Little is known about the signaling pathways that limit airway smooth muscle cell proliferation. In bovine tracheal myocytes, PDGF treatment and Rac1, each of which induce transcription from the cyclin D1 promoter, activate the stress-activated MAP kinases P38alpha and Jun amino-terminal kinase (JNK)-1, consistent with the notion that these MAP kinases play a role in growth regulation. Our preliminary studies show that selective activation of p38 pathway decreases PDGF and Rac1-induced cyclin D1 expression, suggesting that p38 functions a negative regulator of airway smooth muscle cell cycle progression. Further, activation of protein kinase C delta (PKCdelta) also decreases cyclin D1 expression, perhaps via activation of the p38 pathway. Finally, our pilot studies suggest that p38 attenuates transcription from the cyclin D1 promoter by reducing transactivation of the cAMP response element-binding protein (CREB)/ATF-2 response element. These data suggest a model in which growth factor stimulation of airway smooth muscle cells results in the simultaneous activation of both mitogenic and anti-mitogenic pathways, perhaps as a mechanism to prevent excessive cell proliferation. To determine the potential roles of p38 and PKCdelta in the regulation of transcription from the cyclin D1 promoter, we propose the following Specific Aims: 1. Determine the roles of the p38 and JNK MAP kinases in the regulation of cyclin D1 expression in airway smooth muscle cells. We will determine the effects of inhibition and activation of p38 and JNK on transcription from the cyclin D1 promoter, cyclin D1 protein abundance, and cell cycle progression. We will also determine the effects of inhibition and activation of p38 on ERK activation. 2. Determine the role of PKCdelta in the regulation of cyclin D1 expression in airway smooth muscle cells. We will assess PKCdelta translocation and activation following PDGF treatment of bovine tracheal myocytes. We will also determine the effects of inhibition or activation of PKCdelta on p38alpha activity and cyclin D1 expression, and whether p38 is required for PKCdelta-mediated suppression of cyclin D1 promoter activity. To determine the site where PKCdelta enters the p38 pathway, we will examine the requirement of PKCdelta for p38alpha activation by upstream signaling intermediates. 3. Determine the mechanism by which p38 MAP kinase attenuates transcription from the cyclin D1 promoter in airway smooth muscle cells. We will examine the effects of inhibitors and activators of p38 on CREB-1 and ATF-2 phosphorylation, DNA binding and CREB/ATF-2 transactivation. DNA binding proteins will be determined by supershift studies. Finally, we will assess the effects of dominant-negative inhibitors of CREB-1 and ATF-2 on transcription from the cyclin D1 promoter. Insight aired from these studies may shed light on a novel anti- mitogenic signaling pathway that may operate in asthma and other disease states in which abnormal cell proliferation is a feature, and lead to therapeutic interventions.
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