The long-term aim of these studies is to define signal pathways that mediate the mitogenic actions of the neuropeptide, arginine vasopressin (AVP). AVP is mitogenic on cultured fibroblasts and autocrine activation of neuropeptide receptors, including AVP receptors, is proposed to participate in cellular transformation in human small cell lung cancer (SCLC). SCLC cells are noted for synthesizing multiple neuropeptides including AP and gastrin-releasing peptide (GRP) as well as expressing the specific receptors for these mitogenic peptides. Neuropeptide receptors signal through heterotrimeric G proteins which stimulate specific effector enzymes that couple to signal transduction pathways controlling cell growth. Previous studies have highlighted the regulation of Gq and phospholipase Cbeta (PLCbeta) in neuropeptide- stimulated cell growth where PLC beta leads to increased intracellular calcium and activation of protein kinase C (PKC), a proximal activator of the extracellular signal-regulated kinase (ERK) members of the mitogen-activated protein (MAP) kinase family. By contrast, the cJun- terminal kinase (JNK) members of the MAP kinase family are stimulated by neuropeptides in a PKC-independent manner. The dominant G proteins an effector enzymes that mediate neuropeptide-stimulated JNK activity are poorly defined. Also, regulation of the JNKs by oncogenic forms of Ras, Src and Ga proteins as well as by cytotoxic stresses that induce apoptosis hampers clear assignment of the role of JNKs in cell biology. The hypothesis that stimulation of mitogenesis and transformed growth by AVP and other neuropeptides requires activation of specific cJun N- terminal kinases through Gq and G12- regulation of the Src and Tec families of cytosolic tyrosine kinases will be tested. Both AVP- responsive Swiss 3T3 fibroblasts and SCLC cells will be employed to complete these specific aims: 1) determine the JNK isoform expression profile in Swiss 3T3 and SCLC cells. 2) express epitope-tagged JNKs to identify the specific isoforms stimulated by AVP, GTPase-deficient G- alphaq and Galpha12 and activated forms of cytosolic tyrosine kinases. 3) express inhibitory mutants of Src and Tec family protein tyrosine kinases to define their requirement for regulation of JNKs and stimulation of cell growth and transformation by neuropeptides. 4) express inhibitory forms of specific JNK isoforms and their cytosolic targets (cJun, ATF2) to define the requirement of the Jnk pathway in neuropeptide-stimulated cell growth and transformation. Completion of these specific aims will clarify the requirement and mechanism of activation of the JNKs by neuropeptides such as AVP in normal fibroblasts and in specific human cancers such as SCLC. The findings from this proposal may identify specific signaling components involved in the establishment and maintenance of cell transformation for future consideration as novel targets for pharmacologic inhibition.
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