The goal of this proposal is to define the cellular and molecular signaling processes that regulate contractile agonist-induced proliferation of human airway smooth muscle (ASM) cells. This question is critical to an understanding of the pathogenesis of asthma, where, in addition to increased airway hyperreactivity, increased smooth muscle mass is a well documented pathologic finding. Data, recently published by the P.I., has shown that the chronic stimulation of ASM by some, but not all, contractile agonists induces myocyte proliferation even though these agonists are equally potent stimulators of intracellular calcium and phosphoinositide turnover. Since contractile agents signal via G protein-coupled receptors that do not have intrinsic tyrosine kinase activity, the mechanisms of agonist-induced proliferation must be substantially different from those of conventional peptide growth factors. Preliminary data has been generated to show that inhibition of phosphotidylinositol 3-kinase (PtdIns 3-kinase) abolishes both G protein- dependent and growth factor-dependent ASM proliferation. PtdIns 3-kinase has recently been implicated in the Ras/Raf/mitogen-activated protein (MAP) kinase cascade. Thus, PtdIns 3-kinase may play a central role in integrating mitogenic signals from both receptors coupled to G proteins and those coupled to intrinsic tyrosine kinases. Based on this data, and on the recent demonstration of the importance of p21ras in regulating growth of non-transformed fibroblasts, the central hypothesis of this proposal is that agonist-induced ASM cell growth is activated by p21ras and mediated by stimulation of PtdIns 3-kinase and subsequently MAP kinase. To test this hypothesis, in Aim 1, Ras activation will be inhibited by microinjecting antagonists or neutralizing antibodies, and the effects on DNA synthesis will be determined in cells stimulated with agonist. If agonist-induced cell proliferation is dependent on activation of p21ras, then inhibition of p21ras should abolish this response.
In Aim 2, the role of PtdIns 3-kinase regulating Ras activation will be examined: 1) by determining the magnitude and time course of PtdIns 3-kinase activation in response to agonists, in comparison with those that stimulate cell proliferation; 2) by studying the role of PtdIns 3-kinase in mediating ASM cell growth using microinjection of neutralizing antibodies or inhibitory synthetic peptides and measuring agonist-induced DNA synthesis; 3) by directly stimulating PtdIns 3-kinase using microinjection of activating synthetic peptides or of 3-phosphoinositides to determine whether stimulation of PtdIns 3-kinase is sufficient to induce ASM cell growth.
In Aim 3, the role of MAP kinases, which are important downstream effector proteins of p21ras, in stimulating myocyte growth will be examined: 1) by studying the magnitude and time course of MAP kinase activation by agonists in comparison with those that stimulate cell growth; and 2) by measuring agonist-induced MAP kinase activity in cells treated with PtdIns 3-kinase inhibitors. These results will provide information regarding the critical cellular and molecular mechanisms by which agonists induce airway smooth muscle cell growth. By understanding the mechanisms of ASM cell growth, new issues in the pathogenesis of asthma will be addressed and therapeutic measures to prevent these alterations can be developed.
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