Overexpression of Cas-interacting zinc finger protein (CIZ) suppresses proliferation and enhances expression of type I collagen gene in osteoblast-like MC3T3E1 cells

Osteoblasts are the cells which form bone under the regulation not only by hormones and cytokines but also by ECM molecules via their attachment. To obtain insights into the role of intracellular signaling molecules operating to mediate the attachment-related regulation of osteoblastic functions, we investigated in osteoblast-like MC3T3E1 cells the effects of the overexpression of CIZ, a novel signaling protein which interacts with p130Cas. In MC3T3E1 cells, CIZ mRNA is expressed constitutively. Endogenous CIZ was localized in the MC3T3E1 cells with relatively high levels of accumulation at the attachment sites when the cells were cultured on fibronectin, collagen, or BSA. CIZ overexpression increased the number of adhesion plaques and reduced proliferation of the cells compared to that of control cells transfected with an empty vector. Furthermore, CIZ overexpression enhanced type I collagen mRNA expression, the most abundant constituent of bone matrix and a major product of osteoblasts. Analysis of the promoter region of type I collagen gene identified the presence of a consensus CIZ-binding sequence, which indeed conferred responsiveness to CIZ overexpression to a heterologous promoter. These data indicate that CIZ acts as a novel regulatory molecule in controlling osteoblastic function.     

Conditional deletion of gremlin causes a transient increase in bone formation and bone mass

Gremlin is a glycoprotein that binds bone morphogenetic proteins (BMPs) 2, 4, and 7, antagonizing their actions. Gremlin opposes BMP effects on osteoblastic differentiation and function in vitro and in vivo, and its overexpression causes osteopenia. To define the function of gremlin in the skeleton, we generated gremlin 1 (grem1) conditional null mice by mating mice where grem1 was flanked by lox(P) sequences with mice expressing the Cre recombinase under the control of the osteocalcin promoter. grem1 null male mice displayed increased trabecular bone volume due to enhanced osteoblastic activity, because mineral apposition and bone formation rates were increased. Osteoblast number and bone resorption were not altered. Marrow stromal cells from grem1 conditional null mice expressed higher levels of alkaline phosphatase activity. Gremlin down-regulation by RNA interference in ST-2 stromal and MC3T3 osteoblastic cells increased the BMP-2 stimulatory effect on alkaline phosphatase activity, on Smad 1/5/8 phosphorylation, and on the transactivation of the BMP/Smad reporter construct 12xSBE-Oc-pGL3. Gremlin down-regulation also enhanced osteocalcin and Runx-2 expression, Wnt 3a signaling, and activity in ST-2 cells. In conclusion, deletion of grem1 in the bone microenvironment results in sensitization of BMP signaling and activity and enhanced bone formation in vivo.     

MSX2 stimulates chondrocyte maturation by controlling Ihh expression

Several studies indicated that a homeobox gene, Msx2, is implicated in regulation of skeletal development by controlling enchondral ossification as well as membranous ossification. However, the molecular basis by which Msx2 conducts chondrogenesis is currently unclear. In this study, we examined the role of Msx2 in chondrocyte differentiation using mouse primary chondrocytes and embryonic metatarsal explants. Treatment with BMP2 up-regulated the expression of Msx2 mRNA along with chondrocyte differentiation in murine primary chondrocytes. Overexpression of wild-type Msx2 stimulated calcification of primary chondrocytes in the presence of BMP2. We also found that constitutively active Msx2 (caMsx2) enhanced BMP2-dependent calcification more efficiently than wild-type Msx2. Consistently, caMsx2 overexpression up-regulated the expression of alkaline phosphatase and collagen type X induced by BMP2. Furthermore, organ culture experiments using mouse embryonic metatarsals indicated that caMsx2 clearly stimulated the maturation of chondrocytes into the prehypertrophic and hypertrophic stages in the presence of BMP2. In contrast, knockdown of Msx2 inhibited maturation of primary chondrocytes. The stimulatory effect of Msx2 on chondrocyte maturation was enhanced by overexpression of Smad1 and Smad4 but inhibited by Smad6, an inhibitory Smad for BMP2 signaling. These data suggest that Msx2 requires BMP2/Smad signaling for its chondrogenic action. In addition, caMsx2 overexpression induced Ihh (Indian hedgehog) expression in mouse primary chondrocytes. Importantly, treatment with cyclopamine, a specific inhibitor for hedgehogs, blocked Msx2-induced chondrogenesis. Collectively, our results indicated that Msx2 promotes the maturation of chondrocytes, at least in part, through up-regulating Ihh expression.     

Antisense targeting of TGF-beta1 augments BMP-induced upregulation of osteopontin, type I collagen and Cbfa1 in human Saos-2 cells

Despite commonalities in signal transduction in osteoblasts from different species, the role of TGF-beta1 on bone formation remains elusive. In particular, the role of autocrine TGF-beta1 on human osteoblasts is largely unknown. Here we show the effect of TGF-beta1 knock-down on the proliferation and differentiation of osteoblasts induced by BMP2. Treatment with antisense TGF-beta1 moderately increased the rate of cell proliferation, which was completely reversed by the exogenous addition of TGF-beta1. Notably, TGF-beta1 blockade significantly enhanced BMP2-induced upregulation of mRNAs encoding osteopontin, type I collagen and Cbfa1, which was suppressed by exogenous TGF-beta1. Moreover, TGF-beta1 knock-down increased BMP2-induced phosphorylation of Smad1/5 as well as their nuclear import, which paralleled a reduction of inhibitory Smad6. These data suggest autocrine TGF-beta1 antagonizes BMP signaling through modulation of inducible Smad6 and the activity of BMP specific Smad1/5.     

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.     

Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.     

Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two “master” regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations.