Embryonic cartilage formation or chondrogenesis is an early process of mesenchymal conversion leading to the formation of skeletal structures. Precursor cells are recruited into a condensing core that, in turn, differentiates into chondrocytes. This differentiation process is accompanied by the elaboration of characteristic cartilage marker proteins such as collagen II and the chondroitin sulfate proteoglycan, aggrecan. Subsequently, chondrocytes undergo hypertrophic growth, a phase in which they express other marker proteins, including collagen type X and alkaline phosphatase. Molecular controls over chondrogenesis are manifest at many levels including the expression of key growth factors/cytokines and transcription factors. In addition, several lines of evidence indicate that heparan sulfate-bearing molecules of the extracellular matrix appear to promote chondrogenesis. Recently it has been reported that the expression of the heparan sulfate proteoglycan, perlecan, in both embryonic and adult cartilage. Moreover, targeted disruption of the perlecan gene results in abnormal cartilage development in mice. Our lab has found that perlecan rapidly and efficiently induces chondrogenesis in multipotential mouse embryonic fibroblasts and maintains the chondrogenic phenotype of adult human chondrocytes in vitro. This activity appears to be dependent upon both the heparan sulfate and protein constituents of perlecan. Taken together, these studies suggest that perlecan plays an important role in the cascade of events required for cartilage formation and orderly differentiation. We suggest that perlecan provides an organizational matrix that helps coordinate chondrogenesis. The proposed studies will explore the molecular basis of the action of perlecan in the promotion of chondrogenesis. Possibilities include growth factor concentration and presentation, modulation of cell adhesion and alteration of signal transduction cascades and gene expression. Implicit in all of these non-exclusionary possibilities is the interaction of perlecan, alone, or in a complex with one or more cell surface receptors. It is also unclear if perlecan can cooperate with growth factors to drive precursor cells through the complete program of chondrogenesis in vitro and in vivo. We will use a variety of cell biological, biochemical and molecular biological techniques to define the structure-function relationship between perlecan domains and chondrogenic activity. As model system, we will use multipotential mouse chondrogenic precursor cells and maintenance of the chondrogenic phenotype in human normal and exostoses-derived chondrocytes. These studies provide novel opportunities to use perlecan or perlecan fragments as inducers of chondrogenesis for design of therapeutic strategies to replace damaged or surgically-removed cartilage.
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