Ihh/Gli2 Signaling Promotes Osteoblast Differentiation by Regulating Runx2 Expression and Function

Abstract

Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2-but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.

Constitutive Activation of Smoothened Leads to Impaired Developments of Postnatal Bone in Mice

“…Runx2 is considered to induce chondrocyte maturation and enhance chondrocyte proliferation through the direct induction of Ihh expression in the growth plate (Shimoyama et al, 2007). These findings, along with our present data, indicate a close association of reduced Runx2 expression with lowered osteogenic differentiation and mineralization in vitro as well as retarded postnatal development in vivo in the SmoM2;OCN-Cre mutant.…”

“…Ihh-deficient mice showed a lack of endochondral bone formation due to the absence of osteoblasts, and runt-related transcription factor-2 (Runx2) expression was found to be absent in perichondrial cells before vascular invasion (Hilton et al, 2005). Ihh expression was also found to up-regulate the function of Runx2 through Gli2 (Shimoyama et al, 2007). In addition, ectopic Ihh expression in chondrocytes mediates Runx2 induction throughout the perichondrium, although Ihh alone is not sufficient to induce bone formation (Komori, 2012).…”

Sox9 Family Members Negatively Regulate Maturation and Calcification of Chondrocytes through Up-Regulation of Parathyroid Hormone–related Protein

“…HA-tagged Sox9, Sox5, Sox6, Myc-tagged Gli1, Flag-tagged Gli2, and Flagtagged Gli3 expression vectors were used as described previously (Shimoyama et al, 2007;Hata et al, 2008). Myc-tagged dominant-negative Sox9 (aa2-234) and Myc-tagged dominant-negative Gli2 (aa2-968) that lacked the corresponding transcriptional activation domains were also used as described previously (Shimoyama et al, 2007;Hata et al, 2008).…”

“…Myc-tagged dominant-negative Sox9 (aa2-234) and Myc-tagged dominant-negative Gli2 (aa2-968) that lacked the corresponding transcriptional activation domains were also used as described previously (Shimoyama et al, 2007;Hata et al, 2008). The human PTHrP gene promoter including the 1120-base pair 5Ј-flanking region (Gallwitz et al, 2002) and the deletion mutants were ligated into the pGL4.10 vector (Promega, Madison, WI).…”

“…Recombinant adenoviruses carrying Sox5, Sox6, HA-tagged Sox9, a dominant-negative Myc-tagged Sox9, Ihh, Myc-tagged Gli2, or a dominant-negative Gli2 were constructed by homologous recombination between the expression cosmid cassette (pAxCAwt) and the parental virus genome in 293T cells (Riken Cell Bank) using the adenovirus construction kit (Takara) as described previously (Shimoyama et al, 2007;Hata et al, 2008). Mock adenovirus was used as a control adenovirus.…”