Calpain inhibition impairs glycogen syntheses in HepG2 hepatoma cells without altering insulin signaling

Calpains are a family of non-lysosomal cytoplasmatic cysteine proteases. Since calpain 10 (CAPN10), a member of the calpain family of proteases, has been found to represent a putative diabetes susceptibility gene, it was argued that calpains may be involved in the development of type 2 diabetes. The functional role of calpains in insulin signaling and/or insulin action is, however, not clear. We investigated the effects of the calpains 1 and 2 inhibitor PD151746 on insulin signaling and insulin action in human hepatoma G2 cells (HepG2). HepG2 cells were incubated without (-PD) or with (+PD) 5.33 micromol/l PD151746 for different times and then stimulated with 100 nmol/l insulin for 0 (t(0)), 5 (t(5)), 15 (t(15)), 30 (t(30)), 45 (t(45)), and 60 (t(60)) min. After solubilization of the cells, insulin receptor kinase activity, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol-3 kinase (PI3-kinase), PI3-kinase activity, Thr(308) phosphorlyation of Akt, amount of protein tyrosine phosphatase-epsilon (PTPepsilon), and glycogen synthase activity were determined. Incubation with PD151746 resulted in a significant reduction of insulin-stimulated glycogen synthesis compared with cells not pre-incubated with the calpain inhibitor (-PD: t(0), 4.90 +/- 1.20%; t(5), 5.90 +/- 1.02%; t(15), 5.29 +/- 0.95%; t(30), 5.60 +/- 1.10%; t(45), 5.52 +/- 0.90%; t(60), 5.67 +/- 0.97%;+PD: t(0), 4.56 +/- 1.10%; t(5), 6.16 +/- 1.05%; t(15), 7.52 +/- 1.09%; t(30), 7.68 +/- 1.10%; t(45), 8.28 +/- 0.89%; t(60), 7.69 +/- 0.98%; P < 0.05). Incubation with PD151746 significantly increased the protein amount of PTPepsilon in the cells after 12 h (-PD: t(1), 0.85 +/- 0.18 RU (Relative unit); t(8), 0.87 +/- 0.18 RU; t(12), 0.9 +/- 0.13 RU; +PD: t(1), 0.92 +/- 0.21 RU; t(8), 1.1 +/- 0.15 RU; t(12), 1.34 +/- 0.16 RU; P < 0.05). Calpain inhibition with PD151746 had no effect on the insulin stimulation of the investigated insulin signaling parameters. These results in HepG2 cells suggest that calpains play a role in the hepatic regulation of insulin-stimulated glycogen synthesis independent of the PI3-kinase/Akt signaling pathway.     

Regulation of the RAP1/RAF-1/extracellularly regulated kinase-1/2 cascade and prolactin release by the phosphoinositide 3-kinase/AKT pathway in pituitary cells

In pituitary cells, prolactin (PRL) synthesis and release are controlled by multiple transduction pathways. In the GH4C1 somatolactotroph cell line, we previously reported that MAPK ERK-1/2 are a point of convergence between the pathways involved in the PRL gene regulation. In the present study, we focused on the involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the MAPK ERK-1/2 regulation and PRL secretion in pituitary cells. Either specific pharmacological PI3K and Akt inhibitors (LY294002, Akt I, and phosphoinositide analog-6) or Akt dominant-negative mutant (K179M) enhanced ERK-1/2 phosphorylation in unstimulated GH4C1 cells. Under the same conditions, PI3K and Akt inhibition also both increased Raf-1 kinase activity and the levels of GTP-bound (active form) monomeric G protein Rap1, which suggests that a down-regulation of the ERK-1/2 cascade is induced by the PI3K/Akt signaling pathway in unstimulated cells. On the contrary, ERK-1/2 phosphorylation, Raf-1 activity, and Rap1 activation were almost completely blocked in IGF-I-stimulated cells previously subjected to PI3K or Akt inhibition. Although the PRL promoter was not affected by either PI3K/Akt inhibition or activation, PRL release increased in response to the pharmacological PI3K/Akt inhibitors in unstimulated GH4C1 and rat pituitary primary cells. The IGF-I-stimulated PRL secretion was diminished, on the contrary, by the pharmacological PI3K/Akt inhibitors. Taken together, these findings indicate that the PI3K/Akt pathway exerts dual regulatory effects on both the Rap1/Raf-1/ERK-1/2 cascade and PRL release in pituitary cells, i.e. negative effects in unstimulated cells and positive ones in IGF-I-stimulated cells.     

Glimepiride induces proliferation and differentiation of rat osteoblasts via the PI3-kinase/Akt pathway

Glimepiride is a third-generation sulfonylurea agent and is widely used in the treatment of type 2 diabetes mellitus. In addition to the stimulatory effects on pancreatic insulin secretion, glimepiride has also been reported to have extrapancreatic functions including activation of PI3 kinase (PI3K) and Akt in rat adipocytes and skeletal muscle. PI3-kinase and Akt are important signaling molecules in the regulation of proliferation and differentiation in various cells. This study investigated the actions of glimepiride in rat osteoblasts and the role of PI3K/Akt pathway. Cell proliferation was determined by measuring absorbance at 550 nm. Supernatant assay was used for measuring alkaline phosphatase activity. Western blot analysis was used for determining collagen I, insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase expression. We found that glimepiride significantly enhanced proliferation and differentiation of osteoblasts and led to activation of several key signaling molecules including insulin receptor substrate-1/2, PI3K/Akt, and endothelial nitric oxide synthase. Furthermore, a specific inhibitor of PI3K abolished the stimulatory effects of glimepiride on proliferation and differentiation. Taken together, these observations provide concrete evidence that glimepiride activates the PI3K/Akt pathway; and this activation is likely required for glimepiride to stimulate proliferation and differentiation of rat osteoblasts.     

PI3K/Akt信号通路调节心脏功能的研究进展

PI3K/Akt是调节心脏功能的一条重要的信号转导通路,主要通过血管内皮生长因子介导血管生成、抑制心肌细胞凋亡、重构心室、促进细胞能量代谢等方面调节心脏功能。研究发现PI3K/Akt信号通路在内分泌疾病、肾病、肝纤维化、肿瘤及心血管疾病的发生、发展中起着重要作用。本文就PI3K/Akt信号通路调节心脏功能的分子机制作一阐述。     

Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway

The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was recently shown to negatively regulate LPS-induced acute inflammatory responses. We previously observed that the metabolic thiol antioxidant alpha-lipoic acid (LA) inhibits LPS-induced expression of cellular adhesion molecules and adherence of monocytes to human aortic endothelial cells. Here we investigated the mechanism by which LA attenuates LPS-induced monocyte activation in vitro and acute inflammatory responses in vivo. Incubation of human monocytic THP-1 cells with LA induced phosphorylation of Akt in a time- and dose-dependent manner. In cells pretreated with LA followed by LPS, Akt phosphorylation was elevated initially and further increased during incubation with LPS. This LA-dependent increase in Akt phosphorylation was accompanied by inhibition of LPS-induced NF-kappaB DNA binding activity and up-regulation of TNFalpha and monocyte chemoattractant protein 1. Lipoic acid-dependent Akt phosphorylation and inhibition of NF-kappaB activity were abolished by the PI3K inhibitors LY294002 and wortmannin. Furthermore, LA treatment of LPS-exposed C57BL/6N mice strongly enhanced phosphorylation of Akt and glycogen synthase kinase 3beta in blood cells; inhibited the LPS-induced increase in serum concentrations and/or tissue expression of adhesion molecules, monocyte chemoattractant protein 1, and TNFalpha; and attenuated NF-kappaB activation in lung, heart, and aorta. Lipoic acid also improved survival of endotoxemic mice. All of these antiinflammatory effects of LA were abolished by treatment of the animals with wortmannin. We conclude that LA inhibits LPS-induced monocyte activation and acute inflammatory responses in vitro and in vivo by activating the PI3K/Akt pathway. Lipoic acid may be useful in the prevention of sepsis and inflammatory vascular diseases.     

p-Akt和P27的生物学特性及其与鼻咽癌关系的研究进展

蛋白激酶B（PKB／Akt）磷酸化后激活为p-Akt,可以促进细胞存活、抑制细胞凋亡,并调控与细胞周期相关的蛋白,磷酸肌醇3-激酶（P13K）／AKT信号转导通路促进恶性肿瘤转移。P27蛋白作为细胞周期负性调控因子,能抑制各种细胞周期蛋白和激酶的活性。P27表达下降或者缺失,促进了肿瘤的增殖和侵袭。两者都在鼻咽癌的发病和转移中起作用,并对预后有影响。     

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.     

Antitumor activity and drug interactions of proteasome inhibitor Bortezomib in human high-risk myelodysplastic syndrome cells

The purpose of this study was to investigate the antitumor effects and drug interactions of the proteasome inhibitor Bortezomib against high-risk myelodysplastic syndrome (MDS) cells in vitro and in vivo. The high-risk MDS-derived MUTZ-1 cell line and bone marrow mononuclear cells from primary high-risk MDS patients were used to examine antitumor activity and drug interactions for Bortezomib. Apoptotic proteins, including caspase and Bcl-2 family members, as well as the protein FLIP, were studied. Phosphoinositide 3-kinase (PI3K)/Akt and MAPK signaling pathways were also examined. The PI3K inhibitor LY294002 was used to examine the involvement of the PI3K/Akt signaling pathway in the induction of apoptosis. Cytarabine (AraC) and daunorubicin (DNR) were used to test for synergistic effects between Bortezomib and chemotherapeutic agents. SCID mice xenografted with MUTZ-1 cells were used for in vivo study. We found that Bortezomib could induce growth arrest and apoptosis in high-risk MDS cells in vitro and in vivo. The mechanisms were related to decreased activation of the PI3K/Akt survival signaling pathway, but not the MAPK pathway, and involved inhibition of the NF-κB activity and downregulation of the Bcl-2/Bax and FLIPL/FLIPS ratios, triggering the activation of caspase cascades. This phenomenon was inhibited by the PI3K inhibitor LY294002. Bortezomib also acted synergistically with the chemotherapeutic agents AraC and DNR, which are associated with the inhibition of NF-κB activity. Our results demonstrate that Bortezomib can induce growth arrest and apoptosis of high-risk MDS cells and had a synergistic effect with two chemotherapeutic agents. Our findings provide new insights for the treatment of high-risk MDS, using either Bortezomib alone, or in combination with conventional antineoplastic agents.     

An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice

PTEN phosphatase acts as a tumor suppressor by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. It is unclear which downstream components of this pathway are necessary for oncogenic transformation. In this report we show that transformed cells of PTEN^+/− mice have elevated levels of phosphorylated Akt and activated p70/S6 kinase associated with an increase in proliferation. Pharmacological inactivation of mTOR/RAFT/FRAP reduced neoplastic proliferation, tumor size, and p70/S6 kinase activity, but did not affect the status of Akt. These data suggest that p70/S6K and possibly other targets of mTOR contribute significantly to tumor development and that inhibition of these proteins may be therapeutic for cancer patients with deranged PI3K signaling.     

PPIs抑制P13K/Akt／mTOR信号通路逆转胃癌细胞的化疗多药耐药

背景：前期实验显示质子泵抑制剂（PPIs）可抑制空泡型质子泵（V-H＋-ATPases）和多药耐药蛋白P—gP、MRP1表达,增强胃癌细胞的化疗敏感性。目的：探讨PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药与P13K／Akt／mTOR信号通路的关系。方法：应用不同浓度埃索美拉唑或泮托拉唑预处理人胃腺癌细胞敏感株SGC7901和多药耐药株SGC7901／MDR,或以V—H＋-ATPasessiRNA干扰SGC7901／MDR细胞内的V-H＋-ATPases表达,或以雷帕霉素阻断mTOR表达,以蛋白质印迹法检测经不同方式处理的细胞内V—H＋ATPases、P—SP、MRPl蛋白表达以及P13K／Akt/mT0R／HIF—1α信号通路及其信号旁路TSCl／2-Rheb中的相关蛋白表达;以免疫荧光法检测经埃索美拉唑预处理的SGC7901／MDR细胞内的V-H＋ATPases、P—gP蛋白表达和定位。结果：PPIs可呈浓度依赖性地抑制SGC7901／MDR细胞内的V—H＋ATPases、P13K、Akt、roTOR、HIF-1仅、TSCI、TSC2、Rheb、P—gP、MRP1表达以及Akt底物和TSC2磷酸化,改变V-H＋ATPases、P—gP的胞内定位,对SGC7901细胞则无上述影响。以V—H＋-ATPasessiRNA抑制SGC7901／MDR细胞内的V—H＋-ATPases表达,作用与PPIs预处理相似。以雷帕霉素阻断mTOR可呈浓度依赖性地抑制SGC7901／MDR细胞内的HIF-1α、P—gP表达。结论：PPIs抑制空泡型质子泵逆转胃癌细胞化疗多药耐药的机制与抑制P13K／Akt／mTOR信号通路有关。     

Akt和胞外信号调节激酶通路间的信号交流对内质网应激条件下肝癌细胞周期的调控

目的 研究内质网应激介导的磷脂酰肌醇3激酶(PI3K)/Akt和丝裂原活化蛋白激酶(MEK)/胞外信号调节激酶(ERK)途径间的信号交流及其对内质网应激条件下肝癌细胞周期的调控作用.方法 采用PI3K抑制剂LY294002、Akt激活型突变载体myr-Akt和MEK抑制剂U0126分别阻断或激活内质网应激介导的Akt和ERK活化,并利用Western blot和流式细胞技术分析内质网应激条件下PI3K/Akt和MEK/ERK途径间的信号交流及其对肝癌细胞株SMMC-7721、Hep3B和HepG2细胞周期的调控作用.数据处理采用Sperman等级相关分析,P＜0.05为差异有统计学意义.结果 阻断PI3K/Akt明显促进内质网应激介导的MEK/ERK活化,而过度激活PI3K/Akt则抑制内质网应激介导的MEK/ERK活化.阻断MEK/ERK对内质网应激介导的PI3K/Akt活化无影响.持续活化的Akt突变载体myr-Akt和MEK抑制剂U0126均明显抑制了内质网应激诱导的压力细胞G0/G1期阻滞.结论 PI3K/Akt和MEK/ERK信号途径在内质网应激肝癌细胞中存在信号交流,该信号交流对细胞周期起重要调控作用.     

Interleukin 3-dependent survival by the Akt protein kinase

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.     

Cardioprotective stimuli mediate phosphoinositide 3-kinase and phosphoinositide dependent kinase 1 nuclear accumulation in cardiomyocytes

The phosphoinositide 3-kinase (PI3K)/phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K/PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K/PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt⁴⁷³ show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.     

PI3K/Akt/mTOR信号转导通路在肝细胞癌发生发展中的作用

PI3K/Akt/mTOR信号转导通路可以通过调控基因表达,在肝细胞癌肿瘤细胞生成、增殖、转移和凋亡等过程中发挥重要作用。简述了PI3K/Akt/mTOR信号通路的组成及功能,对PI3K/Akt/mTOR通路在肝细胞癌进程中的作用机制及相关抑制剂的研究进展进行了综述,揭示阻断PI3K/Akt/mTOR通路可以成为肝细胞癌治疗新的靶点方向。     

HCV core protein promotes heparin binding EGF‐like growth factor expression and activates Akt

Aims: Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction and is closely associated with the development of human hepatocellular carcinoma (HCC). Among HCV components, core protein is implicated in cell growth regulation, and we previously demonstrated that HCV core protein interacted with 14‐3‐3 protein and activated the kinase Raf‐1 and mitogen‐activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway. In the present study, we investigated the expression levels and function of downstream molecules in the MAPK/ERK signaling pathway in cells expressing HCV core protein. Method: Heparin‐binding EGF‐like growth factor (HB‐EGF) mRNA, in HepG2 cells stably expressing HCV core protein, was detected by RT‐PCR. The soluble HB‐EGF in culture media was measured by heparin agarose chromatography/Western blot analysis. Immunodetection of Akt and IKK and IB, in HeLa cells and HepG2 cells expressing HCV core protein, were performed with neutralizing antibody for HB‐EGF, phospatidylinositol‐3‐kinase [PI(3)K] inhibitor and dominant‐negative mutant of Ras (DN‐Ras). Results: HB‐EGF expression was significantly elevated in cells expressing HCV core protein. HCV core protein activated Akt through the Ras/PI(3)K pathway by autocrine secretion of HB‐EGF. Also, HCV core protein activated IKK through Ras/PI(3)K/Akt pathway by autocrine secretion of HB‐EGF. As the Ras/PI(3)K/Akt pathway is critical in anti‐apoptotic HB‐EGF signaling, we examined the possible role of this pathway in cells expressing HCV core protein. In addition, we investigated the relationship between IB kinases (IKK) and Akt in cells expressing HCV core protein, since IKKs are known to be activated by HCV core protein and by Akt in the presence of potent mitogen. We showed that HCV core protein promoted autocrine secretion of HB‐EGF and activated Akt through the Ras/PI(3)K pathway. This model indicates a new approach to mechanism of proliferation and anti‐apoptosis in HCC. Conclusion: HCV core protein is a potent activator of mitogenic and anti‐apoptotic signaling involved in hepatocarcinogenesis.