Abstract
Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.
Highlights
Bone formation includes two distinct processes: endochondral ossification which requires a cartilage intermediate and intramembranous ossification which forms directly from mesenchymal condensations without cartilage template
The discovery that Osx inhibits the Wnt pathway highlights the potential for novel feedback control mechanisms involved in bone formation [15]
Hypoxia inhibited osteoblast proliferation To examine the effect of hypoxia on osteoblast proliferation, MC3T3 osteoblastic cells were cultured in Alpha Minimum Essential Medium, and maintained for different time points in normoxic (20%O2) or hypoxia (1%O2) condition under a humidified hypoxia incubator
Summary
Bone formation includes two distinct processes: endochondral ossification which requires a cartilage intermediate and intramembranous ossification which forms directly from mesenchymal condensations without cartilage template. Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Osteoblast differentiation from mesenchymal stem cells is controlled by various transcription factors and signaling proteins, including Indian Hedgehog, Runx, Osterix (Osx), and Wnt pathway [1]. Indian Hedgehog is indispensible for endochondral ossification and the initial activation of Runx2 [2]. Runx is required for both endochondral and membranous ossification and needed for mesenchymal cell differentiation into preosteoblasts [3]. Osx was first discovered as a bone morphogenetic protein 2 (BMP-2) inducible gene in mesenchymal stem cells. Osx is required for osteoblast differentiation and bone formation
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