Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells

Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low-density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with beta-glycerophosphate was found to induce a time-dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both beta-glycerophosphate and oxLDL, beta-glycerophosphate's ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of beta-glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, beta-glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both beta-glycerophosphate-induced Osx expression and osteoblast differentiation in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2) -dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185-193, 2008. (c) 2008 Wiley-Liss, Inc.     

Loss of Smad3-mediated negative regulation of Runx2 activity leads to an alteration in cell fate determination

Runx2 is required for osteoblast differentiation but is expressed in certain nonosteoblastic cells without activating the differentiation process, suggesting that its activity is suppressed through a lineage-specific mechanism. Here we report that primary mouse dermal fibroblasts lacking Smad3 can acquire an osteoblast-like phenotype, including activation of Runx2 activity, expression of osteoblast-specific genes, and calcium deposition. We further show that negative regulation of Runx2 activity by Smad3 in dermal fibroblasts is likely mediated by controlling the expression of Msx2, an antagonist of Runx2 in this cellular context. These data support the presence of a novel mechanism for controlling cell fate determination of mesenchymal lineages by preventing differentiation toward the osteoblastic lineage via negative regulation of Runx2 activity.     

Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3β

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4μg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3β on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a >2-fold increase in GSK-3β activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3β activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.     

Osx transcriptional regulation is mediated by additional pathways to BMP2/Smad signaling

Bone morphogenetic protein (BMP)-2 induces Osterix (Osx) in mouse C2C12 cells and chondrocytes. Genetic studies place Osx downstream to the BMP-2/Smad/Runx2 signaling pathway; however, limited information is available on the mediators of Osx expression in osteoblast lineage commitment. Several lines of research implicate the presence of Runx2-independent ossification. Therefore, the purpose of this study was to identify possible mediators of Osx expression beyond the BMP-2/Smad pathway. Using real-time RT-PCR, we showed upregulation of Osx in response to BMP-2 in human mesenchymal stem cells (hMSC). Insulin-like growth factor (IGF)-I upregulated Osx, but not Runx2. Further, IGF-I in combination with BMP-2 was synergistic for Osx, suggesting a pathway beyond Smad signaling. MAPK was tested as a common mediator across BMP-2 and IGF-I signaling pathways. Inhibition of MAPK component ERK1/2 did not affect Runx2 gene expression, but inhibited Osx expression and matrix mineralization. BMP-2-mediated Osx expression was downregulated in response to p38 inhibition. We therefore conclude that during osteogenic lineage progression, in addition to the BMP-2/Smad pathway, IGF-I and MAPK signaling may mediate Osx.     

Teriparatide (1-34 human PTH) regulation of osterix during fracture repair

Based on remarkable success of PTH as an anabolic drug for osteoporosis, case reports of off-label use of teriparatide (1-34 PTH) in patients with complicated fractures and non-unions are emerging. We investigated the mechanisms underlying PTH accelerated fracture repair. Bone marrow cells from 7 days 40 microg/kg of teriparatide treated or saline control mice were cultured and Osx and osteoblast phenotypic gene expression assessed by real-time RT-PCR in these cells. Fractured animals injected daily with either saline or 40 microg/kg of teriparatide for up to 21 days were X-rayed and histological assessment performed, as well as immunohistochemical analyses of the Osx expression in the fracture callus. Osx, Runx2 and osteoblast or chondrocyte phenotypic gene expression was also assessed in fracture calluses. Our data shows that Osx and Runx2 are up-regulated in marrow-derived MSCs isolated from mice systemically treated with teriparatide. Furthermore, these MSCs undergo accelerated osteoblast maturation compared to saline injected controls. Systemic teriparatide treatments also accelerated fracture healing in these mice concomitantly with increased Osx expression in the PTH treated fracture calluses compared to controls. Collectively, these data suggest a mechanism for teriparatide mediated fracture healing possibly via Osx induction in MSCs.     

Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors

Hedgehog and canonical Wnt/beta-catenin signaling are implicated in development of the osteoblast, the bone matrix-secreting cell of the vertebrate skeleton. We have used genetic approaches to dissect the roles of these pathways in specification of the osteoblast lineage. Previous studies indicate that Ihh signaling in the long bones is essential for initial specification of an osteoblast progenitor to a Runx2+ osteoblast precursor. We show here that this is a transient requirement, as removal of Hh responsiveness in later Runx2+, Osx1+ osteoblast precursors does not disrupt the formation of mature osteoblasts. By contrast, the removal of canonical Wnt signaling by conditional removal of the beta-catenin gene in early osteoblast progenitors or in Runx2+, Osx1+ osteoblast precursors results in a similar phenotype: osteoblasts fail to progress to a terminal osteocalcin+ fate and instead convert to a chondrocyte fate. By contrast, stabilization of beta-catenin signaling in Runx2+, Osx1+ osteoblast precursors leads to the premature differentiation of bone matrix secreting osteoblasts. These data demonstrate that commitment within the osteoblast lineage requires sequential, stage-specific, Ihh and canonical Wnt/beta-catenin signaling to promote osteogenic, and block chondrogenic, programs of cell fate specification.