Ultrafine carbon black particles stimulate proliferation of human airway epithelium via EGF receptor-mediated signaling pathway

Exposure to ambient ultrafine particles induces airway inflammatory reactions and tissue remodeling. In this experiment, to determine whether ultrafine carbon black (ufCB) affects proliferation of airway epithelium and, if so, what the mechanism of action is, we studied human primary bronchial epithelial cell cultures. Incubation of cells in the serum-free medium with ufCB increased incorporations of [3H]thymidine and [3H]leucine into cells in a time- and dose-dependent manner. This effect was attenuated by Cu- and Zn-containing superoxide dismutase (Cu/Zn SOD) and apocynin, an inhibitor of NADPH oxidase, and completely inhibited by pretreatment with the epidermal growth factor receptor (EGF-R) tyrosine kinase inhibitors AG-1478 and BIBX-1382, and the mitogen-activated protein kinase kinase inhibitor PD-98059. Transfection of a dominant-negative mutant of H-Ras likewise abolished the effect ufCB. Stimulation with ufCB also induced processing of membrane-anchored proheparin-binding (HB)-EGF, release of soluble HB-EGF into the medium, association of phosphorylated EGF-R and Shc with glutathione- S-transferase-Grb2 fusion protein, and phosphorylation of extracellular signal-regulated kinase (ERK). Pretreatment with AG-1478, [Glu52] Diphtheria toxin, a specific inhibitor of HB-EGF, neutralizing HB-EGF antibody, Cu/Zn SOD, and apocynin each inhibited ufCB-induced ERK activation. These results suggest that ufCB causes oxidative stress-mediated proliferation of airway epithelium, involving processing of HB-EGF and the concomitant activation of EGF-R and ERK cascade.

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Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

Biochemical Journal ◽

10.1042/bj20070688 ◽

2008 ◽

Vol 409 (3) ◽

pp. 751-759 ◽

Cited By ~ 7

Author(s):

Steven T. Russell ◽

Helen L. Eley ◽

Stacey M. Wyke ◽

Michael J. Tisdale

Keyword(s):

Protein Degradation ◽

Kinase Inhibitor ◽

Muscle Protein ◽

Specific Inhibitor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Phosphoinositide 3 Kinase

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser473 within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin–proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB (nuclear factor κB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-κB and that this also allows for a specific synthesis of proteasome subunits.

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Biochemical and Biological Functions of the N-Terminal, Noncatalytic Domain of Extracellular Signal-Regulated Kinase 2

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.249-259.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 249-259 ◽

Cited By ~ 38

Author(s):

Scott T. Eblen ◽

Andrew D. Catling ◽

Marcela C. Assanah ◽

Michael J. Weber

Keyword(s):

Kinase Activity ◽

Mutational Analysis ◽

Kinase Domain ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Transcriptional Responses ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal

ABSTRACT Extracellular signal-regulated kinase 1 (ERK1) and ERK2 are important components in signal transduction pathways involved in many cellular processes, including cell differentiation and proliferation. These proteins consist of a central kinase domain flanked by short N- and C-terminal noncatalytic domains. While the regulation of ERK2 by sequences within the kinase domain has been extensively studied, little is known about the small regions outside of the kinase domain. We performed mutational analysis on the N-terminal, noncatalytic domain of ERK2 in an attempt to determine its role in ERK2 function and regulation. Deleting or mutating amino acids 19 to 25 (ERK2-Δ19-25) created an ERK2 molecule that could be phosphorylated in response to growth factor and serum stimulation in a MEK (mitogen-activated protein kinase kinase or ERK kinase)-dependent manner but had little kinase activity and was unable to bind to MEK in vivo. Since MEK acts as a cytoplasmic anchor for the ERKs, the lack of a MEK interaction resulted in the aberrant nuclear localization of ERK2-Δ19-25 mutants in serum-starved cells. Assaying these mutants for their ability to affect ERK signaling revealed that ERK2-Δ19-25 mutants acted in a dominant-negative manner to inhibit transcriptional signaling through endogenous ERKs to an Elk1-responsive promoter in transfected COS-1 cells. However, ERK2-Δ19-25 had no effect on the phosphorylation of RSK2, an ERK2 cytoplasmic substrate, whereas a nonactivatable ERK (T183A) that retained these sequences could inhibit RSK2 phosphorylation. These results suggest that the N-terminal domain of ERK2 profoundly affects ERK2 localization, MEK binding, kinase activity, and signaling and identify a novel dominant-negative mutant of ERK2 that can dissociate at least some transcriptional responses from cytoplasmic responses.

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The EGF Receptor and HER2 Participate in TNF-α-Dependent MAPK Activation and IL-8 Secretion in Intestinal Epithelial Cells

Mediators of Inflammation ◽

10.1155/2012/207398 ◽

2012 ◽

Vol 2012 ◽

pp. 1-12 ◽

Cited By ~ 18

Author(s):

Humberto B. Jijon ◽

Andre Buret ◽

Christina L. Hirota ◽

Morley D. Hollenberg ◽

Paul L. Beck

Keyword(s):

Epithelial Cells ◽

Egf Receptor ◽

Kinase Inhibitor ◽

Intestinal Epithelial Cells ◽

Specific Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Angiogenic Factor ◽

Intestinal Epithelial ◽

Mapk Activation ◽

Erk Phosphorylation

TNF-αactivates multiple mitogen-activated protein kinase (MAPK) cascades in intestinal epithelial cells (IECs) leading to the secretion of interleukin 8 (IL-8), a neutrophil chemoattractant and an angiogenic factor with tumor promoting properties. As the epidermal growth factor receptor (EGFR) is a known transducer of proliferative signals and a potent activator of MAPKs, we hypothesized that the EGFR participates in TNF-dependent MAPK activation and IL-8 secretion by intestinal epithelial cells (IECs). We show that the EGFR is tyrosine-phosphorylated following treatment of IECs (HT-29 and IEC-6) with TNF-α. This requires EGFR autophosphorylation as it was blocked by the EGFR kinase inhibitor AG1478. Autophosphorylation was also inhibited by both a Src-kinase inhibitor and the metalloproteinase inhibitor batimastat. TNF treatment of IECs resulted in the accumulation of soluble TGF-α; treatment of IECs with batimastat suppressed TGF-αrelease and immunoneutralization of TGF-αresulted in decreased EGFR and ERK phosphorylations. TNF-αtreatment of IECs resulted in an association between EGFR and HER2 and inhibition of HER2 using a specific inhibitor AG879 in combination with AG1478-suppressed TNF-α-dependent ERK phosphorylation and IL-8 release. Downregulation of HER2 via siRNA resulted in a significant decrease in ERK phosphorylation and a 50% reduction in IL-8 secretion.

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JAK2 Activates TFII-I and Regulates Its Interaction with Extracellular Signal-Regulated Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3387-3397.2000 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3387-3397 ◽

Cited By ~ 21

Author(s):

Dae-Won Kim ◽

Brent H. Cochran

Keyword(s):

Transcription Factor ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Serum Stimulation

ABSTRACT TFII-I is a transcription factor that shuttles between the cytoplasm and nucleus and is regulated by serine and tyrosine phosphorylation. Tyrosine phosphorylation of TFII-I can be regulated in a signal-dependent manner in various cell types. In B lymphocytes, Bruton's tyrosine kinase has been identified as a TFII-I tyrosine kinase. Here we report that JAK2 can phosphorylate and regulate TFII-I in nonlymphoid cells. The activity of TFII-I on the c-fospromoter in response to serum can be abolished by dominant negative JAK2 or the specific JAK2 kinase inhibitor AG490. Consistent with this, we have also found that JAK2 is activated by serum stimulation of fibroblasts. Tyrosine 248 of TFII-I is phosphorylated in vivo upon serum stimulation or JAK2 overexpression, and mutation of tyrosine 248 to phenylalanine inhibits the ability of JAK2 to phosphorylate TFII-I in vitro. Tyrosine 248 of TFII-I is required for its interaction with and phosphorylation by ERK and its in vivo activity on the c-fos promoter. These results indicate that the interaction between TFII-I and ERK, which is essential for its activity, can be regulated by JAK2 through phosphorylation of TFII-I at tyrosine 248. Thus, like the STAT factors, TFII-I is a direct substrate of JAK2 and a signal-dependent transcription factor that integrates signals from both tyrosine kinase and mitogen-activated protein kinase pathways to regulate transcription.

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UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53

Biochemical Journal ◽

10.1042/bj20020072 ◽

2002 ◽

Vol 365 (1) ◽

pp. 133-145 ◽

Cited By ~ 96

Author(s):

Nadine CHOUINARD ◽

Kristoffer VALERIE ◽

Mahmoud ROUABHIA ◽

Jacques HUOT

Keyword(s):

Adaptive Response ◽

Human Keratinocytes ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Dominant Negative Mutant ◽

Normal Human Keratinocytes ◽

Mitogen Activated Protein ◽

Normal Human ◽

Induced Apoptosis ◽

Negative Mutant

Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

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Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00326.2001 ◽

2002 ◽

Vol 282 (6) ◽

pp. L1324-L1329 ◽

Cited By ~ 171

Author(s):

Andre Kulisz ◽

Ningfang Chen ◽

Navdeep S. Chandel ◽

Zuohui Shao ◽

Paul T. Schumacker

Keyword(s):

Kinase Inhibitor ◽

Anion Channel ◽

P38 Map Kinase ◽

Proximal Region ◽

Mitogen Activated Protein Kinase ◽

Ros Generation ◽

Dependent Manner ◽

Adaptive Responses ◽

Mitochondrial Ros ◽

Mitogen Activated Protein

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1–5% O2). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a Po 2-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1–2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H2O2 during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H2O2, but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H2O2 or superoxide participates in activating that process.

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Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

Journal of Cell Science ◽

10.1242/jcs.114.8.1579 ◽

2001 ◽

Vol 114 (8) ◽

pp. 1579-1589 ◽

Cited By ~ 2

Author(s):

M. Reyes-Reyes ◽

N. Mora ◽

A. Zentella ◽

C. Rosales

Keyword(s):

Signaling Pathways ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Small Gtpase ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Dependent Manner ◽

Monocytic Leukemia ◽

Monocytic Cells ◽

Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

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HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

Journal of Molecular Endocrinology ◽

10.1530/jme-20-0211 ◽

2021 ◽

Author(s):

Toshihiro Kobayashi ◽

Hitomi Imachi ◽

Kensaku Fukunaga ◽

Jingya Lyu ◽

Seisuke Sato ◽

...

Keyword(s):

Gene Expression ◽

Signaling Pathways ◽

Promoter Activity ◽

Active Form ◽

Specific Inhibitor ◽

Density Lipoprotein ◽

Dominant Negative ◽

Activation Mechanism ◽

Dominant Negative Mutant ◽

Type I

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

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Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00065.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. G886-G898 ◽

Cited By ~ 10

Author(s):

Ann M. Hopkins ◽

Matthias Bruewer ◽

G. Thomas Brown ◽

A’Drian A. Pineda ◽

Julie J. Ha ◽

...

Keyword(s):

Growth Factor ◽

Epithelial Cell ◽

Hepatocyte Growth Factor ◽

Fluorescent Protein ◽

Kinase Inhibitor ◽

Dominant Negative ◽

Dependent Manner ◽

Epithelial Migration ◽

Cell Matrix ◽

Hepatocyte Growth

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, β1-integrin. This was independent of cell-matrix adhesion through β1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with β1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.

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RacG Regulates Morphology, Phagocytosis, and Chemotaxis

Eukaryotic Cell ◽

10.1128/ec.00221-06 ◽

2006 ◽

Vol 5 (10) ◽

pp. 1648-1663 ◽

Cited By ~ 20

Author(s):

Baggavalli P. Somesh ◽

Georgia Vlahou ◽

Miho Iijima ◽

Robert H. Insall ◽

Peter Devreotes ◽

...

Keyword(s):

Rho Gtpases ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Dominant Negative ◽

Dependent Manner ◽

Free System ◽

Mild Phenotype ◽

Particle Uptake ◽

Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

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