Previous investigations have shown that synthesis of high molecular weight hyaluronic acid is not coordinately linked to synthesis of cartilage-type proteoglycan monomer in cultures of newly-differentiating chondrocytes. This data has raised the hypothesis that synthesis of high molecular weight HA is rate- limiting for cartilage extracellular matrix synthesis. As a corollary, it follows that the matrix requires this particular HA to become """"""""functional"""""""" and that its appearance reflects maturation of the matrix. Biosynthetic events leading to high molecular weight HA could involve switches in HA-type (ie. as collagen-type switches from type I to type II), alterations in HA-degrading (hyaluronidase) activity, or changes in de novo synthetic patterns. The resolution of these alternative mechanisms is a crucial step in a complete understanding of cartilage matrix development. The proposed experiments will investigate these alternative mechanism by: 1) isolating and characterizing HA synthesis during de novo chondrogenesis, 2) determining the ability of each type of isolated HA to bind proteoglycan monomer, and the kinetics of this association, 3) determining the pattern of appearance, isoform type, and HA-type specificity of hyaluronidase, and 4) analyzing the ability of pre-chondrocytes and chondrocytes to synthesize HA in a cell-free system. Newly synthesized HA will be isolated from pre- and postchondrogenic cultures of skeletal muscle on demineralized bone and characterized by density, hydrodynamic size, conformation in solution, and ability to promote aggregation with purified proteoglycan monomer. Hyaluronidase activity of these cultures will be determined against a well-characterized, commercial preparation of HA followed by an investigation of the activity against HA-types identified previously. Cell-free synthesis will determine the ability of de novo chondrocytes to synthesize HA- types in the absence of HA-degrading activity. Control tissues will include nonchondrogenic (skeletal muscle grown on collagen gels) and chondrogenic (Swarm rat chondrosarcoma) sources; these tissues will permit a comparative investigation of HA synthetic potential. Data derived from these experiments will provide a detailed description of HA synthesis during chondrogenesis and of the mechanism(s) whereby chondrocytes synthesize HA capable of aggregating with proteoglycan monomers.
| Smith, C I; Webster, E H; Nathanson, M A et al. (1990) Altered patterns of proteoglycan deposition during maturation of the fetal mouse lung. Cell Differ Dev 32:83-96 |
