Sonic Hedgehog Regulates Osteoblast Function by Focal Adhesion Kinase Signaling in the Process of Fracture Healing

Yuu Horikiri,Kenichi Matsumoto,Tatsuo Okui,Naito Kurio,Akira Sasaki,Masahiro Iwamoto,Tsuyoshi Shimo,Rajeev Samant

doi:10.1371/journal.pone.0076785

Yuu Horikiri, Kenichi Matsumoto + Show 6 more

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https://doi.org/10.1371/journal.pone.0076785

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Journal: PloS one	Publication Date: Oct 4, 2013
Citations: 83	License type: CC BY 4.0

Affiliation: Okayama University, Children's Hospital of Philadelphia

Abstract

Several biological studies have indicated that hedgehog signaling plays an important role in osteoblast proliferation and differentiation, and sonic hedgehog (SHH) expression is positively correlated with phosphorylated focal adhesion kinase (FAK) Tyr397. However, the relationship between them and their role in the process of normal fracture repair has not been clarified yet. Immunohistochemical analysis revealed that SHH and pFAK Tyr397 were expressed in bone marrow cells and that pFAK Tyr397 was also detected in ALP-positive osteoblasts near the TRAP-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture. SHH and pFAK Tyr397 were detectable in osteoblasts near the hypertrophic chondrocytes on day 14. In vitro analysis showed that SHH up-regulated the expression of FAK mRNA and pFAK Tyr397 time dependently in osteoblastic MC3T3-E1 cells. Functional analysis revealed that 5 lentivirus encoding short hairpin FAK RNAs (shFAK)-infected MC3T3-E1 cell groups displayed a round morphology and decreased proliferation, adhesion, migration, and differentiation. SHH stimulated the proliferation and differentiation of MC3T3-E1 cells, but had no effect on the shFAK-infected cells. SHH also stimulated osteoclast formation in a co-culture system containing MC3T3-E1 and murine CD11b+ bone marrow cells, but did not affect the shFAK-infected MC3T3-E1 co-culture group. These data suggest that SHH signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture and regulated their proliferation and differentiation, as well as osteoclast formation, via FAK signaling.

Highlights

Fracture healing is a complex physiological process that involves the combination of both intramembranous and endochondral ossification
Immunohistochemical analysis revealed that sonic hedgehog (SHH) and pFAK Tyr397 were expressed in bone marrow cells and that pFAK Tyr397 was detected in ALP-positive osteoblasts near the tartrateresistant acid phosphatase (TRAP)-positive osteoclasts in the fracture site in the ribs of mice on day 5 after fracture
At that time SHH expression was intense in many of these cells (Figure 1E and F), whereas pFAK Tyr397 was weakly expressed in the bone marrow cells (Figure 1G and H)

Summary

Introduction

Fracture healing is a complex physiological process that involves the combination of both intramembranous and endochondral ossification. Sonic hedgehog (Shh) is a 45-kDa potent signaling protein that regulates the proliferation, differentiation, and cellular patterning across a wide range of cell types [2,3]. It has been shown that hedgehog signaling is involved in fracture healing and bone maintenance [4,5]. Mark et al showed that conditional deletion of Ptch selectively in mature osteoblasts enhances hedgehog signaling and leads to increased osteoclastogenesis [9]. They showed that hedgehog signaling indirectly induce osteoclast formation by upregulating parathyroid hormone-related peptide (PTHrP), which promoted receptor activator for nuclear factor κB ligand (RANKL). SHH stimulates osteoclast formation with PTHrP in a co-culture system consisting of ST2 cells and murine CD11b+

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