The role of intraflagellar transport 80 (IFT80) in skeletal development
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Intraflagellar transport proteins (IFT) play important roles in cilia formation and organ development. Partial loss of IFT80 function leads Jeune asphyxiating thoracic dystrophy (JATD) or short-rib polydactyly (SRP) syndrome type III, displaying narrow thoracic cavity and multiple cartilage anomalies. However, it is unknown how IFT80 regulates skeletal development. To understand the role and mechanism by which IFT80 regulates bone and cartilage development in vivo, we generated IFT80 conditional mouse model (IFT80 f/f ). Osteoblast or chondrocyte specific deletion of IFT80 was achieved by crossing IFT80f/f mice with the mice expressing tissue-specific Cre recombinase. We first used Osterix (OSX)-Cre transgenic line to delete IFT80 in osteoblast precursor cells (OPCs). OSX; IFT80 f/f mice display growth retardation and markedly decreased bone mass, suggesting the important role of IFT80 in bone development. Further study shows deletion of IFT80 results in impaired osteoblast differentiation, which is partially rescued by overexpression of BMP2 or Runx2. Loss of IFT80 significantly blocks canonical Hh-Gli signaling, but dramatically elevates non-canonical Hh-Gαi-RhoA-stress fiber signaling. Inhibition of RhoA and ROCK activity partially restores osteogenic differentiation in IFT80 -deficient OPCs by blocking non-canonical Hh-Gαi signaling activation and reduction of Cofilin and MLC2 phosphorylation. Cytochalasin D, an inhibitor of actin polymerization, greatly restores osteoblast differentiation in IFT80 -deficient OPCs via disrupting actin stress fiber and promoting cilia formation and Hh-Gli transduction. Our findings reveal a new mechanism that IFT80 controls canonical Hh-Gli and non-canonical Hh-Gαi-RhoA pathways and actin dynamics during osteoblast differentiation. To define the role and mechanism of IFT80 in chondrocyte and cartilage formation, we generated Col2α1; IFT80 f/f mouse model by crossing IFT80 f/f mice with inducible Col2α1-CreER mice and deleted IFT80 in chondrocyte lineage cells by injection of tamoxifen at embryonic or postnatal stage. Loss of IFT80 in embryonic stage results in short limbs at birth. Histological studies shows that IFT80 -deficient mice have shortened cartilage with marked changes in cellular morphology and organization in the resting, proliferative, pre-hypertrophic, and hypertrophic zones. Moreover, deletion of IFT80 in postnatal stage leads to stunted growth with shortened growth plate but thickened articular cartilage. These findings demonstrate that IFT80 is essential for cartilage formation. We also found that IFT80 silencing impairs cilia formation and chondrogenic differentiation in mouse bone marrow derived stromal cells (BMSCs), and decreases the expression of chondrocyte marker genes - collagen II and aggrecan. Additionally, silencing IFT80 down-regulates Hh signaling activity whereas up-regulates Wnt signaling activity. Overexpression of Gli2 in IFT80 -silenced cells promotes chondrogenesis. In addition, defect of ciliogenesis is also found in the cartilage of IFT80 -deficient mice and primary IFT80 -deficient chondrocytes. Overall, our results demonstrate that loss of IFT80 blocks chondrogenic differentiation by disruption of ciliogenesis and alteration of Hh and Wnt signaling transduction. Together, these results demonstrate that IFT80 plays an important role in regulating the development of bone and cartilage.