CD-RAP is a small chondrocyte-characteristic gene that is co-expressed in cartilage with type II collagen and aggrecan. CD-RAP is co-expressed with type IIB collagen in chondrogenesis, making it an excellent model for chondrocyte-specific gene expression. This grant renewal will focus on continued analysis of CD-RAP gene expression and explore the trans-acting factors and co-regulators necessary for specific expression in chondrocytes and repressed expression in other cells. We have analyzed 12 kb of the CD-RAP gene locus, determined multiple regulatory domains and defined a 183-bp regulatory module that confers tissue specificity of CD-RAP gene expression. Within the 183 bp module, a negative regulatory element was discovered that represses gene expression in non-cartilaginous tissues. Our overall strategy is to analyze in detail the mechanism of transcriptional regulation in the CD-RAP gene, then to test these results in other chondrocyte genes, primarily type II procollagen.
The specific aims of this proposal are to: (1) Determine the mechanism of 183-bp-coordinated tissue specificity: activation in chondrocytes and repression in other tissues, and define the minimum """""""" chondrocyte regulatory module"""""""" (CRM). (2) Determine the role of additional negative/ positive regulatory elements (for example, the deltaEF-1 and USFs and the new domains A, B, C and D), in the control of gene transcription. (3) Screen and analyze transcriptional co-regulators (co-repressors and co-activators) that function with the CRM. (4) Extend this analysis of a single gene to the analysis of the chondrocyte phenotype by (a) indentification of novel cartilage genes regulated by the CRM and (2) Identification of novel motifs for chondrocyte-characteristic genes. The experimental approach will include in vivo studies in transgenic mice and in vitro studies using cell culture of primary chondrocytes and cell lines transfected with recombinant DNA constructs.
In Specific Aim 4, the experimental approach will be complimented by a computational analysis using the technique of phylogenetic mapping to determine conserved regulatory domains within a set of genes. These studies will help to elucidate the mechanism for directing gene transcription in chondrocytes and will provide the basic science data that will be necessary to develop strategies to control chondrogenesis during development, in tissue engineering and repair, and in diseases such as osteoarthritis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR045550-07
Application #
6855802
Study Section
Special Emphasis Panel (ZRG1-GMA-1 (01))
Program Officer
Tyree, Bernadette
Project Start
1998-03-01
Project End
2009-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
7
Fiscal Year
2005
Total Cost
$336,600
Indirect Cost
Name
Washington University
Department
Orthopedics
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Chinzei, N; Brophy, R H; Duan, X et al. (2018) Molecular influence of anterior cruciate ligament tear remnants on chondrocytes: a biologic connection between injury and osteoarthritis. Osteoarthritis Cartilage 26:588-599
Takebe, K; Rai, M F; Schmidt, E J et al. (2015) The chemokine receptor CCR5 plays a role in post-traumatic cartilage loss in mice, but does not affect synovium and bone. Osteoarthritis Cartilage 23:454-61
Patra, Debabrata; DeLassus, Elizabeth; Liang, Guosheng et al. (2014) Cartilage-specific ablation of site-1 protease in mice results in the endoplasmic reticulum entrapment of type IIb procollagen and down-regulation of cholesterol and lipid homeostasis. PLoS One 9:e105674
Rai, Muhammad Farooq; Patra, Debabrata; Sandell, Linda J et al. (2014) Relationship of gene expression in the injured human meniscus to body mass index: a biologic connection between obesity and osteoarthritis. Arthritis Rheumatol 66:2152-64
Patra, Debabrata; DeLassus, Elizabeth; McAlinden, Audrey et al. (2014) Characterization of a murine type IIB procollagen-specific antibody. Matrix Biol 34:154-60
Wu, P; Holguin, N; Silva, M J et al. (2014) Early response of mouse joint tissue to noninvasive knee injury suggests treatment targets. Arthritis Rheumatol 66:1256-65
Wu, Peihui; DeLassus, Elizabeth; Patra, Debabrata et al. (2013) Effects of serum and compressive loading on the cartilage matrix synthesis and spatiotemporal deposition around chondrocytes in 3D culture. Tissue Eng Part A 19:1199-208
Rai, Muhammad Farooq; Patra, Debabrata; Sandell, Linda J et al. (2013) Transcriptome analysis of injured human meniscus reveals a distinct phenotype of meniscus degeneration with aging. Arthritis Rheum 65:2090-101
Patra, Debabrata; Sandell, Linda J (2012) Antiangiogenic and anticancer molecules in cartilage. Expert Rev Mol Med 14:e10
Hashimoto, S; Rai, M F; Janiszak, K L et al. (2012) Cartilage and bone changes during development of post-traumatic osteoarthritis in selected LGXSM recombinant inbred mice. Osteoarthritis Cartilage 20:562-71

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