REGULATION OF GLUCOSE TRANSPORT BY CTAP-III ISOFORMS

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Accumulating evidence suggests that many naturally occurring peptides activate connective tissue cells in vitro and may play important roles in promoting increased secretion of extracellular matrix and increased replication of connective tissue cells in rheumatic diseases. One such peptide, CTAP-III (connective tissue activating peptide-III), is a member of a family of homologous proteins (PF-4, NAP-1, MGSA, GRO, gamma IP-10, 9E3) thought to be important in inflammation, wound healing and neoplasia. We propose to study a vital, and one of the earliest events following addition of CTAP-III to cultured connective tissue cells - namely stimulation of glucose transport. Incubation with CTAP-III for 30 min increases glucose transport by 30-110% ("immediate" effect); a more substantial stimulation (400-800%) ("delayed effect") is evident after 17- 41 h in human fibroblast cell types that are relatively insensitive to insulin. Only the delayed effect appears to require mRNA synthesis, indicating that the effects involve different pathways. Since recent studies indicate that the active form of CTAP-III may be a product of proteolytic processing, designated CTAP-III (des1-15)/NAP-2, the effects of CTAP-III and the des 1-15 form on glucose transport in connective tissue cells will be compared. Both forms will be tested for their ability to stimulate glucose transport in neutrophils, a cell type stimulated to chemotaxis by CTAP-III (des1-15)/NAP-2. The delayed effect on transport is accompanied by increased numbers of GLUT 1 glucose transporters in murine 3T3-F442A fibroblasts. Specific polyclonal antibodies to the glucose transporter combined with immunoblotting and immunoprecipitation of [35S]methionyl-labeled transporters will be used to test whether CTAP-III increases the rate of synthesis or decreases the degradation rate of glucose transporters. Since preliminary data indicate CTAP-III increases GLUT 1 mRNA levels, the ability of CTAP-III to increase the rate of transcription and/or message stability will be tested. If CTAP-III increases the rate of transcription, CAT constructs will be used to start to determine the part of the GLUT 1 gene conferring CTAP-III sensitivity. Whether the immediate effects of CTAP-III isoforms on glucose transport are mediated via an increase in transporter number, increased intrinsic activity, or redistribution of glucose transporters will be tested as will the hypothesis that the effects of CTAP-III on glucose transport are mediated via PGE2 and subsequent production of cAMP. Whether CTAP-III uses a receptor specific for CTAP-III isoforms or ones shared by structural homologues such as NAP-1 and PF-4 will also be determined. In providing insight into the mechanism by which members of the CTAP-III functional and structural families regulate glucose transport, the molecular mechanisms by which locally released growth factors enable specific, localized relatively insulin-insensitive areas of the body to meet increased demands for glucose will be clarified. Understanding the signal transduction mechanisms used by growth factors other than insulin to regulate glucose transport will provide valuable insight into the earliest events involved in rheumatic disease, inflammation and wound healing, and possibly suggest tactics for therapeutic intervention.