Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

We previously described an osteocalcin (OC) fibroblast growth factor (FGF) response element (FRE) DNA binding activity as a target of Msx2 transcriptional regulation. We now identify Ku70, Ku80, and Tbdn100, a variant of Tubedown-1, as constituents of the purified OCFRE-binding complex. Northern and Western blot analyses demonstrate expression of Ku and Tbdn100 in MC3T3E1 osteoblasts. FGF2 treatment regulates Ku, but not Tbdn100, protein accumulation. Gel supershift studies confirm sequence-specific DNA binding of Ku in the OCFRE complex; chromatin immunoprecipitation assays confirm association of Ku and Tbdn100 with the endogenous OC promoter. In the promoter region ؊154 to ؊113, the OCFRE is juxtaposed to OSE2, an osteoblast-specific element that binds Runx2 (Osf2, Cbfa1 (14), key features of OC promoter regulation are remarkably well conserved across mammalian species (3). The osteoblast homeoprotein Msx2 plays a crucial role in the regulation of osteoblast proliferation and differentiation (15). Msx2 and Msx1 are absolutely required for craniofacial bone formation (15). Msx2 controls the timing of osteoblast differentiation, including the temporospatial patterns of OC expression in the calvarium and teeth (12, 16). Msx2 plays a global role in determination of skeletal mass, elegantly demonstrated in murine genetic models; moreover, as highlighted by Maas and colleagues (15), osteogenic effects during development are dependent upon Msx gene dosage, suggesting that stoichiometry is an important feature of Msx2 action. Consistent with this, we (11, 12, 16 -18) and others (2, 19) have shown that Msx2 and Msx1 participate in specific protein-protein interactions that control gene transcription. In the rat OC gene, transcriptional regulation by Msx2 converges on a 42-bp region at nucleotides Ϫ154 to Ϫ113 relative to the transcription initiation site, encompassing an FGF responsive element at nucleotides Ϫ142 to Ϫ136 (17). The OCFRE (OC FGF response element) is a GCAGTCA motif that confers both basal and FGF2-regulated expression of the OC gene in MC3T3E1 calvarial osteoblasts (17,20,21). A DNA binding activity up-regulated by FGF2 in MC3T3E1 cells recognizes this element and is constitutively expressed by MG63 osteosarcoma cells (17,20). Msx2 exerts suppressive actions on the OC promoter in part by inhibiting the OCFRE DNA binding activity present in either MC3T3E1 cells or purified from MG63 cells (17). By contrast, Msx2 does not alter vitamin D receptor-dependent transcription or vitamin D receptor binding to its DNA cognate (17). Inhibition of OCFRE activity is dependent upon key regulatory domains encoded in the Msx2 homeodomain NH 2 -terminal arm and

supporting

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Regulation of Osteocalcin Gene Expression by a Novel Ku Antigen Transcription Factor Complex

Willis¹,

Loewy²,

Charlton-Kachigian³

et al. 2002

“…Two mechanisms have been proposed to explain how Msx2 counteracts Dlx5. In one model, both factors compete for the same binding site in the target promoters (29,30). Alternatively, interactions between Msx2 and Dlx5 homeodomains may inhibit Dlx5 binding to the target promoters (31).…”

Section: Discussionmentioning

confidence: 99%

Molecular Regulation of Matrix Extracellular Phosphoglycoprotein Expression by Bone Morphogenetic Protein-2

Cho¹,

Yoon²,

Woo³

et al. 2009

Matrix extracellular phosphoglycoprotein (MEPE) is mainly expressed in mineralizing tissues, and its C-terminal proteolytic cleavage product is an acidic-serine-asparate-rich-MEPE-associated motif (ASARM) that is a strong regulator of body phosphate metabolism and mineralization. There is sufficient data supporting a role for MEPE protein function in mineralization, however, little is known about the regulation of MEPE gene expression. As bone morphogenetic protein-2 (BMP-2) is one of the most important signals for calvarial mineralization and MEPE expression is higher in mineralized tissues, we attempted to uncover a regulatory circuit between BMP-2 and MEPE expression. Mepe expression is very low in proliferating MC3T3-E1 cells, but is dramatically increased in the mineralization stage and is strongly stimulated by treatment with BMP-2, even in proliferating cells. Overexpression and knockdown experiments of Smads, Dlx5, and Runx2 indicated that they are indispensable mediators of BMP-2-induced Mepe expression. In contrast, Msx2 showed strong inhibition of Mepe transcription. PHEX is an enzyme that prevents the release of the ASARM motif, a mineralization inhibitor, from the MEPE molecule. Thus, the MEPE/PHEX ratio may be a good indicator of mineralization progression because we found that the mRNA ratio and protein levels were low when osteoblasts were actively differentiating to the mineralization stage and the ratio was high when the cells reached the mineralization stage when it is assumed that osteocytes may protect themselves and make a space to survive from the mineralized matrix by releasing the ASARM motif. Collectively, MEPE expression is bone cell-specific and induced by the BMP-2 signaling pathway. In addition, the MEPE/PHEX ratio of the cell could be a very important barometer indicating the progression of tissue mineralization.Mineral homeostasis in the body is critical for healthy bones and teeth, and is generally regulated by the calcium-phosphate balance in the bone and kidney networks. In the past, the vitamin D/parathyroid hormone axis was assumed to be a single major circuit in bone-renal phosphate regulation, but recently, new bone-renal phosphate regulating factors have been identified. The finding of fibroblast growth factor 23 (FGF23), 2 phosphate-regulating genes with homologies to endopeptidases on the X chromosome (PHEX) and matrix extracellular phosphoglycoprotein (MEPE) genes, and their pathophysiological roles in the genetic diseases of mineral metabolism, have provided a great deal of insight into the understanding of bone-and mineral-related diseases. Among these, autosomal-dominant hypophosphatemic rickets (OMIM number 193100) is characterized by renal phosphate wasting, hypophosphatemia, and inappropriately normal 1,25-dihydroxyvitamin D 3 levels (1). Autosomal-dominant hypophosphatemic rickets is caused by a missense mutation of FGF23, which is resistant to proteolysis by PHEX and increases the half-life of full-length phosphaturic FGF23 (2). Second, X-linked hypophosphatemic...

“…However, Msx2 also clearly functions to delay the timing of osteoblast terminal differentiation. This is best exemplified by Msx2-dependent suppression of the os-teocalcin (OC) 1 gene in teeth and calvarial osteoblasts (13)(14)(15)(16). During odontogenesis, where stage-specific gene expression programs are spatially resolved, Msx2 and OC are reciprocally expressed (14).…”

mentioning

confidence: 99%

MINT, the Msx2 Interacting Nuclear Matrix Target, Enhances Runx2-dependent Activation of the Osteocalcin Fibroblast Growth Factor Response Element

Sierra

Cheng

Loewy

et al. 2004

Msx2 promotes osteogenic lineage allocation from mesenchymal progenitors but inhibits terminal differentiation demarcated by osteocalcin (OC) gene expression. Msx2 inhibits OC expression by targeting the fibroblast growth factor responsive element (OCFRE), a 42-bp DNA domain in the OC gene bound by the Msx2 interacting nuclear target protein (MINT) and Runx2/ Cbfa1. To better understand Msx2 regulation of the OC-FRE, we have studied functional interactions between MINT and Runx2, a master regulator of osteoblast differentiation. In MC3T3E1 osteoblasts (with endogenous Runx2 and FGFR2), MINT augments transcription driven by the OCFRE that is further enhanced by FGF2 treatment. OCFRE regulation can be reconstituted in the naïve CV1 fibroblast cell background. In CV1 cells, MINT synergizes with Runx2 to enhance OCFRE activity in the presence of activated FGFR2. The RNA recognition motif domain of MINT (which binds the OCFRE) is required. Runx2 structural studies reveal that synergy with MINT uniquely requires Runx2 activation domain 3. In confocal immunofluorescence microscopy, MINT adopts a reticular nuclear matrix distribution that overlaps transcriptionally active osteoblast chromatin, extensively co-localizing with the phosphorylated RNA polymerase II meshwork. MINT only partially co-localizes with Runx2; however, co-localization is enhanced 2.5-fold by FGF2 stimulation. Msx2 abrogates Runx2-MINT OCFRE activation, and MINT-directed RNA interference reduces endogenous OC expression. In chromatin immunoprecipitation assays, Msx2 selectively inhibits Runx2 binding to OC chromatin. Thus, MINT enhances Runx2 activation of multiprotein complexes assembled by the OCFRE. Msx2 targets this complex as a mechanism of transcriptional inhibition. In osteoblasts, MINT may serve as a nuclear matrix platform that organizes and integrates osteogenic transcriptional responses.Bone formation arises via two overlapping yet distinct mechanisms (1). Endochondral ossification occurs via the calcification and vascularization of an initial cartilaginous template, best exemplified by long bone development and fracture repair. Non-endochondral ossification occurs during development in the flat bones of the skull, in teeth, and in the lateral portion of the clavicles. Direct bone deposition occurs in type I collagenbased extracellular matrix; no cartilage template precedes bone deposition. Both in vivo and in cell culture models, a characteristic gene expression program is elaborated as osteoblasts differentiate and mature from osteoprogenitors (2-4). Although transcription factors crucial to bone development (Runx2/Cbfa1, CBF-␤, Msx2, Msx1, Dlx5, Alx4, Osx, and Sox9) and metabolic/endocrine regulation (multiple nuclear receptors, Runx2/Cbfa1) have been identified, our understanding of the molecular details of stage-specific osteoblast gene expression is rudimentary (5). "Cross-talk" between these factors occurs in the osteoblast nucleus; an integrative model is lacking and sorely needed to better address unmet clinical needs in metabolic...