Caveolin-1 phosphorylation is required for stretch-induced EGFR and Akt activation in mesangial cells

Increased glomerular hydrostatic pressure is an important determinant of glomerulosclerosis and can be modeled in vitro by exposure of mesangial cells (MC) to cyclic mechanical strain. We have recently shown that Akt mediates the stretch-induced production of type I collagen, an important contributor to sclerosis, in MC. Here we studied the upstream mediators of Akt activation. Primary rat MC were exposed to 1 Hz cyclic strain for 10 min, previously shown to induce maximal Akt activation. Neither the integrin inhibitor GRDGSP nor cytoskeletal disruptors had any effect on stretch-induced Akt activation. Akt activation was, however, mediated by transactivation of the epidermal growth factor receptor (EGFR), and this required receptor kinase activity since Akt activation did not occur in cells expressing kinase-dead EGFR (K721A). Src was further shown to be upstream of the EGFR, with its inhibitor SU6656 preventing both EGFR and Akt activation. The membrane microdomains caveolae were found to be required for this signaling to occur. Chemical disruption of caveolae with cyclodextrin or filipin prevented Akt activation, and both EGFR and Akt activation were lost in caveolin-1 (cav-1) knockout MC. The latter was rescued with reexpression of cav-1. Further, Src-mediated phosphorylation of cav-1 on Y14 was required for stretch-induced EGFR and Akt activation, since these were abrogated in MC expressing the nonphosphorylatable cav-1 Y14A mutant. Thus, mechanical strain-induced activation of Akt in MC is independent of integrin activation and the actin cytoskeleton, but depends upon EGFR transactivation. EGFR transactivation requires intact caveolae and the Src-mediated phosphorylation of cav-1 on Y14. These studies define a novel function for cav-1 and caveolae in EGFR transactivation leading to Akt activation by mechanical stress.     

TACE is required for fetal murine cardiac development and modeling

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.     

EGFR signalling is required for Paracentrotus lividus endomesoderm specification

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-α, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axis was also detected, after AG1478 treatment, by the extended distribution of HE and decreased nuclearization of β-catenin in vegetal cells. Moreover, inhibition of EGFR-like reduced ERK phosphorylation, necessary for cell fate specification in the micromeres and their derivates. Taken together these results indicate that EGFR-like activity is required both for A/V axis formation and endomesoderm differentiation.     

PI3K activation is required for PMA-directed activation of cSrc by AFAP-110

Activation of PKC will induce the cSrc binding partner AFAP-110 to colocalize with and activate cSrc. The ability of AFAP-110 to colocalize with cSrc is contingent on the integrity of the amino-terminal pleckstrin homology (PH1) domain, while the ability to activate cSrc is dependent on the integrity of its SH3 binding motif, which engages the cSrc SH3 domain. The outcome of AFAP-110-directed cSrc activation is a change in actin filament integrity and the formation of podosomes. Here, we address what cellular signals promote AFAP-110 to colocalize with and activate cSrc, in response to PKC activation or PMA treatment. Because PH domain integrity in AFAP-110 is required for colocalization, and PH domains are known to interact with both protein and lipid binding partners, we sought to determine whether phosphatidylinositol 3-kinase (PI3K) activation played a role in PMA-induced colocalization between AFAP-110 and cSrc. We show that PMA treatment is able to direct activation of PI3K. Treatment of mouse embryo fibroblast with PI3K inhibitors blocked PMA-directed colocalization between AFAP-110 and cSrc and subsequent cSrc activation. PMA also was unable to induce colocalization or cSrc activation in cells that lacked the p85 and - regulatory subunits of PI3K. This signaling pathway was required for migration in a wound healing assay. Cells that were null for cSrc or the p85 regulatory subunits or expressed a dominant-negative AFAP-110 also displayed a reduction in migration. Thus PI3K activity is required for PMA-induced colocalization between AFAP-110 and cSrc and subsequent cSrc activation, and this signaling pathway promotes cell migration. phorbol 12-myristate 13-acetate; Src; protein kinase C; AFAP-110; phosphatidylinositol 3-kinase; pleckstrin homology domain     

DNA-PKcs is required for activation of innate immunity by immunostimulatory DNA

Bacterial DNA and related synthetic immunostimulatory oligodeoxyribonucleotides (ISS-ODN) stimulate innate immunity. However, the molecular recognition mechanism that initiates signaling in response to bacterial DNA and ISS-ODN has not been identified. Herein, we demonstrate that administration of bacterial DNA and ISS-ODN to mice lacking the catalytic subunit of DNA-PK (DNA-PKcs) and in vitro stimulation of BMDM from these mice result in defective induction of IL-6 and IL-12. Further analysis using BMDM of IKKbeta(-/-) revealed that both DNA-PKcs and IKKbeta are essential for normal cytokine production in response to ISS-ODN or bacterial DNA. ISS-ODN and bacterial DNA activate DNA-PK, which in turn contributes to activation of IKK and NF-kappaB. These results reveal a novel role of DNA-PKcs in innate immune responses and a link between DNA repair and innate immunity.     

NADPH oxidase activation is required for migration by LIGHT in human monocytes

Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM; TR2) (LIGHT), a ligand of herpes virus entry mediator (HVEM), increased reactive oxygen species (ROS) and enhanced the destruction of bacteria in human monocytes. In this study, rhLIGHT was found to increase the expression of the chemokine receptors, chemokine receptor 1 (CCR1) and CCR2, as well as to accelerate the migration activity of human monocytes. Additionally, rhLIGHT was found to increase ROS via NADPH oxidase p47^phox phosphorylation, which was found to be required for LIGHT-induced NF-κB activation, CCR1 and CCR2 expression, migration and IL-8 and TNF-α production. Taken together, these results indicate that NADPH oxidase activation is required for rhLIGHT-induced migration in human monocytes.     

Caspase-4 is required for activation of inflammasomes

IL-1β and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1β and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1β activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.     

PKC is required for activation of ROCK by RhoA in human endothelial cells

Rho/Rho-kinase (ROCK) complex formation is the only proposed mechanism for ROCK activation. Rho/ROCK and PKC can exhibit a convergence of cellular effects such as suppression of endothelial nitric oxide synthase (eNOS) expression. We, therefore, investigated the role of PKC in RhoA/ROCK complex formation and activation linked to eNOS expression in cultured human umbilical vein endothelial cells. We showed that expression of constitutively active RhoA (Rho63) or ROCK (CAT) suppressed eNOS gene expression. This effect of Rho63 but not that of CAT was abolished by phorbol ester-sensitive PKC depletion. Accordingly, depletion or inhibition of PKC prevented ROCK activation by Rho63 without affecting RhoA/ROCK complex formation. Similarly, suppression of eNOS expression and activation of ROCK, but not RhoA by thrombin were prevented by PKC inhibition or depletion. These results indicate that RhoA/ROCK complex formation alone is not sufficient and PKC is required for RhoA-induced ROCK activation leading to eNOS gene suppression.     

Tyrosine dephosphorylation is required for Bak activation in apoptosis

Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.     

Amyloid-beta fibril formation is not necessarily required for microglial activation by the peptides

There is increasing evidence that microglial activation has pathogenic influence on Alzheimer's disease. According to in vitro studies, microglia activated by amyloid-beta (Abeta) peptides have been reported to damage or kill neurons by the release of neurotoxic molecules such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, nitric oxide or reactive oxygen species. Although the relationship between the aggregational state of Abeta peptides and their neurotoxic activities has been well investigated, little is known about the relationship between the aggregational state of Abeta peptides and their ability to induce microglial activation. In the present study, we thus performed both structural and biochemical studies to clarify the relationship between the aggregational state of Abeta peptides and their ability to activate microglia. Our results have shown that, in the presence of interferon-gamma, the Abeta25-35(M(35)Nle) peptide had almost the same potency of activating microglia and producing TNF-alpha as the Abeta25-35 peptide on both protein and mRNA levels, in spite of the fact that former peptide represented much less amyloid fibril formation than the latter in a thioflavine-T fluorometric assay. These results suggest that Abeta fibril formation is not necessarily required for microglial activation by the peptides.     

Interferon-gamma is required in activation of macrophages for tumor cytotoxicity

The participation of interferon-gamma in activation of murine macrophages for tumor cell lysis was investigated. Biochemically macrophage activation factor and interferon-gamma have not been separated. Antiviral titers correlated closely with macrophage activation in antigen- or mitogen-induced spleen cell supernatants. A monoclonal rat antibody that neutralized virus-induced interferon was also found to neutralize interferon-gamma in such supernatants. These monoclonal antibodies were coupled to CH-Sepharose 4B and used for absorption of antiviral activity from mitogen-induced spleen cell supernatants. Absorption of the interferon was paralleled by the reduction of the macrophage-activating capacity of the supernatants. Data from control absorptions supported the specificity of the absorption effect. These results indicate that interferon-gamma is required for activation of macrophages for tumor cell lysis. These results can be interpreted in two ways: (a) the monoclonal antibodies cross-react with interferon-gamma and with a mediator that is required for activation of macrophages for tumor cell lysis or (b) interferon-gamma itself is an essential cofactor for macrophage activation.     

Calcium is required for the mitogenic activation of lymphocytes by periodic acid oxidation.

This investigation of Ca2+ requirements for the mitogenic activation of lymphocytes by periodic acid has shown that oxidation by periodate causes an immediate and transient increase of Ca2+ influx and efflux in oxidized cells. Oxidized lymphocytes maintained in the medium containing 0.2 mM Ca2+ failed to proliferate or to produce IL-2, whereas a 1.4 mM Ca2+ concentration was shown to be sufficient to sustain cellular proliferation and IL-2 secretion. These results indicate that mitogenic activation of lymphocytes by periodic acid oxidation is Ca(2+)-dependent.     

NF-kappaB activation is required for the development of cardiac hypertrophy in vivo

In the present study, we examined whether NF-kappaB activation is required for cardiac hypertrophy in vivo. Cardiac hypertrophy in rats was induced by aortic banding for 1, 3, and 5 days and 1-6 wk, and age-matched sham-operated rats served as controls. In a separate group of rats, an IkappaB-alpha dominant negative mutant (IkappaB-alphaM), a superrepressor of NF-kappaB activation, or pyrrolidinedithiocarbamate (PDTC), an antioxidant that can inhibit NF-kappaB activation, was administered to aortic-banded rats for 3 wk. The heart weight-to-body weight ratio was significantly increased at 5 days after aortic banding, peaked at 4 wk, and remained elevated at 6 wk compared with age-matched sham controls. Atrial natriuretic peptide and brain natriuretic peptide mRNA expressions were significantly increased after 1 wk of aortic banding, reached a maximum between 2 and 3 wk, and remained increased at 6 wk compared with age-matched sham controls. NF-kappaB activity was significantly increased at 1 day, reached a peak at 3 wk, and remained elevated at 6 wk, and IKK-beta activity was significantly increased at 1 day, peaked at 5 days, and then decreased but remained elevated at 6 wk after aortic banding compared with age-matched sham controls. Inhibiting NF-kappaB activation in vivo by cardiac transfection of IkappaB-alphaM or by PDTC treatment significantly attenuated the development of cardiac hypertrophy in vivo with a concomitant decrease in NF-kappaB activity. Our results suggest that NF-kappaB activation is required for the development of cardiac hypertrophy in vivo and that NF-kappaB could be an important target for inhibiting the development of cardiac hypertrophy in vivo.     

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.     

EGFR kinase activity is required for TLR4 signaling and the septic shock response

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4—which signals from both plasma and endosomal membranes—is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-β (IFN-β) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR—as is IRF co-activator β-catenin—through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.     

Reactive nitrogen species is required for the activation of the AMP-activated protein kinase by statin in vivo

The AMP-activated protein kinase (AMPK) is reported to mediate the beneficial effects of statin on the vascular functions, but the biochemical mechanisms are incompletely understood. The aim of the study was to determine how statin activates AMPK. Exposure of confluent bovine aortic endothelial cells to simvastatin (statin) dose-dependently increased phosphorylation of AMPK at Thr(172) and activities of AMPK, which was in parallel with increased detection of both LKB1 phosphorylation at Ser(428) and LKB1 nuclear export. Furthermore, statin treatment was shown to increase protein kinase C (PKC)-zeta activity and PKC-zeta phosphorylation at Thr(410)/Thr(403). Consistently, inhibition of PKC-zeta either by pharmacological or genetic manipulations abolished statin-enhanced LKB1 phosphorylation at Ser(428), blocked LKB1 nucleus export, and prevented the subsequent activation of AMPK. Similarly, in vivo transfection of PKC-zeta-specific small interfering RNA in C57BL/6J mice significantly attenuated statin-enhanced phosphorylation of AMPK-Thr(172), acetyl-CoA carboxylase (ACC)-Ser(79), and LKB1-Ser(428). In addition, statin significantly increased reactive oxygen species, whereas preincubation of mito-TEMPOL, a superoxide dismutase mimetic, abolished statin-enhanced phosphorylation of both AMPK-Thr(172) and ACC-Ser(79). Finally, in vivo administration of statin increased 3-nitrotyrosine and the phosphorylation of AMPK and ACC in C57BL/6J mice but not in mice deficient in endothelial nitric-oxide synthase. Taken together, our data suggest that AMPK activation by statin is peroxynitrite-mediated but PKC-zeta-dependent.     

EGFR signaling is required for regenerative proliferation in the cochlea: conservation in birds and mammals

Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75+ mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75+ mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27(Kip1) (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.