The in vitro autocrine growth of human keratinocytes is principally attributable to the heparin-binding growth factors signaling through the epidermal growth factor receptor; amphiregulin and heparin-binding epidermal growth factor mediate most of this effect. Available evidence suggests that membrane heparan sulfate proteoglycans, such as splice variants of CD44V3, and cell density-dependent interactions with matrix beta1 integrin receptors regulate signaling. Moreover, heparan sulfate structure is modified during differentiation. Under the general hypothesis that the candidate regulatory mechanisms involving these molecules act to provide redundant control of autocrine cytokines, the specific hypotheses are: that endogenous CD44V3 isoforms, amphiregulin or heparin-binding epidermal growth factor, and epidermal growth factor receptor form a trimolecular signaling complex, wherein the CD44V3 heparan sulfate side chains act as multivalent templates to approximate the other reactants and to facilitate receptor oligomerization; that the complex is regulated by heterogeneity of heparan sulfate structure determined by the cell growth status; and that during the transition from migratory to stationary phenotype, relocalization of beta-1 integrins correlates with alterations in cytolocalization and activity of the growth factors, which act to down regulate autocrine growth. The mechanisms by which heparan sulfates contribute to signaling will be investigated by determining binding constants of the cytokines to receptors, under conditions with and without blockade of heparan sulfate synthesis and in cell mutants defective in proteoglycan O-glycanation. Regulation of autocrine growth by heterogeneity of heparan sulfate structure will be tested by analyzing the structural motifs for the heparan sulfate side chains of recombinant and native isoforms of CD44V3 that correlate with growth arrest and with affinity for heparin-binding cytokines. Lastly, the effects of modulating alph-3-beta-1 integrin expression and localization on transcription, cellular distribution, processing, and juxtacrine activity of the membrane anchored cytokines will be characterized. The observation that amphiregulin is over- expressed in hyperproliferative disorders of the epidermis, including psoriasis, suggests that heparin-binding cytokines contribute pathophysiologically to up-regulated keratinocyte growth in neoplastic and non-neoplastic skin diseases. These considerations support the general objective to characterize the mechanisms of their signaling and the manner by which CD44V3 isoforms and beta-1 integrins regulate the process.
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