Role of protein kinase C-η in cigarette smoke extract-induced apoptosis in MRC-5-cells

2014 ◽  
Vol 34 (9) ◽  
pp. 869-877 ◽  
Author(s):  
ES Son ◽  
SY Kyung ◽  
SP Lee ◽  
SH Jeong ◽  
JY Shin ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cigarette Smoke ◽  
Caspase 3 ◽  
Dominant Negative ◽  
Lung Fibroblasts ◽  
Apoptotic Pathway ◽  
Kinase C ◽  
Extrinsic Apoptotic Pathway ◽  
Infected Cells

Cigarette smoke (CS) is a major risk factor for emphysema, which causes cell death in structural cells of the lung by mechanisms that are still not completely understood. We demonstrated previously that CS extract (CSE) induces caspase activation in MRC-5 human lung fibroblasts, activated protein kinase C-η (PKC-η), and translocated PKC-η from the cytosol to the membrane. The objective of this study was to investigate the involvement of PKC-η activation in a CSE-induced extrinsic apoptotic pathway. We determined that CSE increases expression of caspase 3 and 8 cleavage in MRC-5 cells and overexpression of PKC-η significantly increased expression of caspase 3 and 8 cleavage compared with control LacZ-infected cells. In contrast, dominant negative (dn) PKC-η inhibited apoptosis in MRC-5 cells exposed to CSE and decreased expression of caspase 3 and 8 compared with control cells. Exposure to 10% CSE for >8 h significantly increased lactate dehydrogenase release in PKC-η-infected cells compared with LacZ-infected cells. Additionally, PKC-η-infected cells had an increased number of Hoechst 33342 stained nuclei compared with LacZ-infected cells, while dn PKC-η-infected cells exhibited fewer morphological changes than LacZ-infected cells under phase-contrast microscopy. In conclusion, PKC-η activation plays a pro-apoptotic role in CSE-induced extrinsic apoptotic pathway in MRC-5 cells. These results suggest that modulation of PKC-η may be a useful tool for regulating the extrinsic apoptosis of MRC-5 cells by CSE and may have therapeutic potential in the treatment of CS-induced lung injury.

scholarly journals Regulation of Caspase 9 through Phosphorylation by Protein Kinase C Zeta in Response to Hyperosmotic Stress

2005 ◽  
Vol 25 (23) ◽  
pp. 10543-10555 ◽  
Author(s):  
Suzanne C. Brady ◽  
Lindsey A. Allan ◽  
Paul R. Clarke
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Caspase 3 ◽  
Hyperosmotic Stress ◽  
Intrinsic Apoptotic Pathway ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Kinase C ◽  
In Cells

ABSTRACT Caspase 9 is a critical component of the mitochondrial or intrinsic apoptotic pathway and is activated by Apaf-1 following release of cytochrome c from mitochondria in response to a variety of stimuli. Caspase 9 cleaves and activates effector caspases, mainly caspase 3, leading to the demise of the cell. Survival signaling pathways can impinge on this pathway to restrain apoptosis. Here, we have identified Ser144 of human caspase 9as an inhibitory site that is phosphorylated in a cell-free system and in cells in response to the protein phosphatase inhibitor okadaic acid. Inhibitor sensitivity and interactions with caspase 9 indicate that the predominant kinase that targets Ser144 is the atypical protein kinase C isoform zeta (PKCζ). Prevention of Ser144 phosphorylation by inhibition of PKCζ or mutation of caspase 9 promotes caspase 3 activation. Phosphorylation of serine 144 in cells is also induced by hyperosmotic stress, which activates PKCζ and regulates its interaction with caspase 9, but not by growth factors, phorbol ester, or other cellular stresses. These results indicate that phosphorylation and inhibition of caspase 9 by PKCζ restrain the intrinsic apoptotic pathway during hyperosmotic stress. This work provides further evidence that caspase 9 acts as a focal point for multiple protein kinase signaling pathways that regulate apoptosis.

scholarly journals Essential role of protein kinase C ζ in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

2007 ◽  
Vol 176 (7) ◽  
pp. 1049-1060 ◽  
Author(s):  
Kageaki Kuribayashi ◽  
Kiminori Nakamura ◽  
Maki Tanaka ◽  
Tsutomu Sato ◽  
Junji Kato ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Morphological Changes ◽  
Specific Inhibitor ◽  
Squamous Carcinoma ◽  
Inflammatory Cells ◽  
Dominant Negative ◽  
Chemotactic Peptide ◽  
Kinase C ◽  
Pkc Ζ

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

scholarly journals Novel Protein Kinase C δ Isoform Insensitive to Caspase-3

2001 ◽  
Vol 24 (9) ◽  
pp. 973-977 ◽  
Author(s):  
Yoshiaki SAKURAI ◽  
Yoshiaki ONISHI ◽  
Yutaka TANIMOTO ◽  
Harutoshi KIZAKI
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Caspase 3 ◽  
Kinase C ◽  
Novel Protein

scholarly journals Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3227-3241 ◽  
Author(s):  
Kyoungmin Park ◽  
Qian Li ◽  
Christian Rask-Madsen ◽  
Akira Mima ◽  
Koji Mizutani ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Angiotensin Ii ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Serine Phosphorylation ◽  
Kinase C ◽  
Pkc Activation

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.

Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. L403-L411 ◽  
Author(s):  
Sang-Do Lee ◽  
Dong-Soon Lee ◽  
Yong-Gam Chun ◽  
Tae-Sun Shim ◽  
Chae-Man Lim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Cigarette Smoke ◽  
De Novo ◽  
Ex Vivo ◽  
Cigarette Smoke Extract ◽  
Kinase C

We examined the mechanism of endothelin (ET)-1 regulation by cigarette smoke extract (CSE) and the effect of platelets on CSE-induced stimulation of ET-1 gene expression in human and bovine pulmonary artery endothelial cells (PAECs). Our data show that CSE (1%) induces ET-1 gene expression (after 1 h) and ET-1 peptide synthesis (after 4 h) in bovine PAECs. The induction of preproET-1 mRNA level was due to de novo transcription, and new protein synthesis was not required for this induction. The protein kinase C inhibitors staurosporine (10−8mol/l) and calphostin C (10−7mol/l) abolished the induction of ET-1 gene expression by CSE in bovine and human PAECs. Although a lower concentration of platelets (106cells/ml in bovine PAECs; 107cells/ml in human PAECs) did not significantly alter ET-1 gene expression in PAECs, incubation of platelets with CSE (1%) and PAECs produced a significant increase in preproET-1 mRNA and ET-1 peptide compared with the values in the presence of CSE (1%) alone. CSE (1%) induced platelet aggregation and increased the expression of platelet membrane glycoproteins ex vivo. Thus our data suggest that CSE stimulates ET-1 gene expression via PKC in PAECs. CSE and platelets showed a synergistic effect on ET-1 gene expression, possibly through the activation of platelets by CSE.

Epithelial injury induces Egr-1 and Fos expression by a pathway involving protein kinase C and ERK

1999 ◽  
Vol 276 (2) ◽  
pp. G322-G330 ◽  
Author(s):  
Brian K. Dieckgraefe ◽  
Danielle M. Weems
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Erk Activation ◽  
Kinase C

The signaling pathways activated in response to gastrointestinal injury remain poorly understood. Previous work has implicated the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase as a mediator of wound-signal transduction and a possible regulator of epithelial restitution. Monolayer injury resulted in rapid activation of p42 and p44 ERK. Injury-induced ERK activation was blocked by protein kinase C inhibition or by disruption of the cell cytoskeleton. Significant increases in Fos and early growth response (Egr)-1 mRNA levels were stimulated by injury, peaking by 20 min. ERK activation and the induction of Egr-1 mRNA were inhibited in a dose-dependent fashion with PD-98059. Fos mRNA expression was partially blocked by PD-98059. Western blot analysis demonstrated strong expression and nuclear localization of Fos and Egr after wounding. Electrophoretic mobility shift assays demonstrated that nuclear extracts contained a protein that specifically bound double-stranded oligonucleotides containing the Egr consensus binding element. Gel supershift assays demonstrated that the protein-DNA complexes were recognized by anti-Egr antibody. Inhibition of injury-induced ERK activation by PD-98059 or direct interference with Egr by expression of a dominant negative mutant led to significantly reduced in vitro monolayer restitution.

scholarly journals Autophagy and protein kinase C are required for cardioprotection by sulfaphenazole

2010 ◽  
Vol 298 (2) ◽  
pp. H570-H579 ◽  
Author(s):  
Chengqun Huang ◽  
Wayne Liu ◽  
Cynthia N. Perry ◽  
Smadar Yitzhaki ◽  
Youngil Lee ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Enhanced Recovery ◽  
Ischemia Reperfusion ◽  
Dominant Negative ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Kinase C ◽  
Rat Hearts

Previously, we showed that sulfaphenazole (SUL), an antimicrobial agent that is a potent inhibitor of cytochrome P4502C9, is protective against ischemia-reperfusion (I/R) injury (Ref. 15 ). The mechanism, however, underlying this cardioprotection, is largely unknown. With evidence that activation of autophagy is protective against simulated I/R in HL-1 cells, and evidence that autophagy is upregulated in preconditioned hearts, we hypothesized that SUL-mediated cardioprotection might resemble ischemic preconditioning with respect to activation of protein kinase C and autophagy. We used the Langendorff model of global ischemia to assess the role of autophagy and protein kinase C in myocardial protection by SUL during I/R. We show that SUL enhanced recovery of function, reduced creatine kinase release, decreased infarct size, and induced autophagy. SUL also triggered PKC translocation, whereas inhibition of PKC with chelerythrine blocked the activation of autophagy in adult rat cardiomyocytes. In the Langendorff model, chelerythrine suppressed autophagy and abolished the protection mediated by SUL. SUL increased autophagy in adult rat cardiomyocytes infected with GFP-LC3 adenovirus, in isolated perfused rat hearts, and in mCherry-LC3 transgenic mice. To establish the role of autophagy in cardioprotection, we used the cell-permeable dominant-negative inhibitor of autophagy, Tat-Atg5K130R. Autophagy and cardioprotection were abolished in rat hearts perfused with recombinant Tat-Atg5K130R. Taken together, these studies indicate that cardioprotection mediated by SUL involves a PKC-dependent induction of autophagy. The findings suggest that autophagy may be a fundamental process that enhances the heart's tolerance to ischemia.

scholarly journals Caspase-mediated protein kinase C-δ cleavage is necessary for apoptosis of vascular smooth muscle cells

2009 ◽  
Vol 297 (6) ◽  
pp. H2253-H2261 ◽  
Author(s):  
Kaori Kato ◽  
Dai Yamanouchi ◽  
Karla Esbona ◽  
Kentaro Kamiya ◽  
Fan Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Tyrosine Phosphorylation ◽  
Vascular Smooth Muscle Cells ◽  
Caspase 3 ◽  
Kinase C

Apoptotic death of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling and various vascular diseases. We have previously shown that protein kinase C-δ (PKC-δ) plays a critical role in SMC apoptosis. In this study, we tested the importance of PKC-δ proteolytic cleavage and tyrosine phosphorylation within the apoptosis pathway. Using hydrogen peroxide as a paradigm for oxidative stress, we showed that proteolytic cleavage of PKC-δ occurred in SMCs that underwent apoptosis, while tyrosine phosphorylation was detected only in necrotic cells. Furthermore, using a peptide (z-DIPD-fmk) that mimics the caspase-3 binding motif within the linker region of PKC-δ, we were able to prevent the cleavage of PKC-δ, as well as apoptosis. Inhibition of PKC-δ with rottlerin or small-interfering RNA diminished caspase-3 cleavage, caspase-3 activity, cleavage of poly (ADP-ribose) polymerase, cleavage of PKC-δ, and DNA fragmentation, confirming the previously reported role of PKC-δ in initiation of apoptosis. In contrast, z-DIPD-fmk markedly diminished caspase-3 activity, cleavage of PKC-δ, and DNA fragmentation without affecting cleavage of caspase-3 and poly (ADP-ribose) polymerase. Taken together, our data suggest that caspase-3-mediated PKC-δ cleavage underlies SMC apoptosis induced by oxidative stress, and that PKC-δ acts both upstream and downstream of caspase-3.

scholarly journals Activation of IκB Kinase β by Protein Kinase C Isoforms

1999 ◽  
Vol 19 (3) ◽  
pp. 2180-2188 ◽  
Author(s):  
Maria-José Lallena ◽  
María T. Diaz-Meco ◽  
Gary Bren ◽  
Carlos V. Payá ◽  
Jorge Moscat
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Functions ◽  
Kinase C ◽  
Iκb Kinase ◽  
Tnf Α

ABSTRACT The atypical protein kinase C (PKC) isotypes (λ/ιPKC and ζPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-α) and are required for the activation of NF-κB by this cytokine through a mechanism that most probably involves the phosphorylation of IκB. The inability of these PKC isotypes to directly phosphorylate IκB led to the hypothesis that ζPKC may use a putative IκB kinase to functionally inactivate IκB. Recently several groups have molecularly characterized and cloned two IκB kinases (IKKα and IKKβ) which phosphorylate the residues in the IκB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-κB through the activation of the IKKs. We report here that αPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of ζPKC positively modulates IKKβ activity but not that of IKKα, whereas the transfection of a ζPKC dominant negative mutant severely impairs the activation of IKKβ but not IKKα in TNF-α-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKβ, which is entirely dependent on the activity of αPKC but not that of the atypical isoforms. In contrast, the inhibition of αPKC does not affect the activation of IKKβ by TNF-α. Interestingly, recombinant active ζPKC and αPKC are able to stimulate in vitro the activity of IKKβ but not that of IKKα. In addition, evidence is presented here that recombinant ζPKC directly phosphorylates IKKβ in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-κB pathway at the level of IKKβ activation and IκB degradation.

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