p38 and EGF receptor kinase-mediated activation of the phosphatidylinositol 3-kinase/Akt pathway is required for Zn2+-induced cyclooxygenase-2 expression

Cyclooxygenase 2 (COX-2) expression is induced by physiological and inflammatory stimuli. Regulation of COX-2 expression is stimulus and cell type specific. Exposure to Zn2+ has been associated with activation of multiple intracellular signaling pathways as well as the induction of COX-2 expression. This study aims to elucidate the role of intracellular signaling pathways in Zn2+-induced COX-2 expression in human bronchial epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) potently block Zn2+-induced COX-2 mRNA and protein expression. Overexpression of adenoviral constructs encoding dominant-negative Akt kinase downstream of PI3K or wild-type phosphatase and tensin homolog deleted on chromosome 10, an important PI3K phosphatase, suppresses COX-2 mRNA expression induced by Zn2+. Zn2+ exposure induces phosphorylation of the tyrosine kinases, including Src and EGF receptor (EGFR), and the p38 mitogen-activated protein kinase. Blockage of these kinases results in inhibition of Zn2+-induced Akt phosphorylation as well as COX-2 protein expression. Overexpression of dominant negative p38 constructs suppresses Zn2+-induced increase in COX-2 promoter activity. In contrast, the c-Jun NH2-terminal kinase and the extracellular signal-regulated kinases have minimal effect on Akt phosphorylation and COX-2 expression. Inhibition of p38, Src, and EGFR kinases with pharmacological inhibitors markedly reduces Akt phosphorylation induced by Zn2+. However, the PI3K inhibitors do not show inhibitory effects on p38, Src, and EGFR. These data suggest that p38 and EGFR kinase-mediated Akt activation is required for Zn2+-induced COX-2 expression and that the PI3K/Akt signaling pathway plays a central role in this event.     

Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a copper stress.     

Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.     

Pyrroloquinoline quinone rescues hippocampal neurons from glutamate-induced cell death through activation of Nrf2 and up-regulation of antioxidant genes

Pyrroloquinoline quinone (PQQ) has been shown to protect primary cultured hippocampal neurons from glutamate-induced cell apoptosis by scavenging reactive oxygen species (ROS) and activating phosphatidylinositol-3-kinase (PI3K)/Akt signaling. We investigated the downstream pathways of PI3K/Akt involved in PQQ protection of glutamate-injured hippocampal neurons. Western blot analysis indicated that PQQ treatment following glutamate stimulation triggers phosphorylation of glycogen synthase kinase 3β, accompanied by maintenance of Akt activation. Immunostaining and quantitative RT-PCR revealed that PQQ treatment promotes nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), and up-regulates mRNA expression of Nrf2 and the antioxidant enzyme genes, heme oxygenase-1 and glutamate cysteine ligase catalytic in glutamate-injured hippocampal neurons; this is a process dependent on the PI3K/Akt pathway, as evidenced by blocking experiments with PI3K inhibitors. In addition, increased ROS production and decreased glutathione levels in glutamate-injured hippocampal neurons were found to be reduced by PQQ treatment. Collectively, our findings suggest that PQQ exerts neuroprotective activity, possibly through PI3K/Akt-dependent activation of Nrf2 and up-regulation of antioxidant genes. However, the ability of PQQ to scavenge ROS was not totally regulated by PI3K/Akt signaling; possibly it is governed by other mechanisms.     

镁离子对成骨细胞活力和分化的促进作用及其机制研究

目的:研究不同浓度镁离子对成骨细胞活力和分化的影响,并探讨镁基生物材料促进骨再生的机制。方法:分离培养大鼠乳鼠颅骨成骨细胞,之后将细胞分别在DMEM培养基(含有0.8 m M镁离子;对照组)和含有6 m M、10 m M、18 m M镁离子(实验组)的培养基中进行培养,通过MTT法测定细胞活力,ALP活力、茜素红染色法测定成骨细胞的分化,通过western blot法测定不同浓度镁离子组中PI3K/Akt信号通路的表达情况。结果:6 m M、10 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平较对照组明显增加(P0.05),18 m M镁离子组成骨细胞活力、ALP活力、基质矿化水平对照组明显降低(P0.05)。在10 m M镁离子组加入wortmannin后,上述增强的结果受到抑制。结论:6-10 m M镁离子促进成骨细胞的活力和分化,而过高浓度镁离子(18 m M)对成骨细胞的活力和分化具有抑制作用。10 m M镁离子通过激活PI3K/Akt信号通路促进成骨细胞的活力和分化。这项研究为医用镁基生物材料的进一步研究提供了很好的参考作用。     

Higenamine mitigates interleukin-1β-induced human nucleus pulposus cell apoptosis by ROS-mediated PI3K/Akt signaling

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. Higenamine exerts multiple pharmacological properties in inflammation-related disorders. Our study aimed to explore the function of higenamine on interleukin (IL)-1β-caused apoptosis of human nucleus pulposus cells (HNPCs). Cell apoptosis was investigated by TUNEL and flow cytometry. Apoptosis-related biomarkers were determined by qRT-PCR or Western blotting. The protein in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling was measured by Western blotting. We found that higenamine showed little effect on cell apoptosis, but mitigated IL-1β-caused apoptosis in a dose-dependent pattern. Higenamine attenuated IL-1β-induced decrease of Bcl-2 and increase of Bax and cleaved caspase-3. Higenamine did not affect the reactive oxygen species (ROS) level and the PI3K/Akt signaling, but attenuated IL-1β-induced ROS production and inhibition of the PI3K/Akt signaling. IL-1β repressed the activation of the PI3K/Akt pathway, but ROS inhibition using N-acetylcysteine (NAC) rescued this pathway. The PI3K/Akt signaling suppression using LY294002 reversed the inhibitive effect of higenamine on IL-1β-caused apoptosis, and this effect was weakened by ROS inhibition. In conclusion, higenamine attenuates IL-1β-caused apoptosis of HNPCs via ROS-mediated PI3K/Akt pathway.

     

Activation of the PI3K/Akt signaling pathway through P2Y2 receptors by extracellular ATP is involved in osteoblastic cell proliferation

We studied the PI3K/Akt signaling pathway modulation and its involvement in the stimulation of ROS 17/2.8 osteoblast-like cell proliferation by extracellular ATP. A dose- and time-dependent increase in Akt-Ser 473 phosphorylation (p-Akt) was observed. p-Akt was increased by ATPγS and UTP, but not by ADPβS. Akt activation was abolished by PI3K inhibitors and reduced by inhibitors of PI-PLC, Src, calmodulin (CaM) but not of CaMK. p-Akt was diminished by cell incubation in a Ca²⁺-free medium but not by the use of L-type calcium channel blockers. The rise in intracellular Ca²⁺ induced by ATP was potentiated in the presence of Ro318220, a PKC inhibitor, and attenuated by the TPA, a known activator of PKC. ATP-dependent p-Akt was diminished by TPA and augmented by Ro318220 treatment in a Ca²⁺-containing but not in a Ca²⁺-free medium. ATP stimulated the proliferation of both ROS 17/2.8 cells and rat osteoblasts through PI3K/Akt. In the primary osteoblasts, ATP induces alkaline phosphatase activity via PI3K, suggesting that the nucleotide promotes osteoblast differentiation. These results suggest that ATP stimulates osteoblast proliferation through PI-PLC linked-P2Y₂ receptors and PI3K/Akt pathway activation involving Ca²⁺, CaM and Src. PKC seems to regulate Akt activation through Src and the Ca²⁺ influx/CaM pathway.     

AD认知功能障碍的代谢紊乱机制:脑胰岛素抵抗及其介导的PI3K/Akt信号通路受损

阿尔茨海默病(Alzheimer’s disease, AD)是一种以进行性痴呆为主要特征的中枢神经系统退行性疾病,其认知功能障碍可能与Ⅱ型糖尿病(type 2 diabetes, T2DM)诱发的胰岛素抵抗所损伤的PI3K/Akt胰岛素信号级联通路相关。胰岛素是调节机体新陈代谢的重要激素,通过与神经细胞表面的胰岛素受体结合激活PI3K/Akt信号通路,以调控葡萄糖、脂质的代谢。任何中间媒介功能紊乱所导致的脑胰岛素水平和胰岛素敏感性的降低都会损坏PI3K/Akt信号通路,诱发脑能量代谢障碍、Aβ沉积、Tau蛋白过度磷酸化,引起并加重AD认知功能障碍。因此,本文以PI3K/Akt胰岛素信号通路为主线,揭示了T2DM中脑胰岛素抵抗(insulin resistance, IR)与AD之间的复杂机制,旨在加深对脑IR介导的AD病理过程的系统性理解,借此为延缓或治疗AD的认知功能障碍提供理论基础。     

Adenosine triphosphate regulates NADPH oxidase activity leading to hydrogen peroxide production and COX-2/PGE2 expression in A549 cells

Non-small cell lung carcinoma (NSCLC) accounts for most of all lung cancers, which is the leading cause of mortality in human beings. High level of cyclooxygenase-2 (COX-2) is one of the features of NSCLC and related to the low survival rate of NSCLC. However, whether extracellular nucleotides releasing from stressed resident tissues contributes to the expression of COX-2 remains unclear. Here, we showed that stimulation of A549 cells by adenosine 5'-O-(3-thiotriphosphate) (ATPγS) led to an increase in COX-2 gene expression and prostaglandin E(2) (PGE(2)) synthesis, revealed by Western blotting, RT-PCR, promoter assay, and enzyme-linked immunosorbent assay. In addition, ATPγS induced intracellular reactive oxygen species (ROS) generation through the activation of NADPH oxidase. The increase of ROS level resulted in activation of the c-Src/epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)-κB cascade. We also found that activated Akt was translocated into the nucleus and recruited with NF-κB and p300 to form a complex. Thus, activation of p300 modulated the acetylation of histone H4 via the NADPH oxidase/c-Src/EGFR/PI3K/Akt/NF-κB cascade stimulated by ATPγS. Our results are the first to show a novel role of NADPH oxidase-dependent Akt/p65/p300 complex formation that plays a key role in regulating COX-2/PGE(2) expression in ATPγS-treated A549 cells. Taken together, we demonstrated that ATPγS stimulated activation of NADPH oxidase, resulting in generation of ROS, which then activated the downstream c-Src/EGFR/PI3K/Akt/NF-κB/p300 cascade to regulate the expression of COX-2 and synthesis of PGE(2) in A549 cells. Understanding the regulation of COX-2 expression and PGE(2) release by ATPγS on A549 cells may provide potential therapeutic targets of NSCLC.     

Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway during Porcine Circovirus Type 2 Infection Facilitates Cell Survival and Viral Replication

Virus infection activates host cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which regulates diverse cellular activities related to cell growth, survival, and apoptosis. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wasting syndrome, which is an emerging and important swine disease, can transiently induce the PI3K/Akt pathway in cultured cells at an early step during PCV2 infection. Activation of the PI3K/Akt signal was also induced by UV-irradiated PCV2, indicating that virus replication was not required for this induction. Inhibition of PI3K activation leads to reduced virus yield, which is associated with decreased viral DNA replication and lower virus protein expression. However, inhibition of PI3K activation greatly enhanced apoptotic responses as evidenced by the cleavage of poly-ADP ribose polymerase and caspase-3 as well as DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining during the early stage of PCV2 infection. Furthermore, the pancaspase inhibitor zVAD.fmk alleviated the reduction in Akt phosphorylation levels by inhibiting PI3K activation, indicating that the signaling promotes cell survival and thereby favors viral replication. These results reveal that an antiapoptotic role for the PI3K/Akt pathway induced by PCV2 infection to suppress premature apoptosis for improved virus growth after infection, extending our understanding of the molecular mechanism of PCV2 infection.     

Redox Signaling via Oxidative Inactivation of PTEN Modulates Pressure-Dependent Myogenic Tone in Rat Middle Cerebral Arteries

The present study examined the level of generation of reactive oxygen species (ROS) and roles of inactivation of the phosphatase PTEN and the PI3K/Akt signaling pathway in response to an increase in intramural pressure-induced myogenic cerebral arterial constriction. Step increases in intraluminal pressure of cannulated cerebral arteries induced myogenic constriction and concomitant formation of superoxide (O₂ ^.−) and its dismutation product hydrogen peroxide (H₂O₂) as determined by fluorescent HPLC analysis, microscopic analysis of intensity of dihydroethidium fluorescence and attenuation of pressure-induced myogenic constriction by pretreatment with the ROS scavenger 4,hydroxyl-2,2,6,6-tetramethylpiperidine1-oxyl (tempol) or Mito-tempol or MitoQ in the presence or absence of PEG-catalase. An increase in intraluminal pressure induced oxidation of PTEN and activation of Akt. Pharmacological inhibition of endogenous PTEN activity potentiated pressure-dependent myogenic constriction and caused a reduction in NPo of a 238 pS arterial K_Ca channel current and an increase in [Ca²⁺]_i level in freshly isolated cerebral arterial muscle cells (CAMCs), responses that were attenuated by Inhibition of the PI3K/Akt pathway. These findings demonstrate an increase in intraluminal pressure induced increase in ROS production triggered redox-sensitive signaling mechanism emanating from the cross-talk between oxidative inactivation of PTEN and activation of the PI3K/Akt signaling pathway that involves in the regulation of pressure-dependent myogenic cerebral arterial constriction.     

Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes

In a variety of cell types, insulin stimulation elicits the rapid production of H(2)O(2), which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938-21942). In the present work, we explored the potential role of insulin-induced H(2)O(2) generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H(2)O(2) production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3'-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H(2)O(2) was added back to cells that had been pretreated with DPI. Interestingly, the H(2)O(2)-induced activation of Akt was entirely mediated by upstream stimulation of PI 3'-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H(2)O(2). Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H(2)O(2) generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H(2)O(2) may occur through a unique pathway, independent of cellular PI 3'-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H(2)O(2) generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3'-kinase, Akt, and ultimately cellular glucose transport in response to insulin.     

17 beta-estradiol activates PI3K/Akt signaling pathway by estrogen receptor (ER)-dependent and ER-independent mechanisms in endometrial cancer cells

Cellular response to estrogen is mediated both by estrogen receptor (ER) binding to estrogen response element (ERE) and by non-nuclear actions like activation of signal transducing pathways. The main aims are to study if PI3K/Akt signaling pathway can be activated by 17beta-estradiol (E2) via non-nuclear action and to investigate the relationship of the action of E2 and ER in endometrial cancer cells expressing with different status of ER. The levels of phosphorylated Akt (Ser473) (P-Akt) and total Akt were examined by western blot and Akt kinase activity was measured in cells after stimulation with 1 microM E2 at different time points. Inhibitory role of LY294002 on activation of Akt induced by E2 and its estrogen antagonist, ICI182780 were also tested. P-Akt/Akt was used as a measure of activation of Akt. We found that maximum P-Akt/Akt and Akt kinase activity took place at 30 min in Ishikawa cells and 15 min in HEC-1A cells and the activation persisted for at least 2 h after stimulation with 1 microM E2. The activation of Akt elicited gradually with increasing doses of E2. PI3K inhibitor, LY294002, stopped the activating Akt in a dose-dependent manner and 50 microM LY294002 completely blocked the activation of Akt induced by E2. ICI182780 could block the activation of PI3K/Akt in ER-positive Ishikawa cells but not in HEC-1A cells with poor-expressed ER. This study demonstrated that E2 is able to promptly activate PI3K/Akt signal pathway in Ishikawa cells in an ER-dependent manner and ER-independent in HEC-1A cells. Blockage of PI3K/Akt cascade may become a potential and effective way to control endometrial carcinoma, especially in ER-negative cancers, which show no response to endocrinal therapy.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Differential thiol oxidation of the signaling proteins Akt,PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle

Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.     

Activation of Akt/PDK signaling in macrophages upon binding of receptor-recognized forms of alpha2-macroglobulin to its cellular receptor: effect of silencing the CREB gene

Macrophage binding of receptor-recognized forms of alpha2-macrogobulin (alpha2M*) significantly increases cAMP, CREB, and activated CREB. We have now examined the participation of the PI 3-kinase/PDK/Akt/p70s6k signaling cascade in alpha2M*-induced cellular proliferation and also studied the role of CREB in these events. Exposure of cells to alpha2M* caused an approximately 2-fold increase in CREB and its phosphorylation at Ser133, phosphorylation of the regulatory subunit of PI 3-kinase, Akt phosphorylation at Ser473 or Thr308, and phosphorylated 70s6k. Silencing of the CREB gene with dsRNA homologous in sequence to the target gene, markedly reduced the levels of CREB mRNA activation of CREB, PI 3-kinase, Akt, and p70s6k in alpha2M*-stimulated macrophages. We conclude that in murine peritoneal macrophages, alpha2M*-induced increase of cAMP is involved in cellular proliferation and this process is mediated by the PI 3-kinase signaling cascade.