The fundamental purpose of these studies is to define the mechanisms by which insulin-like growth factor I (IGF-I) and its binding protein function together to stimulate human fibroblast replication. The human fibroblast is the principle cell type involved in wound healing and the response to injury in many disease states. In addition it has been used extensively to study cellular aging. Understanding the mechanisms by which growth factors control fibroblast replication is fundamental to understanding the repair process and how this may be altered in aged humans. The form of the IGF binding protein that is secreted by fibroblasts will be purified to homogeneity and its amino acid sequence determined. The physicochemical properties of the protein will be analyzed in detail to determine if post- translational modification(s) occur, and if these modifications result in changes in its biologic activity. Fibroblasts from donors of several ages will be used to determine if donor age influences the replicative response to this protein. Monoclonal antibodies to the type I IGF receptor will be used to determine if IGF-I must bind this receptor for the IGF-I-binding protein complex to stimulate cell growth or if this complex can function independently. To determine the mechanisms by which the IGF-I- binding protein complex stimulates growth, studies will be initiated to determine if the complex is internalized and its intracellular location. The effect of binding protein-IGF-I complex on post-receptor events that are linked to growth stimulation such as, autophosphorylation and c-myc and c-fos induction will be determined. To determine the variables that control synthesis of the binding protein the factors that control the expression of its mRNA in fibroblasts will be determined.
This aim will be accomplished by cloning the fibroblast binding protein gene, determining its cDNA sequence and isolating an appropriate restriction fragments for labelling. If binding protein mRNA expression is shown to be regulated by growth factors the regulation of IGF-I-B mRNA by the same factors will be analyzed. An inhibitory form of the binding protein will be purified and its structure compared to the stimulatory form to determine if they are different. Its effects on fibroblast replication particularly the fibroblast response to IGF-I and the stimulatory form of binding protein will be determined. The results of these studies should provide basic information that is necessary to construct valid hypotheses regarding the role of growth factors in the response to injury and cellular aging.
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