced VDR cistrome overlaps extensively with SMAD3 binding sites, with co-occupancy at numerous cis-regulatory elements identified on a large set of pro-fibrotic genes. Addition of VDR ligand reduces SMAD3 occupancy at co-regulated genes, revealing an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis. These results define a role for VDR as a endocrine checkpoint to modulate the wound healing response in liver, and suggest VDR ligands as a potential therapy for liver fibrosis.

Recent development in the field of gene regulation by nuclear receptors (NRs) have identified a role for cofactors in transcriptional control. While some of the NRassociated proteins serve as coactivators, the effect of the receptor interacting protein 140 (RIP140) on NR transcriptional responses is complex. In this report we have studied the effect of RIP140 on gene regulation by the glucocorticoid receptor (GR). We demonstrate that RIP140 antagonized all GR-mediated responses tested, which included activation through classical GRE, the synergistic effects of glucocorticoids on AP-1 and Pbx1/ HOXB1 responsive elements, as well as gene repression through a negative GRE and cross-talk with NF-B (RelA). This involved the ligand-binding domain of the GR and did not occur when the GR was bound to the antagonist RU486. The strong repressive effect of RIP140 was restricted to glucocorticoid-mediated responses in as much as it slightly increased signaling through the RelA and the Pit-1/Pbx proteins and only slightly repressed signaling through the Pbx1/HOXB1 and AP-1 proteins, excluding general squelching as a mechanism. Instead, this suggests that RIP140 acts as a direct inhibitor of GR function. In line with a direct effect of RIP140 on the GR, we demonstrate a GR-RIP140 interaction in vitro by a glutathione S-transferase-pull down assay. Furthermore, the repressive effect of RIP140 could partially be overcome by overexpression of the coactivator TIF2, which involved a competition between TIF2 and RIP140 for binding to the GR.

Transforming growth factor-␤ (TGF-␤

Inclusion of the Smad binding elements (SBEs) with the VDRE in the heterologous promoter restored synergistic activation. Furthermore, this synergy was dependent on the spacing between VDRE and SBEs. The Smad3-Smad4 heterodimer was found to bind in gel shift assay to two distinct DNA segments of the osteocalcin promoter: ؊1030 to ؊989 (SBE3) and ؊418 to ؊349 (SBE1). Deletion of SBE1, which is proximal to the VDRE, but not the distal SBE3 in this promoter reporter abolished TGF-␤ responsiveness and eliminated synergistic co-activation with vitamin D. Thus the molecular mechanism, whereby Smad3 and VDR mediate cross-talk between the TGF-␤ and vitamin D signaling pathways, requires both a VDRE and a SBE located in close proximity to the target promoter.The biological actions of 1,25-dihydroxyvitamin D 3 (vitamin D) 1 are mediated through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily (1). Although VDR homodimers may play some role, most VDR actions are thought to be mediated via VDR heterodimerization with the retinoid X receptor (RXR) (2). The VDR/RXR heterodimers bind to vitamin D response elements (VDREs) in the promoters of vitamin D target genes (3, 4). VDREs typically consist of at least two copies of the consensus motif, PuG(G/T)TCA, arranged as direct repeats with a three-nucleotide spacing (DR3) (5). In addition to spacing, small differences in the half-site sequence, and the sequence of the flanking extension of the response elements also appear to be important in determining receptor binding efficiency (Ref. 6 and references therein). Upon binding, these receptors can either stimulate or repress transcription of target genes (7), assisted by direct and indirect interaction with other transcriptional regulatory proteins. Such additional factors may be recruited by other regulatory signals, such as TGF-␤, which has been shown to act in synergy with vitamin D (8, 9). Transforming growth factor-␤ (TGF-␤), like activin and bone morphogenetic protein (BMP), signals through the Smad family of intracellular transducing proteins (10). Among the Smads, Smad2 and Smad3 are known to permit signaling by TGF-␤ and activin ligands (11). Once phosphorylated, these Smad proteins heterodimerize with Smad4, the common mediator of TGF-␤ pathways. These complexes translocate to the nucleus where they regulate target genes either by interacting with DNA or with other transcription factors (11,12). Though a palindromic Smad binding element (SBE), GTCTAGAC, was shown to interact with the Smad3 and Smad4 complex, the smaller 4-bp half-site of this SBE DNA sequence, GTCT or AGAC, has been shown to be sufficient to bind these proteins (12, 13).Although Smads can bind to DNA, Smad-DNA binding alone is not always sufficient for gene activation. There are now a growing number of examples in which Smads cooperate with DNA-binding partners to regulate transcription (14). This type of interaction has been demonstrated for the Mix.2, goosecoid and Xvent2 promoters where Smads interact with FAST-1, FAST-2, m...

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Hardeman, E. C. (1998) Mol. Cell. Biol. 18, 6641-6652) was identified in a yeast one-hybrid screen as a protein that binds within an upstream enhancer-containing region of the skeletal muscle-specific gene, TNNI1 (human troponin I slow; hTnI slow ). It has been proposed that hMus-TRD1␣1 may play an important role in fiber-specific muscle gene expression by virtue of its ability to bind to an Inr-like element (nucleotides ؊977 to ؊960) within the hTnI slow upstream enhancer-containing region that is necessary for slow fiber-specific expression. In this study we demonstrate that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1␣1 regulate hTnI slow through the Inr-like element. Co-transfection assays in C2C12 cells and Cos-7 cells demonstrate that hMusTRD1␣1 represses hTnI slow transcription and prevents MEF2C-mediated activation of hTnI slow transcription. Gel shift analysis shows that hMusTRD1␣1 can abrogate MEF2C binding to its cognate site in the hTnI slow enhancer. Glutathione S-transferase pull-down assays demonstrate that hMusTRD1␣1 can interact with both MEF2C and the nuclear receptor co-repressor. The data support the role of hMusTRD1␣1 as a repressor of slow fiber-specific transcription through mechanisms involving direct interactions with MEF2C and the nuclear receptor co-repressor.

The effect of steroid hormones on multiplication of the human polyomavirus BK (BKV) was studied. Physiological concentrations of the synthetic glucocorticoid dexamethasone, progesterone R5020, or estrogen 17,-estradiol enhanced the permissivity of the host cell for BKV, resulting in an up to 11-fold (dexamethasone), 5-fold (progesterone), or 3-fold (17I1-estradiol) higher virus yield. The increase in virus yield in dexamethasone-stimulated cells correlated with enhanced steady-state levels of viral transcripts. The late leader sequence of the BKV control region contains a hormone response unit composed of a nonconsensus glucocorticoid and/or progesterone response element (GRE/PRE) and a fully consensus estrogen response element (ERE). DNA-protein binding studies showed that the glucocorticoid receptor and the progesterone receptor bound to this BKV GRE/PRE-like sequence, while the estrogen receptor could bind to the BKV ERE motif. By transient transfection assays, we were able to show that these sequences can mediate steroid hormone-induced gene expression. However, no cooperative transactivation effect between the BKV GRE/PRElike motif and BKV ERE motif was observed. This BKV hormone response unit may play an important role in vivo by enhancing a productive BKV infection, and perhaps also by reactivating a latent infection, during physiological or pathological conditions accompanied by increased steroid hormone levels.