The long-term goal of the proposed research is to devise therapeutic modalities to enhance cartilage repair in degenerative cartilage disease. The applicants expect that manipulation of signaling initiated by transforming growth factor-beta (TGF-b) will allow them to selectively activate its potent capacity to stimulate cartilage matrix production and the chondrocyte differentiated state, and circumvent the mechanisms that normally restrict or modulate these important activities. This molecular approach could enhance the capacity to replace local damaged cartilage with living cartilage derived from in vitro cell culture, and manipulate gene expression and cellular metabolism in early clinical lesions to slow or prevent degeneration. The proposed research will provide necessary details concerning potential targets in TGF-b signaling in articular chondrocytes by extending the emerging model that TGF-b binding induces formation of heterotypic receptor complexes, activation of type I and II receptor intrinsic kinase activity and initiation of a kinase cascade. Newly developed and characterized antipeptide antibodies to the N- and C-terminal domains of the type II receptor will be used to evaluate the formation of endogenous binary and ternary heterotypic receptor complexes and determine their capacity for signaling selectively for changes in matrix synthesis, differentiated state, and proliferation. This approach is possible because of the unique property of the N-terminal antibody to block affinity labeling of the type I receptor. Receptor immunoprecipitation and 2-dimensional protein mapping will be used to characterize the phosphorylation state of the endogenous type II receptor and its associated proteins and study the requirements for initiation of the putative kinase cascade. The investigators will link these phosphorylation events with the early kinase cascade substrate that they have identified and characterize its cellular localization, biochemistry, affiliation with selective TGF-b-dependent signaling pathways, and potential for kinase activity. The acquired information will thus encompass three important target domains in TGF-b signaling: cell surface receptor complexes, receptor associated proteins/effectors, and post-receptor kinase signaling.Each will be evaluated for sensitivity to the TGF-b -enhancing effects of the protein kinase inhibitor, HA1004, and the microfilament modifier, dihydrocytochalasin B, to identify their sites of interaction with this signaling system and to provide preliminary models for its manipulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042894-04
Application #
2442827
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Project Start
1994-07-29
Project End
1998-09-30
Budget Start
1997-07-01
Budget End
1998-09-30
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Southern California
Department
Orthopedics
Type
Schools of Medicine
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089