The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smyth, D. C.
Right arrow Articles by Richards, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smyth, D. C.
Right arrow Articles by Richards, C. D.
The Journal of Immunology, 2006, 177: 8740-8747.
Copyright © 2006 by The American Association of Immunologists, Inc.

Oncostatin M-Induced IL-6 Expression in Murine Fibroblasts Requires the Activation of Protein Kinase C{delta}1

David C. Smyth, Christine Kerr and Carl D. Richards2

Centre for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Oncostatin M (OSM) is an IL-6/LIF cytokine family member whose role has been identified in a range of biological activities in vitro, including up-regulation of inflammatory gene expression and regulation of connective tissue metabolism. However, the mechanisms through which OSM regulates cellular responses are not completely understood. In this study, we show that activation of the calcium-independent or novel protein kinase C (PKC) isoform PKC{delta} is a critical event during OSM-mediated up-regulation of IL-6 expression in murine fibroblasts. The pan-PKC inhibitor GF109203X (bisindolylmaleimide I) reduced secretion of IL-6; however, use of Go6976, an inhibitor of calcium-dependent PKC enzymes, did not. The PKC{delta}-selective inhibitory compound rottlerin abrogated expression of IL-6 transcript and protein, but only reduced PKC{delta} activity when used at higher concentrations as determined by kinase activity assay, suggesting rottlerin may inhibit IL-6 expression in a PKC{delta}-independent manner. However, silencing of PKC{delta} protein expression, but not the related novel isoform PKC{epsilon}, by use of RNA interference (i.e., small interfering RNA) demonstrated that PKC{delta} is required for murine OSM (mOSM) induction of IL-6 protein secretion. Furthermore, inhibition of PI3K by use of LY294002 reduces expression of IL-6 at both the mRNA and protein level in murine fibroblasts, and we suggest that PI3K is required for activation of PKC{delta}. Knockdown of phosphoinositide-dependent kinases PDK-1 or Akt1 using small interfering RNA strategies did not influence mOSM-induced IL-6 expression, suggesting mOSM uses a PI3K–PKC{delta} pathway of activation independent of these kinases. Our findings illustrate a novel signaling network used by mOSM that may be important for its mediation of inflammatory processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The induction of inflammatory processes involves complex interactions between a variety of cell types, which communicate with each other through surface bound molecules or by elaboration of small soluble factors, including cytokines. Typically, inflammation is potentiated through secretion and bioactivity of the primary mediators IL-1 and TNF-{alpha}, whose activities promote increased vascular endothelial permeability, adhesion molecule up-regulation, and production of inflammatory mediators by connective tissue cells such as fibroblasts within local sites of inflammation (1, 2). Other cytokines associated with inflammatory states include IL-6 and oncostatin M (OSM),3 two members of the IL-6/LIF cytokine family that are derived from numerous cell types and exhibit pleiotropic functions, including contribution to inflammatory responses (3).

Animal models of inflammatory disease or samples derived from human subjects show elevated levels of IL-6 and OSM at sites of inflammation (4, 5), and studies indicate potential therapeutic benefit may be achieved through inhibitory strategies targeting the bioactivity of these cytokines (6). OSM has been proposed to participate during chronic inflammatory processes (7, 8, 9) including rheumatoid arthritis, where its role may be to potentiate pannus formation and cartilage destruction as indicated by studies using adenoviral delivery of murine OSM (mOSM) to murine knee joints (10). OSM may also contribute to chronic inflammatory respiratory diseases (11), though definitive studies proving this role have not been completed.

Our laboratory has been studying the cellular signaling events underlying OSM-mediated regulation of several inflammation-modulating gene products, including IL-6 (8, 12). A functional property OSM shares with other IL-6/LIF cytokines is the capacity to initiate signal transduction by the JAK/STAT and MAPK pathways (13), likely due to shared usage of the common gp130 receptor subunit. In murine systems, OSM acts exclusively through a heterodimer consisting of gp130 and OSMRbeta (14), and the use of this signaling platform might allow for OSM-selective activation of factors such as STAT1 and STAT5 and p38 MAPK (15, 16). However, the activation of JAK/STAT and MAPK signaling is not sufficient to mediate maximal OSM effects, prompting the examination of alternate pathways OSM might use in regulation of inflammatory gene expression.

Protein kinase C (PKC) is a family of serine/threonine kinases that are involved in regulation of cellular processes including growth, migration, and inflammatory responses (17), and several PKC isoforms have been shown to be required for regulation of IL-6 expression (18, 19). PKC{delta} is a calcium-independent, or novel, PKC isoform whose activity is regulated by stimuli including oxidative stress, UV exposure, and inflammatory cytokines such as IFN-{gamma} and IL-1beta (20, 21, 22). PKC{delta} may modulate inflammatory responses, as evidenced by its capability to induce matrix metalloproteinase and chemokine expression in vitro (23, 24), and may play a role during osteoblastic differentiation (25), a process also known to be dependent upon IL-6. PI3K comprises a family of kinases that phosphorylate the 3' hydroxyl group of phosphatidylinositols. PI3K is known to be activated by various inflammatory mediators and growth factors, and its principle downstream kinase Akt regulates cell survival and differentiation and promotes synthesis of inflammatory cytokines and chemokines (26, 27, 28). Therefore, PI3K may be an important signaling intermediate during the initiation and progression of inflammatory diseases. Studies of Kaposi’s sarcoma indicate human OSM induces activation of PI3K. Recently, we identified PI3K as a pathway activated by OSM in murine fibroblasts, where it had an attenuating role during OSM-induced tissue inhibitor of matrix metalloproteinase (TIMP)-1 expression (29). In this study, we assess mOSM signaling in murine fibroblasts, examining the potential role of PKC{delta} and PI3K as novel intracellular components of mOSM signal transduction. Our findings identify PKC{delta} and PI3K as critical contributors to mOSM regulation of IL-6, a model inflammation-associated gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Explanted murine lung fibroblast (MLF) cultures

MLFs were derived from explanted cells of finely minced lung tissue taken from C57BL/6 mice (10–12 wk old; Charles River Breeding Laboratories), and were cultured in Earle’s modified MEM (F-15) supplemented with 10% FBS (Invitrogen Life Technologies), 1% penicillin/streptomycin, 0.5% Fungizone, and 0.03% L-glutamine.

Cytokines, Abs, and pharmacologic inhibitors

Reagents were purchased from Sigma-Aldrich unless otherwise indicated. Recombinant mOSM and murine IL-6/LIF were purchased from R&D Systems. Recombinant human platelet-derived growth factor (PDGF)-AA was purchased from Oncogene Research Products. Rabbit polyclonal antisera for STAT3, Akt, phosphotyrosine 705 STAT3, and phosphoserine 473 Akt, phosphothreonine 308 Akt and mAb for Akt1 were purchased from Cell Signaling Technologies. Rabbit polyclonal antiserum for PKC{delta} and goat polyclonal antiserum for actin were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antiserum for PKC{epsilon} was purchased from Upstate Biotechnology. Mouse mAbs for PKC{delta} and phosphoinositide-dependent kinase-1 (PDK-1) were purchased from BD Transduction Laboratories. HRP-conjugated secondary Abs for rabbit and goat antisera were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. LY294002, rottlerin, GF109203X, and Go6976 were purchased from Calbiochem.

RNA isolation

Fibroblast cultures grown to near confluence in 75-cm2 flasks were stimulated with cytokines as indicated in normal supplemented medium and incubated for 24 h. Where indicated, cultures were subjected to pretreatment with described concentrations of pharmacologic inhibitor reconstituted in DMSO. Occasionally, control cultures were incubated with DMSO alone. Total RNA was extracted from cultures with TRIzol (Invitrogen Life Technologies), according to the manufacturer’s directions. RNA was quantitated by spectrophotometric measurement of absorbance at 260 nm, and RNA quality was assessed by resolving 2–3 µl of sample on 1% agarose gels containing 0.01% ethidium bromide.

Quantitative real-time PCR (TaqMan)

One microgram of reverse-transcribed cDNA derived from C57BL/6 lung fibroblast cells cultured as described was analyzed for IL-6 mRNA expression by semiquantitative real-time PCR (TaqMan) as previously described (30), using custom optimized primer-probe pairs (Applied Biosystems). Gene expression was quantitated relative to GAPDH, where relative expression of the target gene was calculated as 2–ddCt, where dCt is the difference between the threshold cycle (Ct) for the gene of interest and the threshold cycle for GAPDH. In each experiment, the value of the relative expression of the control sample (untreated) was given a value of 1 and the expression of other treatments was plotted relative to the control.

RNA interference

Double-stranded 25-mer RNAs corresponding to target regions of PKC{delta} were generated as Stealth RNA interference oligonucleotides (Invitrogen Life Technologies). Oligonucleotides were constructed based upon the following sequences isolated from murine PKC{delta} (GenBank accession number NM_011103): target 1, 5'-(210)-GCCGTGTTATCCAGATTGTGCTGAT-(224)-3' and target 2, 5'-(1153)-GGACGTGGTGTTGATTGACGATGAT-(1177)-3'. Duplexed RNA molecules based upon these sequences were constructed and were named PKC{delta} 210 and PKC{delta} 1153, respectively. Additionally, scrambled duplexes were generated for both targets (PKC{delta} 210scr and PKC{delta} 1153scr). RNA interference duplex oligomers were also constructed for PKC{epsilon}, PDK-1, and Akt1 as follows: PKC{epsilon} (NM_011104) 5'-(1582)-CAGCACGGAGTGATCTACAGGGATT-(1606)-3'; PDK-1 (NM_011062) 5'(1616)-GAACTCCGACCAGAAGCCAAGAATT-(1640)-3'; and Akt1 (NM_009652) 5'(1510)-CAACATCGTGTGGCAGGATGTGTAT-(1534)-3'. For RNA interference studies, 1 x 105 C57BL/6 cells/well in 24-well culture dishes were transfected with 20 pmol of the indicated RNA interference or scrambled control using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s protocols. For studies using small interfering RNA (siRNA) targeting PKC{delta} as described in this study, PKC{delta} 1153 was the siRNA species used. Following an overnight incubation with the indicated siRNA species, cultures were stimulated with 25 ng/ml mOSM and incubated an additional 24 h. Supernatants from stimulated cultures were collected and used for IL-6 capture ELISA as later described. Culture lysates were also collected and verification of both efficacy and specificity of RNA knockdown was conducted by immunoblot analysis with the indicated Abs.

Immunoblotting

C57BL/6 lung fibroblasts were cultured in 100-mm culture dishes as described and stimulated with cytokines and pharmacologic inhibitors as indicated in Results. Cells were lysed in 50 mM Tris/125 mM NaCl/2 mM EDTA (pH 7.4) containing 1% Triton X-100, 003% aprotinin, 50 mg/ml PMSF, and 0.5 mM sodium orthovanadate. Lysates were collected in 1.5-ml Eppendorf tubes, rocked at 4°C for 45 min, centrifuged at 12,000 x g for 10 min, and frozen at –70°C. Protein concentration was determined by Bio-Rad protein assay and 10-µg quantities were loaded onto 8% SDS-PAGE gels. Proteins were transferred onto Immobilon-NC membranes (Millipore), and subsequently blots were blocked with TBS containing 0.2% Tween 20 and 5% low-fat milk powder. Blots were probed with primary Abs as indicated, followed by incubation with HRP-conjugated secondary Abs, and protein expression was visualized by ECL (Amersham Biosciences) on X-Omat film (Kodak).

ELISA

A total of 1 x 105 C57BL/6 lung fibroblasts was cultured in 24-well dishes to subconfluence (~75%) and stimulated with cytokines or pharmacologic inhibitors as indicated for 24 h in normal supplemented medium as previously described. Conditioned medium was collected and stored at –20°C until time of analysis. IL-6 secretion was measured using mouse IL-6 matched capture and biotinylated detection Ab pairs, with recombinant mouse IL-6 as standard (R&D Systems), according to the manufacturer’s protocols.

Immunoprecipitation and PKC{delta} kinase activity assay

Whole cell lysates generated as described were incubated at 4°C overnight with 2 µg/ml anti-PKC{delta} mAb, followed by 1 h of incubation with 15 µg/ml protein A-agarose. Immune complexes were washed three times in lysis buffer followed by two washes in kinase reaction buffer containing 25 mM Tris (pH 7.5), 0.5 mM EDTA, 5 mM MgCl2, and 0.5 mM DTT. Beads were resuspended in a 20-µl reaction mix containing kinase buffer and 10 µCi of [{gamma}-32P]ATP, and the assay was initiated by addition of 10 µg of histone H1 per reaction. Samples were incubated for 30 min at 37°C with occasional mixing, and the reaction was terminated by addition of 10 µl of 4x Laemmli buffer. Samples were resolved on 12% SDS-PAGE, and gels were dried and exposed to X-Omat film for autoradiography.

Statistics

Statistical analyses were conducted using Sigma Stat (SPSS) applying the Student’s t test except where use of one-way ANOVA is indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
OSM-mediated PI3K activation in murine fibroblasts

It has been shown that human OSM induces activation of PI3K in Kaposi’s sarcoma cell lines; therefore, we examined C57BL/6 lung fibroblasts to determine whether PI3K was activated by OSM in murine systems. Immunoblots of whole cell lysates demonstrated that mOSM treatment induced phosphorylation of Akt (Fig. 1A), although not as potently as PDGF. By contrast, IL-6 stimulation of cells induced little, if any, detectable Akt phosphorylation. Time course analysis of mOSM-stimulated fibroblasts showed maximal Akt phosphorylation at 20 min poststimulation and remained detectable at 45 min, whereas cultures treated with murine IL-6 demonstrated markedly lower phosphorylation of Akt (Fig. 1B). Akt phosphorylation was not detectable at any time point post mOSM or murine IL-6 stimulation in the presence of LY294002, a widely used inhibitor of PI3K activity. Immunoblot analysis of STAT3 showed that mOSM as well as murine IL-6 and murine LIF induced STAT3 tyrosine phosphorylation, and mOSM induction of STAT3 phosphorylation was not affected by LY294002 (Fig. 1C). Subsequently, we examined the role of PI3K activity on regulation of IL-6.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 1. PI3K-dependent Akt phosphorylation in murine fibroblasts is induced by OSM. A, C57BL/6 lung fibroblasts (MLF) were cultured in 100-mm dishes to near confluence in serum-containing medium, followed by 3 h of incubation in serum-free conditions before stimulation. For stimulation, cells were either left untreated or treated with 25 ng/ml mOSM, 25 ng/ml murine IL-6, or 25 ng/ml PDGF-AA. Following a 15-min stimulation, whole cell lysates were collected and 10 µg per sample was denatured in 4x Laemmli sample buffer, resolved on 8% SDS-PAGE, and subjected to immunoblotting for phosphoserine 473 Akt (p-ser473Akt) or total Akt (Akt). B, MLF cultured as described in A were either left untreated or treated with 25 ng/ml recombinant mOSM or murine IL-6 (mIL-6) over the indicated times, following a 15-min pretreatment with DMSO vehicle or 20 µM PI3K inhibitor LY294002 (LY). Cell lysates were collected and resolved by SDS-PAGE followed by immunoblotting for phosphoserine 473 Akt (p-ser473Akt) or total Akt (Akt). C, MLF were cultured as described and left untreated or stimulated with mOSM, murine IL-6, murine LIF, or mOSM in the presence of 15-min 20 mM LY294002 pretreatment. Cell lysates were collected and resolved by SDS-PAGE followed by immunoblotting for phosphotyrosine 705 STAT3 (p-tyr705STAT3) or total STAT3 (STAT3).

 
Effects of PI3K inhibition on IL-6 expression

Our laboratory has shown that mOSM is a more potent inducer of gene products including IL-6 and eotaxin-1 relative to other gp130 cytokines in murine fibroblast cell lines. As we had determined that PI3K inhibition affected OSM signaling in C57BL/6 fibroblasts, we assayed IL-6 gene expression induced by OSM following LY294002 treatment. mOSM potently up-regulated IL-6 mRNA levels in C57BL/6 lung fibroblasts as determined by quantitative real-time PCR (TaqMan) (Fig. 2A), consistent with our previous findings. LY294002 treatment reduced levels of IL-6 mRNA transcription, and protein secretion was reduced by PI3K inhibition in a dose-responsive manner, which was statistically significant at inhibitor concentrations of 5 µM (Fig. 2B).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		FIGURE 2. Effects of PI3K inhibition by LY294002 on OSM-induced IL-6 mRNA and protein expression in murine fibroblasts. A, C57BL/6 lung fibroblasts (MLF) were either left untreated or treated with 25 ng/ml mOSM for 24 h, in the presence or absence of LY294002 pretreatment, or with 25 ng/ml mIL-6. One microgram of reverse-transcribed cDNA was subjected to TaqMan for quantitation of IL-6 with normalization to GAPDH. Data presented are the fold increase in mRNA expression relative to unstimulated control, with error bars corresponding to SD. *, p < 0.01 compared with mOSM-stimulated controls. B, MLF were either left untreated or treated with 25 ng/ml mOSM for 24 h, in presence or absence of LY294002 pretreatment at the indicated concentrations. Each assay condition was performed in quadruplicate. Conditioned medium was collected and subjected to capture ELISA for IL-6. Error bars represent SD. *, p < 0.01 compared with mOSM-stimulated controls.

 
Pharmacologic inhibition of PKC{delta} results in decreased expression of IL-6

Studies using human gastric cell lines indicated a potential requirement for activation of PKC family enzymes in the regulation of IL-6 by OSM (31). Furthermore, several isoforms of PKC have been shown to be dependent upon PI3K activation. Therefore, we examined the effects of PKC inhibition on IL-6 expression in murine fibroblasts. Use of the pan-specific PKC inhibitor GF109203X reduced mOSM-induced IL-6 secretion in C57BL/6 lung fibroblasts as measured by ELISA (Fig. 3A). To determine whether specific PKC isozymes contributed to mOSM regulation of IL-6, we assessed the effects of the Ca2+-dependent PKC inhibitor Go6976 and the PKC{delta}-selective inhibitor rottlerin. Go6976 did not inhibit mOSM-stimulated IL-6 secretion when used at 20 nM (Fig. 3B). However, rottlerin pretreatment inhibited mOSM-induced IL-6 secretion in a dose-responsive manner, which was statistically significant at a concentration of 3 µM, the IC50 concentration for rottlerin, and at concentrations as low as 1 µM in C57BL/6 lung fibroblasts (Fig. 3C).


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIGURE 3. Effects of PKC inhibition upon OSM-induced IL-6 protein expression in murine fibroblasts. A, C57BL/6 lung fibroblasts (MLF) were cultured in 24-well dishes and either left untreated or treated with 25 ng/ml mOSM for 24 h following a 30-min pretreatment with DMSO (Control) or 7.5 µM GF109203X (GF). Each assay condition was conducted in quadruplicate. Conditioned medium was collected and subjected to capture ELISA for IL-6. Error bars represent SD. *, p < 0.01 compared with mOSM-stimulated controls. B, MLF were cultured as described in A and either left untreated or treated with 25 ng/ml mOSM for 24 h following pretreatment with DMSO (Control), 5 µM rottlerin (rott) or 20 nM Go6976 (Go). Conditioned medium was collected from quadruplicate assays and IL-6 was measured by capture ELISA. Error bars represent SD. *, p < 0.01 compared with mOSM-stimulated controls. C, MLF were cultured as described and either left untreated or treated with 25 ng/ml mOSM in the presence or absence of a 15-min pretreatment with rottlerin at the indicated concentrations. Conditioned medium was collected from quadruplicate assays and IL-6 was measured by capture ELISA. Error bars represent SD. *, p < 0.01 compared with mOSM-stimulated controls.

 
PKC{delta} is activated in C57BL/6 lung fibroblasts stimulated with mOSM

To verify the involvement of PKC{delta} during mOSM-induced IL-6 expression, we conducted kinase activity assays using C57BL/6 lung fibroblasts. Expression of PKC{delta} was confirmed by immunoblotting of immunoprecipitated protein from whole cell lysates (Fig. 4A), and activation of PKC{delta} was assessed by kinase activity assay using histone H1 as a substrate. mOSM stimulation resulted in a marked increase of PKC{delta}-mediated histone H1 phosphorylation (Fig. 4A). IL-6 also induced PKC{delta} activation. mOSM-stimulated PKC{delta} activity was inhibited by LY294002 pretreatment as indicated by reduced histone H1 phosphorylation compared with mOSM stimulation alone. Rottlerin pretreatment did not affect PKC{delta} activity at 5 µM concentrations; however, higher concentrations (10 and 15 µM) inhibited PKC{delta} in a dose-dependent manner. The observed effects of LY294002 were not due to a lack of PKC{delta} enzyme, as evidenced by immunoblotting of the membrane used for kinase activity (Fig. 4A). Immunoblotting from input lysate used for kinase activity reactions demonstrated LY294002 reduction of Akt phosphorylation (Fig. 4B). Whole cell lysates from cultures stimulated with mOSM and pretreated with rottlerin showed partial inhibition of Akt phosphorylation, indicating the possibility that rottlerin affects the activity of kinase enzymes other than PKC{delta}. We did not observe coimmunoprecipitation of Akt or MAPK enzymes ERK1/2 and p38 with PKC{delta} following mOSM stimulation (data not shown).


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 4. PKC{delta} is activated by OSM in C57BL/6 lung fibroblasts through a mechanism involving PI3K activation. A, C57BL/6 lung fibroblasts (MLF) were cultured as described and either left untreated or treated with 200 nM PMA, 25 ng/ml mIL-6, 25 ng/ml mOSM (left). Cultures were preincubated with DMSO vehicle, and additional mOSM-stimulated cultures were pretreated for 15 min with 20 µM LY294002 or 5 µM rottlerin. MLF were pretreated for 15 min with the indicated concentrations of rottlerin and subsequently treated with 25 ng/ml mOSM (right). Following a 5-min stimulation, cells were harvested and ~750 µg of whole cell lysates were immunoprecipitated at 4°C overnight with monoclonal anti-PKC{delta} Ab, followed by a 1-h incubation with protein A-agarose. Immune complexes were subjected to kinase activity assay using histone H1 as a substrate in presence of 10 µCi per reaction of [{gamma}-32P]ATP. Reactions were stopped with 4x Laemmli buffer, resolved on 12% SDS-PAGE and proteins were transferred to nitrocellulose. Determination of PKC{delta} loading was performed by immunoblotting using anti-PKC{delta} Ab (top) while PKC{delta} kinase activity was assessed by autoradiograph detection of {gamma}-32P-labeled histone (bottom). B, A portion of MLF whole cell lysates from experiments described were 8% SDS-PAGE and subjected to immunoblotting for phosphoserine 473 Akt (p-ser473Akt) or total Akt (Akt).

 
RNA interference targeting PKC{delta} inhibits IL-6 secretion

Because PKC{delta}-independent effects mediated by rottlerin have also been suggested in prior studies, we used RNA interference strategies to selectively abrogate PKC{delta} expression in C57BL/6 lung fibroblasts and subsequently examined IL-6 production in response to mOSM stimulation. Forty-eight hour incubation with the PKC{delta} 1153 target siRNA dramatically reduced PKC{delta} protein expression as evidenced by immunoblot analysis (Fig. 5A). Neither scrambled 1153 control siRNA nor random oligomer controls affected PKC{delta} expression, indicating specificity of the 1153 target oligomer. Expression of PKC{epsilon}, another novel PKC isoform, was not affected by PKC{delta}-targeted siRNA (Fig. 5B). Cells transfected with PKC{delta} 1153 and subsequently stimulated with mOSM for 24 h demonstrated significant reduction of IL-6 mRNA levels, consistent with effects observed in GF109203X-treated cell cultures or cultures treated with rottlerin (Fig. 5C). OSM-induced IL-6 secretion was also affected by PKC{delta} 1153 siRNA treatment (Fig. 5D). IL-6 expression was not reduced in cell cultures that were either mock-transfected (Lipofectamine alone) or pretreated with the 1153 scrambled siRNA or random RNA oligomer controls.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. Selective attenuation of PKC{delta} expression by RNA interference. A, MLF cultures in 24-well dishes (1 x 105 cells/well) were transfected with Lipofectamine alone (Mock), PKC{delta} targeting RNA duplex (PKC{delta} 1153), scrambled control (PKC{delta} 1153scr), or irrelevant intermediate guanine plus cytosine (G+C) content (Med G+C) of RNA duplex. Following overnight incubation cells were either left untreated or stimulated with 25 ng/ml mOSM for 24 h. Whole cell lysates were collected in 4x Laemmli buffer and subjected to SDS-PAGE and immunoblotting for PKC{delta} (top) or actin (bottom). B, MLF cultures (1 x 105 cells/well, 24-well dish) were either left untreated or transfected with Lipofectamine alone (Mock), PKC{delta} 1153 scrambled control (PKC{delta} 1153scr), or PKC{delta} 1153 target duplex (PKC{delta} 1153). Following a 48-h incubation, whole cell lysates were collected, resolved by SDS-PAGE and subsequently immunoblotted for PKC{delta} (top) and PKC{epsilon} (bottom). C, MLF were cultured in 12-well dishes (2.5 x 105 cells/well) were either left untreated or stimulated for 24 h with 25 ng/ml mOSM in presence of indicated pretreatments. GF109203X (7.5 µM) and rottlerin (5 µM) pretreatments were administered 15 min before stimulation. For cultures subjected to RNA interference, either duplex or Lipofectamine vehicle were administered overnight before mOSM treatment as indicated and described in A. Following stimulation total RNA was isolated using a Qiagen RNEasy microextraction kit according to the manufacturer’s instructions. One microgram of reverse-transcribed cDNA was subjected to TaqMan for quantitation of IL-6 with normalization to GAPDH. Data are presented from triplicate experiments as the fold increase in mRNA expression relative to unstimulated control. Error bars correspond to SD. *, p < 0.05 compared with mOSM-stimulated controls. D, MLF cultures in 24-well dishes (1 x 105 cells/well) were transfected with Lipofectamine alone (Mock), PKC{delta} targeting RNA duplex (PKC{delta} 1153), scrambled control (PKC{delta} 1153scr), or irrelevant intermediate G+C content (Med G+C) of RNA duplex. Following overnight incubation, cells were either left untreated ({square}) or stimulated with 25 ng/ml mOSM for 24 h ({blacksquare}). Conditioned medium was tested in the capture ELISA for quantification of IL-6. The experiment was conducted in triplicate. *, p < 0.01 as assessed by one-way ANOVA among transfected mOSM-stimulated controls.

 
PKC{delta}, but not PKC{epsilon} or PI3K-dependent kinases PDK-1 or Akt1, is required for IL-6 expression

To further assess the requirement for PKC{delta} during mOSM-induced IL-6 expression, we examined the effects of RNA interference knockdown of PKC{epsilon}, a closely related PKC isoform. Additionally, as our results indicated a dependence of PI3K for both IL-6 expression and PKC{delta} activity, we also examined whether PDK-1, known to phosphorylate various PKC enzymes, or Akt1, a ubiquitous downstream target of PI3K-PDK-1 activity, contributed to OSM-induced effects mediated through PKC{delta}. mOSM-induced IL-6 secretion by C57BL/6 MLF was also not affected by PKC{epsilon} siRNA pretreatment (Fig. 6A) and ablation of PKC{epsilon} expression by siRNA did not influence PKC{delta} kinase activity as demonstrated in Fig. 6B. PDK-1-targeted RNA interference did not affect mOSM-induction of IL-6 (Fig. 6A), and we observed an apparent increase in PKC{delta} activity in cells depleted of PDK-1 (Fig. 6B). Akt1 knockdown using RNA interference effectively reduced expression and activity assessed by Thr308 phosphorylation. However, it did not influence mOSM-induced IL-6 secretion (Fig. 6C). The siRNA treatment targeting PCK{epsilon}, PDK-1, or Akt1 did not affect PKC{delta} protein levels. These results indicate a requirement for PI3K–PKC{delta} in mediating mOSM stimulation of IL-6 gene expression in murine fibroblast cell lines that is independent of PDK-1 and Akt1.


Figure 6
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 6. Inhibition of PKC{delta}, but not PKC{epsilon}, PDK-1 or Akt1, reduces IL-6 expression. A, MLF cultures in 24-well dishes (1 x 105 cells/well) were either left untreated (Control) or transfected with Lipofectamine alone (Mock), PKC{delta} target siRNA (PKC{delta} 1153), PKC{epsilon} target siRNA (PKC{epsilon} 1582), PDK-1 target siRNA (PDK-1 1616), or scrambled PKC{delta} siRNA control (Scramble). Following overnight incubation transfected cultures were treated with 25 ng/ml mOSM for 24 h, and conditioned medium was measured for IL-6 secretion by capture ELISA. Assay was conducted in triplicate. *, p < 0.01 as assessed by one-way ANOVA among transfected mOSM-stimulated cultures. B, MLF were cultured in 100-mm dishes to ~75% confluence and subsequently either left untreated (Control) or transfected with Lipofectamine alone (Mock) or the indicated siRNA oligomers. Following overnight incubation, cells were stimulated for 5 min with 25 ng/ml mOSM and whole cell lysates were collected. Fifteen micrograms of lysates was separated by 8% SDS-PAGE and subjected to immunoblotting with Abs to the indicated proteins. The remaining lysate (750 µg of protein) was subsequently used to assess PKC{delta} activity by kinase activity assay as previously described. C, MLF were cultured in 100-mm dishes and transfected with siRNA oligomers as indicated (top). Following overnight incubation, cultures were stimulated for 5 min with 25 ng/ml mOSM and whole cell lysates were collected, separated by 8% SDS-PAGE, and probed for activated Akt1 (p-thr308Akt) or total Akt (Akt). MLF were cultured in 24-well dishes (1 x 105 cells/well) as previously described and transfected with siRNA oligomers as indicated (bottom). Following overnight incubation, cultures were stimulated with 25 ng/ml mOSM for 24 h, and conditioned medium (conducted in triplicate) was collected and measured for IL-6 secretion by capture ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our results indicate that OSM induces both PKC{delta} and PI3K/Akt activation in murine fibroblasts, two pathways used in addition to the JAK/STAT and MAPK signaling mechanisms previously established in other studies (29, 30). We show that mOSM-mediated maximal up-regulation of IL-6 expression in murine fibroblasts is dependent upon a pathway involving the activity of PKC{delta}. Additionally, we indicate that PI3K activity is selectively induced by OSM, and in explant cultures of MLFs PI3K is an important pathway mediating IL-6 up-regulation. These findings reveal a novel and mOSM-specific signal transduction pathway in murine fibroblasts, which is involved in IL-6 gene regulation in vitro.

Previously, we had demonstrated that expression of a variety of gene products, including IL-6 and proinflammatory mediators, such as MCP-1 and eotaxin-1, are more potently induced by mOSM relative to other gp130 cytokine family members (30, 32). Several studies have also shown that OSM is uniquely involved in the activation of a variety of gene products associated with inflammatory responses, including plasminogen activator (33), cyclooxygenase-2 (34), and several matrix metalloproteinases (35). Additionally, when transiently overexpressed in vivo, OSM is capable of inducing substantial inflammation that includes immune cell infiltration, fibroblast hyperplasia and alterations of the extracellular matrix network within connective tissue in mouse models (10, 36). The broad spectrum of activities mediated by OSM likely reflects its stimulation of multiple signaling pathways, some of which may regulate discrete patterns of gene expression. Our studies presented here suggest that activation of PI3K and PKC{delta} contribute to OSM-induced effects, and further investigation is required to assess the activity this putative pathway in other cell types that respond to OSM.

PKC{delta} belongs to the novel, or diacylglycerol-dependent and calcium-independent family of PKC isoforms. Evidence indicating PKC{delta} contributes to OSM regulation of osteoblastic factor expression from human osteosarcoma cell lines (25) along with results presented in this study suggest further examination of the role of PKC{delta} in OSM-mediated gene regulatory events is merited, particularly due to our indication that PKC{delta} is an important molecule mediating mOSM-dependent up-regulation of inflammation-associated factors such as IL-6. The potency of inhibition of IL-6 gene expression by rottlerin suggested that PKC{delta} may be an essential intracellular factor for mOSM-mediated up-regulation of IL-6. Several studies have called into question the specificity of rottlerin for PKC{delta} (37, 38, 39), and we have shown that at concentrations sufficient for inhibition of IL-6 expression rottlerin does not directly affect PKC{delta} activity in C57BL/6 fibroblasts. Furthermore, our data support the findings of one published report in which the authors showed rottlerin inhibited activation of Akt (40). Nonetheless, ablation of PKC{delta} expression using RNA interference clearly shows that PKC{delta} is required for induction of IL-6 expression by OSM, as evidenced by reduction of both mRNA levels and protein secretion. PKC{delta} has been shown to influence the activation of several signaling intermediates known to regulate expression of IL-6, including components of the JAK/STAT and p38 MAPK pathways (41, 42, 43). Whether PKC{delta} regulates these kinase cascades following OSM stimulation and its influence upon transcriptional regulation through these pathways is at present unclear and requires further investigation.

Human OSM has been shown in studies using Kaposi’s sarcoma tumor cells to induce activation of PI3K (44). We also demonstrate that PI3K is involved in regulation of IL-6 expression in early passage explanted murine fibroblast cultures stimulated with mOSM, and may be required in the activation of PKC{delta} mediated by mOSM, as shown through kinase activity assay of C57BL/6 lung fibroblast lysates. A number of kinase enzymes, notably the serine/threonine kinases PDK-1, PKB/Akt, and several PKC isoforms, are known to be recruited to the plasma membrane and activated through interaction with 3'-phosphorylated phosphatidylinositol (45). PKC{delta} activity has been shown to be indirectly dependent upon PI3K-induced activated phosphatidylinositols (46), findings which correlate to our observations that PI3K inhibition attenuates PKC{delta} activity. Interestingly, our observations that RNA interference-mediated knockdown of PDK-1 did not reduce IL-6 expression and actually increased PKC{delta} activity contradict previously published statements implicating the requirement of PDK-1 for PKC{delta} activity (47). Although the reason for increased PKC{delta} activity is not clear, PDK-1 and PKC{delta} may possibly compete for access to molecules such as 3'-phosphatidylinositols. Additionally, our findings using siRNA to deplete Akt1 indicate that Akt1 does not influence mOSM-induced IL-6 expression. Furthermore, we have not observed any direct association between PKC{delta} and Akt by coimmunoprecipitation (data not shown), which collectively suggests that mOSM-stimulated PKC{delta} activation, while dependent upon PI3K, occurs independently of PDK-1 and Akt1. Our results demonstrating the requirement for activation of PI3K and PKC{delta} during OSM-induced IL-6 gene expression in MLFs has led us to pursue whether PI3K-associated pathways contribute to regulation of additional OSM gene targets in murine connective tissue cells. In conclusion, our findings indicate that OSM-mediated activation of PKC{delta} contributes to the ability of OSM to regulate expression of IL-6, and suggest that the induction of PKC{delta} and PI3K activity is critical for the generation of inflammatory responses associated with this cytokine.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding from the Canadian Institutes for Health Research and the Arthritis Society of Canada. Back

2 Address correspondence and reprint requests to Dr. Carl D. Richards, Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, Michael G. DeGroote Centre for Learning and Discovery, Room 4020, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. E-mail address: richards{at}mcmaster.ca Back

3 Abbreviations used in this paper: OSM, oncostatin M; mOSM, murine OSM; MLF, murine lung fibroblast; PKC, protein kinase C; PDK-1, phosphoinositide-dependent kinase-1; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; Ct, threshold cycle. Back

Received for publication February 15, 2005. Accepted for publication September 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Pettipher, E. R., G. A. Higgs, B. Henderson. 1986. Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc. Natl. Acad. Sci. USA 83: 8749-8753. [Abstract/Free Full Text]
  2. Saklatvala, J.. 1986. Tumour necrosis factor {alpha} stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature 322: 547-549. [Medline]
  3. Taga, T., T. Kishimoto. 1997. gp130 and the interleukin-6 family of cytokines. Annu. Rev. Immunol. 15: 797-819. [Medline]
  4. Schindler, R., J. Mancilla, S. Endres, R. Ghorbani, S. C. Clark, C. A. Dinarello. 1990. Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75: 40-47. [Abstract/Free Full Text]
  5. Manicourt, D. H., P. Poilvache, A. Van Egeren, J. P. Devogelaer, M. E. Lenz, E. J. Thonar. 2000. Synovial fluid levels of tumor necrosis factor {alpha} and oncostatin M correlate with levels of markers of the degradation of crosslinked collagen and cartilage aggrecan in rheumatoid arthritis but not in osteoarthritis. Arthritis Rheum. 43: 281-288. [Medline]
  6. Carroll, G., M. Bell, H. Wang, H. Chapman, J. Mills. 1998. Antagonism of the IL-6 cytokine subfamily: a potential strategy for more effective therapy in rheumatoid arthritis. Inflamm. Res. 47: 1-7. [Medline]
  7. Hui, W., M. Bell, G. Carroll. 1996. Oncostatin M (OSM) stimulates resorption and inhibits synthesis of proteoglycan in porcine articular cartilage explants. Cytokine 8: 495-500. [Medline]
  8. Richards, C. D., A. Agro. 1994. Interaction between oncostatin M, interleukin 1 and prostaglandin E2 in induction of IL-6 expression in human fibroblasts. Cytokine 6: 40-47. [Medline]
  9. Modur, V., M. J. Feldhaus, A. S. Weyrich, D. L. Jicha, S. M. Prescott, G. A. Zimmerman, T. M. McIntyre. 1997. Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules. J. Clin. Invest. 100: 158-168. [Medline]
  10. Langdon, C., C. Kerr, M. Hassen, T. Hara, A. L. Arsenault, C. D. Richards. 2000. Murine oncostatin M stimulates mouse synovial fibroblasts in vitro and induces inflammation and destruction in mouse joints in vivo. Am. J. Pathol. 157: 1187-1196. [Abstract/Free Full Text]
  11. O’Hara, K. A., M. A. Kedda, P. J. Thompson, D. A. Knight. 2003. Oncostatin M: an interleukin-6-like cytokine relevant to airway remodelling and the pathogenesis of asthma. Clin. Exp. Allergy 33: 1026-1032. [Medline]
  12. Brown, T. J., J. M. Rowe, J. W. Liu, M. Shoyab. 1991. Regulation of IL-6 expression by oncostatin M. J. Immunol. 147: 2175-2180. [Abstract]
  13. Heinrich, P. C., I. Behrmann, G. Muller-Newen, F. Schaper, L. Graeve. 1998. Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem. J. 334: (Pt. 2):297-314. [Medline]
  14. Ichihara, M., T. Hara, H. Kim, T. Murate, A. Miyajima. 1997. Oncostatin M and leukemia inhibitory factor do not use the same functional receptor in mice. Blood 90: 165-173. [Abstract/Free Full Text]
  15. Li, W. Q., F. Dehnade, M. Zafarullah. 2001. Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway. J. Immunol. 166: 3491-3498. [Abstract/Free Full Text]
  16. Wang, Y., O. Robledo, E. Kinzie, F. Blanchard, C. Richards, A. Miyajima, H. Baumann. 2000. Receptor subunit-specific action of oncostatin M in hepatic cells and its modulation by leukemia inhibitory factor. J. Biol. Chem. 275: 25273-25285. [Abstract/Free Full Text]
  17. Newton, A. C.. 1995. Protein kinase C: structure, function, and regulation. J. Biol. Chem. 270: 28495-28498. [Free Full Text]
  18. Fima, E., G. Shahaf, T. Hershko, R. N. Apte, E. Livneh. 1999. Expression of PKC{eta} in NIH-3T3 cells promotes production of the pro-inflammatory cytokine interleukin-6. Eur. Cytokine Netw. 10: 491-500. [Medline]
  19. Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2: 449-456. [Medline]
  20. Gschwendt, M.. 1999. Protein kinase C{delta}. Eur. J. Biochem. 259: 555-564. [Medline]
  21. Carpenter, L., D. Cordery, T. J. Biden. 2001. Protein kinase C{delta} activation by interleukin-1beta stabilizes inducible nitric-oxide synthase mRNA in pancreatic beta-cells. J. Biol. Chem. 276: 5368-5374. [Abstract/Free Full Text]
  22. Deb, D. K., A. Sassano, F. Lekmine, B. Majchrzak, A. Verma, S. Kambhampati, S. Uddin, A. Rahman, E. N. Fish, L. C. Platanias. 2003. Activation of protein kinase C{delta} by IFN-{gamma}. J. Immunol. 171: 267-273. [Abstract/Free Full Text]
  23. Liu, J. F., M. Crepin, J. M. Liu, D. Barritault, D. Ledoux. 2002. FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway. Biochem. Biophys. Res. Commun. 293: 1174-1182. [Medline]
  24. Jedrzkiewicz, S., H. Nakamura, E. S. Silverman, A. D. Luster, N. Mansharamani, K. H. In, G. Tamura, C. M. Lilly. 2000. IL-1beta induces eotaxin gene transcription in A549 airway epithelial cells through NF-{kappa}B. Am. J. Physiol. 279: L1058-L1065.
  25. Chipoy, C., M. Berreur, S. Couillaud, G. Pradal, F. Vallette, C. Colombeix, F. Rédini, D. Heymann, F. Blanchard. 2004. Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKC{delta} and STAT3. J. Bone Miner. Res. 19: 1850-1861. [Medline]
  26. Kitaura, J., K. Asai, M. Maeda-Yamamoto, Y. Kawakami, U. Kikkawa, T. Kawakami. 2000. Akt-dependent cytokine production in mast cells. J. Exp. Med. 192: 729-740. [Abstract/Free Full Text]
  27. Knall, C., G. S. Worthen, G. L. Johnson. 1997. Interleukin 8-stimulated phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc. Natl. Acad. Sci. USA 94: 3052-3057. [Abstract/Free Full Text]
  28. Kim, K. W., M. L. Cho, M. K. Park, C. H. Yoon, S. H. Park, S. H. Lee, H. Y. Kim. 2005. Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor {kappa}B-dependent pathway in patients with rheumatoid arthritis. Arthritis Res. Ther. 7: R139-R148. [Medline]
  29. Tong, L., D. Smyth, C. Kerr, J. Catterall, C. D. Richards. 2004. Mitogen-activated protein kinases Erk1/2 and p38 are required for maximal regulation of TIMP-1 by oncostatin M in murine fibroblasts. Cell. Signal. 16: 1123-1132. [Medline]
  30. Langdon, C., C. Kerr, L. Tong, C. D. Richards. 2003. Oncostatin M regulates eotaxin expression in fibroblasts and eosinophilic inflammation in C57BL/6 mice. J. Immunol. 170: 548-555. [Abstract/Free Full Text]
  31. Ding, S. Z., C. H. Cho, S. K. Lam. 2000. Regulation of interleukin 6 production in a human gastric epithelial cell line MKN-28. Cytokine 12: 1129-1135. [Medline]
  32. Langdon, C., J. Leith, F. Smith, C. D. Richards. 1997. Oncostatin M stimulates monocyte chemoattractant protein-1- and interleukin-1-induced matrix metalloproteinase-1 production by human synovial fibroblasts in vitro. Arthritis Rheum. 40: 2139-2146. [Medline]
  33. Hamilton, J. A., T. Leizer, D. S. Piccoli, K. M. Royston, D. M. Butler, M. Croatto. 1991. Oncostatin M stimulates urokinase-type plasminogen activator activity in human synovial fibroblasts. Biochem. Biophys. Res. Commun. 180: 652-659. [Medline]
  34. Bernard, C., R. Merval, M. Lebret, P. Delerive, I. Dusanter-Fourt, S. Lehoux, C. Créminon, B. Staels, J. Maclouf, A. Tedgui. 1999. Oncostatin M induces interleukin-6 and cyclooxygenase-2 expression in human vascular smooth muscle cells: synergy with interleukin-1beta. Circ. Res. 85: 1124-1131. [Abstract/Free Full Text]
  35. Catterall, J. B., S. Carrere, P. J. Koshy, B. A. Degnan, W. D. Shingleton, C. E. Brinckerhoff, J. Rutter, T. E. Cawston, A. D. Rowan. 2001. Synergistic induction of matrix metalloproteinase 1 by interleukin-1{alpha} and oncostatin M in human chondrocytes involves signal transducer and activator of transcription and activator protein 1 transcription factors via a novel mechanism. Arthritis Rheum. 44: 2296-2310. [Medline]
  36. Kerr, C., C. Langdon, F. Graham, J. Gauldie, T. Hara, C. D. Richards. 1999. Adenovirus vector expressing mouse oncostatin M induces acute-phase proteins and TIMP-1 expression in vivo in mice. J. Interferon Cytokine Res. 19: 1195-1205. [Medline]
  37. Kayali, A. G., D. A. Austin, N. J. Webster. 2002. Rottlerin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial oxidative phosphorylation. Endocrinology 143: 3884-3896. [Abstract/Free Full Text]
  38. Susarla, B. T., M. B. Robinson. 2003. Rottlerin, an inhibitor of protein kinase C{delta} (PKC{delta}), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKC{delta}-independent. J. Neurochem. 86: 635-645. [Medline]
  39. Tapia, J. A., R. T. Jensen, L. J. Garcia-Marin. 2006. Rottlerin inhibits stimulated enzymatic secretion and several intracellular signaling transduction pathways in pancreatic acinar cells by a non-PKC-{delta}-dependent mechanism. Biochim. Biophys. Acta 1763: 25-38. [Medline]
  40. Leitges, M., W. Elis, K. Gimborn, M. Huber. 2001. Rottlerin-independent attenuation of pervanadate-induced tyrosine phosphorylation events by protein kinase C-{delta} in hemopoietic cells. Lab. Invest. 81: 1087-1095. [Medline]
  41. Jain, N., T. Zhang, W. H. Kee, W. Li, X. Cao. 1999. Protein kinase C{delta} associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J. Biol. Chem. 274: 24392-24400. [Abstract/Free Full Text]
  42. García-Fernández, L. F., A. Losada, V. Alcaide, A. M. Álvarez, A. Cuadrado, L. González, K. Nakayama, K. I. Nakayama, J. M. Fernández-Sousa, A. Muñoz, J. M. Sánchez-Puelles. 2002. Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C{delta}. Oncogene 21: 7533-7544. [Medline]
  43. Igarashi, M., H. Wakasaki, N. Takahara, H. Ishii, Z. Y. Jiang, T. Yamauchi, K. Kuboki, M. Meier, C. J. Rhodes, G. L. King. 1999. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J. Clin. Invest. 103: 185-195. [Medline]
  44. Soldi, R., A. Graziani, R. Benelli, D. Ghigo, A. Bosia, A. Albini, F. Bussolino. 1994. Oncostatin M activates phosphatidylinositol-3-kinase in Kaposi’s sarcoma cells. Oncogene 9: 2253-2260. [Medline]
  45. Maffucci, T., M. Falasca. 2001. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein cooperative mechanism. FEBS Lett. 506: 173-179. [Medline]
  46. Toker, A., M. Meyer, K. K. Reddy, J. R. Falck, R. Aneja, S. Aneja, A. Parra, D. J. Burns, L. M. Ballas, L. C. Cantley. 1994. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J. Biol. Chem. 269: 32358-32367. [Abstract/Free Full Text]
  47. Le Good, J. A., W. H. Ziegler, D. B. Parekh, D. R. Alessi, P. Cohen, P. J. Parker. 1998. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281: 2042-2045. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
S. P. Kastl, W. S. Speidl, C. Kaun, K. M. Katsaros, G. Rega, T. Afonyushkin, V. N. Bochkov, P. Valent, A. Assadian, G. W. Hagmueller, et al.
In Human Macrophages the Complement Component C5a Induces the Expression of Oncostatin M via AP-1 Activation
Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): 498 - 503.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
R. D. Ramnath, J. Sun, S. Adhikari, L. Zhi, and M. Bhatia
Role of PKC-{delta} on substance P-induced chemokine synthesis in pancreatic acinar cells
Am J Physiol Cell Physiol, March 1, 2008; 294(3): C683 - C692.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Smyth, D. C.
Right arrow Articles by Richards, C. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Smyth, D. C.
Right arrow Articles by Richards, C. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS