Diabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF- Morroniside manufacture promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes and increased FOXO1 DNA binding activity in diabetic fracture calluses (< 0.05). These results suggest that diabetes-enhanced TNF- increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF- dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage. Osteopenia associated with decreased bone mineral density is an important complication of type 1diabetes.1,2,3,4,5 The effect of osteopenia is thought to significantly enhance the risk of fractures as evidenced by increased fractures of the long bones of diabetics.6,7,8 Clinical studies have reported delayed union or increased fracture healing time in diabetic subjects compared with matched controls.9,10,11 Similar findings of impaired or delayed fracture healing have been reported in multiple animal models.12,13,14 Normal fracture repair is dependent INPP4A antibody on the coordinated expression of cytokines that initiate and regulate the fracture healing process including the production and removal of cartilage coupled with bone formation and remodeling.15 Diabetes has been shown to enhance expression of receptor activator for nuclear factor kB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor- (TNF-) that stimulate formation of osteoclasts and are responsible for resorption of mineralized cartilage and bone.16,17,18 Under some conditions diabetes has been shown to increase osteoclastogenesis.18,19,20,21,22 Diabetes-enhanced osteoclast formation is thought to contribute to diabetic osteopenia in adults as well as in acute Charcot arthropathy, a complication of diabetic neuropathy that increases bone fragility and in diabetic fracture healing.2,23,24 In both conditions increased osteoclastogenesis is linked to increased expression of pro-resorptive factors including RANKL, M-CSF, and TNF-.24 One of the mechanisms by which diabetes may impair fracture healing is through increased levels of TNF-. 16 Increased TNF- is thought to contribute to a number of diabetic complications including microangiopathy and neuropathy, cardiovascular diseases, retinopathy, and increased inflammation associated with infection and periodontitis.25,26 Although nuclear factor B is typically associated with TNF-induced inflammation, 27 it Morroniside manufacture is also likely that other transcription factors play an important role. Because we previously demonstrated that forkhead box 01 (FOXO1) mediated the pro-apoptotic effects of TNF- and TNF–induced pro-apoptotic gene expression,28 the experiments described below were undertaken to determine whether TNF- contributed to impaired fracture healing and whether FOXO1 could potentially regulate mRNA levels of pro-osteoclastogenic factors induced by TNF- stimulated mRNA levels of factors in chondrocytic cells that were pro-osteoclastogenic or pro-inflammatory, which was mediated in part by FOXO1. Morroniside manufacture These studies provide new insight into diabetes impaired fracture healing and support a previously unrecognized role for TNF- and FOXO1 in mediating this untoward response. Materials and Methods Induction of Type 1 Diabetes The research was conducted in conformity with all Federal and U.S. Department of Agriculture guidelines, as well as an Institutional Animal Care and Use Committee approved protocol. Studies were done on 8-week-old, male CD-1 mice purchased from Charles River Laboratories (Wilmington, MA). Diabetes was induced by intraperitoneal injection of streptozotocin (40 mg/kg) (Sigma, St. Louis, MO) in 10 mmol/L citrate buffer daily for 5 days.33 Normoglycemic control mice were treated with vehicle alone, 10 mmol/L citrate buffer. Venous blood obtained from the tail was assessed for glucose levels (Accu-Chek, Roche Diagnostics, Indianapolis, IN) and mice were considered to be diabetic when blood glucose levels exceeded 250 mg/dl. Glycosylated hemoglobin levels were measured by Glyco-tek affinity chromatography (Helena Laboratories, Beaumont, TX) at the time of euthanasia.