Sonic hedgehog regulates osteoblast function by focal adhesion kinase signaling in the process of fracture healing

PLoS One. 2013 Oct 4;8(10):e76785. doi: 10.1371/journal.pone.0076785. eCollection 2013.

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) Tyr(397). 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 Tyr(397) were expressed in bone marrow cells and that pFAK Tyr(397) 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 Tyr(397) 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 Tyr(397) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Adhesion / genetics
  • Cell Differentiation / genetics
  • Cell Line
  • Cell Movement / genetics
  • Cell Proliferation
  • Focal Adhesion Protein-Tyrosine Kinases / genetics
  • Focal Adhesion Protein-Tyrosine Kinases / metabolism*
  • Fracture Healing / physiology*
  • Gene Expression
  • Gene Expression Regulation
  • Gene Knockdown Techniques
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism*
  • Male
  • Mice
  • Osteoblasts / cytology
  • Osteoblasts / metabolism*
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism
  • Parathyroid Hormone-Related Protein / metabolism
  • Parathyroid Hormone-Related Protein / pharmacology
  • RNA Interference
  • Rib Fractures / genetics
  • Rib Fractures / metabolism
  • Rib Fractures / pathology
  • Signal Transduction*

Substances

  • Hedgehog Proteins
  • Parathyroid Hormone-Related Protein
  • Focal Adhesion Protein-Tyrosine Kinases

Grants and funding

The work was supported by the programs Grants-in-Aid for Young Scientists (A) [to T.S.] and Grants-in-Aid for Exploratory Research (to T.S. and A.S.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.