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Exercise dependent gene regulation in cartilage

ABSTRACT: Objective. Exercise is crucial for maintaining cartilage integrity and providing prophylaxis against the onset of osteoarthritis (OA). Here we systematically examined the exercise driven transcriptional activation/repression of genes from healthy articular cartilage to dissect the metabolic pathways responsible for its potential benefits. Methods. Healthy Sprague-Dawley rats were either not exercised (control) or subjected to daily exercise (low intensity treadmill walking) for 2, 5, or 15 days. Subsequently, articular cartilage from distal femoral heads was harvested, RNA extracted and the transcriptome-wide regulation of gene expression analyzed by Affymetrix microarray chips. Kyoto Encyclopedia of Genes and Genome (KEGG) database was used to identify the metabolic pathways regulated by exercise. Results. Microarray analysis of articular cartilage from exercised rats revealed two fold or greater up or down regulation of 926 transcripts for up to 15 days, as compared to control rat cartilage. The KEGG pathway analysis revealed that exercise transcriptionally activates/suppresses genes that encode proteins involved in regulating 123 metabolic pathways. These pathways control the biosynthesis of molecules associated with extracellular matrix, intermediate metabolites, cell signaling, cell growth and differentiation, cytoskeletal rearrangements, and inflammation including those associated with OA. Conclusions. Our findings highlight that exercise is a robust transcriptional regulator of a wide array of genes in the healthy cartilage. The actions of exercise collectively regulate metabolic pathways that are critical for strengthening cartilage while attenuating detrimental effects of proteolytic enzymes and inflammatory pathways to provide prophylaxis against inflammatory joint diseases.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE74898 | GEO | 2015/11/10

SECONDARY ACCESSION(S): PRJNA301749

REPOSITORIES: GEO

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Project description:Objective. Exercise is crucial for maintaining cartilage integrity and providing prophylaxis against the onset of osteoarthritis (OA). Here we systematically examined the exercise driven transcriptional activation/repression of genes from healthy articular cartilage to dissect the metabolic pathways responsible for its potential benefits. Methods. Healthy Sprague-Dawley rats were either not exercised (control) or subjected to daily exercise (low intensity treadmill walking) for 2, 5, or 15 days. Subsequently, articular cartilage from distal femoral heads was harvested, RNA extracted and the transcriptome-wide regulation of gene expression analyzed by Affymetrix microarray chips. Kyoto Encyclopedia of Genes and Genome (KEGG) database was used to identify the metabolic pathways regulated by exercise. Results. Microarray analysis of articular cartilage from exercised rats revealed two fold or greater up or down regulation of 926 transcripts for up to 15 days, as compared to control rat cartilage. The KEGG pathway analysis revealed that exercise transcriptionally activates/suppresses genes that encode proteins involved in regulating 123 metabolic pathways. These pathways control the biosynthesis of molecules associated with extracellular matrix, intermediate metabolites, cell signaling, cell growth and differentiation, cytoskeletal rearrangements, and inflammation including those associated with OA. Conclusions. Our findings highlight that exercise is a robust transcriptional regulator of a wide array of genes in the healthy cartilage. The actions of exercise collectively regulate metabolic pathways that are critical for strengthening cartilage while attenuating detrimental effects of proteolytic enzymes and inflammatory pathways to provide prophylaxis against inflammatory joint diseases. Sprague Dawley rats (n=5 per group, 12-14 weeks old females, Harlan Labs, IN) were housed at 5 animals/cage in individually ventilated cages with sterile Aspen bedding, and standard environmental enrichment. The cages were maintained at 12-h light/12-h dark cycle at 21Â°C in a pathogen free unit. All rats had ad libitum access to water and food and allowed normal cage activity. Animals randomly assigned to each group were either not exercised (Controls/Group I) or exercised (treadmill walking, 12 m/min for 45 min daily) for 2 (Group II), 5 (Group III), or 15 (Group IV) days13. Articular cartilage on the condyles of distal femurs were carefully dissected on ice under a stereomicroscope (10â15 mg/femur), immediately placed on dry ice and pulverized (3x30 seconds at 2500 RPM) in a Mikrodismembrator S (Sartorius, Germany). RNA was extracted using Trizol reagent (Invitrogen, CA), and RNA quality verified by a Bioanalyzer 2100 (Agilent, CA)13. The Whole Transcript (WT) cDNA Synthesis and Amplification Kit and WT Terminal Labeling Kit (Affymetrix, CA) were used for cDNA synthesis and labeling from 300 ng RNA template as recommended by the manufacturers. Labeled samples from 3 different rats from each group were hybridized to Affymetrix GeneChip Rat Gene 1.0 ST Array and scanned with GeneChip Scanner 3000 7G System (Microarray Shared Resource Facility, OSU Comprehensive Cancer Center)13.

Project description:As the most common degenerative joint disease, osteoarthritis (OA) contributes significantly to pain and disability during aging. Several genes of interest involved in articular cartilage damage in OA have been identified. However, the direct causes of OA are poorly understood. Evaluating the public human RNA-seq dataset showed that Cbfβ, (subunit of a heterodimeric Cbfβ/Runx1,Runx2, or Runx3 complex) expression is decreased in the cartilage of patients with OA. Here, we found that the chondrocyte-specific deletion of Cbfβ in tamoxifen-induced Cbfβf/fCol2α1-CreERT mice caused a spontaneous OA phenotype, worn articular cartilage, increased inflammation, and osteophytes. RNA-sequencing analysis showed that Cbfβ deficiency in articular cartilage resulted in reduced cartilage regeneration, increased canonical Wnt signaling and inflammatory response, and decreased Hippo/YAP signaling and TGF-β signaling. Immunostaining and western blot validated these RNA-seq analysis results. ACLT surgery-induced OA decreased Cbfβ and Yap expression and increased active β-catenin expression in articular cartilage, while local AAV-mediated Cbfβ overexpression promoted Yap expression and diminished active β-catenin expression in OA lesions. Remarkably, AAV-mediated Cbfβ overexpression in knee joints of mice with OA showed the significant protective effect of Cbfβ on articular cartilage in the ACLT OA mouse model. Overall, this study, using loss-of-function and gain-of-function approaches, uncovered that low expression of Cbfβ may be the cause of OA. Moreover, Local admission of Cbfβ may rescue and protect OA through decreasing Wnt/β-catenin signaling, and increasing Hippo/Yap signaling and TGFβ/Smad2/3 signaling in OA articular cartilage, indicating that local Cbfβ overexpression could be an effective strategy for treatment of OA. Using unbiased genome-wide RNA-seq data from Cbfβf/f;Col2α1-Cre hip joint articular cartilage and Cbfβf/f;Aggrecan-cre knee joint articular cartilage and their controls, we examined Cbfβ-mediated transcriptional targets for articular cartilage regeneration in OA.

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Exercise dependent gene regulation in cartilage

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