The Use Of Genetic Models To Define The Role Of Beta-catenin In Post-natal Growth
Regis J Okeefe, Professor And Associate Chair For Academ
Orthopaedicsuniversity Of Rochester
Grant 5R01AR053717-02 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SBSR
Abstract: ?-catenin has emerged as a critical regulator of endochondral bone formation. However, the ability to study the role of ?-catenin in growth and development is severely limited by the embryonic or early lethality that occurs in all cartilage-related gene deletions that result in loss or gain of ?-catenin function. This proposal defines the role of ?-catenin in post-natal growth and development and uses an innovative approach that permits us to temporally control tissue specific conditional gene deletion in mice. Preliminary data establish that delivery of tamoxifen to Col2a1-Cre+/-ERT2 transgenic mice results in tissue specific gene recombination. Aim 1 characterizes the temporal expression of Cre-recombinase in murine cartilage and assesses the role of ?-catenin gain of function on post-natal growth and development. Aim 1A consists of a focused set of experiments that complete a characterization of the pattern of gene recombination following delivery of tamoxifen and optimizes its administration using Col2a1-Cre+/-ERT2; ROSA26R +/- mice. Aim 1B examines the phenotype of ?-catenin+/fiox(exon3); Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in expression of a constitutively active ?-catenin in cartilage. Aim 2 characterizes the role of ?-catenin loss of function on post-natal growth and development and uses two complementary models. ICAT is an intracellular protein that competitively binds ?-catenin and inhibits signaling. Preliminary data show delayed chondrocyte maturation and runting in Col2a1-ICAT transgenic mice, which have inhibition of ?-catenin signaling throughout development and during post-natal growth. Aim 2A characterizes the phenotype of Col2a1-ICAT transgenic mice. Aim 2B characterizes the phenotype of ?-cateninfloxKO/ floxKO; Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in loss of ?-catenin expression in cartilage. Aim 3 uses an in vitro approach and defines mechanisms involved in the interdependence of ?-catenin and BMP signaling on VEGF gene expression during terminal chondrocyte maturation. Preliminary data show that induction of VEGF requires both BMP and ?-catenin signaling. Aim 3A examines regulation of BMP-2, 4, and 6 expressions in chondrocytes by ?-catenin. Aim 3B defines the cooperative induction of the VEGF gene by BMP-2 and ?-catenin. Aim 3C defines critical Smad-?-catenin interactions on the VEGF-A promoter. Altogether, these experiments will establish the concept that regulation of key maturation associated genes by ?-catenin requires the presence of BMP co-signals that act to enhance the rate of chondrocyte differentiation. The findings will establish ?-catenin and its cooperative effects with BMPs as an essential signal for chondrocyte hypertrophy, completion of endochondral bone formation, and regulation of VEGF expression. The proposed studies will provide critical new insights regarding cartilage diseases including chondrodysplasia, bone repair, and osteoarthritis
Project start date: 2007-09-01
Project end date: 2012-08-31
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Grants awarded to Regis J Okeefe
Prostaglandin Signaling In Wear Debris-induced Osteolysis
Regis J Okeefe, Professor And Associate Chair For Academ
Orthopaedicsuniversity Of Rochester
Grant 5R01AR046545-08 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: ZRG1
Abstract: This is a competitive renewal of a grant proposal that during the initial funding period defined the importance of RANKL in the osteolysis that occurs around total joint replacements. There are currently more than 400,000 arthroplasties performed annually in the U.S. to treat this condition. Since up to 20% of arthroplasties require revision surgery due to aseptic loosening, this complication of arthritic treatment constitutes a major source of the morbidity, and represents billions of dollars in health care costs. During the last funding period, our work defined the interfascial membrane synovial fibroblast as a major source of RANKL. Preliminary data show that RANKL expression is dependent upon the stimulation of COX-2 and PGE2 production by Ti particles. Moreover, our findings suggest that the EP4 receptor is involved in the induction of RANKL in synovial fibroblasts. Aim 1 defines the signaling mechanisms through which particulate debris stimulate COX-2 in synovial fibroblasts and initiates intracellular signals leading to the expression of RANKL. Our hypothesis is that the transcription factor NFicB initiates the synovial fibroblast response to particles and is necessary for COX-2 induction. Aim 2 assesses in vitro responses to particles in synovial fibroblast from mice lacking the various EP receptors and examines the ability of these cells to stimulate osteoclastogenesis in co-cultures with osteoclast precursors. We anticipate that the EP4 receptor will be required for RANKL induction and osteoclast formation by Ti treated synovial fibroblasts. Finally, Aim 3 uses an in vivo model of calvarial bone loss in mice lacking EP receptor signaling to definitively address signals involved in RANKL induction, osteoclast formation, gene expression, and osteolysis. The role of the synovial fibroblast will be confirmed by conditional deletion of the EP4 receptor in fibroblasts. Thus through a series of highly integrated in vitro and in vivo experiments, the competitive renewal advances the findings of the initial funding period. The use of transgenic models and state of the art methods will characterize the sequential events of NFicB activation, COX-2 and PGES1 expression, PGE2 secretion, EP4 receptor binding, and RANKL expression in SF as a critical event in osteolysis
Keywords: cytolysis, inflammation, osteoclast, pathologic bone resorption, prosthesis biological signal transduction, biomaterial interface interaction, cytokine, fibroblast, gene expression, macrophage, nuclear factor kappa beta, postoperative complication, prostaglandin endoperoxide synthase, receptor binding RNase protection assay, gene targeting, genetically modified animal, laboratory mouse, southern blotting, tissue /cell culture, transfection /expression vector, western blotting
Project start date: 1999-12-01
Project end date: 2010-06-30
The Use Of Genetic Models To Define The Role Of Beta-catenin In Post-natal Growth
Regis J Okeefe, Professor And Associate Chair For Academ
Orthopaedicsuniversity Of Rochester
Grant 5R01AR053717-02 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SBSR
Abstract: ?-catenin has emerged as a critical regulator of endochondral bone formation. However, the ability to study the role of ?-catenin in growth and development is severely limited by the embryonic or early lethality that occurs in all cartilage-related gene deletions that result in loss or gain of ?-catenin function. This proposal defines the role of ?-catenin in post-natal growth and development and uses an innovative approach that permits us to temporally control tissue specific conditional gene deletion in mice. Preliminary data establish that delivery of tamoxifen to Col2a1-Cre+/-ERT2 transgenic mice results in tissue specific gene recombination. Aim 1 characterizes the temporal expression of Cre-recombinase in murine cartilage and assesses the role of ?-catenin gain of function on post-natal growth and development. Aim 1A consists of a focused set of experiments that complete a characterization of the pattern of gene recombination following delivery of tamoxifen and optimizes its administration using Col2a1-Cre+/-ERT2; ROSA26R +/- mice. Aim 1B examines the phenotype of ?-catenin+/fiox(exon3); Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in expression of a constitutively active ?-catenin in cartilage. Aim 2 characterizes the role of ?-catenin loss of function on post-natal growth and development and uses two complementary models. ICAT is an intracellular protein that competitively binds ?-catenin and inhibits signaling. Preliminary data show delayed chondrocyte maturation and runting in Col2a1-ICAT transgenic mice, which have inhibition of ?-catenin signaling throughout development and during post-natal growth. Aim 2A characterizes the phenotype of Col2a1-ICAT transgenic mice. Aim 2B characterizes the phenotype of ?-cateninfloxKO/ floxKO; Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in loss of ?-catenin expression in cartilage. Aim 3 uses an in vitro approach and defines mechanisms involved in the interdependence of ?-catenin and BMP signaling on VEGF gene expression during terminal chondrocyte maturation. Preliminary data show that induction of VEGF requires both BMP and ?-catenin signaling. Aim 3A examines regulation of BMP-2, 4, and 6 expressions in chondrocytes by ?-catenin. Aim 3B defines the cooperative induction of the VEGF gene by BMP-2 and ?-catenin. Aim 3C defines critical Smad-?-catenin interactions on the VEGF-A promoter. Altogether, these experiments will establish the concept that regulation of key maturation associated genes by ?-catenin requires the presence of BMP co-signals that act to enhance the rate of chondrocyte differentiation. The findings will establish ?-catenin and its cooperative effects with BMPs as an essential signal for chondrocyte hypertrophy, completion of endochondral bone formation, and regulation of VEGF expression. The proposed studies will provide critical new insights regarding cartilage diseases including chondrodysplasia, bone repair, and osteoarthritis
Project start date: 2007-09-01
Project end date: 2012-08-31