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Cartilage-Specific Fibronectin Isoform
Department of Veterinary Sciences
Lameness is the primary variable limiting athletic careers of most performance horses. Medical advances in rheumatology will directly benefit the equine industry. Fibronectin is one of the most important molecules through which cells interact with their surrounding environment. We have discovered that 50-80% of the fibronectin in mammalian articular cartilage is a novel type which is not found in any other tissue of the body. Experiments will test the hypothesis that the unique structure and restricted dimerization of this fibronectin variant influences cartilage matrix organization and cell/matrix interactions that regulate chondrocyte function. The long-term goal of this project is to learn more about the function of fibronectin in normal cartilage and how the 10-20 fold increase in fibronectin that occurs in osteoarthritic lesions contributes to the pathogenesis of this disease. New knowledge about cartilage and osteoarthritis will lead to improved diagnostic and therapeutic strategies for joint disease.
2009 Project Description
Outputs included biomedical research experiments on articular cartilage, osteoarthritis, and the extracellular matrix protein fibronectin. Graduate and undergraduate students were instructed and mentored. Events for the reporting and distribution of results included seminars, lectures, workshops, and publications. Products included 27 publications, over 9300 submissions of cDNA sequences to the GenBank database, and the graduation of 9 students.
Mammalian cartilage contains an unique isoform of fibronectin that is not present in any other body tissues. It is designated (V+C)- fibronectin and results from an alternative RNA splicing pattern that deletes not only the variable (v) region, but also nucleotides that would normally encode protein segments III-15 and I-10. This internal in-frame deletion of 771 nucleotides significantly limits the ability of (V+C)- fibronectin to heterodimerize with other native v-region splice variants and results in a fibronectin structure within the extracellular matrix of cartilage that is not found in other tissues.
The hypothesis that was tested is that the unique primary structure and restricted dimerization of (V+C)- fibronectin influences cartilage matrix organization and cell/matrix interactions that regulate the differentiated phenotype of chondrocytes.
In objective 1, a comparison was made between the (V+C)- and V+C+ isoforms of fibronectin to investigate any differential effects on cell adhesion and cell migration. Clonal chinese hamster ovary (CHO) cell lines with stable transfection of specific integrin heterodimers were studied in parallel to compare binding to cartilage and plasma fibronectin. The results demonstrated that some cell surface receptors have different binding affinities for (V+C)- fibronectin compared to other isoforms. Peptide inhibitors of integrin binding and specific blocking monoclonal antibodies were used in an effort to identify which specific receptors are responsible for the differential binding affinities.
In objective 2, cell culture models were used to determine if contact with (V+C)- fibronectin affects chondrocyte gene expression and maintenance of the differentiated phenotype of chondrocytes. Microarray-based transcriptional profiling was used to identify genes that are differentially expressed by primary articular chondrocytes cultured for either 6 or 24 hours in serum free medium supplemented with dimeric fibronectin from either cartilage or plasma. Expression patterns in selected genes of high scientific interest by the microarray experiments were further evaluated by quantitative reverse transcription - polymerase chain reaction (RT/PCR) amplification.
Mienaltowski, M.J., Huang, L., Frisbie, D.D., McIlwraith, C.W., Stromberg, A.J., Bathke, A., and MacLeod, J.N. (2009) Transcriptional profiling differences for articular cartilage and repair tissue in equine joint surface lesions. BMC Medical Genomics 14(2):60.
Wade, C.M., Giulotto, E., Sigurdsson, S., Zoli, M., Gnerre, S., Imsland, F., Lear, T.L., Adelson, D.L., Bailey, E., Bellone, R.R., Blocker, H., Distl, O., Edgar, R.C., Garber, M., Leeb, T., Mauceli, E., MacLeod, J.N., Penedo, M.C., Raison, J.M., Sharpe, T., Vogel, J., Andersson, L., Antczak, D.F., Biagi, T., Binns, M.M., Chowdhary, B.P., Coleman, S.J., Della Valle, G., Fryc, S., Guerin, G., Hasegawa, T., Hill, E.W., Jurka, J., Kiialainen, A., Lindgren, G., Liu, J., Magnani, E., Mickelson, J.R., Murray, J., Nergadze, S.G., Onofrio, R., Pedroni, S., Piras, M.F., Raudsepp, T., Rocchi, M., Roed, K.H., Ryder, O.A., Searle, S., Skow, L., Swinburne, J.E., Syvanen, A.C., Tozaki, T., Valberg, S.J., Vaudin, M., White, J.R., Zody, M.C.; Broad Institute Genome Sequencing Platform; Broad Institute Whole Genome Assembly Team, Lander, E.S., and Lindblad-Toh, K. (2009) Genome sequence, comparative analysis, and population genetics of the domestic horse. Science 326:865-867.
Mienaltowski, M.J., Huang, L., Frisbie, D.D., McIlwraith, C.W., Stromberg, A.J., Bathke, A., and MacLeod, J.N. (2008) Transcriptional differences between articular chondrocytes and cells that populate repair tissue within full thickness articular lesions. Plant and Animal Genome XVI, W153.
Coleman, S.J., Zeng, Z., Mienaltowski, M., Liu, J., and MacLeod, J.N. (2009) Analysis of Equine Gene Structural Annotation by RNA Sequencing. Plant and Animal Genome XVII, W152.
Liu, J., Wang, K., Zeng, Z., Coleman, S.J., MacLeod, J.N., and Prins, J. (2009) Map RNA-seq Short Reads for Splice Junction Discovery, 17th Annual International Conference on Intelligent Systems for Molecular Biology (ISMB/ECCB).
MacLeod, J.N., Coleman, S.J., and Liu, J. (2009) Analyses of the equine mRNA transcriptome using RNA-seq data. Eigth International Equine Genome Workshop.
MacLeod, J.N., Zeng, Z., Coleman, S.J., and Liu, J. (2009) Analysis of Equine Gene Expression by RNA Sequencing. Plant and Animal Genome XVII, W153.
Tremblay, M., Huang, L., Zhu, W., Mienaltowski, M.J., Bathke, A., and MacLeod, J.N. (2009) Ontology of genes with stable and constitutive expression across eleven equine tissues. Plant and Animal Genome XVII, P539.
Coleman, S., Wang, K., Zeng, Z., Mienaltowski, M., Liu, J., MacLeod, J. (2009) Equine Gene Structure Annotation by RNA Sequencing. Journal of Equine Veterinary Science, 29(5), 319 320.