Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblastsecreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE 2 )), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the -galactosidase reporter gene (pOPG5.9gal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9gal reporter construct led to 39-, 7-, and 16-fold increases in -galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE 2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE 2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequencespecific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.Bone growth, development, and maintenance in mammals is a highly regulated process. The level of bone mass is dependent on the balance between bone formation and resorption. At the cellular level, this balance involves the coordinate regulation and interaction of the component cell types: bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and their formation and function are under the control of an osteoblast-specific transcription factor known as core binding factor a1 (Cbfa1) or osteoblast specific factor 2 (Osf2) 1 (1-3). Cbfa1 has been shown to regulate the expression of genes that characterize the osteoblast phenotype, including osteocalcin, osteopontin, type I collagen, bone sialoprotein, and collagenase-3, by binding to DNA sequence elements called OSE 2 that are present in the control regions of these genes (1, 4 -6). Cbfa1 knock out animals completely lack bones because of a maturational arrest of osteoblasts and die soon after delivery (2, 3). Further, haploinsufficiency of the Cbfa1 gene product in mice and humans heterozygous for the Cbfa...
Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] in an in vivo preclinical surrogate model of psoriasis.
Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1--38) (0.01--20 microg/100 g) continuous infusion for 1--24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 microg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not different from the Sham-parathyroidectomized rats, and osteoclast number was 3-fold of the vehicle control and 1.7-fold of the Sham-parathyroidectomized rats. In the distal femur, RANKL mRNA expression was increased (27-fold) and OPG mRNA expression was decreased (4.6-fold). The changes in RANKL and OPG mRNA levels were rapid (as early as 1 h), dose dependent, and sustained over a 24-h period that was examined. Immunohistochemical evaluation of bone sections confirmed that OPG level was reduced in proximal tibial metaphysis upon PTH infusion. Circulating OPG protein level was also decreased by 32% when compared with the parathyroidectomized control. The expression of genes that mark the osteoblast phenotype was significantly decreased [osteoblast specific transcription factor (2.3-fold), osteocalcin (3-fold), bone sialoprotein (2.8-fold), and type I collagen (5-fold)]. These results suggest that the catabolic effect of PTH infusion in vivo in this well-established resorption model is associated with a reciprocal expression of OPG/RANKL and a co-ordinate decrease in the expression of bone formation-related genes. We propose that the rapid and sustained increase in RANKL and decrease in OPG initiate maintain and favor the cascade of events in the differentiation/recruitment and activation of osteoclasts.
Transforming growth factor- (TGF-) regulates osteoclastogenesis and osteoclast survival, in part through the induction of osteoprotegerin (OPG), a protein known to inhibit osteoclast formation and function. To explore the molecular basis of TGF- regulation of OPG expression, we evaluated the effects of TGF- on osteoclast formation, OPG protein secretion, mRNA expression, and gene transcription. The marked inhibitory effect of TGF- on osteoclast differentiation was confirmed in a co-culture model utilizing murine stromal/osteoblastic BALC cells and bone marrow hematopoietic precursors. This inhibition in osteoclast differentiation was preceded by a decrease in RANKL mRNA expression (5-fold) and a reciprocal increase in OPG mRNA (6.1-fold) and protein The interaction of osteoclast precursors with cells of the osteoblast lineage is essential for their differentiation to form mature, bone-resorbing osteoclasts. Molecules mediating this interaction include RANK ligand (RANKL) 1 (also known as osteoclast differentiation factor, tumor necrosis factor-related activation-induced cytokine, and OPG ligand) (1, 2) that is expressed on the osteoblast/stromal cell surface and the cognate receptor, receptor activator of NF-B (RANK) (3, 4), expressed on hematopoietic precursor cells. The interaction of RANKL with RANK initiates a cascade of signaling events (4 -7) that result in the differentiation of these precursors to form tartrate-resistant acid phosphatase-positive multinucleated osteoclasts that are capable of resorbing bone. OPG, a secreted glycoprotein of the tumor necrosis factor receptor superfamily, acts as a decoy receptor and blocks the interaction between RANKL and RANK, thus inhibiting osteoclast differentiation. OPG has also been shown to inhibit the activity and survival of osteoclasts in vitro and bone resorption in vivo (8 -13).The effects of a number of hormones, growth factors, and cytokines that modulate osteoclast differentiation are mediated by the osteoblasts. Many of these agents have now been shown to exert their actions by regulating OPG and/or RANKL expression in osteoblasts (1, 14 -20). TGF- is one such cytokine that plays a major role in the regulation of bone formation and resorption (21). It has been shown to inhibit the formation of osteoclast-like cells in long term human marrow cultures (22) and to inhibit bone resorption in fetal rat long bone cultures (23). More recently, TGF- has been shown to induce OPG expression in osteoblastic cells and to inhibit osteoclast differentiation and survival (17,19). However it is not known whether TGF- directly affects the transcriptional activity of the OPG gene. To further investigate the molecular basis of TGF- effects on OPG production, we analyzed the effect of TGF- on osteoclast formation, OPG protein secretion, mRNA expression, and gene transcription. In order to analyze the mechanism by which TGF- stimulates OPG gene transcription, we have characterized the TGF--responsiveness of a 5.9-kb fragment of the human OPG promoter that we recen...
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