Activation of the acquired immune response reduces coupled bone formation in response to a periodontal pathogen.

Osteoimmunolgy involves the interaction of the immune system with skeletal elements. This interaction can lead to the formation of osseous lesions. To investigate how the acquired immune response could contribute to osteolytic lesions, we injected the periodontal pathogen Porphyromonas gingivalis adjacent to calvarial bone with or without prior immunization against the bacterium. Activation of the acquired immune response increased osteoclastogenesis and decreased coupled bone formation. The latter was accompanied by an increase in nuclear translocation of the transcription factor FOXO1 in vivo, increased apoptosis of bone-lining cells measured by the TUNEL assay and number of activated caspase-3 positive cells and a decrease in bone lining cell density. Further studies were conducted with MC3T3 osteoblastic cells. Apoptosis and increased FOXO1 DNA binding activity were induced when a combination of cytokines was tested, IL-beta, TNF-alpha, and IFN-gamma. Knockdown of FOXO1 by small interfering RNA significantly reduced cytokine stimulated apoptosis, cleaved caspase-3/7 activity and decreased mRNA levels of the proapoptotic genes, TNF-alpha, FADD, and caspase-3, -8, and -9. These results indicate that activation of the acquired immunity by a periodontal pathogen reduces the coupling of bone formation and resorption. This may occur by enhancing bone lining cell apoptosis through a mechanism that involves increased FOXO1 activation. These studies give insight into inflammatory bone diseases such as periodontal disease and arthritis were the formation of lytic lesions occurs in conjunction with deficient bone formation and activation of an acquired immune response.

Diabetes-enhanced tumor necrosis factor-alpha production promotes apoptosis and the loss of retinal microvascular cells in type 1 and type 2 models of diabetic retinopathy.

Retinal microvascular cell loss plays a critical role in the pathogenesis of diabetic retinopathy. To examine this further, type 1 streptozotocin-induced diabetic rats and type 2 Zucker diabetic fatty rats were treated by intravitreal injection of the tumor necrosis factor-specific inhibitor pegsunercept, and the impact was measured by analysis of retinal trypsin digests. For type 2 diabetic rats, the number of endothelial cells and pericytes positive for diabetes-enhanced activated caspase-3 decreased by 81% and 86%, respectively, when treated with pegsunercept (P < 0.05). Similarly, the number of diabetes-enhanced terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive endothelial cells and pericytes decreased by 81% and 67% respectively when treated with pegsunercept (P < 0.05). Diabetes-increased activated caspase-3- and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive microvascular cell numbers were both reduced by 81% and 80%, respectively, in pegsunercept-treated type 1 diabetic rats (P < 0.05). Inhibition of tumor necrosis factor reduced type 1 diabetes-enhanced pericyte ghost formation by 87% and the number of type 2 diabetes-enhanced pericyte ghosts by 62% (P < 0.05). Similarly, increased acellular capillary formation caused by type 1 and type 2 diabetes was reduced by 68% and 67%, respectively, when treated with pegsunercept (P < 0.05). These results demonstrate a previously unrecognized role of tumor necrosis factor-alpha in promoting the early pathogenesis of diabetic retinopathy leading to loss of retinal microvascular cells and demonstrate the potential therapeutic benefit of modulating its 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) -alpha 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-alpha, interleukin (IL) -1alpha, and IL-6. In addition, cotransfection of ST18 and a TNF-alpha or IL-1alpha 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.

Diabetes enhances mRNA levels of proapoptotic genes and caspase activity, which contribute to impaired healing.

We previously reported that after a bacteria-induced wound in the scalp, type 2 diabetic (db/db) mice had higher levels of apoptosis of fibroblasts and bone-lining cells that are critical for healing compared with normoglycemic controls. To investigate mechanisms by which this might occur, RNA profiling and caspase activity was measured after inoculation of Porphyromonas gingivalis. Diabetes caused a more than twofold induction of 71 genes that directly or indirectly regulate apoptosis and significantly enhanced caspase-8, -9, and -3 activity. The functional significance of diabetes-induced apoptosis was studied by treating diabetic mice with a pancaspase inhibitor, z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone). Inhibiting apoptosis significantly improved several parameters of healing, including fibroblast density, enhanced mRNA levels of collagen I and III, and increased matrix formation. Improvements were also noted in bone, with an increase in the number of bone-lining cells and new bone formation. Thus, diabetes-enhanced apoptosis represents an important mechanism through which healing is impaired, and this can be explained, in part, by diabetes-increased expression of proapoptotic genes and caspase activity.

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<0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P<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<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.

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-alpha 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.

Tumor necrosis factor-alpha mediates diabetes-enhanced apoptosis of matrix-producing cells and impairs diabetic healing.

Diabetics suffer increased infection followed by increased apoptosis of fibroblasts and bone-lining cells during the healing process. To investigate a potential mechanism, we inoculated Porphyromonas gingivalis into the scalp of type 2 diabetic (db/db) or control mice and inhibited tumor necrosis factor alpha (TNF-alpha) with etanercept. Mice were euthanized at the early phase of infection (21 hours) or during the peak repair of the bacteria-induced wound (8 days). At 21 hours, TNF-alpha inhibition significantly reduced fibroblast apoptosis and caspase-3 activity in both diabetic and normoglycemic mice (P < 0.05). During healing etanercept reduced fibroblast apoptosis and caspase-3 activity by almost 50% in diabetic but not normoglycemic mice (P < 0.05). Concomitantly, etanercept significantly increased fibroblast number by 31% and new matrix formation by 72% in diabetic mice. When bone was examined during healing, administration of the TNF-alpha blocker reduced apoptosis of bone-lining cells by 53%, increased their number by 48%, and enhanced new bone formation by 140% in the diabetic group (P < 0.05). The degree of connective tissue and osseous healing stimulated in the diabetic mice by anti-TNF-alpha treatment was within the range that is physiologically relevant. This enhanced healing may in part be explained by block-ing TNF-alpha-induced apoptosis of critical matrix-producing cells.