Abstract
Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.
Highlights
Somatostatin is a neuropeptide that inhibits the secretion of many hormones, that acts as a neurotransmitter, and that negatively regulates cell proliferation [1]
In Chinese hamster ovary (CHO)-K1 cells, cell proliferation is stimulated through an neuronal NOS (nNOS)/Nitric oxide (NO)/cGMP-dependent pathway elicited by cholecystokinin following protein-tyrosine phosphatase SHP-2 activation of nNOS by dephosphorylation at tyrosyl residues [41]
Treatment of CHO/SST5 cells with 10 nM RC-160 strongly antagonized sodium nitroprusside (Ϫ68 Ϯ 1.5%), L-arginine (Ϫ60 Ϯ 4.5%), and nNOS (Ϫ95 Ϯ 2%)-induced cell proliferation. These results demonstrated that 1) cell proliferation of CHO/SST5 cells was induced by endogenous and exogenous NO donors and that 2) treatment of CHO/SST5 cells with RC-160 strongly inhibited nNOS/NO-induced cell proliferation
Summary
Somatostatin is a neuropeptide that inhibits the secretion of many hormones, that acts as a neurotransmitter, and that negatively regulates cell proliferation [1]. Three isoforms of nitric-oxide synthase (NOS) that generate NO from L-arginine have been characterized and named according to the cell type or conditions under which they were first identified Both endothelial NOS and neuronal NOS (nNOS) are constitutively expressed, whereas expression of inducible NOS requires transcriptional activation. In CHO-K1 cells, cell proliferation is stimulated through an nNOS/NO/cGMP-dependent pathway elicited by cholecystokinin following protein-tyrosine phosphatase SHP-2 activation of nNOS by dephosphorylation at tyrosyl residues [41]. Parallel work from the laboratory demonstrated that treatment of CHO-K1 cells stably expressing the SST5 receptor with the somatostatin analog RC-160 results in a marked reduction of cell proliferation induced by cGMP and cGMP-dependent protein kinase [11]. Molecular mechanisms involved in such regulation and subsequent inhibition of cell proliferation were investigated
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