Abstract
The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone during endochondral ossification remains poorly understood. One of the driving forces is hypoxia. As a master regulator of hypoxia, hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1α cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1α on Wnt pathway are not well understood. In this study, our quantitative RT-PCR results revealed that the expression of a Wnt antagonist Sclerostin (Sost) was upregulated in osteoblasts during hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine (DFO), a HIF-1α activator, led to further increase of Sost expression, suggesting that HIF-1α may activate Sost expression. The regulation of Sost gene expression by HIF-1α was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1α, and found that the inhibition of HIF-1α by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1α, transient transfection assay was performed and showed that HIF-1α activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1α, we generated a series of deletion mutants of Sost constructs. It was demonstrated that Sost-260 was the minimal region of Sost promoter for HIF-1α activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1α-mediated Sost reporter activation. Gel shift assay showed that HIF-1 bound to the promoter sequence of Sost directly. These findings support our hypothesis that HIF-1α activates Sost expression. This study provides a novel molecular mechanism through which HIF-1α inhibits Wnt signaling 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
We asked if the effect of hypoxia on Sost expression is related to Hypoxiainducible factor-1a (HIF-1a)
Our recent observations have indicated that hypoxia/HIF-1a inhibit osteoblast proliferation, and that HIF-1a has a synergistic effect with Osx on the inhibition of Wnt pathway [23]
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 developmental process involving the osteoblast differentiation from mesenchymal stem cells. The observation that Osx inhibits the Wnt pathway highlights the potential for novel feedback control mechanisms involved in bone formation [3]. Replacing the avascular cartilage template with highly vascularized bone is the key step of endochondral ossification. During endochondral bone formation, chondrocytes model the growth plate at the long bone distal ends and become hypertrophic and hypoxic. Growth plate chondrocytes go through well-ordered and regulated phases of cell proliferation, differentiation, and apoptosis [4,5]. Differentiation is followed by hypertrophic chondrocyte death, blood vessel invasion, and replacement of the cartilage matrix with a trabecular bone matrix. The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone remains poorly understood. It has been speculated that the hypoxia in the chondrocytes imposes energetic limitations on the cells as they evolve from a proliferative to a terminally differentiated state [8]
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