IFT80 Is Required for Fracture Healing Through Controlling the Regulation of TGF-β Signaling in Chondrocyte Differentiation and Function

Abstract

Primary cilia are essential cellular organelles that are anchored at the cell surface membrane to sense and transduce signaling. Intraflagellar transport (IFT) proteins are indispensable for cilia formation and function. Although major advances in understanding the roles of these proteins in bone development have been made, the mechanisms by which IFT proteins regulate bone repair have not been identified. We investigated the role of the IFT80 protein in chondrocytes during fracture healing by creating femoral fractures in mice with conditional deletion of IFT80 in chondrocytes utilizing tamoxifen inducible Col2α1‐CreER mice. Col2α1creIFT80f/f mice had smaller fracture calluses than IFT80f/f (control) mice. The max‐width and max‐callus area were 31% and 48% smaller than those of the control mice, respectively. Col2α1creIFT80f/f mice formed low‐density/porous woven bony tissue with significantly lower ratio of bone volume, Trabecular (Tb) number and Tb thickness, and greater Tb spacing compared to control mice. IFT80 deletion significantly downregulated the expression of angiogenesis markers‐VEGF, PDGF and angiopoietin and inhibited fracture callus vascularization. Mechanistically, loss of IFT80 in chondrocytes resulted in a decrease in cilia formation and chondrocyte proliferation rate in fracture callus compared to the control mice. Meanwhile, IFT80 deletion downregulated the TGF‐β signaling pathway by inhibiting the expression of TGF‐βI, TGF‐βR, and phosphorylation of Smad2/3 in the fracture callus. In primary chondrocyte cultures in vitro, IFT80 deletion dramatically reduced chondrocyte proliferation, cilia assembly, and chondrogenic gene expression and differentiation. Collectively, our findings demonstrate that IFT80 and primary cilia play an essential role in fracture healing, likely through controlling chondrocyte proliferation and differentiation, and the TGF‐β signaling pathway. © 2019 American Society for Bone and Mineral Research.

Type II collagen-positive progenitors are major stem cells to control skeleton development and vascular formation

“…To test whether Col2+ cells contribute to chondrocyte differentiation and blood vessel formation at postnatal stages, we created a closed femoral fracture with intramedullary nail fixation model in 10-week-old mice as described 19 to observe fracture healing. We first induced tdTomato expression in Col2+ cells in Col2-creERT; tdTomato mice with TM injection at P3 and then subjected the mice to fracture at 10 weeks of age before harvesting tissues after 2 weeks for fracture healing.…”

“…AC chondrocytes was isolated as previously described 19 . Briefly, 4-week old Col2-cre; tdTomato mice were euthanized at postnatal day 28 (P28).…”

“…Closed femoral fractures with intramedullary nail fixation were created in 10-week-old mice (Col2-creERT;tdTomato or Col2-creERT;DTA fl/fl ;tdTomato) as described 19 . Briefly, closed fractures were generated by a three-point blunt guillotine driven by a dropped weight, which creates a uniform transverse fracture of the femur.…”

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells

“…The chondrogenic medium consisted of low-glucose DMEM (Gibco, Carlsbad, CA, USA) supplemented with TGF-1 (Sigma-Aldrich, St. Louis, MO, USA) 10ng/mL, L-Ascorbate -2-phosphate (Sigma-Aldrich, St. Louis, MO, USA) 50M, Insulin (Sigma-Aldrich, St. Louis, MO, USA) 6.25g/mL. The cells were stained with Alcian blue 8-GX overnight [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 ] to detect the chondrogenic differentiation.…”

Intraflagellar Transport Proteins as Regulators of Primary Cilia Length

“…∼3 Teves et al, 2015;Liu et al, 2020 EndotheliaIn vitro Primary endothelial cells, E15.5 ∼0.85 Abdul-Majeed andNauli, 2011 …”