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.     

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.     

IL-10 synergistically enhances GM-CSF-induced CCR1 expression in myelomonocytic cells

CC chemokine receptor 1 (CCR1) has been implicated in inflammation. The present study examined the signaling mechanisms that mediate GM-CSF/IL-10-induced synergistic CCR1 protein expression in monocytic U937 cells. GM-CSF alone markedly increased both the mRNA and protein expression of CCR1. IL-10 augmented GM-CSF-induced CCR1 protein expression with no effect on mRNA expression. PD098059 and U0126 (two MEK inhibitors), and LY294002 (a PI3K inhibitor) inhibited GM-CSF/IL-10-induced CCR1 gene and protein expression. PD098059, U0126, and LY294002 also attenuated chemotaxis of GM-CSF/IL-10-primed U937 cells in response to MIP-1alpha. Immunoblotting studies show that GM-CSF alone induced ERK2 phosphorylation; whereas, IL-10 alone induced p70(S6k) phosphorylation in U937 cells. Neither cytokine when used alone induced PKB/Akt phosphorylation. Combined GM-CSF/IL-10 treatment of U937 cells induced phosphorylation of ERK2, p70(S6k), and PKB/Akt. PD098059 and U0126 completely abrogated ERK2 phosphorylation; whereas, LY294002 completely blocked PKB/Akt and p70(S6k) phosphorylation. Our findings indicate that IL-10 may potentiate GM-CSF-induced CCR1 protein expression in U937 cells via activation of PKB/Akt and p70(S6k).     

Sphingosine-1-phosphate induces COX-2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1-phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX-2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX-2 expression by S1P in VSMCs remain unclear. Western blotting and RT-PCR analyses showed that S1P induced the expression of COX-2 mRNA and protein in a time- and concentration-dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen-activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX-2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by a selective NF-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB promoter activity was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB pathways was essential for S1P-induced COX-2 gene expression. Understanding the mechanisms involved in S1P-induced COX-2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis.     

Paeoniflorin diminishes ConA-induced IL-8 production in primary human hepatic sinusoidal endothelial cells in the involvement of ERK1/2 and Akt phosphorylation

Liver diseases are closely associated with elevated levels of interleukin-8 (IL-8), suggesting the ability to inhibit IL-8 production could enhance the treatment of liver diseases. Paeoniflorin is a major active constituent of dried Paeoniae Radix Alba root (Baishao in Chinese) which is widely used in China to treat liver diseases. We examined the effects and underlying mechanisms of paeoniflorin on IL-8 production in primary human hepatic sinusoidal endothelial cells (HHSECs). Concanavalin A (ConA) at 20 μg/mL produced a 5.2-fold increase in IL-8 mRNA by 8 h, and a 14.2-fold rise in IL-8 levels by 16 h. Inhibition of MEK (ERK kinase) and extracellular signal-regulated kinase (ERK) by PD98059 and U0126, or inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 blocked both ConA-induced IL-8 mRNA expression and IL-8 secretion. Paeoniflorin reduced ConA-induced IL-8 mRNA expression and IL-8 release by 57.9% and 52.8%, respectively, and also decreased ConA-stimulated phosphorylation of ERK1/2 and Akt, suggesting paeoniflorin inhibits IL-8 expression and release by inhibiting the ERK1/2 and Akt pathways. Combining paeoniflorin with U0126 or LY294002 at low doses showed supra-additive inhibition of not only phospho-ERK1/2 and phospho-Akt by 46.4% and 35.0%, but also IL-8 release by 42.4% and 36.1% and IL-8 mRNA expression by 43.5% and 31.8%, respectively. In conclusion, paeoniflorin most likely contributes to the therapy for liver disease by exerting anti-inflammatory effects on HHSECs through blocking IL-8 secretion via downregulation of ERK1/2 and Akt phosphorylation.     

Different modulation of TRAIL-induced apoptosis by inhibition of pro-survival pathways in TRAIL-sensitive and TRAIL-resistant colon cancer cells

Epithelial cells can be manipulated to undergo apoptosis depending on the balance between pro-survival and apoptotic signals. We showed that TRAIL-induced apoptosis may be differentially regulated by inhibitors of MEK ERK (U0126) or PI3K/Akt (LY294002) pathway in TRAIL-sensitive (HT-29) and TRAIL-resistant (SW620) human epithelial colon cancer cells. U0126 or LY294002 significantly enhanced TRAIL-induced apoptosis in HT-29 cells, but not in SW620 cells. We report a different regulation of the level of an anti-apoptotic Mcl-1 protein under MEK/ERK or PI3K/Akt pathway inhibition and suggest the mechanisms involved. A special attention was paid to the role of the ERK1/2, Akt, and glycogen synthase kinase 3beta.     

PAR2 triggers IL-8 release via MEK/ERK and PI3-kinase/Akt pathways in GI epithelial cells

Proteinase-activated receptor-2 (PAR2) plays pro-inflammatory roles in many organs including the gastrointestinal (GI) tract. To clarify the downstream pro-inflammatory signaling of PAR2 in the GI tract, we examined interleukin-8 (IL-8) release and the underlying cellular signaling following PAR2 stimulation in human colorectal cancer-derived HCT-15 cells and human gastric adenocarcinoma-derived MKN-45 cells. A PAR2-activating peptide, but not a PAR2-inactive scrambled peptide or a PAR1- activating peptide, caused IL-8 release in these GI epithelial cells. The PAR2-triggered IL-8 release was suppressed by inhibitors of MEK (U0126) or PI3-kinase (LY294002), and PAR2 stimulation indeed activated the downstream kinases, ERK and Akt. U0126 blocked the phosphorylation of ERK, but not Akt, and LY294002 blocked the phosphorylation of Akt, but not ERK. Together, PAR2 triggers IL-8 release via two independent signaling pathways, MEK/ERK and PI3-kinase/Akt, suggesting a role of PAR2 as a pro-inflammatory receptor in the GI tract.     

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.     

Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line,HaCaT

Interleukin 15 (IL-15) is a potent stimulator of proliferation and an inhibitor of apoptosis in lymphocytes. We attempted to elucidate the mechanism of IL-15 function in HaCaT keratinocytes. We found that 5-bromo-2(')-deoxyuridine incorporation increased in a dose-dependent manner with IL-15. This was blocked by MEK inhibitor U0126 or PI 3-K inhibitor LY294002. ERK1/2 and Akt phosphorylation by IL-15 were detected in a dose- and time-dependent manner. U0126 and LY294002 abolished ERK1/2 and Akt phosphorylation, respectively. DNA fragmentation and Annexin V binding accompanied by UVB-induced apoptosis were reduced by 30-50% with IL-15. Taken together, IL-15 induced cellular proliferation and had an anti-apoptotic effect on keratinocytes, in which ERK1/2 and Akt phosphorylation played crucial roles. The signal transduction pathways of IL-15 in keratinocytes were partially elucidated; they share a substantial part with growth signals induced by EGF. These results suggest a therapeutic approach to inflammatory skin diseases by controlling these signals.     

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.     

Hypoxia/Reoxygenation Stimulates Proliferation Through PKC-Dependent Activation of ERK and Akt in Mouse Neural Progenitor Cells

Cerebral ischemia increases neural progenitor cell proliferation and neurogenesis. However, the precise molecular mechanism is poorly understood. The present study was undertaken to determine roles of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt and their signaling pathways in neural progenitor cells exposed to hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion. Neural progenitor cells were isolated from postnatal mouse brain. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of MEK, but not by LY294002, a specific inhibitor of PI3K, whereas the Akt activation was blocked by LY294002, but not by U0126. Reoxygenation following 4-h hypoxia stimulated cell proliferation, which was dependent on ERK and Akt activation. Inhibitors of growth factor receptor (AG1478) and Src (PP2) and the antioxidant N-acetylcysteine did not affect activation of ERK and Akt, while the Ras and Raf inhibitors inhibited activation of ERK, but not Akt. PKC inhibitors inhibited both ERK and Akt activation. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt survival signaling pathways through a PKC-dependent mechanism. These pathways may be responsible for the repair process during ischemia/reperfusion.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Human neutrophil peptides (HNP) kill microorganisms but also modulate immune responses through upregulation of the chemokine IL-8 by activation of the nucleotide P2Y(6) receptor. However, the intracellular signaling mechanisms remain yet to be determined. Human lung epithelial cells (A549) and monocytes (U937) were stimulated with HNP in the absence and presence of the specific kinase inhibitors for Src, extracellular signal-regulated kinase-1 and -2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinases (JNK), and Akt. HNP induced a rapid phosphorylation of the kinases in both cell types associated with a dose-dependent, selective production of IL-8 among 10 cytokines assayed. The HNP-induced IL-8 production was blocked by the Src tyrosine kinase inhibitor PP2, MEK1/2 inhibitor U0126, and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002, but not by the JNK inhibitor SP600125 in both cell types. Treatment with the p38 inhibitor SB203580 attenuated the HNP-induced IL-8 production only in monocytes. Blockade of Src kinase blunted HNP-induced phosphorylation of the ERK1/2 and Akt but not p38 in monocytes. In contrast, Src inhibition had no effect on phosphorylation of the other kinases in the lung epithelial cells. We conclude that the activation of ERK1/2 and PI3K/Akt pathways is required for HNP-induced IL-8 release which occurs in a Src-independent manner in lung epithelial cells, while is Src-dependent in monocytes.     

Pertussis toxin activates tyrosine kinase signaling cascade in myelomonocytic cells: a mechanism for cell adhesion

Pertussis toxin (PTX) has recently been shown to specifically bind to CD14 to promote myelomonocytic cell adhesion to serum. The present study investigated the signaling mechanisms responsible for PTX-induced differentiated U937 cell adhesion. PTX-induced myelomonocytic cell adhesion was blocked by genistein or tyrphostin-47 (two protein tyrosine kinase inhibitors), LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor), or PD098059 (a mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor). PTX induced a rapid tyrosine phosphorylation of several discrete cytoplasmic proteins, which could be inhibited by genistein or tyrphostin 47. In addition, PTX induced phosphorylation of Akt and of ERK2, which could be completely blocked by LY294002 and PD098059, respectively, and by genistein or tyrphostin 47 as well. All of these PTX-induced signaling events could be reproduced using purified PTX B-oligomer (PTX-B) alone. Our data show that PTX can activate tyrosine kinase signaling cascade, including the downstream PI3K and ERK/MAPK pathways, in myelomonocytic cells to induce cell adhesion to serum.