Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA

The hormone bombesin(BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) actthrough specific GRP receptors (GRP-R) to affect multiple cellularfunctions in the gastrointestinal tract; the intracellular signalingpathways leading to these effects are not clearly defined. Previously,we demonstrated that the human gastric cancer SIIA possesses GRP-R andthat BBS stimulates activator protein-1 (AP-1) gene expression. Thepurpose of our present study was to determine the signaling pathwaysleading to AP-1 induction in SIIA cells. A rapid induction ofc-jun and jun-B gene expression was noted afterBBS treatment; this effect was blocked by specific GRP-R antagonists,indicating that BBS is acting through the GRP-R. The signaling pathwaysleading to increased AP-1 gene expression were delineated using phorbol12-myristate 13-acetate (PMA), which stimulates protein kinase C(PKC)-dependent pathways, by forskolin (FSK), which stimulates proteinkinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSKstimulated jun-B expression but produced only minimalincreases of c-jun mRNA and AP-1 binding activity.Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKCinhibitors) inhibited the induction of c-jun andjun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor)exhibited only minimal effects. Pretreatment with tyrphostin-25, aprotein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediatedinduction of c-jun and jun-B, but the effect wasnot as pronounced as with H-7. Collectively, our results demonstratethat BBS acts through its receptor to produce a rapid induction of bothc-jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction ofAP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimalinvolvement of PKA.

     

Hydrogen peroxide induces GADD153 in Jurkat cells through the protein kinase C-dependent pathway

Growth arrest and DNA damage-inducible gene 153 (GADD153) is a CCAAT/enhancer binding protein (C/EBP) related gene and is induced in response to various stimuli including DNA damaging agents, UV irradiation, and serum starvation. In this study, we investigated which intracellular signals contribute to the expression of GADD153 mRNA in Jurkat cells in response to oxidative stress using several kinds of kinase inhibitors. GADD153 mRNA expression was immediately enhanced following hydrogen peroxide exposure and was significantly inhibited by treatment with H-7, staurosporin, and Ro-31-8220. In particular, rottlerin, a PKCdelta specific inhibitor, markedly attenuated hydrogen peroxide-induced GADD153 mRNA expression even at 1 microM. Treatment with a potent PKC activator, phorbol-12-myristate-13-acetate (PMA), augmented GADD153 mRNA in Jurkat cells in the presence of hydrogen peroxide, although PMA alone induced GADD153 mRNA marginally. Hydrogen peroxide significantly enhanced the AP-1 binding activity of the nuclear extract from Jurkat cells to the GADD153 AP-1 binding site. AP-1 binding activity was suppressed by rottlerin treatment. These findings indicate that PKC, especially PKCdelta, plays an important role in the induction of GADD153 mRNA following oxidative stress.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism

Metformin is widely used as a hypoglycemic agent for the treatment of type 2 diabetes. Both metformin and rotenone, an inhibitor of respiratory chain complex I, suppressed glucose-6-phosphatase (G6pc), a rate limiting enzyme of liver glucose production, mRNA expression in a rat hepatoma cell line accompanied by a reduction of intracellular ATP concentration and an activation of AMP-activated protein kinase (AMPK). When yeast NADH-quinone oxidoreductase 1 (NDI1) gene was introduced into the cells, neither inhibition of ATP synthesis nor activation of AMPK was induced by these agents. Interestingly, in contrast to rotenone treatment, G6pc mRNA down-regulation was observed in the NDI1 expressing cells after metformin treatment. Since NDI1 can functionally complement the complex I under the presence of metformin or rotenone, our results indicate that metformin induces down-regulation of G6pc expression through an inhibition of complex I and an activation of AMPK-independent mechanism.