Research Accomplishment Reports 2007

Cartilage-Specific Fibronectin Isoform

J.N. MacLeod
Department of Veterinary Sciences

 

Project Description

Mammalian cartilage contains a 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 being 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 is being 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 are being studied in parallel to compare binding to cartilage and plasma fibronectin. Initial results have indeed demonstrated different profiles, strongly suggesting that 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 are being used in an effort to identify which specific receptors are responsible for the differential binding affinities.

In objective 2, cell culture models are being 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 is being 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 are further evaluated by quantitative reverse transcription - polymerase chain reaction (RT/PCR) amplification.

Impact

Arthritis is a major health problem in humans and animals. Although fibronectin levels are markedly increased in cartilage lesions within osteoarthritic joints, neither the function of fibronectin in normal cartilage nor its role in osteoarthritis is well understood. Fibronectin has been shown to be an important molecule through which cells in other tissues interact with their surrounding environment, primarily by binding to cell surface integrin receptors. This project will determine whether the function of fibronectin in joint cartilage includes the direct regulation of gene expression within chondrocytes, the cells that are responsible for synthesizing and maintaining cartilage throughout life.

Publications

Perelygin, A., Lear, T., Astakhova, N., Coleman, S., MacLeod, J., Rios, J., Adelson, D., and Brinton, M. 2007. Characterization of equine innate immunity genes potentially involved in West Nile virus (WNV) susceptibility. Proc. Plant and Animal Genome XV Conference.( P601).

Coleman, S.J., Clinton, R., and MacLeod, J.N. 2007. Construction of a master gene list for a 9322 feature equine cDNA microarray. Plant and Animal Genome XV Conference (P587).

Mienaltowski, M.J., Huang, L., Stomberg, S.J., and MacLeod, J.N. 2007. Transcriptional profiling of equine articular chondrocytes to study postnatal maturation. Plant and Animal Genome XV Conference (P604).

Zhu, W., Huang, L., Saunders, C.P., Bathke, A., Stromberg, A.J. and MacLeod, J.N. 2007. Identification of genes with a cartilage-restricted pattern of expression. Plant and Animal Genome XV Conference (P605).

Huang, L., Zhu, W., Saunders, C., MacLeod, J., Zhou, M., Stromberg, A.J., and Bathke, A. 2007. A novel application of quantile regression for identification of cartilage biomarkers in equine microarray data. Proc. American Statistical Association.

Coleman, S.J., Gong, G., Gaile, D.P., Chowdhary, B.P., Bailey, E., Liu, L., and MacLeod, J.N. (2007). Evaluation of COMPASS as a Comparative Mapping Tool for ESTs Using Horse Radiation Hybrid Maps, Animal Genetics, 38: 294-302.