Estrogen-related receptor α regulates osteoblast differentiation via Wnt/ß-catenin signaling.

Based on its homology to the estrogen receptor and its roles in osteoblast and chondrocyte differentiation, the orphan nuclear receptor estrogen-related receptor α (ERRα (ESRRA)) is an intriguing therapeutic target for osteoporosis and other bone diseases. The objective of this study was to better characterize the molecular mechanisms by which ERRα modulates osteoblastogenesis. Experiments from multiple systems demonstrated that ERRα modulates Wnt signaling, a crucial pathway for proper regulation of bone development. This was validated using a Wnt-luciferase reporter, where ERRα showed co-activator-dependent (peroxisome proliferator-activated receptor gamma co-activator 1α, PGC-1α) stimulatory effects. Interestingly, knockdown of ERRα expression also enhanced WNT signaling. In combination, these data indicated that ERRα could serve to either activate or repress Wnt signaling depending on the presence or absence of its co-activator PGC-1α. The observed Wnt pathway modulation was cell intrinsic and did not alter ß-catenin nuclear translocation but was dependent on DNA binding of ERRα. We also found that expression of active ERRα correlated with Wnt pathway effects on osteoblastic differentiation in two cell types, consistent with a role for ERRα in modulating the Wnt pathway. In conclusion, this work identifies ERRα, in conjunction with co-activators such as PGC-1α, as a new regulator of the Wnt-signaling pathway during osteoblast differentiation, through a cell-intrinsic mechanism not affecting ß-catenin nuclear translocation.

Wnt3-frizzled 1 chimera as a model to study canonical Wnt signaling.

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low-density lipoprotein receptor-related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF-luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF-luciferase activation. DKK-1 protein blocks upregulation of the TCF-luciferase reporter by the Wnt3-Fz1 chimera, suggesting involvement of LRP in Wnt3-Fz1 signaling. Expression of a Wnt3-Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3-Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt-Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases.

A cell-based Dkk1 binding assay reveals roles for extracellular domains of LRP5 in Dkk1 interaction and highlights differences between wild-type and the high bone mass mutant LRP5(G171V).

Dkk1 is a secreted antagonist of the LRP5-mediated Wnt signaling pathway that plays a pivotal role in bone biology. Because there are no well-documented LRP5-based assays of Dkk1 binding, we developed a cell-based assay of Dkk1/LRP5 binding using radioactive (125)I-Dkk1. In contrast to LRP6, transfection of LRP5 alone into 293A cells resulted in a low level of specific binding that was unsuitable for routine assay. However, co-transfection of LRP5 with the chaperone protein MesD (which itself does not bind Dkk1) or Kremen-2 (a known Dkk1 receptor), or both, resulted in a marked enhancement of specific binding that was sufficient for evaluation of Dkk1 antagonists. LRP5 fragments comprising the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, each inhibited Dkk1 binding, with mean IC(50)s of 10 and 196 nM, respectively. The extracellular domain of Kremen-2 ("soluble Kremen") was a weaker antagonist (mean IC(50) 806 nM). We also found that cells transfected with a high bone mass mutation LRP5(G171V) had a subtly reduced level of Dkk1 binding, compared to wild type LRP5-transfected cells, and no enhancement of binding by MesD. We conclude that (1) LRP5-transfected cells do not offer a suitable cell-based Dkk1 binding assay, unless co-transfected with either MesD, Kremen-2, or both; (2) soluble fragments of LRP5 containing either the third and fourth beta-propellers plus the ligand binding domain, or the first beta-propeller, antagonize Dkk1 binding; and (3) a high bone mass mutant LRP5(G171V), has subtly reduced Dkk1 binding, and, in contrast to LRP5, no enhancement of binding with MesD.

A small molecule inhibitor of the Wnt antagonist secreted frizzled-related protein-1 stimulates bone formation.

Canonical Wnt signaling has been demonstrated to increase bone formation, and Wnt pathway components are being pursued as potential drug targets for osteoporosis and other metabolic bone diseases. Deletion of the Wnt antagonist secreted frizzled-related protein (sFRP)-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit sFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 440,000 drug-like compounds was screened for inhibitors of human sFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T-cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to sFRP-1 with a K(D) of 0.35 microM in a tryptophan fluorescence quenching assay. This compound also selectively inhibited sFRP-1 with an EC(50) of 3.9 microM in the cell-based functional assay. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads (WAY-316606) bound to sFRP-1 with a K(D) of 0.08 microM and inhibited it with an EC(50) of 0.65 microM. Moreover, this compound increased total bone area in a murine calvarial organ culture assay at concentrations as low as 0.0001 microM. This work demonstrates the feasibility of developing small molecules that inhibit sFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

Parathyroid hormone synergizes with non-cyclic AMP pathways to activate the cyclic AMP response element.

Parathyroid hormone (PTH) activates multiple signaling pathways following binding to the PTH1 receptor in osteoblasts. Previous work revealed a discrepancy between cAMP stimulation and CRE reporter activation of truncated PTH peptides, suggesting that additional signaling pathways contribute to activation of the CRE. Using a CRE-Luciferase reporter containing multiple copies of the CRE stably transfected into the osteoblastic cell line Saos-2, we tested the ability of modulators of alternative pathways to activate the CRE or block the PTH-induced activation of the CRE. Activators of non-cyclic AMP pathways, that is, EGF (Akt, MAPK, JAK/STAT pathways); thapsigargin (intracellular calcium pathway); phorbol myristate acetate (protein kinase C, PKC pathway) induced minor increases in CRE-luciferase activity alone but induced dramatic synergistic effects in combination with PTH. The protein kinase A (PKA) inhibitor H-89 (10 microM) almost completely blocked PTH-induced activation of the CRE-reporter. Adenylate cyclase inhibitors SQ 22536 and DDA had profound and time-dependent biphasic effects on the CRE response. The MAPK inhibitor PD 98059 partially inhibited basal and PTH-induced CRE activity to the same degree, while the PKC inhibitor bisindolylmaleimide (BIS) had variable effects. The calmodulin kinase II inhibitor KN-93 had no significant effect on the response to PTH. We conclude that non-cAMP pathways (EGF pathway, calcium pathway, PKC pathway) converge on, and have synergistic effects on, the response of a CRE reporter to PTH.

Immortalized mouse articular cartilage cell lines retain chondrocyte phenotype and respond to both anabolic factor BMP-2 and pro-inflammatory factor IL-1.

Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM-Sv/HP and MM-Sv/HP-2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q-PCR) were performed on cells in monolayer culture and cells embedded in a three-dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP-2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro-inflammatory IL-1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP-13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP-2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL-1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase-cleaved aggrecan fragments. In summary, MM-Sv/HP and MM-Sv/HP-2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology.

Regulation of secreted Frizzled-related protein-1 by heparin.

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.

Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function.

Secreted frizzled-related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP-1 mutants and sFRP-1/sFRP-2 chimeras were generated. These mutants were characterized in an optimized T-cell factor (TCF)-luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP-1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73-86 within the second loop of the CRD of sFRP-1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP-1/sFRP-2 chimeras with the netrin domain of sFRP-1 replaced by corresponding sFRP-2 sequences showed 40-70% loss of Wnt antagonist function. The sFRP-1/sFRP-2 chimera with the replacement of C-terminal 19 amino acids of sFRP-1 with 11 amino acids of sFRP-2 resulted in 70% loss of activity indicating that carboxyl-terminal region of sFRP-1 is important for its Wnt inhibitory activity. The structure-function analysis studies of sFRP-1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function.

Structure-based mutation analysis shows the importance of LRP5 beta-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling.

A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Wnts with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf 1 (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5-G171V) induces beta-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity. HBM variant suppresses Dkk1 function and this results in reduced inhibition of TCF activity as compared to that with LRP5. Structural analysis of LRP5 revealed that the HBM mutation lies in the 4th blade of the first beta-propeller domain. To elucidate the functional significance and consequence of the LRP5-G171V mutation in vitro, we took a structure-based approach to design 15 specific LRP5 point mutations. These included (a) substitutions at the G171 in blade 4, (b) mutations in blades 2-6 of beta-propeller 1, and (c) mutations in beta-propellers 2, 3 and 4. Here we show that substitutions of glycine at 171 to K, F, I and Q also resulted in HBM-like activity in the presence of Wnt1 and Dkk1. This indicates the importance of the G171 site rather than the effect of specific amino acid modification to LRP5 receptor function. Interestingly, G171 equivalent residue mutations in other blades of beta-propeller 1 (A65V, S127V, L200V, A214V and M282V) resulted in LRP5-G171V-like block of Dkk1 function. However G171V type mutations in other beta-propellers of LRP5 did not result in resistance to Dkk1 function. These results indicate the importance of LRP5 beta-propeller 1 for Dkk1 function and Wnt signaling. These data and additional comparative structural analysis of the LRP5 family member LDLR suggest a potential functional role of the first beta-propeller domain through intramolecular interaction with other domains of LRP5 wherein Dkk1 can bind. Such studies may also lead to a better understanding of the mechanisms underlying the reduced function of Dkk1-like inhibitory ligands of LRP5 with HBM-like mutations and its relationship to increased bone density phenotypes.

The orphan receptor tyrosine kinase Ror2 promotes osteoblast differentiation and enhances ex vivo bone formation.

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.

Immortalized cell lines from mouse xiphisternum preserve chondrocyte phenotype.

Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA.

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis.

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 pre-osteocytes, 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 E2 (PGE2) 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)-beta1 treatment of pre-osteocytes 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-beta1 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.

Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.

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 beta-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 beta-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-beta signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22).

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.

Identification of pathway-selective estrogen receptor ligands that inhibit NF-kappaB transcriptional activity.

Inflammation is now recognized as a key component in a number of diseases such as atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease. The transcription factor NF-kappaB has been shown to be involved in both the early and late stages of the inflammatory-proliferative process. In this report, we describe the identification of the pathway-selective estrogen receptor (ER) ligand, WAY-169916, that inhibits NF-kappaB transcriptional activity but is devoid of conventional estrogenic activity. This pathway-selective ligand does not promote the classic actions of estrogens such as stimulation of uterine proliferation or ER-mediated gene expression, but is a potent antiinflammatory agent, as demonstrated in the HLA-B27 transgenic rat model of inflammatory bowel disease. Our results indicate the potential utility of pathway-selective ER ligands such as WAY-169916 in the treatment of chronic inflammatory diseases.

Structure-based design of estrogen receptor-beta selective ligands.

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.

Regulated expression of sFRP-1 protein by the GeneSwitch system.

The GeneSwitch system is a mifepristone-inducible expression system that provides exceptionally low uninduced and high-induced protein expression in mammalian cells. We have developed an adenovirus recombinant containing GeneSwitch protein driven by the GAL4-tk promoter, as well as recombinants containing sFRP-1 and luciferase reporter under the control of the GAL4-E1b promoter. Luciferase activity in A549 cells infected with the GeneSwitch and Luciferase viruses is very low in ethanol-treated cells, while the level of luciferase activity increases 200-fold in cells treated with mifepristone. Conditional expression of functional sFRP-1 is demonstrated in A549, human osteoblast, and CHO cell lines by either the co-infection of cells with sFRP-1 and GeneSwitch viruses or the infection of GeneSwitch expressing cell lines with sFRP-1 virus and subsequent treatment with mifepristone. The expression of sFRP-1 is seen as early as 4 h post-mifepristone treatment, reaching the highest levels at 20 h. The sFRP-1 protein is present in conditioned media, and the protein is functional based upon its ability to inhibit the Wnt-mediated activation of TCF-Luciferase reporter activity. The regulated expression of sFRP-1 utilizing adenovirus vectors provides an opportunity to address the contribution of sFRP-1 in the regulation of stem cell differentiation, maturation, and their function by modulating the Wnt signaling.

Molecular determinants of ER alpha and ER beta involved in selectivity of 16 alpha-iodo-7 beta estradiol.

The two known estrogen receptors, ER alpha and ER beta, are hormone inducible transcription factors that have distinct roles in regulating cell proliferation and differentiation. The natural ligand, 17 beta-estradiol (E2), binds with high affinity to both ER alpha and ER beta. However, a close analogue, 16 alpha-iodo-17 beta-estradiol (16 alpha IE2) showed about 10-fold selectivity for ER alpha over ER beta. From X-ray studies, it has been shown that the ligand-binding domains (LBD) of the two receptors are strikingly similar, and that only two changes fall within the binding cavity (ER alpha Leu384 to ER beta Met336, and ER alpha Met421 to ER beta Ile373). To understand the molecular basis for the ER alpha selectivity of 16 alpha IE2, mutants and chimeras of ER alpha and ER beta were generated, and ligand-binding and transactivation functions were studied. The ER alpha Leu384 Met mutant behaved like ER alpha WT in the presence of 16 alpha IE2; whereas the profile of the ER alpha Met421 Ile mutant was similar to that of ER beta WT. The ER beta mutant Ile373 Met behaved like ER alpha with 16 alpha IE2. The results clearly demonstrate the role of ER alpha Met421 in the ER alpha selectivity of 16 alpha IE2.