Hypoxia regulates osteoblast gene expression

Vascular disruption secondary to fracture creates a hypoxic gradient of inj ury wherein the oxygen tension at the center of the wound is very low, In v ivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and ost eoblasts. In order to begin to dissect this complex system, we have examine d the effects of hypoxia on isolated osteoblast gene expression in vitro. U nderstanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair a nd reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production a nd turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed t o hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h, Northern analysis was used to analyze the expression pa tterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibito r of metalloproteinase-l. We have demonstrated a marked elevation of TGF-be ta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor T GF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor w as substantially upregulated within 6 h, In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differential ly, upregulated. Finally, we have demonstrated that the expression of an in hibitor of extracellular matrix turnover, the tissue inhibitor of metallopr oteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulat ors. (C) 2001 Academic Press.