Rottlerin, a PKC isozyme-selective inhibitor, affects signaling events and cytokine production in...

Rottlerin, a PKC isozyme-selective inhibitor, affects signalingevents and cytokine production in human monocytes

Ewa Kontny, Mariola Kurowska, Katarzyna Szczepanska, and Włodzimierz MaslinskiDepartment of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland

Abstract: The implication of select protein kinaseC (PKC) isoenzymes in cytokine production byhuman monocytes was investigated using an isozyme-selective inhibitor of PKC, rottlerin. We found thatlipopolysaccharide (LPS) triggers cytosol-to-mem-brane translocation of PKCa and d isoenzymes,whereas phorbol ester (PMA) induces translocationof several PKC isoforms. Moreover, we show that inLPS- and PMA-stimulated monocytes rottlerin af-fects several cellular responses. (1) At low (15 mM)concentration it blocks translocation of PKCd,diminishes DNA binding activity of AP-1 transcrip-tion factor, and attenuates cytokine production[tumor necrosis factor a (TNF-a) G interleukin-1b(IL-1b)]. (2) At high (50 mM) concentration itprevents translocation of PKCa, and subsequentlyinhibits ERK1/ERK2 phosphorylation, DNA bind-ing activities of AP-1 and nuclear factor-kB tran-scription factors, and the production of both testedcytokines. Thus, we propose that cytosol-to-mem-brane translocation of PKCa and PKd isoenzymesmay represent early steps in the signaling cascadesthat lead to TNF-a and IL-1b production in humanmonocytes. J. Leukoc. Biol. 67: 249–258; 2000.

Key Words: TNF-a· IL-1b · MAP kinases (ERK1/2) · transcrip-tion factors · AP-1 · NF-kB

INTRODUCTION

Monocytes play a critical role in inflammation by releasingoxygen metabolites, lysosomal enzymes, arachidonic acid me-tabolites, and pro-inflammatory cytokines, such as tumornecrosis factor a (TNF-a) and interleukins (IL-1, IL-6, IL-8).Pro-inflammatory cytokines play a crucial role in the immuneresponse, hematopoiesis, and inflammation. However, in somepathological conditions they may exert harmful effects. Forexample, in rheumatoid arthritis (RA), the continuous produc-tion of pro-inflammatory cytokines mediates chronic inflamma-tory responses that result in connective tissue damage [1],whereas in septic shock syndrome, resulting from the hostexposure to bacterial lipopolysaccharide (LPS), pro-inflamma-tory cytokines, especially TNF-a, mediate adverse effects ofLPS [2].

The monocyte-activating agents that induce the productionof pro-inflammatory cytokines include: LPS, a surface compo-nent of gram-negative bacteria released on host infection, the

antigen-antibody complexes and cytokines formed during im-mune response, as well as phorbol esters [the activators ofclassical and novel isoenzymes of the protein kinase C (PKC)family], used in in vitro studies. LPS is the most potent activatorof pro-inflammatory cytokine synthesis in monocytes. Cellularactivation by low concentrations of LPS (,1 µg/mL) requires itsbinding to CD14 surface glycoprotein [3]. However, a highconcentration of LPS can stimulate cells in a CD14-independent manner, probably as a result of LPS binding toother receptor molecules (CD11/CD18 and Toll-like receptor 2)[4, 5]. The molecular mechanism that transduces LPS receptor-mediated signals to the nucleus is not fully understood. It hasbeen shown that a number of src family tyrosine kinases (e.g.,lyn, hck, frg) [6–8], the mitogen-activated protein kinases(MAPK) ERK1/ERK2 [9–12], stress-activated kinases p38[13–15], and JNK [16, 17], as well as PKC [10, 18, 19] becomeactivated in LPS-stimulated monocytes and macrophages.However, recent studies question the importance of src kinasesin LPS-triggered signaling leading to pro-inflammatory cyto-kine production by showing that macrophages from hck-/-frg-/-

lyn-/- knockout mice produce normal levels of TNF-a, IL-1b,and IL-6 in response to LPS stimulation [20]. In contrast, theactivities of MAP kinases and PKC are still consideredimportant for these responses. This statement is based on datashowing that: (1) the selective inhibitors of ERKs damp TNF-aand IL-1b synthesis by blocking transcription of genes encod-ing these cytokines [11, 17], whereas the selective inhibitors ofp38 and JNK affect mostly their translation or act at theposttranscriptional events [13, 17], and (2) the selectiveinhibitors of PKC also potently reduce production of thesecytokines [12, 19]. Despite these reports, little is known aboutthe role of individual PKC isoenzymes in these processes.

The PKC family comprises at least 12 closely relatedisoenzymes. Differences in their structure, cofactor require-ment, substrate specificity, tissue distribution, and subcellularlocalization suggest that in a given cell type PKC may functionin an isozyme-specific manner [21, 22]. Cells of monocyte/macrophage lineage express not only the classical (Ca21-dependent) isoenzymes a, bI, and bII, but also the novel(Ca21-independent) isoenzymes d and e, and the atypicalisoenzyme z [12, 23–25]. Data describing the contribution of

Correspondence: Włodzimierz Maslinski, Department of Pathophysiologyand Immunology, Institute of Rheumatology, Spartanska 1, 02-637 Warsaw,Poland. E-mail: [email protected].

Received June 1, 1999; revised October 20, 1999; accepted October 21,1999.

Journal of Leukocyte Biology Volume 67, February 2000 249

the respective PKC isoenzymes to the regulation of cellfunctions are scarce. Regarding human monocytes and mono-cytic cell lines, it has been shown that: (1) different PKC areresponsible for the stimulation (PKCb) and inhibition (PKCaand e) of the Fcg-receptor-mediated intracellular killing ofbacteria [25], (2) PKCa is required for superoxide anionproduction [26], (3) PKC b and d are essential for thedifferentiation into macrophages [27–29], whereas (4) PKCdand e are implicated in the signaling pathways leading toapoptosis [30]. The role of individual PKC isoenzymes in theinduction of pro-inflammatory cytokine synthesis only recentlygot some attention. For example, there are reports indicatingthat in LPS-stimulated murine macrophages PKCb activationis important for the production of TNF-a [31] and that PKCapositively regulates transcription of TNF-a and IL-1b mRNAs[32].

In our previous studies we reported the important role of PKCactivation for the synthesis of pro-inflammatory cytokines(TNF-a, IL-1b, and IL-6) in human monocytes [12, 33] andsuggested that activation of both the classical (a and bII) andthe novel (d and e) PKC isoenzymes may represent critical stepsin the induction of signaling cascade resulting in the synthesisof these cytokines [12]. In this study, we have further investi-gated the role of PKC isoenzymes in the regulation of pro-inflammatory cytokine synthesis in these cells through the useof the isoenzyme-selective PKC inhibitor, rottlerin.

We report that rottlerin inhibits LPS-triggered cytosol-to-membrane translocation of PKCa and PKCd, several signalingevents important for the cytokine response, and production ofTNF-a and IL-1b. Therefore, we propose that cytosol-to-membrane translocation of a and d isoenzymes of PKC mayrepresent early and critical events in the signaling pathwaysthat lead to the production of TNF-a and IL-1b in humanmonocytes.

MATERIALS AND METHODS

Cell culture and treatment

Monocytes were separated from peripheral blood mononuclear cells of healthyadult volunteers by adherence to plastic surface, as described previously [12].Cells were cultured in RPMI-1640 medium supplemented with 1% fetal calfserum, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and2.5 3 10-5 M b-mercaptoethanol (GIBCO). Cells were stimulated with 5 µg/mLof LPS (Escherichia coli 055:B5; Difco), or with 1 nM phorbol 12-myristate13-acetate (PMA; Sigma, St. Louis, MO). Inhibitor of PKC, rottlerin (Calbio-chem, La Jolla, CA), was added at low (15 µM) or high (50 µM) concentrations30 min before stimuli.

The cell viability, assayed by trypan blue exclusion, exceeded 80% after21-h incubation in the presence or absence of stimuli and 15–50 µM rottlerin.A higher concentration of rottlerin ($70 µM) was toxic to the cells.

Cytokine detection using enzyme-linkedimmunosorbent assay (ELISA)and Western blotting

Cytokine production was assayed after 21 h of cell culture. The concentration ofcytokines was measured in both culture supernatants and cell lysates,representing therefore total cytokine production, using specific ELISA, aspreviously described [12]. Briefly, goat polyclonal neutralizing antibodiesspecific to human TNF-a or IL-1b (R & D Systems) were used as capture,whereas cytokine-specific rabbit polyclonal antibodies (anti-TNF-a from

Genzyme; anti-IL-1b from Sigma) were used as detection antibodies, followedby horseradish peroxidase-conjugated goat anti-rabbit immunoglobulins (GAR-POD) and O-phenylenediamine dihydrochloride (both from Sigma) as asubstrate. Recombinant human TNF-a and IL-1b standards were from R & DSystems. Optical density was measured at 492 nm using an automatic ELISAreader (LP 400, Diagnostics Pasteur). The detection limit was 4 pg/mL forTNF-a and 15 pg/mL for IL-1b.

For Western blotting, proteins present in culture supernatants were acetoneprecipitated at 220°C overnight, resuspended in Laemmli buffer, and boiledfor 5 min. Proteins were separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) denaturing gel followed byblotting onto a membrane (PVDF, Bio-Rad). Cytokines were detected usinganti-TNF-a or IL-1b antibodies (detecting antibodies used in ELISA, seeabove), followed by application of GAR-POD and an enhanced chemilumines-cence (ECL) system (Amersham).

Determination of PKC isoenzymes

Expression of PKC isoenzymes was analyzed in the cytosolic and membraneprotein fractions according to the method previously described [12]. Thecytosolic fraction was prepared by lysis of the cells in 20 mM Tris-HCl buffer,pH 7.5, containing 2 mM EDTA, 10 mM EGTA, 1 mM dithiothreitol, 1 mMphenylmethylsulfonyl fluoride, 100 µg/mL digitonin, 10 µg/mL leupeptin, and10 µg/mL pepstatin A (all from Sigma). The remaining protein pellets weretreated with the lysis buffer supplemented with 1% Triton X-100 (Sigma) toobtain the membrane fraction. Protein concentration was measured using aBio-Rad Protein Assay kit (Bio-Rad) and adjusted to the same level in all testedsamples. Proteins were acetone precipitated, boiled in Laemmli buffer, andseparated on 8% SDS-PAGE gel. After blotting, the PVDF membranes weretreated with the rabbit polyclonal antibodies specific to PKC a, bII, or d

isoenzymes (Calbiochem), followed by GAR-POD and ECL detection systems.The specificity of primary antibody binding was assured by pre-incubation ofthe antibody with 0.5 µg/mL of isoenzyme-specific peptide (Calbiochem) for 10min before use. Human recombinant PKC a, bII, and d isoenzymes (Calbio-chem) were used as the positive controls.

Detection of MAP kinases

Mitogen-activated protein kinases (MAP kinases; ERK1/ERK2) were detectedusing the PhosphoPlus p44/p42 MAP kinase antibody kit (New EnglandBioLabs), according to the manufacturer’s protocol. The phosphorylation statusof MAP kinases was determined by application of antibody that recognizes Thr202/Tyr 204 of phosphorylated ERK1 and ERK2 kinases or with antibody thatdetects total ERK1/ERK2 (phosphorylation-state independent) levels.

Estimation of DNA-binding activity of AP-1 andNF-kB transcription factors by the electromobilitygel-shift assay (EMSA)

The nuclear protein extracts were prepared by a micropreparation technique[34]. After normalization of protein contents, samples were stored at 270°Cuntil use. The AP-1, NF-kB, and Oct-1 oligonucleotides (Santa Cruz) wereend-labeled, using T4 polynucleotide kinase and [g-32P]ATP (both reagentsfrom Amersham). Gel-shift assay was performed according to the methodpreviously described [12]. Briefly, the binding of nuclear proteins (2.5µg/sample) to 32P-labeled probes (0.1 ng) was carried out for 1 h, at 4°C, in asolution of 10 mM HEPES, pH 7.9, 4% Ficoll 400, 0.125% bovine serumalbumin, 1 mM EDTA, 100 mM KCl, 4 mM MgCl2, 0.02% NP-40, 0.83 mM2-mercaptoethanol (all reagents from Sigma), and 2 µg poly(dI)-poly(dC)(Pharmacia). The protein-DNA complexes were resolved for 2 h at 250 V on a5% non-denaturing polyacrylamide gel in TBE buffer supplemented with0.02% NP-40. The specificity of probe binding was controlled by adding100-fold excess of unlabeled probe. The gels were dried and autoradiographedon X-ray films (Amersham).

Densitometric analysis

The products of Western blotting and EMSA were densitometrically scannedusing a Microcomputer Image Quant Device (Molecular Dynamics).

250 Journal of Leukocyte Biology Volume 67, February 2000 http://www.jleukbio.org

Statistical analysis

Results are expressed as means 6 SEM. The paired Student’s t test was appliedto evaluate the effect of the stimuli and an inhibitor. Probability values less than0.05 were considered statistically significant.

RESULTS

Translocation of PKC isoenzymes in LPS- orPMA-stimulated monocytes

It is well documented that on activation PKC isoenzymestranslocate from the cytosol to the particulate cellular fractions(e.g., plasma membrane, nuclear envelope) [35]. Therefore, wecompared the distribution of PKC isoforms among the cytosolicand membrane protein fractions isolated from the cells stimu-lated with LPS or PMA (Fig. 1). In PMA-treated monocytes

cytosol-to-membrane translocation of PKCa, bII, and d isoen-zymes occurred in early time points (5–15 min) and was stillevident after 30 min of stimulation (PKCa and bII). Bycontrast, LPS triggered weaker and transient translocation ofonly PKCa and d isoenzymes (from 5 to 30 min, with themaximum reached 15 min after stimulation). In addition, onLPS stimulation PKCe did not translocate to membrane(data not shown), whereas PMA triggered this event [12].These results indicate that for LPS-triggered responsescytosol-to-membrane translocation of PKCa and d may beimportant.

Rottlerin prevents cytosol-to-membranetranslocation of PKCd and PKCa isoenzymes

Rottlerin is an inhibitor of PKC that exerts different IC50 valuesfor PKCd (3–6 µM) and PKCa (30–42 µM) [36]. Because theabove data come from in vitro studies where purified enzymeswere used, we investigated whether rottlerin shows similarselectivity for PKCd and a isoenzymes present in monocytes.Monocytes were pretreated for 30 min with either a 15 or 50 µMconcentration of rottlerin, followed by 15-min stimulation of thecells with PMA or LPS. The concentrations of rottlerin werechosen according to the reported IC50 values for PKCd andPKCa. The higher concentrations of rottlerin exerted a toxiceffect on cell viability (see Materials and Methods) andtherefore were not used in this study. Both in PMA- andLPS-treated cells, low concentration of rottlerin (15 µM) almostcompletely inhibits cytosol-to-membrane translocation of onlyPKCd isoenzyme, whereas higher concentration (50 µM) of thisinhibitor also blocks cytosol-to-membrane translocation ofPKCa (Fig. 2). By contrast, PMA-triggered translocation ofneither PKCbII nor PKCe was affected by rottlerin (data notshown). Therefore, we confirm that also in our cell system it ispossible to discriminate between PKCd and PKCa isoenzymes,using a different concentration of rottlerin.

Rottlerin differentially affects production of TNF-aand IL-1b

Based on our observations that LPS triggers cytosol-to-membrane translocation of PKCa and PKCd, although rottlerindiscriminates between these isoenzymes, we further investi-

Fig. 1. LPS triggers cytosol-to-membrane translocation of PKCa and d

isoenzymes. Monocytes were stimulated with LPS (5 µg/mL) or PMA (1 nM) forthe indicated time period. The cytosolic (C) and membrane (M) protein fractionswere isolated from the cells as described in Materials and Methods. Proteins (10µg/lane) were tested for the presence of PKC a, bII, and d isoenzymes byWestern blotting. Control, nonstimulated cells. This is a representativeexperiment selected from three performed.

Fig. 2. Rottlerin inhibits translocation of PKCd and a isoenzymes in a dose-dependent manner. Monocytes pretreated for 30 min with either a low (15 µM) or high(50 µM) concentration of rottlerin, were stimulated for 15 min with PMA (1 nM) or LPS (5 µg/mL). Proteins (40 µg/lane) of cytosolic (C) and membrane (M) fractionswere tested for the presence of PKCa and d using Western blotting. Control, nonstimulated cells. This is a representative experiment selected from three performed.

Kontny et al. PKC isoforms and pro-inflammatory cytokine synthesis 251

gated whether rottlerin exerts any effect on pro-inflammatorycytokine production (Fig. 3). As determined by ELISA,spontaneous production of TNF-a and IL-1b in nonstimulatedmonocytes was low (124 6 39 and 352 6 100 pg/mL,respectively). In LPS-stimulated cells production of bothTNF-a (1011 6 173 pg/mL) and IL-1b (2503 6 551 pg/mL)was higher than in PMA-treated cells (878 6 165 and 1876 6533 pg/mL, respectively). Rottlerin at 15 µM strongly inhibitsboth LPS- and PMA-induced production of TNF-a (72 and87% of inhibition, respectively) and at 50 µM almost com-pletely blocks production of this cytokine (96 and 94% ofinhibition, respectively). By contrast, rottlerin exerts a weakerinhibitory effect on IL-1b production. At 15 µM, the inhibitorreduces LPS- and PMA-triggered production of IL-1b by 48and 59%, respectively. Treatment of LPS- and PMA-stimulatedmonocytes with the higher concentration (50 µM) of rottlerin

results in inhibition of IL-1b production by 87 and 82%,respectively (Fig. 3A). Similar differences were noted whenWestern blotting was used to evaluate the presence of bothtested cytokines in cell culture supernatants (Fig. 3B). It shouldbe pointed out that, by contrast to the non-glycosylated rhIL-1bstandard expressed in E. coli, LPS-triggered expression ofIL-1b appeared as a broad band (Fig. 3B), probably due todifferent glycosylation of IL-1b protein [37].

Based on a dose-dependent potency of rottlerin to inhibit theenzymatic activity of PKCa and PKCd isoenzymes [36], andsimilar concentration-dependent selectivity of this inhibitor toprevent translocation of these isoforms (Fig. 2), our resultssuggest that although the translocation (activity?) of both PKCdand a isoenzymes are required for the optimal production ofTNF-a and IL-1b, their contributions to the synthesis of aparticular cytokine are not equal.

Fig. 3. Rottlerin inhibits production ofTNF-a and IL-1b in a dose-dependentmanner. Monocytes pretreated with indi-cated concentrations of rottlerin werestimulated with LPS (5 µg/mL) or PMA(1 nM) for 21 h. (A) Total production ofTNF-a and IL-1b was determined byELISA, as described in Materials andMethods. Results are expressed as thepercentage of cytokine levels in cellcultures not exposed to rottlerin buttreated with LPS or PMA, respectively,and represent the mean 6 SEM of 15experiments. Statistical comparison be-tween cells pretreated with rottlerin andnot treated, is shown as a P value: *P 5

0.002; **P , 0.0001. (B) Expression ofTNF-a and IL-1b proteins in culture supernatants determined by Western blotting, as described in Materials and Methods. Standard, recombinanthuman TNF-a or IL-1b (10 ng/lane), respectively; control, nonstimulated cells; Ro, rottlerin. This is a representative experiment selected from threeperformed.


Rottlerin inhibits LPS- or PMA-triggeredphosphorylation of MAP kinases (ERK1/ERK2)

There is evidence that ERK-type MAP kinases are involved inthe signaling pathway leading to the production of TNF-a andIL-1b in LPS-treated cells [11, 15, 17]. The phosphorylation ofERKs in tyrosine/threonine residues is a prerequisite for theactivation of these enzymes. In some systems these events arePKC-dependent [38]. Therefore, we investigated the effect ofrottlerin on the phosphorylation of ERK1/ERK2 in mono-cytes stimulated with LPS or PMA (Fig. 4). In nonstim-ulated monocytes the expression of phosphorylated ERKswas weak or undetectable, whereas LPS and more stronglyPMA significantly raised the levels of phosphorylated forms ofERKs (both ERK1 and ERK2 become phosphorylated onstimulation; Fig. 4B). The results, based on densitometricscanning (Fig. 4A), show that rottlerin only at high concentra-tion (50 µM) significantly inhibits LPS- or PMA-triggeredphosphorylation of ERKs, by 85 and 74%, respectively. Bycontrast, the expression of the non-phosphorylated form ofERKs, represented by ERK2 because the expression of non-phosphorylated ERK1 was weak or undetectable, was similar innonstimulated and stimulated cells (Fig. 4). Moreover, rottlerindid not change significantly the expression of non-phosphory-lated ERK2 (Fig. 4A).

Therefore these results show that rottlerin inhibits phosphor-ylation of ERK1/ERK2 only at the concentration that prevents

earlier translocation of both PKCd and PKCa (Fig. 2), andsuggest that these events are associated.

Rottlerin inhibits LPS- or PMA-induced bindingactivity of AP-1 transcription factor

AP-1 and NF-kB transcription factors are potent regulators oftranscription of genes encoding TNF-a and IL-1b [39, 40].Therefore, we also investigated the effect of rottlerin onDNA-binding activity of these transcription factors in mono-cytes stimulated with LPS or PMA.

In nonstimulated cells the DNA-binding activity of AP-1 wasweak or undetectable but it was significantly induced in LPS-and PMA-treated cells (Fig. 5B). Rottlerin affected AP-1binding activity in a dose-dependent manner (Fig. 5A): at 15µM it inhibited LPS- and PMA-triggered levels by 37 and 47%,respectively, whereas at 50 µM it almost completely blocked theresponses in the cells treated with LPS and PMA (91 and 88%of inhibition, respectively).

These results show a limited correlation between inhibitedtranslocation of PKCd by a low concentration of rottlerin, andpartial loss of DNA-binding activity of AP-1. In contrast, thereis a significant correlation between high rottlerin concentration-triggered blockade of both PKCd and PKCa isoenzyme translo-cation, ERK1/ERK2 phosphorylation, and almost complete lossof DNA-binding activity of AP-1.

B

Fig. 4. Rottlerin inhibits LPS- or PMA-induced phosphorylation of MAPkinases (ERK1/ERK2) Monocytes pretreated with 15 or 50 µM rottlerin werestimulated with LPS (5 µg/mL) for 60 min or with PMA (1 nM) for 30 min. Totalprotein fraction was isolated and the presence of phosphorylated and non-phosphorylated forms of MAP kinases was determined in the same samples byWestern blotting, as described in Materials and Methods. (A) The mean 6 SEM

of seven (LPS) or five (PMA) experiments. The bands representing MAPkinases were scanned densitometrically. Phosphorylated MAP kinases areexpressed as the percentage of ERK1 1 ERK2 levels in the cells not exposed torottlerin but treated with LPS or PMA, respectively. Non-phosphorylatedMAPK are expressed as the percentage of ERK2 level in the cells that wereneither stimulated nor treated with rottlerin. P values, showing differencesbetween the cells treated versus not treated with rottlerin, are shown above thebars: *P 5 0.001; **P , 0.0001. (B) The representative Western blot. Position ofERK1 and ERK2 isoenzymes is indicated. Control, nonstimulated cells; Ro, rottlerin.


Rottlerin reduces LPS- or PMA-induced bindingactivity of NF-kB transcription factor

Although the DNA-binding activity of NF-kB transcriptionfactor was clearly seen in nonstimulated monocytes (Fig. 6B),stimulation with either LPS or PMA raised it several times.Rottlerin at 15 µM does not affect LPS-induced NF-kB bindingand only slightly diminishes PMA-triggered activity (18% ofinhibition). By contrast, a 50 µM concentration of the inhibitorsignificantly diminishes DNA-binding activity of NF-kB inLPS-stimulated cells (67% of inhibition). However, even such ahigh concentration of rottlerin only moderately reduces bindingactivity of NF-kB in PMA-stimulated cells (32% of inhibition).The discrepancy may result from the differences in signalingevents triggered by PMA and LPS. Despite this, the aboveresults indicate that, at least in LPS-treated cells, rottlerinimportantly reduces DNA-binding activity of NF-kB only whenused at the concentration that blocks the upstream events: (1)translocation of PKCd and PKCa isoenzymes (Fig. 2) and (2)ERK1/ERK2 phosphorylation (Fig. 4).

By contrast to the inhibitory action of rottlerin on DNA-binding activities of AP-1 and NF-kB transcription factors, thisinhibitor has no effect on the binding of nuclear proteins toOct-1 (housekeeping sequence) probe (Fig. 7). Moreover,DNA-binding activity of Oct-1 was similar in nonstimulatedand stimulated monocytes (Fig. 7).

DISCUSSION

We have previously shown that in human monocytes activationof PKC represents a critical event in LPS-triggered signalingcascade leading to pro-inflammatory cytokine production [12,33]. In this study we found that LPS, a potent inducer of TNF-aand IL-1b synthesis, triggers the cytosol-to-membrane translo-cation of PKCa and d, but not of bII or e isoenzymes. Bycontrast, PMA, which induces suboptimal production of testedcytokines, triggers translocation of all these PKC isoforms (Fig.1 [12]). Thus, for the optimal, LPS-induced production ofTNF-a and IL-1b, the translocation of PKCa and d isoenzymesseems to be important. Although we did not test the enzymaticactivity of PKCa and d isoforms, several observations suggestthat in human monocytes cytosol-to-membrane translocation ofthese isoenzymes reflects their enzymatic activities: (1) thetime-course of PKCa and PKCd translocation (Fig. 1) corre-sponds to the reported elevation of total PKC activity inLPS-treated cells [19], and (2) PMA-triggered cytosol-to-membrane translocation of PKCa was reported to be accompa-nied by the shift of PKC enzymatic activity from the cytosol tomembrane in these cells [23]. Regarding the contribution ofPKC isoforms to cytokine production, there are reports suggest-ing the importance of PKCb and PKCa in LPS-inducedresponses in murine peritoneal macrophages [31] and murine

Fig. 5. Rottlerin affects LPS- or PMA-induced DNA-binding activity of AP-1 transcription factor. Monocytes pretreated for 30 min with indicated concentrations ofrottlerin were stimulated with LPS (5 µg/mL) or PMA (1 nM) for 2 h. Nuclear protein fraction was isolated and applied for EMSA analysis, using AP-1 consensusoligonucleotides (see Materials and Methods for details). (A) Mean 6 SEM of six experiments. The bands representing proteins that bind the AP-1 consensus site weredensitometrically scanned. Results are expressed as the percentage of AP-1 binding level in the cells not exposed to rottlerin but treated with LPS or PMA,respectively. P values, showing differences between the cells treated versus not treated with rottlerin, are shown above the bars: *P 5 0.01; **P , 0.001. (B)Representative autoradiograph. Specificity of the binding was tested by: (1) excluding protein samples (no proteins) and (2) adding 100-fold excess of unlabeledprobe (cold AP-1). Control, nonstimulated cells; Ro, rottlerin.


macrophage cell lines [32], respectively. In human monocytes,the LPS-triggered activation of PKCz was reported [41].However, the effect of PKCz activation on pro-inflammatorycytokine production has not been tested.

Elucidation of the role of a particular PKC isoenzyme incellular responses is complicated by the concomitant expres-sion of several isoenzymes in a given cell type as well as by thelack of isoenzyme-specific activators and inhibitors. In thisstudy we applied a partly isoenzyme-selective PKC inhibitor,rottlerin. This compound was reported to show some preferencefor PKCd (IC50 5 3–6 µM), both in vitro [36] and in vivo [42].Higher concentrations of rottlerin are required to inhibit theactivities of other PKC isoenzymes (e.g., for PKCa IC50 530–42 µM) [36]. We confirmed the selectivity of rottlerin byshowing that this inhibitor abrogates both LPS- and PMA-triggered cytosol-to-membrane translocation of either PKCdalone or both PKCd and PKCa, when used at 15 or 50 µM,respectively (Fig. 2). Because rottlerin inhibits activities ofPKC isoforms by competing with ATP for binding to the enzymemolecule [36], it is likely that in LPS- and PMA-treated humanmonocytes this inhibitor not only prevents translocation oftested PKC isoforms, but also reduces their enzymatic activi-ties. However, this suggestion should be confirmed in furtherstudies. In addition, rottlerin was reported to suppress also thecalmodulin-dependent kinase III (CaM-kinase III) as effec-tively as PKCd [36, 43]. Because CaM-kinase III selectivelyphosphorylates and thereby inactivates eEF-2 elongation factor[43], its inhibition by rottlerin should accelerate protein

synthesis. However, in this study rottlerin inhibited but did notenhance the production of TNF-a and IL-1b in a dose-dependent manner (Fig. 3). Thus it is likely that the selectivityof rottlerin toward PKCd and a isoenzymes accounts for itseffects on the cytokine synthesis. It is interesting to note wefound that low (15 µM) concentration of rottlerin more potentlyreduces LPS-triggered production of TNF-a than IL-1b (<70%versus <50% of inhibition, respectively). Similar differenceswere observed in PMA-treated cells (Fig. 3A). High (50 µM)concentration of rottlerin almost completely blocked the produc-tion of TNF-a and IL-1b (.90% and .80%, respectively).Taking into account selective inhibition of PKC isoenzymetranslocation by different doses of rottlerin (Fig. 2) andmechanisms of inhibitory action of rottlerin [36], these resultssuggest that production of TNF-a depends mostly on PKCdtranslocation (activity?), whereas both PKCd and PKCa equallycontribute to IL-1b synthesis. This assumption is consistentwith a previous report that overexpression of a dominant-negative version of PKCa in the murine macrophage cell linestrongly inhibits LPS-induced production of IL-1a, whereas itis less effective in reducing TNF-a synthesis [32].

Next we have investigated the effect of rottlerin on the othersignaling events critical for cytokine production, but lyingdownstream to PKC activation, and found that this inhibitorsimilarly affects several signaling events triggered by eitherPMA or LPS.

It is generally accepted that in LPS-stimulated monocytesthe activation of ERK1/ERK2 MAP kinases is required for

Fig. 6. Rottlerin diminishes LPS- or PMA-induced DNA-binding activity of NF-kB transcription factor. See the legend to Figure 5 for the explanation. Experimentswere done using NF-kB consensus oligonucleotides. (A) The mean 6 SEM of four experiments. P values, showing differences between the cells treated versus nottreated with rottlerin, are shown above the bars: *P 5 0.02; **P 5 0.01. (B) The representative autoradiograph. Specificity of the binding was controlled by:excluding protein samples (no proteins) and by adding 100-fold excess of unlabeled probe (cold NF-kB).


transcription of genes encoding TNF-a and IL-1b [11, 17]. Inmany systems, the ERK pathway is activated via the stimulationof Ras, which in turn activates the serine/threonine kinase Raf.Raf kinase phosphorylates and thereby activates the dual-specific MAP kinase kinases, MEK1 and MEK2, that finallyphosphorylate ERK1 and ERK2 on two critical tyrosine andthreonine residues. The latter event is a prerequisite forenzymatic activity of ERK1/ERK2 [38]. Both Raf-1 [9] andMEK [11] were reported to be activated in LPS-stimulatedmurine macrophages and human monocytes, respectively. More-over, it was suggested that PKC phosphorylates and activatesRaf-1. The latter suggestion is supported by the observationthat phorbol esters, potent PKC activators, also trigger Raf-1and ERK1/ERK2 activation [38]. Our results are consistentwith this notion (Fig. 4B). More recent data support theimplication of different PKC isoenzymes in the phorbol ester-induced activation of the ERKs pathway in different cell types.For example, in COS1 and NIH3T3 cell lines the activation ofPKCd was reported to be sufficient for the activation of MEKand ERKs in c-Raf-dependent but Ras-independent manner[44], whereas in CHO cells three PKC isoforms (a, bI, and g)were shown to mediate this event [45]. In this study we reportthat rottlerin blocks both LPS- and PMA-triggered phosphoryla-tion of ERKs, only when used at high (50 µM) concentration(Fig. 4). Because this concentration of rottlerin affects cytosol-to-membrane translocation of both PKCd and a isoforms, it islikely that both of these isoenzymes participate in the activationof ERKs in our system. Although we cannot exclude the

possibility that rottlerin may directly affect ERKs phosphoryla-tion, recent data [46] show that in pheochromocytoma (PC12)cells treated with neurogenic agents, this inhibitor affects MAPkinase cascade by blocking enzymatic activity of PKCd.According to this report, PKCd has been found to functioneither downstream from or parallel with c-Raf, but upstream ofMEK. Further downstream, ERK1/ERK2 contribute to theinduction of transcriptional activity of AP-1 transcription factorthat bind to a common recognition sequence within thepromoters and enhancers of many genes [38, 47, 48].

Transcription of human genes encoding TNF-a and IL-1b isa complex process that requires participation of several tran-scription factors, including AP-1 [39, 40]. Our results show thatDNA-binding activity of AP-1 in LPS- or PMA-stimulatedmonocytes is inhibited by rottlerin in a dose-dependent man-ner. Rottlerin at 15 µM partially reduces (,40%) AP-1 bindingactivity (Fig. 5) without affecting the phosphorylation of ERKs(Fig. 4). It is interesting that almost complete inhibition(,90%) of AP-1 binding requires a higher (50 µM) concentra-tion of rottlerin (Fig. 5). The same high concentration ofrottlerin also affects the phosphorylation of ERKs (Fig. 4).These results indicate that in LPS- or PMA-stimulated mono-cytes AP-1 binding activity is only partly dependent on theERKs pathway. Thus, other MAP kinases (e.g., JNK, p38) mayalso participate in the regulation of AP-1 activity. The sameconclusion was reported for other cell types [38, 48].

NF-kB, a ubiquitous transcription factor, is one of the majorintracellular mediators of LPS-induced responses [2]. It is well

Fig. 7. Rottlerin does not influence DNA-binding activity of Oct-1 transcription factor See the legend to Figure 5 for the explanation. Experiments were done usingOct-1 consensus oligonucleotides. (A) The mean 6 SEM of three experiments. Results are expressed as the percentage of Oct-1 binding level in the control cells,which were neither stimulated nor treated with rottlerin. The differences between the control cells, the cells stimulated with LPS or PMA in the presence or absenceof rottlerin were statistically insignificant. (B) The representative autoradiograph. Specificity of the binding was controlled by excluding protein samples (no proteins)and by adding 100-fold excess of unlabeled probe (cold Oct-1).


documented that NF-kB positively regulates transcription ofgenes encoding TNF-a and IL-1b [39, 40]. In resting cells,dimeric NF-kB are complexed to I-kB inhibitory protein, whichmasks the NF-kB nuclear localization signal. On cell stimula-tion, I-kB is phosphorylated by I-kB kinases, then ubiquiti-nated and proteolytically degraded, allowing NF-kB dimers totranslocate to the nucleus and bind to consensus DNA se-quences [49]. Although the regulation of I-kB kinase complexactivity is not fully understood, MEKK-1 (an upstream kinaseinvolved in the activation of ERK and JNK cascades) and PKCzare likely candidates to directly activate I-kB kinase complex[49]. Data showing the requirement for ERK pathway inFcg-receptor-triggered activation of NF-kB in human mono-cytic cell line [50] support this suggestion. Moreover, in somecell systems MAP kinases and PKC may regulate NF-kBindependently of I-kB degradation. For example, TNF-a-activated p38 and ERK kinases increase transactivation capac-ity of the NF-kB p65 subunit in the murine fibrosarcoma cellline [51], whereas PKCa regulates export of IkBa from thenucleus to cytosol, thereby facilitating the inactivation ofNF-kB in IL-1a-stimulated hepatocytes [52]. It is also welldocumented that NF-kB activity could be strongly induced byPMA [47]. Altogether, these data show that transcriptionalactivity of NF-kB are regulated by PKC and MAP kinases. Theresults of our study are in agreement with this notion. Consis-tently, LPS-triggered DNA-binding activity of NF-kB wasreduced (in ,70%) by a 50 µM concentration of rottlerin (Fig.6). Moreover, the same concentration of the inhibitor blockssuch upstream events as: (1) the cytosol-to-membrane transloca-tion of PKCd and a isoenzymes (Fig. 2), and (2) the phosphory-lation of ERK1/ERK2 (Fig. 4). However, PMA-triggered bind-ing activity of NF-kB was only moderately reduced by a 50 µMconcentration of rottlerin (Fig. 6). The discrepancy between theability of rottlerin to reduce NF-kB binding activity in LPS- orPMA-stimulated cells may result from the differences in thesignaling pathways triggered by these stimuli (see the Introduc-tion and Fig. 1).

Regarding the role of PKC, MAP kinases, and NF-kBactivation in the signaling pathways leading to cytokine synthe-sis, there is a report showing that overexpression of a dominant-negative version of PKCa in the murine macrophage cell linestrongly affects LPS-induced production of IL-1a and TNF-a,without inhibiting either the phosphorylation of JNK and p38kinases or the activation of NF-kB [32]. However, the contribu-tion of the ERK pathway has not been tested. In this study,using different concentrations of rottlerin, we were able to blockcytosol-to-membrane translocation of either PKCd alone orboth PKCd and a isoenzymes. Thus, in agreement with thelatter report [32], we confirmed the importance of PKCa for thepro-inflammatory cytokine synthesis also in human monocytes.In addition, our results suggest the implication of PKCd inthese cytokine responses.

In summary, we report that in human monocytes LPS triggerscytosol-to-membrane translocation of PKCa and PKCd iso-forms, whereas rottlerin, a partial isozyme-selective inhibitor ofPKC, prevents these events at the concentrations relevant toreported IC50 values for these isoenzymes. It is important tonote we found that rottlerin also affects some downstream

events critical for cytokine synthesis, and inhibits production ofTNF-a and IL-1b. The sequence of events affected by rottlerinsuggests that production of TNF-a is dependent mostly onPKCd translocation (activation?) and AP-1 activity, whereasIL-1b synthesis depends also on PKCa translocation (activa-tion?) and requires subsequent activation of ERK1/ERK2 andNF-kB. However, further studies are needed for formal proof ofthese suggestions.

ACKNOWLEDGMENTS

This work was supported by grant P6 P04B 01709 from theCommittee of Scientific Research (Warsaw, Poland). We acknowl-edge Mrs. Grazyna Luszczykiewicz and Mrs. Iwona Janicka fortechnical assistance.

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