FOXO1 Plays an Important Role in Enhanced Microvascular Cell Apoptosis and Microvascular Cell Loss in Type 1 and Type 2 Diabetic Rats

OBJECTIVE: To investigate early events leading to microvascular cell loss in diabetic retinopathy. RESEARCH DESIGN AND METHODS: FOXO1 was tested in vivo by DNA binding activity and by nuclear translocation in microvascular cells in retinal trypsin digests. In vivo studies were undertaken in STZ-induced diabetic rats and Zucker diabetic fatty rats using the tumor necrosis factor (TNF)-specific blocker, pegsunercept, or by inhibiting FOXO1 with RNAi. Microvascular cell apoptosis, formation of pericyte ghosts, and acellular capillaries were measured. Upstream and downstream effects of high-glucose–induced FOXO1 were tested on rat microvascular endothelial cells (RMECs) by small-interfering RNA (siRNA) in vitro. RESULTS: DNA binding or nuclear translocation of FOXO1, which was reduced by TNF inhibition, was elevated in type 1 and type 2 diabetic retinas. Diabetes stimulated microvascular cell apoptosis; pericyte ghost and acellular capillary development was inhibited by FOXO1 siRNA. High glucose in vitro decreased FOXO1 phosphorylation and DNA binding activity and decreased Akt phosphorylation in RMECs. High-glucose–stimulated FOXO1 DNA binding activity was mediated through TNF-α and formation of reactive oxygen species (ROS), while inhibitors of TNF and ROS and FOXO1 siRNA reduced high-glucose–enhanced RMEC apoptosis. The caspase-3/7 activity and capacity of high glucose to increase mRNA levels of several genes that regulate RMEC activation and apoptosis were knocked down by FOXO1 siRNA. CONCLUSIONS: FOXO1 plays an important role in rat retinal microvascular cell loss in type 1 and type 2 diabetic rats and can be linked to the effect of high glucose on FOXO1 activation.National Institutes of Health (R01DE17732, R01DE07559, R01EY014702); Massachusetts Lions Eye Research Organizatio

Chemokine Expression Is Upregulated in Chondrocytes in Diabetic Fracture Healing

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P \u3c 0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P \u3c 0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect

FOXO1 Modulates Osteoblast Differentiation

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P \u3c 0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced the MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells

Impaired Wound Healing in Mouse Models of Diabetes Is Mediated by TNF-α Dysregulation and Associated With Enhanced Activation of Forkhead Box O1 (FOXO1)

Aims/hypothesis The role of TNF-α in impaired wound healing in diabetes was examined by focusing on fibroblasts. Methods Small excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-α was specifically inhibited by pegsunercept. Results Diabetic wounds had increased TNF-α, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p \u3c 0.05). TNF-α inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-α was blocked (p  \u3c 0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-α enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-α also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown. Conclusions/interpretation These studies indicate that TNF-α dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-α induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis

Diabetes-enhanced fox 01 activation induces retinal microvascular cell loss and is mediated by TNF-[alpha] in experimental diabetic retinopathy

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact [email protected] (D.Sc.)--Henry M. Goldman School of Dental Medicine, 2007 (Periodontology and Oral Biology).Includes bibliography: leaves 141-158.Microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy, an important cause of blindness. To investigate molecular mechanisms responsible for diabetes-increased microvascular cell death animal models of both type 1 and type 2 diabetes were used to investigate the role of elevated TNF-[alpha] and FOXO1 activity in the retinas of diabetic rats. Inhibition of TNF by pegsunercept in vivo significantly reduced diabetes-enhanced FOXO1 activation and microvascular cell apoptosis in both type 1 and type 2 diabetes. Pegsunercept also reduced the formation of pericyte ghosts and acellular capillaries; characteristic lesions of early diabetic retinopathy in both type l and type 2 diabetes. The relationship between high glucose, TNF-[alpha] and FOXO1 in retinal microvascular endothelial cells was further investigated in vitro. High glucose induced apoptosis that was TNF dependent through a mechanism that invoIved activation of FOXO1. By siRNA and focused microarrays several classes of genes that regulate endothelial cells were induced by TNF-[alpha] and high glucose in vitro and many of the genes induced by high glucose were FOXO1 dependent. Intravitreal injection of FOXO1 siRNA in vivo significantly reduced diabetes enhanced apoptosis of retinal microvascular cells and subsequent pericyte ghost formation and acellular capillary formation in type 1 model of diabetes. FOXO1 silencing in vivo reduced retinal microvascular cell apoptosis in type 2 model also. These results demonstrate a previously unrecognized role for FOXO1 and TNF-α in promoting the early pathogenesis of diabetic retinopathy and demonstrate the potential therapeutic benefit of modulating their activity

The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts

Suppression of tumorigenicity 18 (ST18) and the homologues neural zinc-finger protein-3 (NZF3) and myelin transcription factor 3 (Myt3) are transcription factors with unknown function. Previous studies have established that they repress transcription of a synthetic reporter construct consisting of the consensus sequence AAAGTTT linked to the thymidine kinase promoter. In addition, ST18 exhibits significantly reduced expression in breast cancer and breast cancer cell lines. We report here for the first time evidence that ST18 mediates tumor necrosis factor (TNF) -α induced mRNA levels of proapoptotic and proinflammatory genes in fibroblasts by mRNA profiling and silencing with ST18 small interfering RNA (siRNA). Gene set enrichment analysis and mRNA profiling support this conclusion by identifying several apoptotic and inflammatory pathways that are down-regulated by ST18 siRNA. In addition, ST18 siRNA reduces TNF-induced fibroblast apoptosis and caspase-3/7 activity. Fibroblasts that overexpress ST18 by transient transfection exhibit significantly increased apoptosis and increased expression of TNF-α, interleukin (IL) -1α, and IL-6. In addition, cotransfection of ST18 and a TNF-α or IL-1α reporter construct demonstrates that ST18 overexpression in fibroblasts significantly enhanced promoter activity of these genes. Taken together, these studies demonstrate that the transcription factor ST18/NZF3 regulates the mRNA levels of proapoptotic and proinflammatory genes in revealing a previously unrecognized function.—Yang, J., Siqueira, M. F., Behl, Y., Alikhani, M., and Graves, D. T. The transcription factor ST18 regulates proapoptotic and proinflammatory gene expression in fibroblasts

FOXO1 Modulates Osteoblast Differentiation

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P \u3c 0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced the MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells