Chinipardaz, Zahra
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Publication Diabetes Impairs Fracture Healing Through Foxo1-Mediated Downregulation of IFT80 and Disruption of Ciliogenesis(2021-12-21) Chinipardaz, ZahraObjective: Impaired fracture healing in diabetic patients remains a clinical challenge. Diabetes adversely affects chondrocytes, osteoblasts and their progenitors through various mechanisms such as increased inflammatory cytokine expression, enhanced reactive oxygen species production and reduction in growth factor expression during fracture healing. Primary cilia have been shown to play an indispensable role in osteoblast differentiation and functions. Recent studies have reported the adverse effect of diabetes on primary cilia of several organs such as the pancreas and kidney. However, the impact of diabetes on osteoblast primary cilia during fracture healing and the underlying pathological mechanism have not been well defined. We previously showed that IFT80 protein is essential for primary cilia formation and osteoblast differentiation during bone development. Moreover, diabetes upregulates Foxo1 expression, leading to defective fracture healing. Meanwhile, deletion of Foxo1 in chondrocytes can rescue the effect of diabetes-induced impaired fracture healing. Thus, this study tested a hypothesis that primary cilia in osteoblasts are essential for osteogenesis during fracture healing, and diabetes-induced Foxo1 upregulation impairs bone formation through disruption of primary cilia formation in osteoblasts. Methods: To test whether diabetes impairs fracture healing through disruption of primary cilia in osteoblasts, mice with IFT80 deletion in osteoblasts utilizing OSXcretTAIFT80f/f mice model in normoglycemic and streptozotocin-induced type 1 diabetic mice were created and analyzed. To investigate if Foxo1 mediates the effect of diabetes on ciliogenesis, mice with Foxo1 deletion in osteoblasts (OSXcretTAFoxo1f/f) were generated and analyzed in normoglycemic and streptozotocin-induced type 1 diabetic mice. OSXcretTA mice (normoglycemic and diabetic) were considered as control in both studies. A closed femur fracture model was induced in all groups at age 12 weeks old. Femur callus specimens at day 21 post-fracture were obtained to assess osteoblast ciliogenesis by immunostaining, bone formation using microCT and histological analysis. Callus specimens at day 35 post-fracture were used to investigate the mechanical strength of calluses. In-vitroexaminations were performed to explore ciliogenesis and osteogenesis of primary osteoblasts with IFT80 or Foxo1 deletion exposed to advanced glycation end products (AGEs) or unmodified BSA (as control). ChIP, gain of function assays and luciferase activity analysis were carried out to assess if Foxo1 transcriptional factor regulates IFT80 gene expression. All data are presented as the mean ± standard deviation. Statistical significant was determined by ANOVA at P Results: Micro-CT and histological analysis exhibited significantly less bone volume with more porous woven bone in the fracture callus of diabetic OSXcretTAmice, normoglycemic and diabetic OSXcretTAIFT80f/fmice compared to normoglycemic control mice (P0.05). Immunofluorescent images demonstrated a dramatic decrease of cilia number in bone lining cells of fracture callus specimens obtained from normoglycemic OSXcretTAIFT80f/f, diabetic OSXcretTAand diabetic OSXcretTAIFT80f/fmice (14%, 9% and 6%, respectively) compared to those from the control mice (51%) (PIn-vitro studies showed that AGEs treatment and IFT80 deletion significantly reduced cilia number and differentiation of osteoblasts. Interestingly, the adverse effect of diabetes was reversed in diabetic mice with osteoblast-specific deletion of Foxo1 (diabetic OSXcretTAFoxo1f/f). Micro-CT and histological analysis showed a significant reduction in the bone volume of diabetic OSXcretTAmice compared to the control mice (normoglycemic OSXcretTA). However, when Foxo1 was deleted in diabetic mice, the bone volume was significantly restored (P>0.05). Consistently, the expression of osteoblast markers was dramatically downregulated in diabetic OSXcretTAmice but restored considerably in diabetic mice with Foxo1 deletion. Strikingly, diabetes-reduced cilia formation in bone lining cells was rescued in diabetic OSXcretTAFoxo1f/f mice to a level comparable to control mice (P>0.05). In-vitro, AGEs treatment significantly reduced the mineralization and ciliogenesis in osteoblasts, which were dramatically reversed by Foxo1 ablation. ChIP results showed that the interaction between Foxo1 and IFT80 promoter dramatically increased in AGEs treated osteoblasts compared to the unmodified BSA treated cells, and overexpression of Foxo1 resulted in 2.2-fold downregulation of IFT80 gene expression level. Furthermore, the IFT80 promoter-driven luciferase activity was significantly inhibited in osteoblasts treated with AGEs compared to the cells treated with unmodified BSA. Conclusions: Our results show that primary cilia in osteoblasts are essential for osteogenesis during fracture healing. Diabetes-enhanced Foxo1 activity reduced ciliogenesis in osteoblasts resulting in defective fracture healing. Ablation of Foxo1 rescued diabetes- impaired fracture healing through restoring ciliogenesis.