The objective of this proposal is to characterize the signaling pathways that mediate the growth-promoting effects of IGF-I and IGF-binding protein-5 (IGFBP-5), and the growth-inhibiting effects of TGF-beta1 and IGFBP-on human intestinal smooth muscle cells. The experimental approach utilizes normal human intestinal muscle cell during growth in culture, which recapitulates the rapid growth of muscle tissue in the developing or inflamed intestine. Previous studies have shown that the levels of IGF-I and IGFBP-5 produced by growing cells are high during the proliferative phase and decline with the approach of confluence, whereas the levels of TGF-beta1 an IGFBP-3 are highest after the cells attain confluence. The hypotheses that underlie the Specific Aims of the current proposal are that the growth factors and binding proteins activate stimulatory and inhibitory signaling pathways that regulate: (i) the expression of IGF-I receptors; (ii) the production of growth factors and binding proteins, and their ability to promote or inhibit growth; and (iii) the progress of the cell cycle from the G 1 to the S phase.
The Specific Aims are to: (1) characterize the regulation of IGF-I receptor expression by IGF-I, TGF-beta1 IGFBP-5, and IGFBP-3 during culture; (2) characterize the signaling pathways that mediate the stimulatory effects of IGF-I and IGFBP-5, and inhibitory effects ofTGF-B1 and IGFBP-3 on cell growth and the production of growth factors and binding proteins; and (3) identify the signals that regulate cell cycle progression from the G1 to S phase.
Each Specific Aim i s supported by substantial preliminary data. IGF-I and IGFBP-5 are linked by reciprocal pathways that regulate their production, growth-promoting effects, and ability to inhibit IGF-I receptor expression. The pathways include activation of p42/44 MAP kinase and PI 3-kinase by IGF-I receptor tyrosine kinase, an activation of p38 MAP kinase by IGFBP-5 receptor serine kinase. TGF-beta1 and IGFBP-3 activate TGF-beta1 receptor serine kinase leading to Smad2 phosphorylation and inhibition of IGF-I receptor phosphorylation. IGF-I stimulates cyclin Dl expression, cdk4 activity, and pRb phosphorylation, and TGF-beta1 stimulates the cdk inhibitor, p15INK4B. The studies provide an analysis of the cellular mechanisms that determine initiation and cessation of muscle growth.
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