HIF1A adenovirus

Bone is a highly dynamic tissue that is constantly undergoing formation and resorption. Through RNA sequencing analysis, the researchers found that specific genes involved in regulating osteoblast differentiation are regulated by HIF-1α and HIF-2α, but in slightly different ways. Increased HIF-1α expression inhibits osteoblast differentiation by inhibiting RUNX2 function through upregulating Twist2 gene expression, which has been confirmed in Hif1a conditional knockout (KO) mice. Ectopic expression of HIF-1α by adenoviral transduction increased RANKL expression and activity, while knockdown of Hif1a expression by siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no significant effect on osteoblast-mediated osteoclast activation. This unexpected result was elucidated by the upregulation of HIF-2α after Hif1a overexpression, which demonstrated that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, and was verified by experiments in which HIF-2α was knocked down after HIF-1α overexpression. These findings suggest that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation and affects osteoclast formation by regulating RANKL secretion (mediated by HIF-2α).

Ectopic expression of HIF-1α by adenoviral transduction system showed that the differentiation of primary calvarial preosteoblasts was inhibited. HIF-1α overexpression reduced the expression of osteoblast marker genes Ocn and Runx2 (Figure 1a), and ALP and ARS staining showed that mineralization and calcification nodule formation were inhibited (Figure 1b). BMP-2-induced regeneration of calvarial bone defect model confirmed the inhibitory effect of HIF-1α on osteoblast differentiation (Figure 1c). Implantation of Ad-Hif1a in the calvarial defect area significantly delayed BMP-2-induced bone regeneration (Figure 1c). Knockdown of Hif1a by RNAi increased the expression of osteoblast marker genes (Figure 1d), mineralization and calcification nodule formation (Figure 1e). In addition, HIF-1α overexpression significantly reduced RUNX2 activity (Figure 2f). In the current RNA sequencing data (Figure 1f), Twist2 is usually upregulated by HIF-1α and HIF-2α overexpression. Similar to the effects of HIF-2α, HIF-1α overexpression upregulated Twist2 but not Twist1 (Figure 1g). The regulatory cis-element of the Twist2 promoter contains a putative HIF-1α binding sequence 5′-(A/G)CGTG-3′ (Figure 1h). In addition, siRNA-mediated silencing of Hif1a in primary cultured calvarial osteoblasts confirmed that Hif1a regulates Twist2 expression (Figure 1i). Knockdown of Twist2 with specific siRNA partially restored the decrease in Ocn and Runx2 caused by Hif1a overexpression (Figure 1j). These data suggest that HIF-1α and HIF-2α have similar effects on osteoblast differentiation by regulating the TWIST2-RUNX2-OCN axis.

Figure 1. HIF-1α inhibits osteoblast differentiation via the TWIST2-RUNX2-OCN axis. (Lee S Y, et al., 2024)