Restoration of articular cartilage defects will require a temporal sequence of directed cell movement, chondrocyte proliferation and extracellular matrix synthesis. The interaction between two signal transduction pathways, namely, the C kinase and A kinase pathways, may govern the cessation of chondrocyte proliferation which in turn modulates the chondrogenic phenotype. We propose that cross-talk signalling between the C kinase and A kinase pathways, dampens the activity of cAMP-dependent protein kinase leading to reduced proteoglycan synthesis during mitogen- stimulated proliferation of articular chondrocytes in monolayer culture. Furthermore, we propose that since increased accumulation of cAMP, in general, results in increased levels of proteoglycan synthesis, a mechanisms exists to down regulate the expression of the A kinase catalytic unit to enable the chondrocyte to respond to additional stimulatory agonist signals. In order to address these hypotheses, the following objectives and specific aims of this project will be examined experimentally: 1) We will evaluate the requirements for successful transduction of intracellular signals via amplification of the C kinase and A kinase pathways in proliferating and non-proliferating chondrocytes; 2) A determination of the pattern of phosphorylated intracellular substrates via cross-talk signalling between C kinase and A kinase will be made using the mitogens fibroblast growth factor (bFGF) and insulin-like-growth factor -I (IGF-I); 3) We will identify by 2-D gel electrophoresis the characteristics of the 5 intracellular substrates known to be phosphorylated in chondrocytes via activation of A kinase and furthermore evaluate whether 3 low MW substrates (18-25 kD) are GTP-binding proteins and potentially products of an endogenous ras gene in chondrocytes; 4) we intend to assess the regulatory subunit/catalytic subunit ratio of A kinase after accumulation of cAMP by measuring the mRNA transcription of the C subunit 5) Finally, we will integrate the studies at the fundamental level of signal transduction pathways in chondrocytes with those designed to assess the pathophysiological development of osteoarthritis in the partially- meniscectomized rabbit (project #1) and in the development of degenerative disc disease in the sand rat (project #4).
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