Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/ AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (؊97 to ؊93). By chromatin immunoprecipitation, recruitment of ␤-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of ␤-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-␤ signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.During development of the skeleton and formation of bone tissue, several morphogenic growth factor and hormone signaling pathways impinge upon transcriptional regulators to induce the osteogenic phenotype (1, 2). The challenge is to identify how developmental cues and regulatory factors are integrated to accommodate the requirements for biological control of cell differentiation and tissue formation. Here, we have addressed the interaction of two key signals for osteogenesis: the WNT pathway, which contributes to the development of skeletal structures (3, 4), and the transcription factor RUNX2 (CBFA1/AML3), which is required for embryonic bone formation (5, 6).WNT signaling comprises a family of 19 secreted glycoproteins that have functions related to cell specification, formation of the body plan, cell growth, differentiation and apoptosis (7,8). WNT proteins function through Frizzled receptors, which transduce the signal through either the canonical ␤-catenin pathway or non-canonical pathway (7, 8). Activation of the Frizzled receptor complex results in inhibition of a phosphorylation cascade that stabilizes intracellular ␤-catenin levels. ␤-Catenin is subsequently translocated to the nucleus to form a transcriptionally active heterodimeric ␤-catenin TCF 2...

We present the structure-based optimization of a series of estrogen receptor-beta (ERbeta) selective ligands. X-ray cocrystal structures of these ligands complexed to both ERalpha and ERbeta are described. We also discuss how molecular modeling was used to take advantage of subtle differences between the two binding cavities in order to optimize selectivity for ERbeta over ERalpha. Quantum chemical calculations are utilized to gain insight into the mechanism of selectivity enhancement. Despite only two relatively conservative residue substitutions in the ligand binding pocket, the most selective compounds have greater than 100-fold selectivity for ERbeta relative to ERalpha when measured using a competitive radioligand binding assay.

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the preosteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E 2 (PGE 2 ) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-b1 treatment of preosteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE 2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-b1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.

inflammation ͉ therapy

No abstract

Ad2 VAI gene strongly competes for transcription with VAII gene in vitro. It has been suggested that this competition may be a basis for the large excess of VAI gene transcription in virus infected cells at late times. We have studied the effect of the DNA sequence perturbations of the intragenic promoter of the VAI gene on transcription of VAII gene at the level of viral chromosome. Several Ad5 mutants with mutations in the promoter of VAI gene were constructed and transcription of their VAI and VAII genes were analyzed in the infected cells. It was found that transcription of VAII gene increased dramatically when either Box A or Box B promoter sequences of VAI gene were mutated or when the entire VAI gene was replaced by a DNA segment with an unrelated DNA sequence. Thus, at late times, active transcription of VAI gene appears to partially repress transcription of VAII gene. Those mutants which synthesized large quantities of VAII RNA only grew more slowly yielding a titer which was 1/10 of that of their parent but 5 to 6 fold higher than that of an AdS mutant lacking both VAI and VAII genes. INTRODUCTIONCells infected by adenoviruses (Ad) 2 or 5 synthesize large amounts of two low molecular weight RNAs designated virus-associated RNAs I and II (VAI and VAII RNAs, ref. 1,2). The genes coding for these RNAs are located between 29.0 and 31.0 map units (m.u.) on the conventional adenoviral map (2,3,4). The VA RNA genes are transcribed by RNA polymerase III (2,5). The nucleotide sequences of the two VA RNAs and the DNA sequences that encode the RNAs have been determined (6,7,8). The VAI RNA is heterogenous both at 5' and 3' ends and it is 157 to 162 nucleotides long. Transcription of VAI gene is initiated at two closely located sites on the genome separated by three nucleotides (VAI-A and VAI-G spe-

Ror2 is a receptor tyrosine kinase, the expression of which increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test whether Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 overexpression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSCs) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSCs as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator activated receptor gamma. Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human preosteocytic cell line by stable transfection also promoted further differentiation, as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion, and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by short hairpin RNA essentially abrogated dexamethasone-induced mineralization of hMSCs. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, using a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.

Posttranslational processing and subcellular localization of the HCV core protein are critical steps involved in the assembly of hepatitis C virus (HCV). In this study, both of these events were investigated by in vitro translation and transient COS-1 cell transfection of core protein expression constructs. Mutations at amino acid residues 173 to 174 and 191 to 192 disrupted processing events at the two putative cleavage sites in the C-terminal hydrophobic region of the core protein, indicating that these residues are implicated in the pathway of core protein maturation. As a result, two forms of core protein, C173 and C191, were detected by immunoblotting. Indirect immunofluorescence experiments showed that core proteins C173 and C191, when produced from HCV full-length protein or various polyprotein precursors, displayed a cytoplasmic localization. The C173 species, however, was translocated to the nucleus when expressed in the absence of C191. These findings indicate that preferential cleavage may occur during core protein maturation and that the association of the C191 with the C173 species may contribute to the distinct subcellular distribution of core protein. This may provide a possible mechanism for the control of the diverse biological functions of core protein during HCV replication and assembly.