IGF-1 activates hEAG K(+) channels through an Akt-dependent signaling pathway in breast cancer cells: role in cell proliferation

Previous work from our laboratory has shown that human ether à go-go (hEAG) K(+) channels are crucial for breast cancer cell proliferation and cell cycle progression. In this study, we investigated the regulation of hEAG channels by an insulin-like growth factor-1 (IGF-1), which is known to stimulate cell proliferation. Acute applications of IGF-1 increased K(+) current-density and hyperpolarized MCF-7 cells. The effects of IGF-1 were inhibited by hEAG inhibitors. Moreover, IGF-1 increased mRNA expression of hEAG in a time-dependent manner in parallel with an enhancement of cell proliferation. The MCF-7 cell proliferation induced by IGF-1 is inhibited pharmacologically by Astemizole or Quinidine or more specifically using siRNA against hEAG channel. Either mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K) are known to mediate IGF-1 cell proliferative signals through the activation of extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, respectively. In MCF-7 cells, IGF-1 rapidly stimulated Akt phosphorylation, whereas IGF-1 had little stimulating effect on Erk 1/2 which seems to be constitutively activated. The application of wortmannin was found to block the effects of IGF-1 on K(+) current. Moreover, the inhibition of Akt phosphorylation by the application of wortmannin or by a specific reduction of Akt kinase activity reduced the hEAG mRNA levels. Taken together, our results show, for the first time, that IGF-1 increases both the activity and the expression of hEAG channels through an Akt-dependent pathway. Since a hEAG channel is necessary for cell proliferation, its regulation by IGF-1 may thus play an important role in IGF-1 signaling to promote a mitogenic effect in breast cancer cells.     

Resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A–PTEN signaling network

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin-like growth factor (IGF)-1-stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant-negative mitogen-activated protein kinase kinase 1 (MKK1) strengthened resveratrol’s inhibition of the basal or IGF-1-stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)–Akt were implicated in resveratrol-inactivated Erk1/2-dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant-negative PP2A rendered resistance to resveratrol’s suppression of the basal or IGF-1-stimulated phospho-Erk1/2 and cell adhesion, whereas expression of wild-type PP2A enhanced resveratrol’s inhibitory effects. Overexpression of wild-type PTEN or dominant-negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol’s inhibition of the basal or IGF-1-stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol’s inhibitory effects on the basal and IGF-1-induced inhibition of PP2A–PTEN, activation of Akt–Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A–PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.     

Two different signal transduction pathways are implicated in the regulation of initiation factor 2B activity in insulin-like growth factor-1-stimulated neuronal cells

Eukaryotic initiation factor eIF-2B plays an important role in translation regulation and has been suggested to be implicated in the increased protein synthesis promoted in response to growth factors. We have used primary cultured neurons to delineate the signaling pathways by which insulin-like growth factor-1 (IGF-1), which plays a critical role in the survival of neuronal cells, promotes eIF-2B and protein synthesis activation. Treatment of cortical neurons with IGF-1 (100 ng/ml) for 30 min stimulates [(3)H]methionine incorporation, and a parallel increase in eIF-2B activity was observed. Wortmannin and LY294002 reversed both effects, indicating that phosphatidylinositol 3-kinase mediates IGF-1-induced protein synthesis and eIF-2B activation. IGF-1 induced glycogen synthase kinase-3 (GSK-3) inactivation in a phosphatidylinositol 3-kinase-dependent fashion because it is inhibited by wortmannin and LY294002. By using GSK-3 immunoprecipitated from untreated and IGF-1-treated cells, we demonstrate the phosphorylation of eIF-2B coincident with its inactivation. The treatment of cortical neurons with IGF-1 also promoted the activation of mitogen-activated protein kinase (MAPK). The MAPK-activating kinase (MEK) inhibitor PD98059 inhibited MAPK activation and reversed IGF-1-induced protein synthesis and eIF-2B activation. These findings suggest that IGF-1-induced eIF-2B activation on neurons is promoted through phosphatidylinositol 3-kinase and GSK-3 kinase, and we report an IGF-1-induced MEK/MAPK activation pathway implicated in eIF-2B activation.     

Mitogen- and stress-activated protein kinase 1 mediates activation of Akt by ultraviolet B irradiation

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.     

Differential signaling by insulin receptor substrate 1 (IRS-1) and IRS-2 in IRS-1-deficient cells.

Mice made insulin receptor substrate 1 (IRS-1) deficient by targeted gene knockout exhibit growth retardation and abnormal glucose metabolism due to resistance to the actions of insulin-like growth factor 1 (IGF-1) and insulin (E. Araki et al., Nature 372:186-190, 1994; H. Tamemoto et al., Nature 372:182-186, 1994). Embryonic fibroblasts and 3T3 cell lines derived from IRS-1-deficient embryos exhibit no IGF-1-stimulated IRS-1 phosphorylation or IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity but exhibit normal phosphorylation of IRS-2 and Shc and normal IRS-2-associated PI 3-kinase activity. IRS-1 deficiency results in a 70 to 80% reduction in IGF-1-stimulated cell growth and parallel decreases in IGF-1-stimulated S-phase entry, PI 3-kinase activity, and induction of the immediate-early genes c-fos and egr-1 but unaltered activation of the mitogen-activated protein kinases ERK 1 and ERK 2. Expression of IRS-1 in IRS-1-deficient cells by retroviral gene transduction restores IGF-1-stimulated mitogenesis, PI 3-kinase activation, and c-fos and egr-1 induction in proportion to the level of reconstitution. Increasing the level of IRS-2 in these cells by using a retrovirus reconstitutes IGF-1 activation of PI 3-kinase and immediate-early gene expression to the same degree as expression of IRS-1; however, IRS-2 overexpression has only a minor effect on IGF-1 stimulation of cell cycle progression. These results indicate that IRS-1 is not necessary for activation of ERK 1 and ERK 2 and that activation of ERK 1 and ERK 2 is not sufficient for IGF-1-stimulated activation of c-fos and egr-1. These data also provide evidence that IRS-1 and IRS-2 are not functionally interchangeable signaling intermediates for stimulation of mitogenesis despite their highly conserved structure and many common functions such as activating PI 3-kinase and early gene expression.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Chemokine receptors CXCR-1/2 activate mitogen-activated protein kinase via the epidermal growth factor receptor in ovarian cancer cells

Ovarian cancer typically disseminates widely in the abdomen, a characteristic that limits curative therapy. The mechanisms that promote ovarian cancer cell migration are incompletely understood. We studied model SK-OV-3 ovarian cancer cells and observed robust expression of the alpha chemokine receptors CXCR-1 and CXCR-2. Interleukin-8 (IL-8) treatment caused shape changes in the cells, with membrane ruffling and formation/retraction of thin actin-like projections, as detected by time-lapse microscopy. Stimulation of the CXCR-1/2 receptors by human interleukin 8 (IL-8) rapidly activated the p44/42 mitogen-activated protein (extracellular signal-regulated kinase (Erk1/2)) kinase pathway. Treatment of SK-OV-3 cells with the inhibitors genestein and herbimycin A indicated that tyrosine kinases were involved in the IL-8 activation of Erk1 and Erk2. Of note, IL-8 induced transient phosphorylation of the epidermal growth factor (EGF) receptor and its association with the adaptor molecules Shc and Grb2. This transactivation of the EGF receptor was dependent on intracellular Ca(2+) mobilization. Furthermore AG1478, a specific inhibitor of the EGF receptor kinase, blocked Erk1 and Erk2 activation. c-Src kinase was not involved in the IL-8-mediated phosphorylation of the EGF receptor, but was critical for Shc phosphorylation and downstream Erk1/2 kinase activation. These results suggest important "cross-talk" between chemokine and growth factor pathways that may link signals of cell migration and proliferation in ovarian cancer.     

Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) by tert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2.     

Macrophage-colony-stimulating factor-induced activation of extracellular-regulated kinase involves phosphatidylinositol 3-kinase and reactive oxygen species in human monocytes

We previously reported that activation of the phosphatidylinositol (PI) 3-kinase pathway was important in M-CSF-induced monocyte survival. Because M-CSF also induces activation of the mitogen-activated protein (MAP) kinase extracellular-regulated kinase (Erk), we focused on dissecting the mechanism used by M-CSF to induce Erk activation in human monocytes. We found that, in addition to the MAP/Erk kinase inhibitor PD098059, the PI 3-kinase inhibitors LY294002 and wortmannin both suppressed Erk activation in M-CSF-treated monocytes, suggesting that 3-phosphorylated products of PI 3-kinase played a role in Erk activation. Investigating the biochemical pathways regulated by PI 3-kinase to activate Erk, we found that, in response to M-CSF, normal human monocytes induced reactive oxygen species (ROS), which were suppressed by the PI 3-kinase inhibitor wortmannin but not by the solvent control DMSO or the MAP/Erk kinase inhibitor PD098059. We next found that, in the absence of M-CSF, ROS could induce Erk activation in human monocytes. Exogenous H(2)O(2) induced Erk activation in human monocytes, which was suppressed by exogenous catalase. To determine whether ROS induced by M-CSF played a role in Erk activation, we found that N-acetylcysteine and diphenyleneiodonium both suppressed Erk activation in M-CSF-treated monocytes. Erk activation by M-CSF also seemed to play a role in cellular survival in monocytes. These data suggest that, in M-CSF-stimulated human monocytes, PI 3-kinase products and ROS production play a role in Erk activation and monocyte survival.     

Stimulation of C2C12 myoblast growth by basic fibroblast growth factor and insulin-like growth factor 1 can occur via mitogen-activated protein kinase-dependent and -independent pathways.

It is now well-recognized that the mitogen-activated protein (MAP) kinase cascade facilitates signaling from an activated tyrosine kinase receptor to the nucleus. In fact, an increasing number of extracellular effectors have been reported to activate the MAP kinase cascade, with a significant number of cellular responses attributed to this activation. We set out to explore how two extracellular effectors, basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1), which have both been reported to activate MAP kinase, generate quite distinct cellular responses in C2C12 myoblasts. We demonstrate here that bFGF, which is both a potent mitogen and inhibitor of myogenic differentiation, is a strong MAP kinase agonist. By contrast, IGF-1, which is equally mitogenic for C2C12 cells but ultimately enhances the differentiated phenotype, is a weak activator of the MAP kinase cascade. We further demonstrate that IGF-1 is a potent activator of both insulin receptor substrate IRS-1 tyrosyl phosphorylation and association of IRS-1 with activated phosphatidylinositol 3-kinase (PI 3-kinase). Finally, use of the specific MAP kinase kinase inhibitor, PD098059, and wortmannin, a PI 3-kinase inhibitor, suggests the existence of an IGF-1-induced, MAP kinase-independent signaling event which contributes to the mitogenic response of this factor, whereas bFGF-induced mitogenesis appears to strongly correlate with activation of the MAP kinase cascade.     

Differential activation of protein kinase B and p70(S6)K by glucose and insulin-like growth factor 1 in pancreatic beta-cells (INS-1)

It has been shown that IGF-1-induced pancreatic beta-cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic beta-cells, INS-1, suggested that PI3K was not necessary for glucose-induced beta-cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70(S6K)), suggested that a major part of glucose-dependent beta-cell proliferation requires activation of mammalian target of rapamycin/p70(S6K), independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced beta-cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70(S6K) signal transduction pathways for a full commitment to glucose-induced pancreatic beta-cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.     

Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin-Like Growth Factor 1 Signaling

Abstract: The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 m M K⁺ to one containing 5 m M K⁺. IGF-1 protected granule neurons from apoptosis in medium containing 5 m M K⁺. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K⁺, and failed to potentiate cell death in the presence of 5 m M K⁺. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15–20%) was observed at 1 m M and was half-maximal at 45 m M . The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Beta 3- and alpha1-adrenergic Erk1/2 activation is Src- but not Gi-mediated in Brown adipocytes

A novel signaling pathway for mediation of beta(3)-adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G(i)-proteins and Gbetagamma-subunits, distinct from the classical cAMP pathway of beta-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express beta(3)-adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both beta(3) receptors and alpha(1) receptors but not via alpha(2) receptors. Forskolin induced Erk1/2 activation similarly to beta(3) activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G(i)-pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that Gbetagamma activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both beta(3)- and alpha(1)-induced Erk1/2 activation. Thus, the proposed novel G(i) pathway for beta(3) mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed beta(3) receptors examined here. Here, the beta(3) signal is mediated classically via cAMP/protein kinase A. beta(3) and alpha(1) signals converge at Src, which thus mediates Erk1/2 activation in both pathways.     

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression     

The role of cAMP-dependent signaling in receptor-recognized forms of alpha 2-macroglobulin-induced cellular proliferation

Ligation of alpha(2)-macroglobulin receptors by receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) activates various signaling cascades and promotes cell proliferation. It also elevates cAMP in murine peritoneal macrophages. We now report that a significant elevation of cAMP-response element-binding protein (CREB) occurs in alpha(2)M*-stimulated cells, and this effect is potentiated by isobutylmethylxanthine, dibutyryl-cAMP, or forskolin. An alpha(2)M* concentration-dependent rapid increase in phosphorylated CREB at Ser(133) also occurred, a necessary event in its activation. Inhibition of Ca(2+)/calmodulin kinase, protein kinases A and C, tyrosine kinases, ribosomal S6 kinase, farnesyl transferase, extracellular signal-regulated kinases 1/2, phosphatidylinositol 3-kinase, or p38 mitogen-activated protein kinase markedly reduce alpha(2)M*-induced phosphorylation of CREB, indicating a role for the p21(ras)-dependent and phosphatidylinositol 3-kinase signaling pathways in regulating CREB activation by alpha(2)M*. Finally, silencing the CREB gene by transfecting cells with a homologous gene sequence double-stranded RNA drastically reduced the expression of CREB and blocked the ability of alpha(2)M* to promote macrophage cell division. We conclude that cAMP-dependent signal transduction as well as other signaling cascades are essential for alpha(2)M*-induced cell proliferation.