High Levels of Tumor Necrosis Factor-α Contribute to Accelerated Loss of Cartilage in Diabetic Fracture Healing
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090148
ISSN1525-2191
AutoresJazia A. Alblowi, Rayyan A. Kayal, Michelle Siqueria, Erin McKenzie, Nanarao Krothapalli, Jody McLean, Jason Conn, Barbara S. Nikolajczyk, Thomas A. Einhorn, Louis C. Gerstenfeld, Dana T. Graves,
Tópico(s)Bone health and treatments
ResumoDiabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. Microarray and gene set enrichment analysis revealed an up-regulation of gene sets related to inflammation, including tumor necrosis factor (TNF) signaling in the diabetic group, when cartilage is being replaced by bone on day 16, but not on days 12 or 22. This change coincided with elevated osteoclast numbers and accelerated removal of cartilage in the diabetic group (P < 0.05), which was reflected by smaller callus size. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss, and number of TNF-α and receptor activator for nuclear factor kB ligand positive chondrocytes were significantly reduced (P < 0.05). The transcription factor forkhead box 01 (FOXO1) was tested for mediating TNF stimulation of osteoclastogenic and inflammatory factors in bone morphogenetic protein 2 pretreated ATDC5 and C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-α, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1α, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF-α promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes in vivo and increased FOXO1 DNA binding activity in diabetic fracture calluses (P < 0.05). These results suggest that diabetes-enhanced TNF-α increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF-α dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage. Diabetes interferes with fracture repair; therefore, we investigated mechanisms of impaired fracture healing in a model of multiple low-dose streptozotocin-induced diabetes. Microarray and gene set enrichment analysis revealed an up-regulation of gene sets related to inflammation, including tumor necrosis factor (TNF) signaling in the diabetic group, when cartilage is being replaced by bone on day 16, but not on days 12 or 22. This change coincided with elevated osteoclast numbers and accelerated removal of cartilage in the diabetic group (P < 0.05), which was reflected by smaller callus size. When diabetic mice were treated with the TNF-specific inhibitor, pegsunercept, the number of osteoclasts, cartilage loss, and number of TNF-α and receptor activator for nuclear factor kB ligand positive chondrocytes were significantly reduced (P < 0.05). The transcription factor forkhead box 01 (FOXO1) was tested for mediating TNF stimulation of osteoclastogenic and inflammatory factors in bone morphogenetic protein 2 pretreated ATDC5 and C3H10T1/2 chondrogenic cells. FOXO1 knockdown by small-interfering RNA significantly reduced TNF-α, receptor activator for nuclear factor kB ligand, macrophage colony-stimulating factor, interleukin-1α, and interleukin-6 mRNA compared with scrambled small-interfering RNA. An association between FOXO1 and the TNF-α promoter was demonstrated by chromatin immunoprecipitation assay. Moreover, diabetes increased FOXO1 nuclear translocation in chondrocytes in vivo and increased FOXO1 DNA binding activity in diabetic fracture calluses (P < 0.05). These results suggest that diabetes-enhanced TNF-α increases the expression of resorptive factors in chondrocytes through a process that involves activation of FOXO1 and that TNF-α dysregulation leads to enhanced osteoclast formation and accelerated loss of cartilage. 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In addition a potential downstream mechanism, FOXO1-mediated gene expression was further investigated in vitro. The results indicate that TNF-α plays a significant role in diabetes-enhanced osteoclastogenesis and accelerated cartilage degradation in vivo. Moreover, TNF–α in vitro stimulated mRNA levels of factors in chondrocytic cells that were pro-osteoclastogenic or pro-inflammatory, which was mediated in part by FOXO1. These studies provide new insight into diabetes impaired fracture healing and support a previously unrecognized role for TNF-α and FOXO1 in mediating this untoward response. 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.33Kayal RA Alblowi J McKenzie E Krothapalli N Silkman L Gerstenfeld L Einhorn TA Graves DT Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment.Bone. 2009; 44: 357-363Crossref PubMed Scopus (111) Google Scholar 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 (Supplemental Table 1, see http://ajp.amjpathol.org). Treatment of a group of diabetic animals with the TNF-α inhibitor pegsunercept by intraperitoneal injection (4 mg/kg) every 3 days was performed starting on day10 after fracture until the time for euthanasia. Treatment with pegsunercept resulted in no significant differences in glycated hemoglobin level (P > 0.05) (Supplemental Table 1, A and B, see http://ajp.amjpathol.org). A simple transverse closed fracture of the tibia or femur was performed on male mice that were diabetic for at least 3 weeks beforehand as previously described.16Kayal RA Tsatsas D Bauer MA Allen B Al-Sebaei MO Kakar S Leone CW Morgan EF Gerstenfeld LC Einhorn TA Graves DT Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity.J Bone Miner Res. 2007; 22: 560-568Crossref PubMed Scopus (197) Google Scholar, 34Gerstenfeld LC Wronski TJ Hollinger JO Einhorn TA Application of histomorphometric methods to the study of bone repair.J Bone Miner Res. 2005; 20: 1715-1722Crossref PubMed Scopus (125) Google Scholar, 35Gerstenfeld LC Alkhiary YM Krall EA Nicholls FH Stapleton SN Fitch JL Bauer M Kayal R Graves DT Jepsen KJ Einhorn TA Three-dimensional reconstruction of fracture callus morphogenesis.J Histochem Cytochem. 2006; 54: 1215-1228Crossref PubMed Scopus (138) Google Scholar Briefly, a 27 gauge spinal needle was inserted for fixation into the marrow cavity of the long bone. After closure of the incision, a fracture was created by blunt trauma. Fractures that were not consistent with standardized placement criteria (mid-diaphyseal) or grossly comminuted were excluded. For tibial fractures, days 12, 16, and 22 were examined and for femoral fractures, days 10 and 16 were studied. Day 12 in the tibial and day 10 in the femoral fracture are roughly equivalent time points in each when collagen is the predominant tissue in the fracture callus.16Kayal RA Tsatsas D Bauer MA Allen B Al-Sebaei MO Kakar S Leone CW Morgan EF Gerstenfeld LC Einhorn TA Graves DT Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity.J Bone Miner Res. 2007; 22: 560-568Crossref PubMed Scopus (197) Google Scholar, 33Kayal RA Alblowi J McKenzie E Krothapalli N Silkman L Gerstenfeld L Einhorn TA Graves DT Diabetes causes the accelerated loss of cartilage during fracture repair which is reversed by insulin treatment.Bone. 2009; 44: 357-363Crossref PubMed Scopus (111) Google Scholar Animals were euthanized at day 12, 16, and 22 time points for the mRNA profiling and day 10 and 16 time points for all other analysis. Tibias or femurs were harvested and the soft tissue was carefully removed. After euthanasia, tibial fracture calluses from 12, 16, and 22 day groups were carefully dissected by removing all muscle and non-callus tissue and were immediately frozen in liquid nitrogen. Total RNA was extracted with Trizol (Life Technologies, Rockville, MD) from pulverized frozen tissue and further purified by an RNeasy MinElute cleanup kit (Qiagen, Valencia, CA). The concentration and integrity of the extracted RNA was verified by 260 nm/280 nm spectrophotometry and denaturing agarose gel electrophoresis with ethidium bromide staining. mRNA profiling was performed by using a PGA Mouse v1.1 array. Microarray probe preparation, hybridization, and reading of fluorescent intensity were performed by the Massachusetts General Hospital Microarray Core Facility (Cambridge, MA). All slides were co-printed with an internal “alien” sequence that has no sequence homologues in the mouse genome. Slide printing, array labeling, hybridization, and slide reading were performed at the Massachusetts General Hospital Genomics Core Facility as previously described.36Wang K Vishwanath P Eichler GS Al-Sebaei MO Edgar CM Einhorn TA Smith TF Gerstenfeld LC Analysis of fracture healing by large-scale transcriptional profile identified temporal relationships between metalloproteinase and ADAMTS mRNA expression.Matrix Biol. 2006; 25: 271-281Crossref PubMed Scopus (46) Google Scholar All slides were quality control tested and contained appropriate positive and negative control sequences for data analysis. The alien gene in these studies serves as both a genome-extrinsic sequence and serves as a universal in-spot reference. In the experiments reported here all microarrays were printed with an alien 70 mer probe that was co-printed with each gene specific probe such that the alien was at a final concentration of 10% of the murine gene oligonucleotide. The data were normalized by scaling all individual intensities such that the mean total intensities were the same for all comparative samples within a single array and across replicates. Using the intensity reading, background was calculated locally per spot and subtracted from the intensity measurement of each hybridized spot. The ratio of normal over alien was first calculated. Using this ratio, all outliers for each gene were discarded. The standard log10 of diabetic versus normal and diabetic versus alien for each spot was calculated and the distribution of the log ratios was obtained from combined replicates per time point. The data are combined using the geometric mean of four replicate ratios. Microarray data were analyzed by using gene set enrichment analysis (GSEA) described in Subramanian et al.37Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (28593) Google Scholar Genes were first ranked based on the correlation between their expression and the class distinction. An enrichment score was then calculated, which reflects the degree to which a gene is over-represented at the extremes (top or bottom) of the entire ranked list. Statistical significance (P value) and false discovery rate (FDR) of that enrichment score was then calculated. This statistical analysis determines whether a gene set is significantly up-regulated or down-regulated in the experimental sample compared with a control. Gene sets with a FDR less than 25% and a nominal P value less than 0.05 were considered significant.37Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (28593) Google Scholar Genes that contributed to a significant difference in pathways related to inflammation were identified by the GSEA software and further screened for significance by Student’s t-test (P ≤ 0.05) and for a threshold change of 1.5 increase or decrease in the diabetic versus normoglycemic microarray values. For selected genes mRNA levels obtained with microarrays were validated by real-time PCR by using primers and probe sets purchased from Applied Biosystems (Foster City, CA). For a given experiment RNA from six to nine specimens were combined and TaqMan reagents were used for first-strand cDNA synthesis and amplification. Results were normalized with an 18S ribosomal primer and probe set. Each experiment was performed three times and the results from the three separate experiments were combined to derive mean values. The level of a given gene was set relative to the value obtained for the normoglycemic control animals on day 12. Femoral samples from day 10 and 16 groups were fixed for 72 hours in cold 4% paraformaldehyde and decalcified for 2 weeks by incubation in cold Immunocal (Decal Corporation, Congers, NY). The internal fixation pin was removed after decalcification and femurs were embedded in paraffin, sectioned at 5 μm and histomorphometric measurements were performed as described in Gerstenfeld et al.34Gerstenfeld LC Wronski TJ Hollinger JO Einhorn TA Application of histomorphometric methods to the study of bone repair.J Bone Miner Res. 2005; 20: 1715-1722Crossref PubMed Scopus (125) Google Scholar Sections stained with safranin-O/fast green and H&E were used to measure cartilage area and callus size, respectively, by computer assisted image analysis by using Image ProPlus software (Media Cybernetics, Silver Spring, MD) as previously described.38He H Liu R Desta T Leone C Gerstenfeld L Graves D Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss.Endocrinology. 2004; 145: 447-452Crossref PubMed Scopus (159) Google Scholar The number of multinucleated, tartrate-resistant acid phosphatase positive cells lining bone or cartilage was counted to assess osteoclasts.38He H Liu R Desta T Leone C Gerstenfeld L Graves D Diabetes causes decreased osteoclastogenesis, reduced bone formation, and enhanced apoptosis of osteoblastic cells in bacteria stimulated bone loss.Endocrinology. 2004; 145: 447-452Crossref PubMed Scopus (159) Google Scholar There were six to eight specimens per group and measurements were made blindly by one examiner, with the results confirmed by a second examiner. Sections from formalin-fixed, paraffin-embedded samples were deparaffinized and antigen retrieval was performed by immersion in 10 mmol/L sodium citrate (pH 6.0) for 5 minutes at 95°C. Specimens were quenched with 3% hydrogen peroxide and blocked with avidin-biotin (Vector Laboratories, Burlingame, CA) and nonimmune serum matching the secondary antibody. Sections were incubated with primary antibodies at 4°C degree overnight, followed by rinsing and incubation with biotin labeled secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Goat polyclonal anti-murine TNF-α and anti-RANKL antibodies as well as matched control IgG were purchased from Santa Cruz Biotechnology. An additional negative control was performed consisting of primary antibody incubated with excess blocking peptide. Immune complexes were localized with an avidin-biotin horseradish peroxidase kit from Vector Laboratories, visualized with the chromogen 3, 3′-diaminobenzidine (Zymed Laboratories, Inc., South San Francisco, CA) and counterstained with hematoxylin. Analysis of the cartilage was performed by comparison with safranin-O/fast green stained serial sections and the number of positive proliferative or hypertrophic zone chondrocytes were counted at ×1000 magnification and expressed as the percentage that were immunopositive. The expression of genes of interest in osteoblasts/mesenchymal cells in newly forming bone, fibroblasts in the callus capsule, and hypertrophic and proliferative chondrocytes was also assessed by using a scale from 0 to 5. This analysis was performed at ×1000 magnification in a minimum of 20 fields per callus by using the following scale: 0 = no immunopositive cells; 1 = <10% of the cells of interest were lightly immunostained; 2 = <10% moderate to darkly immunostained; 3 = 10 to 25% moderate to darkly immunostained; 4 = 25 to 40% moderate to darkly immunostained; and 5 = greater than 40% of the cells of interest were darkly immunostained. For each data point n = 6 per group. Analysis was done blindly by one examiner with the results confirmed by a second blinded examiner. Sections were prepared as described above and incubated overnight with anti-FOXO1 antibody (Santa Cruz Biotechnology) or matched negative control antibody. Primary antibody to FOXO1 was detected by a Cy5 tagged secondary antibody. Propidium iodide nuclear stain was included in the mounting media. FOXO1 nuclear translocation was detected by confocal laser scanning microscopy at a focal plane that bisected the nuclei (Axiovert-100M, Carl Zeiss, Thornwood, NY). Cy5, propidium iodide, and phase contrast images (original magnification, ×400) of the same field were digitally captured. The entire cartilage area in nonoverlapping fields was analyzed and the percentage of chondrocytes with unambiguous nuclear translocation
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