Neuronal nitric oxide synthase: a substrate for SHP-1 involved in sst2 somatostatin receptor growth inhibitory signaling.

Somatostatin receptor sst2 is an inhibitory G protein-coupled receptor, which inhibits normal and tumor cell growth by a mechanism involving the tyrosine phosphatase SHP-1. We reported previously that SHP-1 associates transiently with and is activated by sst2 and is a critical component for sst2 growth inhibitory signaling. Here, we demonstrate that in Chinese hamster ovary cells expressing sst2, SHP-1 is associated at the basal level with the neuronal nitric oxide synthase (nNOS). Following sst2 activation by the somatostatin analog RC-160, SHP-1 rapidly recruits nNOS tyrosine dephosphorylates and activates it. The resulting NO activates guanylate cyclase and inhibits cell proliferation. Coexpression of a catalytically inactive SHP-1 mutant with sst2 blocks RC-160-induced nNOS dephosphorylation and activation, as well as guanylate cyclase activation. In mouse pancreatic acini, RC-160 treatment reduces nNOS tyrosine phosphorylation accompanied by an increase of its activity. By opposition, in acini from viable motheaten (mev/mev) mice, which express a markedly inactive SHP-1, RC-160 has no effect on nNOS activity. Finally, expression of a dominant-negative form of nNOS prevents both RC-160-induced p27 up-regulation and cell proliferation inhibition. We therefore identified nNOS as a novel SHP-1 substrate critical for sst2-induced cell-growth arrest.

Signal transduction of somatostatin receptors negatively controlling cell proliferation.

Somatostatin acts as an inhibitory peptide of various secretory and proliferative responses. Its effects are mediated by a family of G-protein-coupled receptors (sst1-5) that can couple to diverse signal transduction pathways such as inhibition of adenylate cyclase and guanylate cyclase, modulation of ionic conductance channels, and protein dephosphorylation. The five receptors bind the natural peptide with high affinity but only sst2, sst5 and sst3 bind the short synthetic analogues. Somatostatin negatively regulates the growth of various normal and tumour cells. This effect is mediated indirectly through inhibition of secretion of growth-promoting factors, angiogenesis and modulation of the immune system. Somatostatin can also act directly through sst receptors present on target cells. The five receptors are expressed in various normal and tumour cells, the expression of each receptor being receptor subtype and cell type specific. According to the receptor subtypes, distinct signal transduction pathways are involved in the antiproliferative action of somatostatin. Sst1, 4 and 5 modulate the MAP kinase pathway and induce G1 cell cycle arrest. Sst3 and sst2 promote apoptosis by p53-dependent and -independent mechanisms, respectively.

Somatostatin receptors.

Somatostatin is a neuropeptide produced by neuroendocrine, inflammatory and immune cells in response to different stimuli. Somatostatin inhibits various cellular functions including secretions, motility and proliferation. Its action is mediated by five specific somatostatin receptors (sst1-sst5) which belong to the G protein-coupled receptor family. The five receptors bind the natural peptide with high affinity but only sst2, sst5 and sst3 bind the short synthetic analogues used to treat patients with neuroendocrine tumors. The five receptors are expressed in various normal and tumor cells, the expression of each receptor being receptor subtype and cell-type specific. In neuroendocrine tumors, sst2 is highly expressed whereas in advanced pancreatic adenocarcinoma as well as high-grade colorectal carcinomas, its expression is lost. Each receptor subtype is coupled to different signal transduction pathways through G protein-dependent and -independent mechanisms. The synthesis of selective agonists for each receptor and the recent development of genetic animal models with selective deletion of receptor subtype provide tools for establishing some of the biological roles of the receptors. sst1, 2 and 5 mediate inhibition of GH secretion whereas sst2 and sst5 mediate inhibition of glucagon secretion and insulin secretion, respectively.

Overexpression of the FGF-2 24-kDa isoform up-regulates IL-6 transcription in NIH-3T3 cells.

We have isolated NIH-3T3 cell lines overexpressing the nuclear 24-kDa isoform of fibroblast growth factor (FGF)-2 and characterized its regulatory effect on the expression of interleukin-6 (IL-6) in these cells. The clone pRF5 expressing the highest level was able to grow in 1% serum medium to a high saturation density and acquired a radioresistance advantage. In pRF5 and another clone pRF1, IL-6 RNA levels were markedly increased. Studies with IL-6 promoter constructs revealed that IL-6 gene up-regulation occurred at the transcriptional level and did not involve the AP-1 binding site. Exogenously added 18-kDa isoform of FGF-2 (100 ng/ml) produced down-regulation of IL-6 involving an AP-1 binding site, thus suggesting a receptor-independent pathway for the intracellular 24-kDa isoform.