PI3K信号通路通过Skp2、p27调节肝癌细胞的增殖

探讨磷脂酰肌醇3-激酶(PI3K)信号通路调节肝癌细胞增殖的机制.用LY294002特异性阻断PI3K信号通路后,人肝癌细胞(SMMC-7721)的增殖明显被抑制.RT-PCR及蛋白质印迹结果显示,LY294002增加了p27蛋白的表达,但不影响p27的mRNA表达.在LY294002处理的细胞中转入p27的RNAi质粒以干扰p27蛋白的表达后,肝癌细胞的增殖能力可部分恢复.放线菌酮(Chx)处理实验表明,阻断PI3K信号通路使p27蛋白的半衰期延长,稳定性增加.进一步研究发现,LY294002可抑制介导p27蛋白降解的关键分子Skp2的mRNA表达,还可缩短Skp2蛋白的半衰期,降低Skp2蛋白的稳定性.但在SMMC-7721中分别转染PI3K下游重要靶分子Akt的持续激活和失活突变体,却并不影响p27蛋白的表达.这些结果表明,PI3K信号通路在转录及翻译后水平调节Skp2的表达而影响p27蛋白的降解,从而调节肝癌细胞的增殖,但Akt并没有参与这种调节.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression

Following a fibrogenic stimulus, the hepatic stellate cell (HSC) undergoes a complex activation process associated with increased cell proliferation and excess deposition of type I collagen. The focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway is activated by platelet-derived growth factor (PDGF) in several cell types. We investigated the role of the FAK-PI3K-Akt pathway in HSC activation. Inhibition of FAK activity blocked HSC migration, cell attachment, and PDGF-induced PI3K and Akt activation. Both serum- and PDGF-induced Akt phosphorylation was inhibited by LY294002, an inhibitor of PI3K. A constitutively active form of Akt stimulated HSC proliferation in serum-starved HSCs, whereas LY294002 and dominant-negative forms of Akt and FAK inhibited PDGF-induced proliferation. Transforming growth factor-beta, an inhibitor of HSC proliferation, did not block PDGF-induced Akt phosphorylation, suggesting that transforming growth factor-beta mediates its antiproliferative effect downstream of Akt. Expression of type I collagen protein and alpha1(I) collagen mRNA was increased by Akt activation and inhibited when PI3K activity was blocked. Therefore, FAK is important for HSC migration, cell attachment, and PDGF-induced cell proliferation. PI3K is positioned downstream of FAK. Signals for HSC proliferation are transduced through FAK, PI3K, and Akt. Finally, expression of type I collagen is regulated by the PI3K-Akt signaling pathway.     

IGF-I-induced oligodendrocyte progenitor proliferation requires PI3K/Akt, MEK/ERK, and Src-like tyrosine kinases

Insulin-like growth factor-I (IGF-I) is required for the growth of oligodendrocytes, although the underlying mechanisms are not fully understood. Our aim was to investigate the role of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK1), and Src family tyrosine kinases in IGF-I-stimulated proliferation of oligodendrocyte progenitors. IGF-I treatment increased the proliferation of cultured oligodendrocyte progenitors as determined by measuring incorporation of [(3)H]-thymidine and bromodeoxy-uridine (BrdU). IGF-I stimulated a transient phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK1) and extracellular signal-regulated kinases (ERK1/2) (targets of MEK1), as well as a rapid and sustained activation of Akt (a target of PI3K). Furthermore, inhibitors of PI3K (LY294002 and Wortmannin), MEK1 (PD98059 and U0126), and Src family tyrosine kinases (PP2) decreased IGF-I-induced proliferation, and blocked ERK1/2 activation. LY294002, Wortmannin and PP2 also blocked Akt activation. To further determine whether Akt is required for IGF-I stimulated oligodendrocyte progenitor proliferation, cultures were infected with adenovirus vectors expressing dominant-negative mutants of Akt or treated with pharmacological inhibitors of Akt. All treatments reduced IGF-I-induced oligodendrocyte progenitor proliferation. Our data indicate that stimulation of oligodendrocyte progenitor proliferation by IGF-I requires Src-like tyrosine kinases as well as the PI3K/Akt and MEK1/ERK signaling pathways.     

低浓度哇巴因对负鼠肾小管上皮细胞增殖的影响

目的:用低血清培养液来模拟肾脏供血不足的营养不良状态,研究低浓度哇巴因对低血清培养下OK细胞(负鼠肾小管上皮细胞)增殖的影响。方法:用低浓度哇巴因(1-30n M)处理0.2%血清培养下OK细胞,MTT实验和Brdu掺入法检测哇巴因对OK细胞增殖的影响;Western blot检测Akt和ERK1/2的磷酸化水平;用LY294002和PD98059分别抑制PI3K/Akt和ERK1/2蛋白激酶活性,观察抑制PI3K/Akt和ERK1/2对哇巴因促进OK细胞增殖的影响。结果:低浓度哇巴因(1-30n M)促进OK细胞的增值,上调OK细胞中Akt和ERK1/2磷酸化水平。用LY294002和PD98059特异抑制Akt和ERK1/2的活化能够抑制哇巴因的促增殖作用。结论:低浓度哇巴因(1-10n M)能够促进OK细胞的增值,PI3K/Akt和ERK1/2信号通路参与哇巴因对OK细胞促增殖作用的调节。     

Phosphatidylinositol 3-kinase/Akt auto-regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts

It has been reported that platelet-derived growth factor (PDGF)-BB stimulates the synthesis of interleukin (IL)-6 in osteoblasts. In the present study, we investigated whether the phosphatidylinositol 3-kinase (PI3K)/Akt is involved in the PDGF-BB-induced IL-6 synthesis in osteoblast-like MC3T3-E1 cells. PDGF-BB markedly induced the phosphorylation of Akt and GSK-3beta. Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, significantly amplified the synthesis of IL-6 by PDGF-BB. The PDGF-BB-induced GSK-3beta phosphorylation was suppressed by the Akt inhibitor. The IL-6 synthesis stimulated by PDGF-BB was markedly enhanced by LY294002 and wortmannin, inhibitors of PI3K. Wortmannin and LY294002 suppressed the PDGF-BB-induced phosphorylation of Akt and GSK-3beta. Taken together, these results strongly suggest that PI3K/Akt negatively regulates the PDGF-BB-stimulated IL-6 synthesis in osteoblasts.     

Prolonged high-glucose exposure decreased SREBP-1/FASN/ACC in Schwann cells of diabetic mice via blocking PI3K/Akt pathway

Abnormal lipid metabolism and SREBP-1 downregulation are reported to be involved in the pathogenesis of diabetic peripheral neuropathy (DPN). In the current study, the relationship between PI3K/Akt signaling pathway and SREBP-1 expression was explored in Schwann cells of DPN. The phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression were inhibited in the sciatic nerves of diabetic mice versus those of normal mice, accompanied with the atrophy of nerve fiber and the irregular myelin sheath. High concentration glucose suppressed phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression in cultured Schwann cell (RSC96 cell) in vitro, and 25 mmol/L glucose was enough to lead to the maximum inhibitory effect. The time-course effect of high glucose showed that Akt phosphorylation gradually decreased with the extension of stimulation time. Somewhat differently, short-term high-glucose exposure enhanced SREBP-1 expression and prolonged high-glucose stimulation reduced the SREBP-1 expression in RSC96 cells. Similarly, prolonged high-glucose stimulation also downregulated FASN messenger RNA (mRNA), ACC mRNA, intracellular triglyceride, and cholesterol. LY294002 suppressed Akt activation followed by the decreased SREBP-1, FASN, ACC, triglyceride, and cholesterol. Contrarily, the PI3K/Akt signaling pathway agonist insulin pretreatment avoided prolonged high-glucose stimulation-blocked Akt activation and increased SREBP-1, FASN, and ACC expression in the levels of protein and mRNA in RSC96 cells. The knockdown of SREBP-1 by shRNA prevented insulin-induced enhanced FASN, ACC mRNA expression, triglyceride, and cholesterol in high-glucose-treated RSC96 cells. In conclusion, prolonged high-glucose exposure inhibits the SREBP-1/FASN/ACC expression in the Schwann cells of DPN via the blockage of the PI3K/Akt signaling pathway.     

Glimepiride Promotes Osteogenic Differentiation in Rat Osteoblasts via the PI3K/Akt/eNOS Pathway in a High Glucose Microenvironment

Our previous studies demonstrated that glimepiride enhanced the proliferation and differentiation of osteoblasts and led to activation of the PI3K/Akt pathway. Recent genetic evidence shows that endothelial nitric oxide synthase (eNOS) plays an important role in bone homeostasis. In this study, we further elucidated the roles of eNOS, PI3K and Akt in bone formation by osteoblasts induced by glimepiride in a high glucose microenvironment. We demonstrated that high glucose (16.5 mM) inhibits the osteogenic differentiation potential and proliferation of rat osteoblasts. Glimepiride activated eNOS expression in rat osteoblasts cultured with two different concentrations of glucose. High glucose-induced osteogenic differentiation was significantly enhanced by glimepiride. Down-regulation of PI3K P85 levels by treatment with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor) led to suppression of P-eNOS and P-AKT expression levels, which in turn resulted in inhibition of RUNX2, OCN and ALP mRNA expression in osteoblasts induced by glimepiride at both glucose concentrations. ALP activity was partially inhibited by 10 µM {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Taken together, our results demonstrate that glimepiride-induced osteogenic differentiation of osteoblasts occurs via eNOS activation and is dependent on the PI3K/Akt signaling pathway in a high glucose microenvironment.     

Insulin-like growth factor-I-induced androgen receptor activation is mediated by the PI3K/Akt pathway in C2C12 skeletal muscle cells

Although insulin-like growth factor-I (IGF-I) and androgen receptor (AR) are well known effectors of skeletal muscle, the molecular mechanism by which signaling pathways integrating AR and IGF-I in skeletal muscle cells has not been previously examined. In this study, the role of PI3K/Akt on IGF-I-induced gene expression and activation of AR in skeletal muscle cells was investigated. C2C12 cells were treated with IGF-I in the absence or presence of inhibitors of PI3K/Akt pathway (LY294002 and Wortmannin). Inhibition of the PI3K/Akt pathway with LY294002 or Wortmannin led to a significant decrease in IGF-I-induced AR phosphorylation and total AR protein expression. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by LY294002 or Wortmannin. Confocal images showed that IGF-I-induced AR translocation from cytosol to nucleus was inhibited significantly in response to treatment with LY294002 or Wortmannin. The present results suggest that modulating effect of IGF-I on AR gene expression and activation in C2C12 mouse skeletal muscle cells is mediated at least in part by the PI3K/Akt pathway.     

Growth hormone-stimulated insulin-like growth factor-1 expression in rainbow trout (Oncorhynchus mykiss) hepatocytes is mediated by ERK, PI3K-AKT, and JAK-STAT

Growth hormone (GH) initiates many of its growth-promoting actions by binding to GH receptors (GHR) and stimulating the synthesis and secretion of insulin-like growth factor-1 (IGF-1) from the liver and other sites. In this study, we used hepatocytes isolated from rainbow trout as a model system in which to determine the molecular signaling events of GH in fish. GH directly stimulated the phosphorylation of ERK, protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), JAK2, and STAT5 in hepatocytes incubated in vitro. Activation of ERK, Akt, JAK2, and STAT5 was rapid, occurring within 5-10 min, and was concentration dependent. GH-induced ERK activation was completely blocked by the ERK pathway inhibitor, U0126, and the JAK2 inhibitor, 1,2,3,4,5,6-hexabromocyclohexane (Hex), and was partially blocked by the PI3K inhibitor LY294002. GH-stimulated Akt activation was completely blocked by LY294002 and Hex, but was not affected by U0126; whereas, STAT5 activation by GH was blocked only by Hex, and was not affected by either U0126 or LY294002. GH stimulated hepatic expression of IGF-1 mRNA as well as the secretion of IGF-1, effects that were partially or completely blocked by U0126, LY294002, and Hex. These results indicate that GHR linkage to the ERK, PI3K/Akt, or STAT pathways in trout liver cells requires activation of JAK2, and that GH-stimulated IGF-1 synthesis and secretion is mediated through the ERK, PI3K/Akt, and JAK-STAT pathways.     

PI3K/Akt pathway mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells through regulation of SREBP-1 and TGF-β1

Previous studies have shown that high glucose stimulates renal SREBP-1 gene expression and increases renal tubular cells lipid metabolism, however, the mechanisms remain elusive. In the present study we demonstrated that PI3K/Akt pathway was activated in human renal proximal tubular cell line (HKC) exposed to high glucose accompanied with up-regulation of SREBP-1, TGF-β1, lipid droplets deposits and extracellular matrix production. Inhibition of PI3K/Akt pathway by chemical LY294002 or specific short hairpin RNA (shRNA) vector prevented SREBP-1 and TGF-β1 up-regulation, as well as ameliorated HKC cells lipogenesis and extracellular matrix accumulation. These findings indicate that PI3K/Akt pathway potentially mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells.     

A positive role of the PI3-K/Akt signaling pathway in PC12 cell differentiation

Activation of phosphatidylinositol 3-kinase (PI3-K) is considered to be a key event upon stimulation of cells with growth factors. Akt is known to be a downstream target of PI3-K when it is activated by nerve growth factor (NGF). NGF induces cell differentiation of PC12 cells as indicated by neurite outgrowth. In order to investigate the role of PI3-K/Akt in NGF-induced differentiation of PC12 cells, we generated cells ectopically expressing constitutively activated (CA), wild type (WT) and dominant negative (DN) forms of Akt. NGF-induced neurite outgrowth was greatly accelerated in the cells expressing CA-Akt, and dramatically inhibited in those expressing DN-Akt. Pre-treatment with an Akt inhibitor, ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine], inhibited NGF-induced Akt phosphorylation as well as neurite outgrowth but did not markedly affect the activities of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The PI3-K inhibitors wortmannin and LY294002 blocked NGF-induced Akt phosphorylation as well as neurite outgrowth. These results indicate that PI3-K/Akt is a positive regulator of NGF-induced neuronal differentiation in PC12 cells.     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.     

The PI3 kinase-Akt pathway mediates Wnt3a-induced proliferation

Wnt3a activates proliferation of fibroblasts cells via activation of both extracellular signal-regulated kinase (ERK) and Wnt/beta-catenin signaling pathways. In this study, we show that the phosphatidyl inositol 3 kinases (PI3K)-Akt pathway is also involved in the Wnt3a-induced proliferation. Akt was activated within 30 min by Wnt3a in NIH3T3 cells. By Wnt3a treatment, activated Akt was transiently accumulated in nucleus although beta-catenin was accumulated in the nucleus of cells in a prolonged manner. The Wnt3a-induced Akt activation was not affected by siRNA-mediated reduction of beta-catenin, indicating that Wnt3a-induced Akt activation may occur independently of beta-catenin. The Wnt3a-induced Akt activation was abolished by pre-treatment with PI3K inhibitor, LY294002 and Wortmanin, but not by MEK inhibitor, U0126, indicating that Wnt3a activates Akt via PI3K. The growth and proliferation induced by Wnt3a were blocked by treatments of the PI3K inhibitors. Furthermore, Wnt3a-induced proliferation was blocked by Akt siRNA. These results reveal that the PI3K-Akt pathway mediates the Wnt3a-induced growth and proliferation of NIH3T3 cells.     

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr³⁰⁸), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser⁴¹⁷), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser²⁵⁶). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.