Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

Hydrogen peroxide and ADP-ribose induce TRPM2-mediated calcium influx and cation currents in microglia

Microglial cells are the host macrophages in the central nervous system and respond to brain injury and various neurological diseases. In this process, microglial cells undergo multiple morphological and functional changes from the resting cell toward a fully activated, phagocyting tissue macrophage. In culture, bacterial lipopolysaccharide (LPS) is a frequently used tool to induce this activation. By using calcium-imaging and patch-clamp techniques, we investigated the effect of hydrogen peroxide (H₂O₂), which is released by macrophagic cells themselves, on the intracellular calcium concentration and ion currents in cultured rat microglia. Application of 0.1–5 mM H₂O₂ for several minutes induced small responses in untreated cells but a large calcium influx and cation current in LPS-treated cells. In both untreated and LPS-treated microglia, internal perfusion of ADP-ribose (ADPR) via the patch pipette elicited large cation currents. Both stimuli, H₂O₂ and ADPR, have been reported to activate the recently cloned nonselective cation channel TRPM2. RT-PCR analysis from cultured rat glial and neuronal cells confirmed a strong expression of TRPM2 in rat microglia but not in astrocytes and cerebellar granule cells. In situ hybridizations from mouse brain showed a distribution of TRPM2, which is compatible with the expression in microglial cells. In conclusion, we describe here a novel calcium influx pathway in microglia coupled to hydrogen peroxide and ADPR and provide evidence that this pathway involves TRPM2. The increased sensitivity to H₂O₂ in LPS-stimulated cells suggests a role for TRPM2 in the calcium signaling of activated microglia. nonselective cation channel; transient receptor potential channel; H₂O₂; activated microglia     

Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

Phosphorylation of GRK2 by PKA augments GRK2-mediated phosphorylation, internalization, and desensitization of VPAC2 receptors in smooth muscle

The smooth muscle of the gut expresses mainly G(s) protein-coupled vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptors (VPAC(2) receptors), which belong to the secretin family of G protein-coupled receptors. The extent to which PKA and G protein-coupled receptor kinases (GRKs) participate in homologous desensitization varies greatly among the secretin family of receptors. The present study identified the novel role of PKA in homologous desensitization of VPAC(2) receptors via the phosphorylation of GRK2 at Ser(685). VIP induced phosphorylation of GRK2 in a concentration-dependent fashion, and the phosphorylation was abolished by blockade of PKA with cell-permeable myristoylated protein kinase inhibitor (PKI) or in cells expressing PKA phosphorylation-site deficient GRK2(S685A). Phosphorylation of GRK2 increased its activity and binding to G betagamma. VIP-induced phosphorylation of VPAC(2) receptors was abolished in muscle cells expressing kinase-deficient GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. VPAC(2) receptor internalization (determined from residual (125)I-labeled VIP binding and receptor biotinylation after a 30-min exposure to VIP) was blocked in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A) or by PKI. Finally, VPAC(2) receptor degradation (determined from residual (125)I-labeled VIP binding and receptor expression after a prolonged exposure to VIP) and functional VPAC(2) receptor desensitization (determined from the decrease in adenylyl cyclase activity and cAMP formation after a 30-min exposure to VIP) were abolished in cells expressing GRK2(K220R) and attenuated in cells expressing GRK2(S685A). These results demonstrate that in gastric smooth muscle VPAC(2) receptor phosphorylation is mediated by GRK2. Phosphorylation of GRK2 by PKA enhances GRK2 activity and its ability to induce VPAC(2) receptor phosphorylation, internalization, desensitization, and degradation.     

Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway.

It is well established that insulin and serum stimulate gene expression at the level of mRNA translation in animal cells, and previous studies have mainly focused on the initiation process. Here we show that, in Chinese hamster ovary cells expressing the human insulin receptor, insulin causes decreased phosphorylation of elongation factor eEF-2 and that this is associated with stimulation of the rate of peptide-chain elongation. eEF-2 is phosphorylated by a very specific Ca 2+/calmodulin-dependent protein kinase (eEF-2 kinase) causing its complete inactivation. The decrease in eEF-2 phosphorylation induced by insulin reflects a fall in eEF-2 kinase activity. Rapamycin, a macrolide immunosuppressant which blocks the signalling pathway leading to the stimulation of the 70/85 kDa ribosomal protein S6 kinases, substantially blocks the activation of elongation, the fall in eEF-2 phosphorylation and the decrease in eEF-2 kinase activity, suggesting that p7O S6 kinase (p70s6k) and eEF-2 kinase may tie on a common signalling pathway. Wortmannin, an inhibitor of phosphatidylinositide-3-OH kinase, had similar effects. eEF-2 kinase was phosphorylated in vitro by purified p70s6k but this had no significant effect on the in vitro activity of eEF-2 kinase.     

RACK1 mRNA translation is regulated via a rapamycin-sensitive pathway and coordinated with ribosomal protein synthesis

RACK1 has been shown to interact with several proteins, this suggesting that it may play a central role in cell growth regulation. Some recent articles have described RACK1 as a component of the small ribosomal subunit. To investigate the relationship between RACK1 and ribosome, we analyzed RACK1 mRNA structure and regulation. Translational regulation was studied in HeLa cells subjected to serum or amino acid deprivation and stimulation. The results show that RACK1 mRNA has a 5' terminal oligopyrimidine sequence and that its translation is dependent on the availability of serum and amino acids in exactly the same way as any other vertebrate ribosomal protein mRNA.     

Hydrogen peroxide stimulates the epithelial sodium channel through a phosphatidylinositide 3-kinase-dependent pathway

Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H(2)O(2)). In the cell-attached configuration, H(2)O(2) significantly increased ENaC open probability (P(o)) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H(2)O(2) are required to elevate intracellular H(2)O(2), probably because catalase, the enzyme that promotes the decomposition of H(2)O(2) to H(2)O and O(2), is highly expressed in A6 cells. The effect of H(2)O(2) on ENaC P(o) was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H(2)O(2) levels are critical to produce the effect. However, H(2)O(2) did not directly activate ENaC in inside-out patches. The effects of H(2)O(2) on ENaC P(o) and amiloride-sensitive Na(+) current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H(2)O(2) elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P(3), these data suggest that H(2)O(2) stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P(3).     

hPER3 promotes adipogenesis via hHSP90AA1-mediated inhibition of Notch1 pathway

The period circadian regulator 3 (PER3) has been reported to play a negative role in human immortalized bone marrow-derived Scp-1 cells (iBMSCs) and patient adipose-derived stromal cells (PASCs) or a negative/positive role in mice adipogenesis. However, human PER3 (hPER3) was identified as a positive regulator of human adipose tissue-derived stromal cells (hADSCs) adipogenesis in this study. Silencing or overexpression of hPER3 in hADSCs inhibited and promoted adipogenesis in vitro. In vivo, the overexpression of hPER3 increased high-fat diet-induced inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) forms, increasing systemic glucose intolerance and insulin resistance. Molecularly, hPER3 does not interact with hPPARγ, but represses Notch1 signaling pathway to enhance adipogenesis by interacting with hHSP90AA1, which is able to combine with the promoter of hNotch1 and inactivate its expression. Thus, our study revealed hPER3 as a critical positive regulator of hADSCs adipogenesis, which was different from the other types of cells, providing a critical role of it in treating obesity.Subject terms: Molecular biology, Obesity     

Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic beta-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic beta-cell lines (betaTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a beta-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gbetagamma subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and beta-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic beta-cells.     

GRK2 negatively regulates IGF‐1R signaling pathway and cyclins' expression in HepG2 cells

G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin‐like growth factor 1 receptor (IGF‐1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF‐1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF‐1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF‐1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF‐1R signaling pathway and cyclins' expression. J. Cell. Physiol. 228: 1897–1901, 2013. © 2013 Wiley Periodicals, Inc.     

Proinflammation effect of Mst1 promotes BV-2 cell death via augmenting Drp1-mediated mitochondrial fragmentation and activating the JNK pathway

Inflammation has been increasingly studied as part of the pathophysiology of neurodegenerative diseases. Mammalian Ste20-like kinase 1 (Mst1), a key factor of the Hippo pathway, is connected to cell death. Unfortunately, little study has been performed to detect the impact of Mst1 in neuroninflammation. The results indicated that Mst1 expression was upregulated because of LPS treatment. However, the loss of Mst1 sustained BV-2 cell viability and promoted cell survival in the presence of LPS treatment. Molecular investigation assay demonstrated that Mst1 deletion was followed by a drop in the levels of mitochondrial fission via repressing Drp1 expression. However, Drp1 adenovirus transfection reduced the protective impacts of Mst1 knockdown on mitochondrial stress and neuronal dysfunction. Finally, our results illuminated that Mst1 affected Drp1 content and mitochondrial fission in a JNK-dependent mechanism. Reactivation of the JNK axis inhibited Mst1 knockdown-mediated neuronal protection and mitochondrial homeostasis. Altogether, our results indicated that Mst1 upregulation and the activation of JNK-Drp1-mitochondrial fission pathway could be considered as the novel mechanism regulating the progression of neuroninflammation. This finding would pave a new road for the treatment of neurodegenerative diseases via modulating the Mst1-JNK-Drp1-mitochondrial fission axis.     

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCβ2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCβ2/Ca2+ signal transduction in endothelial cells.     

Hydrogen peroxide regulates glucose‐regulated protein 78 expression via a cyclooxygenase‐2 dependent mechanism

This study was designed to investigate the effect of hydrogen peroxide on the expression of endoplasmic reticulum stress marker glucose‐regulated protein 78 (GRP78) in endothelial cells and reveals the possible role of cyclooxygenase in this effect. The porcine endothelial cell line was cultured in 1640 medium. Western blot and immunocytochemistry were used to detect the expression of GRP78. The caspase‐12 activity was analyzed with the immune fluorescence method. The results showed that after the endothelial cells were incubated with 250 μM of hydrogen peroxide for 12 h, apoptosis increased, which was antagonized by the cyclooxygenase‐2 inhibitor nimesulide or the nonselective cyclooxygenase inhibitor aspirin, but not by the cyclooxygenase‐1 inhibitor piroxicam. The expression of GRP78 was induced in endothelial cells after exposure to hydrogen peroxide for 12 h. The overexpression of GRP78 was inhibited by nimesulide and aspirin, but not by piroxicam. There are no significant differences in caspase‐12 activity among all groups. The present study provides evidence that hydrogen peroxide induced GRP78 overexpression in endothelial cells by a mechanism involving cyclooxygenase‐2‐dependent pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:279–285, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20336     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis.

Protein phosphatases 2C (PP2Cs) exhibit diverse regulatory functions in signalling pathways of animals, yeast and plants. ABI1 is a PP2C of Arabidopsis that exerts negative control on signalling of the phytohormone abscissic acid (ABA). Characterisation of the redox sensitivity of ABI1 revealed a strong enzymatic inactivation by hydrogen peroxide (H2O2) which has recently been implicated as a secondary messenger of ABA signalling. H2O2 reversibly inhibited ABI1 activity in vitro with an IC(50) of approximately 140 microM in the presence of physiological concentrations of glutathione. In addition, ABI1 was highly susceptible to inactivation by phenylarsine oxide (IC(50)=3-4 microM) indicative for the facile oxidation of vicinal cysteine residues. Thus, H2O2 generated during ABA signalling seems to inactivate the negative regulator of the ABA response.     

Endothelin-1 impairs glucose transporter trafficking via a membrane-based mechanism

Endothelin-1 (ET-1) disrupts insulin-regulated glucose transporter GLUT4 trafficking. Since the negative consequence of chronic ET-1 exposure appears to be independent of signal disturbance along the insulin receptor substrate-1/phosphatidylinositol (PI) 3-kinase (PI3K)/Akt-2 pathway of insulin action, we tested if ET-1 altered GLUT4 regulation engaged by osmotic shock, a PI3K-independent stimulus that mimics insulin action. Regulation of GLUT4 by hyperosmotic stress was impaired by ET-1. Because of the mutual disruption of both insulin- and hyperosmolarity-stimulated GLUT4 translocation, we tested whether shared signaling and/or key phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated cytoskeletal events of GLUT4 trafficking were targets of ET-1. Both insulin and hyperosmotic stress signaling to Cbl were impaired by ET-1. Also, plasma membrane PIP2 and cortical actin levels were reduced in cells exposed to ET-1. Exogenous PIP2, but not PI 3,4,5-bisphosphate, restored actin structure, Cbl activation, and GLUT4 translocation. These data show that ET-1-induced PIP2/actin disruption impairs GLUT4 trafficking elicited by insulin and hyperosmolarity. In addition to showing for the first time the important role of PIP2-regulated cytoskeletal events in GLUT4 regulation by stimuli other than insulin, these studies reveal a novel function of PIP2/actin structure in signal transduction.