Src Activation of NF-B Augments IL-1–Induced Nitric Oxide Production in Mesangial Cells
Diane I. Jalal* and
Bruce C. Kone*,,
Departments of * Internal Medicine and Integrative Biology, Pharmacology and Physiology, The University of Texas Medical School at Houston; and the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, Houston, Texas
Address correspondence to: Dr. Bruce C. Kone, Departments of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, 6431 Fannin, MSB 1.150, Houston, TX 77030. Phone: 713-500-6502; Fax: 713-500-6497; E-mail:bruce.c.kone{at}uth.tmc.edu
Received for publication July 7, 2005.
Accepted for publication October 20, 2005.
NF-B is a critical transcription factor that is involved inglomerulonephritis and inflammatory host responses and a criticaltransactivator of the inducible nitric oxide (NO) synthase genein mesangial cells. The Src protein tyrosine kinases (SFK) areinvolved in several signaling pathways and have been proposedto mediate cytokine activation of NF-B in a few cell types.However, the specific involvement of SFK in IL-1 induction ofNO production has not been clearly established. Accordingly,pharmacologic and molecular tools were used to clarify thisissue in cultured murine mesangial cells. The SFK antagonist4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine(PP2) dramatically inhibited IL-1–mediated induction ofendogenous NO production as measured by the Griess reaction,as well as the induction of NF-B p50/p65 DNA-binding activityin gel shift assays and the activity of an NF-B–responsivepromoter–reporter construct transiently transfected intothe cells. Immunoprecipitation and immunoblotting with anti-IBand anti-phosphotyrosine antibodies revealed that PP2 also inhibitedIL-1–stimulated tyrosine phosphorylation of IB, a requisitestep in NF-B activation in this signaling cascade. In agreementwith the pharmacologic inhibition studies, siRNA directed againstc-Src specifically limited c-Src protein expression and inhibitedIL-1–mediated induction of NF-B DNA-binding activity,whereas control siRNA had no effect. Conversely, overexpressionof constitutively active c-Src augmented basal and IL-1–mediatedinduction of NF-B DNA-binding activity and NO production. Thus,SFK play a key role in IL-1–induced NO production in mesangialcells and do so via tyrosine phosphorylation of IB and consequentNF-B activation.
Nitric oxide (NO) is an important molecular mediator of numerousphysiologic processes in virtually every organ. In the kidney,NO plays prominent roles in the homeostatic regulation of glomerular,vascular, and tubular function. During glomerular inflammation,NO and cytokines such as IL-1 and TNF- from resident and infiltratingcells act in concert to sustain, promote, or limit glomerulardamage. NO that is produced by these cells leads to activationor silencing of genes that encode antioxidant defense enzymes,matrix-metabolizing enzymes, proinflammatory mediators, andsignaling mechanisms (1). In human glomerulonephritis, induciblenitric oxide synthase (iNOS) gene expression has been describedin glomerular mesangial cells, as well as in local and infiltratingmacrophages (2,3). Mesangial cells contribute prominently tothe pathogenesis of glomerulonephritis, in part by producinga variety of cytokines and NO via iNOS, and there is evidencefor participation of iNOS-generated NO in the induction, progression,or protection of several types of experimental and human glomerulonephritis.Proinflammatory stimuli, such as IL-1 (4), activate iNOS genetranscription in mesangial cells through a complex network ofsignaling pathways and inducible transcription factors, includingc-Jun N-terminal kinase (JNK) and p38 mitogen-activated proteinkinase (5), cAMP response element binding protein (CREB) (4),CCAAT-enhancer binding protein (C/EBP) (4), and NF-B (6). Ofthese, NF-B is considered the most potent transactivator ofthe iNOS gene and has been the subject of more intense investigation.
NF-B is a pivotal transcription factor that regulates genesthat control multiple immune and inflammatory responses, aswell as cell proliferation and apoptosis (7). NF-B is activatedin several forms of experimental and human glomerulonephritis,including immune complex kidney disease (8–11), crescenticglomerulonephritis (12), and lupus nephritis (9), and it activatesiNOS in these settings. A variety of inflammatory stimuli, suchas IL-1 (13,14), bacterial lipopolysaccharide and IFN- (14),and oxidative stress (15) activate this transcription factorin mesangial cells. NF-B consists of a group of five proteins,namely c-Rel, RelA (p65), RelB, NF-B1 (p50 and p105), and NF-B2(7). In the resting state, NF-B is sequestered in the cytoplasmthrough a tight association with specific inhibitory proteins,called inhibitors of NF-B (IB). The most comprehensively studiedpathway of NF-B activation by agents such as cytokines, endotoxin,and phorbol esters consists of IB phosphorylation at Ser-32or Ser-36 by the IB kinase (IKK) complex, which targets IB forubiquitination and degradation through the ubiquitin-26S proteasomalpathway (16,17), allowing nuclear translocation of NF-B, wherethe transcription factor binds to specific DNA sequences locatedin the promoter regions of many proinflammatory genes. The majorityof stimuli that lead to NF-B activation act through this classicalpathway (16,18,19).
Two other pathways have been described to activate NF-B in distinctcell types. The first pathway has been described in lymphocytesthat were subjected to oxidant stress (20,21) or pervanadatetreatment (22) and involves phosphorylation of Tyr-42 but notSer-32 or Ser-36 of IB. The dissociation of tyrosine-phosphorylatedIB from NF-B in this setting is not the result of degradationby the 26S proteasome (23). This pathway seems to be specificfor IB, because Tyr-42 is not conserved in other IB family members.The exact protein tyrosine kinase(s) or phosphatase(s) involvedin this pathway remains unknown, but members of the Src familyof nonreceptor tyrosine kinases (SFK), such as p56Lck, ZAP-70,and SFK (24–27), have been implicated. Moreover, Tyr-42phosphorylation of IB occurs within a consensus binding sitefor the p85 subunit of phosphatidylinositol 3-kinase and seemsto stabilize its affinity for p85 (28). SFK have also been implicatedin the regulation of cytokine-mediated NF-B activation in Thelper 2 cells (25) and bone marrow macrophages (24). The secondatypical pathway occurs in cells that are exposed to short-wavelengthultraviolet radiation and involves IB degradation by the proteasomalpathway without phosphorylation of Ser-32, Ser-36, or Tyr-42of IB (29,30). The mechanisms for this alternative pathway ofIB degradation are unknown.
IL-1 signaling in mesangial cells activates multiple pathwaysthat are involved in iNOS expression—p38 mitogen-activatedprotein kinase (31), JNK/stress-activated protein kinase (SAPK)(31), and phosphatidylinositol 3-kinase (32)—and thesecan be inhibited by pharmacologic blockade in an NF-B–independentmanner (31). We have also shown that NF-B plays a complex rolein transcriptional regulation of iNOS in murine mesangial cells(13,14). In this report, we sought to determine additional signalingevents in the NF-B pathway that are critical to iNOS activationin these cells. We demonstrate that IL-1–mediated inductionof iNOS-mediated NO production in murine mesangial cells proceedsthrough SFK tyrosine phosphorylation of IB and consequent activationof NF-B.
Cell Culture and Reagents
Mouse mesangial cells (American Type Culture Collection CRL-1927,Manassas, VA) were maintained in Ham’s F-12 plus DMEMsupplemented with 2 mM l-glutamine, and 5% FBS. Mouse recombinantIL-1 was obtained from R&D Systems (Minneapolis, MN). TheSFK inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine(PP2 [33]) was obtained from Calbiochem (San Diego, CA). Polyclonalantibodies that recognize NF-B p65 and p50, as well IB, andan NF-B consensus oligomer (5'-AGTTGAGGGGACTTTCCCAGGC-3'; consensussequence boldface) were obtained from Santa Cruz Biotechnologies(Santa Cruz, CA). Lipofectamine 2000 reagent was purchased fromInvitrogen (Carlsbad, CA). The Dual Luciferase Reporter Assaysystem and the luciferase vectors pGL3-Basic and pRL-SV40 werefrom Promega (Madison, WI). The protein estimation kit was purchasedfrom Bio-Rad (Hercules, CA). The Silencer siRNA TransfectionKit, siPORT Amine Transfection Reagent, and predesigned siRNAtargeting exon 5 of murine SFK protein tyrosine kinase (ID#160390)were purchased from Ambion (Austin, TX). Monoclonal anti–c-Srcantibody was obtained from Dharmacon (Chicago, IL).
Plasmids
The NF-B promoter–reporter construct p36B(–)(NF-B)3-luc,which contains three tandem copies of the NF-B–bindingelement (GGGGACTCTCCC) upstream of the SV40 early promoter sequenceand fused to the coding sequence for the luciferase gene, wasprovided by Dr. Bharat Aggarwal (The University of Texas M.D.Anderson Cancer Center, Houston, TX). We have used this constructin our previous publication (14). Constitutively active chickenSFK (34) subcloned into pCMV5 (35), designated here pCMV5–c-Src,was provided by Dr. Jeffrey Frost (University of Texas HealthScience Center at Houston, Houston, TX).
Nitrite Assays
Mesangial Cells were seeded in 24-well plates and transientlytransfected in some experiments or treated with inhibitors inother experiments as indicated in the text or figure legends,and then treated with IL-1 at a dose of 10 ng/ml for 24 h. Themedium then was collected, and the nitrite concentration wasdetermined with the Griess Reagent system (Promega) accordingto the manufacturer’s protocol.
Transient Transfections
Mesangial cells were seeded in 24-well plates and grown to 90to 95% confluence in complete medium without antibiotics andtransfected the next day using Lipofectamine 2000 reagent accordingto the manufacturer’s protocol and a total of 1 µg/wellof plasmid DNA. The amount of DNA was kept constant by addingappropriate amounts of the parental empty expression vector.Transfection efficiencies were normalized by co-transfectionwith 10 ng/well of the Renilla luciferase expression plasmidpRL-SV40. Twenty-four hours after transfection, complete mediumwas added with vehicle or IL-1. Twenty-four hours later, celllysates were prepared and firefly and Renilla luciferase activitieswere measured in 100 µl of lysate samples as describedpreviously by our laboratory (36). In some experiments, as indicatedin the text and figure legends, PP2 (10 µM/ml) was addedafter transfection for 24 h before the cell lysates were prepared.This concentration of PP2 was chosen on the basis of its effectivenessin inhibiting tyrosine kinase activities in previously publishedstudies (26,37).
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared from time-paired vehicle andIL-1–treated mesangial cells as detailed in our previouswork (36,38). NF-B element double-stranded oligonucleotideswere end-labeled with -32ATP (3000 Ci/mmol) using T4 polynucleotidekinase. Binding reactions were performed in 20 µl of solutionfor 30 min at room temperature by incubating 10 µg ofnuclear extract protein with duplex DNA probe (2 x 105 cpm inreaction buffer [13 mM HEPES (pH 7.9), 65 mM NaCl, 0.14 mM EDTA,1 mM MgCl2, 1 mM dithiothreitol, 8% glycerol, and 50 µg/mlpoly(dI-dC)]). For supershift assays, antibodies (2 µg)that are specific for NF-B p65 or p50 were added to the bindingreaction and incubated on ice for 10 min before the additionof the labeled probe. Aliquots of the reaction were resolvedin 5% native polyacrylamide gels in 0.5x Tris-borate EDTA buffer.The gels were dried and exposed to x-ray film with an enhancingscreen at –70°C to detect the DNA–protein andDNA–protein–antibody complexes. Experiments werereplicated a minimum of three times as indicated in the figurelegends.
Immunoprecipitation and Immunoblot Analysis of Total and Tyrosine-Phosphorylated IB
The procedures followed were generally described in our previouswork (39). In brief, stimulated mesangial cells were harvestedand lysed in lysis buffer (20 mM Tris-HCl [pH 8.0], 1.5 mM MgCl2,0.2 mM EDTA, 25% Glycerol, and 0.5 mM PMSF). These lysates thenwere precleared with 20 µl/ml protein A/G agarose beads(Santa Cruz) for 1 h at 4°C. After brief centrifugation,the supernatant was added to anti-IB antibody (0.2 µg/ml)lysis buffer overnight at 4°C, followed by the additionof protein A/G agarose beads and incubation for 1 h at 4°C.Immunoprecipitates were washed four times in lysis buffer andresuspended in SDS sample buffer, boiled for 5 min, and analyzedon 4 to 20% SDS-PAGE gels. The proteins were electrophoreticallytransferred to polyvinylidene difluoride membranes (Hybond-ECL;Amersham, Piscataway, NJ) and probed with anti-IB for identificationof total IB levels or with anti-phosphotyrosine antibodies (0.2µg/ml) for identification of tyrosine phosphorylated IBovernight as indicated in the text and figure legends. The blotswere washed extensively with TBST solution that contained 50mM Tris (pH 8.0), 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20.The antigen–antibody complexes were detected by the ECLprotocol using horseradish peroxidase–conjugated donkeyanti-rabbit IgG as secondary antibody.
c-Src Silencing by RNA Interference
Cultured murine mesangial cells were grown in complete mediumuntil 90% confluent. Before transfection, the medium was changedto serum- and antibiotic-free medium (Opti-MEM). The c-Src–specificor control siRNA was combined with siPORT Amine TransfectionReagent, and the cells were transfected according to the recommendedprotocol with siRNA (50 nM final concentration). Six hours aftertransfection, the medium was changed, and 48 h were allowedbefore IL-1 treatment. Successful, specific gene knockdown wasdetermined by immunoblot analysis of c-Src and glyceraldehyde-3-phosphatedehydrogenase protein levels.
Statistical Analyses
Quantitative data are presented as mean ± SEM and wereanalyzed either by paired t test or ANOVA as appropriate. Significancewas assigned at P < 0.05.
SFK Antagonist PP2 Inhibits IL-1–Mediated Induction of Endogenous NO Production and iNOS Expression
We showed previously that iNOS is the only NOS isoform expressedin these cells and that IL-1–stimulated nitrite productioncorrelates exclusively with iNOS transcription, iNOS gene expression,and iNOS-generated NO (14,36,38). Accordingly, we used IL-1–stimulatednitrite production as a convenient index of iNOS gene regulation.To determine whether SFK activity contributes to the regulationof iNOS-generated NO production in mesangial cells, we measurednitrite production induced by treatment with IL-1 (10 ng/ml)in the presence of vehicle or PP2, a potent and specific SFKinhibitor (40). As shown in Figure 1, IL-1–stimulatedmesangial cells that had been treated with 10 µM/ml PP2exhibited nitrite levels that were approximately half that ofIL-1–stimulated controls. From the same cells used forthe nitrite determinations, we also analyzed iNOS protein expression.As seen in Figure 2A, no iNOS was apparent in the absence ofIL-1 regardless of treatment with vehicle or PP2. iNOS was sharplyinduced by IL-1, and this was substantially blunted by co-administrationof PP2, in agreement with the nitrite results.
Figure 1. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine (PP2) inhibits induction of IL-1–induced nitric oxide (NO) production in mesangial cells. Cells were treated with vehicle or the Src family inhibitor PP2 (10 µM) together with IL-1 for 24 h. Nitrite concentrations in the culture supernatants were measured in triplicate by the Griess reaction. Basal nitrite levels were negligible. *P < 0.05 versus IL-1 + vehicle; n = 3.
Figure 2. PP2 inhibits IL-1 induction of NF-B DNA-binding activity and transactivation potential. (A) Cytosolic and nuclear extracts were prepared from mesangial cells that had been treated with vehicle, IL-1, PP2, or IL-1 + PP2. Cytosolic extracts were immunoblotted (IB), with intervening membrane stripping, with antibodies directed against inducible NO synthase (iNOS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nuclear extracts were subjected to electrophoretic mobility shift assay (EMSA) with a B-specific consensus oligomer. Sequence-specific DNA–NF-B complexes I and II are indicated. Blots are representative of three observations. (B) Supershift assay in which antibodies directed against NF-B p50 or p65, or nonimmune IgG were included in the binding reactions performed as in A. The supershift bands "SS" are designated. (C) Mesangial cells were transiently co-transfected with p36B(–)(NF-B)3-luc, which contains three tandem copies of the B binding element, together with the Renilla luciferase expression plasmid pRL-SV40. The cells were treated with IL-1 and vehicle or PP2 for 24 h. The luciferase activity then was measured in cell lysates. *P < 0.05 versus IL-1 alone; n = 5.
PP2 Inhibits IL-1–Mediated Induction of NF-B DNA-Binding Activity and the Activity of an NF-B–Responsive Promoter
NF-B is known to play a major role in cytokine induction ofthe murine iNOS promoter. To determine whether SFK participatein this signaling pathway, we tested the effects of PP2 treatmenton NF-B DNA-binding activity and the activity of an NF-B–responsivepromoter–reporter construct in mesangial cells. Gel shiftstudies demonstrated two sequence-specific B DNA–proteincomplexes in nuclear extracts under basal conditions (Figure 2A).Both complexes were upregulated after IL-1 treatment, butthis response was markedly greater for complex I. Moreover,this complex was supershifted by antibodies to either the p50or the p65 subunits of NF-B (Figure 2B), indicating that thecomplex contained p50/p65 heterodimers. PP2 treatment of thecells significantly reduced the intensity of complex I, reflectingreduced NF-B DNA-binding activity, in the nuclear extracts thatwere prepared from IL-1–stimulated mesangial cells (Figure 2A).In keeping with this observation, PP2-treated cells exhibitedapproximately 40% lower rates of IL-1–stimulated activityof the NF-B promoter–reporter compared with vehicle-treatedcontrols (Figure 2C). Collectively, these results suggest thatSFK activity is critical for the DNA-binding properties andtransactivation potential of NF-B and iNOS induction in IL-1–stimulatedmesangial cells.
PP2 Inhibits IL-1–Stimulated Tyrosine Phosphorylation of IB
To determine whether tyrosine phosphorylation of IB is importantfor IL-1 activation of NF-B, we measured protein levels of totalIB and phosphotyrosine-IB in mesangial cell lysates from control,IL-1 + vehicle–treated, PP2-treated, and IL-1 + PP2–treatedmesangial cells. Low levels of total IB were present in thecontrol and PP2-treated cells, whereas comparably high levelswere present in the IL-1–treated cells in the presenceor absence of PP2. Phosphotyrosine-IB, detected by immunoprecipitationof total IB followed by immunoblotting with anti-phosphotyrosineantibody, was evident in control cells, strongly induced inIL-1–treated cells, at control levels in the PP2-treatedcells, and less than control levels in the IL-1 + PP2–treatedcells (Figure 3). These results were consistent with the variationsin NF-B DNA-binding activity (Figure 2A).
Figure 3. PP2 inhibits tyrosine phosphorylation of IB. Aliquots of whole-cell lysate protein were immunoprecipitated (intraperitoneally) with anti-IB Ab, followed by immunoblotting (IB) with an anti-phosphotyrosine Ab (anti–Tyr-P) to detect tyrosine phosphorylation on IB. The blots were stripped and reprobed with an anti-IB antibody for estimation of total IB levels. A representative (n = 3) blot is shown.
siRNA Knockdown of c-Src Protein Expression in Mesangial Cells Inhibits IL-1–Mediated Induction of NF-B DNA-binding Activity
Mesangial cells were transfected with either siRNA targetingc-Src or an equimolar amount of negative control siRNA and thentreated with IL-1. Western blots of cytosolic extracts showedthat c-Src levels in cells that were treated with IL-1 aloneor with IL-1 + negative control siRNA were similar. However,cells that were transfected with c-Src siRNA revealed substantiallyless c-Src protein (Figure 4A). In contrast, glyceraldehyde-3-phosphatedehydrogenase protein levels, used as a specificity control,were invariant across the samples (Figure 4A). Electrophoreticmobility shift assay that was performed on nuclear extractsrevealed no effects of the negative control siRNA on IL-1–inducedNF-B DNA-binding activity but diminished IL-1 induction of NF-BDNA-binding activity in c-Src siRNA transfected cells comparedwith controls (Figure 4B), in keeping with the pharmacologicinhibition studies with PP2 (Figure 2A).
Figure 4. siRNA knockdown of c-Src inhibits IL-1–stimulated NF-B DNA-binding activity. Mesangial cells were transfected with control siRNA or c-Src–specific siRNA and then exposed to vehicle or IL-1 for 24 h. (A) Immunoblots of cytosolic extracts were prepared and probed with an anti–c-Src antibody, then stripped and reprobed with an anti-GAPDH antibody. (B) Nuclear extracts were assayed for NF-B DNA binding activity by EMSA. Blots are representative of three observations.
c-Src Overexpression Augments Basal and IL-1–Mediated Induction of NF-B DNA-Binding Activity and NO Production
Immunoblots of cytosolic extracts from cells that were transfectedwith pCMV5–c-Src encoding constitutively active c-Srcrevealed the expected overexpression of c-Src compared withthe vector-transfected cells regardless of treatment with vehicleor IL-1 (Figure 5A). Gel shift assays demonstrated increasedNF-B DNA-binding activity both in the basal state and in responseto IL-1 in the cells that overexpressed constitutively activec-Src as compared with empty vector-transfected controls (Figure 5A).Similarly, overexpression of constitutively active c-Srcby transient transfection resulted in higher expression of iNOSprotein (Figure 5A) and rates of nitrite generation (Figure 5B)under both basal and IL-1–treated conditions comparedwith controls. The increment was greater in the IL-1–treatedcells.
Figure 5. Overexpression of c-Src augments basal and IL-1–stimulated NF-B DNA-binding activity and NO production. Twenty-four hours after transient transfection with constitutively active c-Src expression plasmid or empty vector, mesangial cells were treated with vehicle or IL-1. Twenty-four hours later, cytosolic extracts were immunoblotted (IB), with intervening membrane stripping, with antibodies directed against iNOS, c-Src, and GAPDH, and nuclear extracts were assayed for NF-B DNA binding activity by EMSA (A), and the medium supernatant was assayed for nitrite concentration (B). *P < 0.05 versus control vector + vehicle; #P < 0.05 versus control vector + IL-1; n = 4.
Resting mesangial cells produce low basal levels of inflammatorymediators, including NO, but soluble factors that are producedby inflammatory cells that invade the glomerulus or by circulatingfactors can activate iNOS and stimulate NO production. The signalingmechanisms that are activated by cytokines and lead to inductionof iNOS gene transcription are complex and incompletely defined.In this report, we explored SFK as an upstream component thatleads to activation of NF-B, a transcription factor that isknown to be a strong transactivator of the iNOS gene in multiplecell types, including mesangial cells. Through pharmacologicand molecular manipulation of SFK activity and expression, wefound that SFK significantly activate tyrosine phosphorylationof IB, NF-B DNA-binding activity and transactivation potential,iNOS protein expression, and iNOS-generated NO production inmesangial cells. Cells that were treated with the SFK inhibitorPP2 exhibited notable blunting of iNOS gene expression and NOproduction, NF-B DNA-binding activity, and NF-B–dependentpromoter activity in response to IL-1. In agreement with theseresults, siRNA knockdown of c-Src resulted in similar bluntingof IL-1 induction of NF-B DNA-binding activity. Conversely,overexpression of constitutively active c-Src in mesangial cellsaugmented basal and IL-1–mediated induction of iNOS, NOgeneration, and NF-B–binding activity. These actions correlatedwith SFK-mediated tyrosine phosphorylation of IB. Collectively,these results more clearly define a new pathway for iNOS induction,involving cytokine activation of SFK, consequent IB tyrosinephosphorylation, and NF-B activation, that ultimately resultsin induction of iNOS-generated NO production.
To date, studies that have evaluated the functional regulationof IB tyrosine phosphorylation and NF-B regulation have beenrestricted to a few cell types, including T cells (22,25), bonemarrow–derived macrophages (24), and epithelial cells(21,37). Abu-Amer et al. (24) demonstrated in bone marrow–derivedmacrophages that SFK tyrosine phosphorylation was essentialin TNF-–mediated activation of NF-B through a mechanismthat involved tyrosine phosphorylation of IB but did not involveIB degradation. Similarly, Natarajan et al. (41) showed thatNF-B activation in human endothelial cells that were subjectedto hypoxia/reperfusion injury required tyrosine phosphorylationof IB, which in turn did not induce IB proteolysis. In Th2 cells,the SFK member Lck was shown to be important for IL-1–inducedNF-B activation (25) by a mechanism that did not involve IBdegradation (25). Huang et al. (37), however, examined the involvementof these proteins in TNF-–induced cyclooxygenase-2 expressionand found that SFK-mediated activation of NF-B was associatedwith rapid degradation of IB. Hence, the exact mechanism throughwhich SFK participates in relaying cytokine-induced cell signalingthat leads to NF-B activation seems to be cell type and stimulusspecific.
The role of NF-B in iNOS transcriptional control is complex.The iNOS 5'-flanking region contains at least two B enhancerelements that bind NF-B and mediate strong transactivation ofthe iNOS gene (42). In addition, we previously demonstratedthat NF-B p65 physically and functionally interacts with histonedeacetylase-2 (14) and STAT3 (13) in mesangial cells to augmentand inhibit, respectively, NF-B–mediated transactivationof the iNOS gene and endogenous NO production in cultured glomerularmesangial cells. In the case of STAT3, the mechanism(s) didnot impair the ability of NF-B to bind its cognate elementsin the iNOS promoter and did not require STAT3 itself to binddirectly to the promoter. These transcription factor interactionsseem to be nuclear events downstream of SFK and IB actions,although it remains plausible that SFK-mediated tyrosine phosphorylationof other target proteins, including STAT3 itself (43), may governthese protein–protein interactions and the ultimate degreeof NF-B–mediated induction of the iNOS gene. For example,if both IB (shown here) and STAT3 (shown in other settings [43])were targets of SFK-mediated tyrosine phosphorylation, thenthe degree of NF-B activity might depend on whether SFK-mediatedtyrosine phosphorylation of STAT3 impaired or accentuated itsbinding interaction with NF-B p65. Further studies will testthis hypothesis. Furthermore, that PP2 was effective in blockingNF-B activation indicates that the tyrosine kinase activityof SFK was important for the effect. These results differ fromthose of Funokoshi-Tago et al. (26), who found that SFK tyrosinekinase activity was not required for IL-1–induced NF-Bactivation in a glioblastoma cell line, apparently because ofa physical and functional interaction between SFK and IB kinase-.
Because IL-1 triggered tyrosine phosphorylation of IB, it isreasonable to conclude that IL-1 acts through both serine andtyrosine phosphorylation of IB to regulate NF-B–mediatedcell signaling. Given that c-Src overexpression in mesangialcells also significantly increased basal NO production and NF-BDNA-binding activity, it seems that c-Src–mediated tyrosinephosphorylation alone is necessary and sufficient to activateNF-B and consequently the iNOS gene in these cells. Furtherstudies will be needed to elucidate the exact mechanism involvedin this process. Similar findings were observed in a human glioblastomacell line in which overexpression of SFK induced a moderateincrease in NF-B activity in the absence of cytokine stimulation(26). This is not unique to c-Src, as overexpression of othersignaling components in the IL-1–dependent pathway, suchas TRAF6, have been reported to induce NF-B activation in theabsence of cytokine stimulation (44).
In the resting state, NF-B typically is bound to various IBproteins. Only IB in this family of proteins has a tyrosineresidue at position 42 (28,41). This association between NF-Band IB may allow NF-B to respond specifically to certain stimulithat induce tyrosine phosphorylation, allowing cell type–andstimulus-specific cell signal modulation. Likewise, iNOS transcriptionhas cell- and stimulus-specific controls. This diversity ofregulatory mechanisms provides considerable versatility in activating,sustaining, and inactivating potent inducible effectors suchas NF-B and iNOS. That the inhibition of NO production and NF-Bactivation by PP2 and c-Src knockdown and the basal and IL-1–stimulatedactivation of NO production by overexpressed c-Src were so prominentindicates that the data reported here add another key componentto the regulatory schemes for these proteins.
Acknowledgments
This work was supported by National Institutes of Health grantsRO1 DK50745, P50 GM38529, and T32 DK061929; the Department ofDefense "T5" grant; and endowment funds from The James T. andNancy Willerson Chair to B.C.K.
We thank Dr. Bharat Aggarwal (The University of Texas MD AndersonCancer Center, Houston, TX) and Dr. Jeffrey Frost (The Universityof Texas Medical School, Houston, TX) for generous gifts ofplasmids used in this study. We also thank Drs. Zhang, Yu, andZou, as well as Teresa Kuncewicz for assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Pfeilschifter J, Beck KF, Eberhardt W, Huwiler A: Changing gears in the course of glomerulonephritis by shifting superoxide to nitric oxide-dominated chemistry.
Kidney Int61
:809
–815,2002[CrossRef][Medline]
Wang JS, Tseng HH, Shih DF, Jou HS, Ger LP: Expression of inducible nitric oxide synthase and apoptosis in human lupus nephritis.
Nephron77
:404
–411,1997[Medline]
Furusu A, Miyazaki M, Abe K, Tsukasaki S, Shioshita K, Sasaki O, Miyazaki K, Ozono Y, Koji T, Harada T, Sakai H, Kohno S: Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis.
Kidney Int53
:1760
–1768,1998[CrossRef][Medline]
Eberhardt W, Pluss C, Hummel R, Pfeilschifter J: Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells.
J Immunol160
:4961
–4969,1998[Abstract/Free Full Text]
Guan Z, Baier LD, Morrison AR: p38 mitogen-activated protein kinase down-regulates nitric oxide and up- regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta.
J Biol Chem272
:8083
–8089,1997[Abstract/Free Full Text]
Beck KF, Sterzel RB: Cloning and sequencing of the proximal promoter of the rat iNOS gene activation of NF-kappaB is not sufficient for transcription of the iNOS gene in rat mesangial cells.
FEBS Lett394
:263
–267,1996[CrossRef][Medline]
Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle.
Cell109
[Suppl]:S81
–S96,2002[CrossRef][Medline]
Kim JH, Ha IS, Hwang CI, Lee YJ, Kim J, Yang SH, Kim YS, Cao YA, Choi S, Park WY: Gene expression profiling of anti-GBM glomerulonephritis model: The role of NF-kappaB in immune complex kidney disease.
Kidney Int66
:1826
–1837,2004[CrossRef][Medline]
Tsukinoki T, Sugiyama H, Sunami R, Kobayashi M, Onoda T, Maeshima Y, Yamasaki Y, Makino H: Mesangial cell Fas ligand: Upregulation in human lupus nephritis and NF-kappaB-mediated expression in cultured human mesangial cells.
Clin Exp Nephrol8
:196
–205,2004[CrossRef][Medline]
Gomez-Guerrero C, Lopez-Franco O, Suzuki Y, Sanjuan G, Hernandez-Vargas P, Blanco J, Egido J: Nitric oxide production in renal cells by immune complexes: Role of kinases and nuclear factor-kappaB.
Kidney Int62
:2022
–2034,2002[CrossRef][Medline]
Lopez-Franco O, Suzuki Y, Sanjuan G, Blanco J, Hernandez-Vargas P, Yo Y, Kopp J, Egido J, Gomez-Guerrero C: Nuclear factor-kappaB inhibitors as potential novel anti-inflammatory agents for the treatment of immune glomerulonephritis.
Am J Pathol161
:1497
–1505,2002[Abstract/Free Full Text]
Sakai N, Wada T, Furuichi K, Iwata Y, Yoshimoto K, Kitagawa K, Kokubo S, Kobayashi M, Takeda S, Kida H, Kobayashi K, Mukaida N, Matsushima K, Yokoyama H: p38 MAPK phosphorylation and NF-kappaB activation in human crescentic glomerulonephritis.
Nephrol Dial Transplant17
:998
–1004,2002[Abstract/Free Full Text]
Yu Z, Kone BC: The STAT3 DNA-binding domain mediates interaction with NF-kappaB p65 and inducible nitric oxide synthase transrepression in mesangial cells.
J Am Soc Nephrol15
:585
–591,2004[Abstract/Free Full Text]
Yu Z, Zhang W, Kone BC: Histone deacetylases augment cytokine induction of the iNOS gene.
J Am Soc Nephrol13
:2009
–2017,2002[Abstract/Free Full Text]
Kang BP, Frencher S, Reddy V, Kessler A, Malhotra A, Meggs LG: High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism.
Am J Physiol Renal Physiol284
:F455
–F466,2003[Abstract/Free Full Text]
DiDonato JA, Mercurio F, Karin M: Phosphorylation of I kappaB alpha precedes but is not sufficient for its dissociation from NF-kappaB.
Mol Cell Biol15
:1302
–1311,1995[Abstract]
DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S, Karin M: Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation.
Mol Cell Biol16
:1295
–1304,1996[Abstract]
Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr: Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappaB alpha: A mechanism for NF-kappaB activation.
Mol Cell Biol13
:3301
–3310,1993[Abstract/Free Full Text]
Henkel T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah Y, Baeuerle PA: Rapid proteolysis of I kappaB-alpha is necessary for activation of transcription factor NF-kappaB.
Nature365
:182
–185,1993[CrossRef][Medline]
Takada Y, Mukhopadhyay A, Kundu GC, Mahabeleshwar GH, Singh S, Aggarwal BB: Hydrogen peroxide activates NF-kappaB through tyrosine phosphorylation of I kappaB alpha and serine phosphorylation of p65: Evidence for the involvement of I kappaB alpha kinase and Syk protein-tyrosine kinase.
J Biol Chem278
:24233
–24241,2003[Abstract/Free Full Text]
Fan C, Li Q, Ross D, Engelhardt JF: Tyrosine phosphorylation of I kappaB alpha activates NF kappaB through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation.
J Biol Chem278
:2072
–2080,2003[Abstract/Free Full Text]
Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB, Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P, Baeuerle PA, Peyron JF: Tyrosine phosphorylation of I kappaB-alpha activates NF-kappaB without proteolytic degradation of I kappaB-alpha.
Cell86
:787
–798,1996[CrossRef][Medline]
Imbert V, Rupec RA, Livolsi A, Pahl HL, Traenckner EB, Mueller-Dieckmann C, Farahifar D, Rossi B, Auberger P, Baeuerle PA, Peyron JF: Tyrosine phosphorylation of IkappaB-a activates NF-kappaB without proteolytic degradation of IkappaB-a.
Cell86
:787
–798,1996[CrossRef][Medline]
Abu-Am Y, Ross FP, McHugh KP, Livolsi A, Peyron JF, Teitelbaum SL: Tumor necrosis factor-alpha activation of nuclear transcription factor-kappaB in marrow macrophages is mediated by c-Src tyrosine phosphorylation of Ikappa Balpha.
J Biol Chem273
:29417
–29423,1998[Abstract/Free Full Text]
al-Ramadi BK, Welte T, Fernandez-Cabezudo MJ, Galadari S, Dittel B, Fu XY, Bothwell AL: The Src-protein tyrosine kinase Lck is required for IL-1-mediated costimulatory signaling in Th2 cells.
J Immunol167
:6827
–6833,2001[Abstract/Free Full Text]
Funakoshi-Tago M, Tago K, Andoh K, Sonoda Y, Tominaga S, Kasahara T: Functional role of c-Src in IL-1-induced NF-kappaB activation: c-Src is a component of the IKK complex.
J Biochem (Tokyo)137
:189
–197,2005[Abstract/Free Full Text]
Livolsi A, Busuttil V, Imbert V, Abraham RT, Peyron JF: Tyrosine phosphorylation-dependent activation of NF-kappaB. Requirement for p56 LCK and ZAP-70 protein tyrosine kinases.
Eur J Biochem268
:1508
–1515,2001[Medline]
Beraud C, Henzel WJ, Baeuerle PA: Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-kappaB activation.
Proc Natl Acad Sci U S A96
:429
–434,1999[Abstract/Free Full Text]
Bender K, Gottlicher M, Whiteside S, Rahmsdorf HJ, Herrlich P: Sequential DNA damage-independent and -dependent activation of NF-kappaB by UV.
EMBO J17
:5170
–5181,1998[CrossRef][Medline]
Li N, Karin M: Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms.
Proc Natl Acad Sci U S A95
:13012
–13017,1998[Abstract/Free Full Text]
Guan Z, Buckman SY, Springer LD, Morrison AR: Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells.
J Biol Chem274
:36200
–36206,1999[Abstract/Free Full Text]
Donaldson A, Daphna-Iken D, Tetsuka T, Morrison AR: Interleukin-1 beta activates PI 3-kinase in renal mesangial cells.
Biochem Biophys Res Commun227
:289
–293,1996[CrossRef][Medline]
Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA: Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.
J Biol Chem271
:695
–701,1996[Abstract/Free Full Text]
Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA: A target for Src in mitosis.
Nature368
:871
–874,1994[CrossRef][Medline]
Tran NH, Frost JA: Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition.
J Biol Chem278
:11221
–11226,2003[Abstract/Free Full Text]
Gupta AK, Kone BC: CCAAT/enhancer binding protein-beta trans-activates murine nitric oxide synthase 2 gene in an MTAL cell line.
Am J Physiol276
:F599
–F605,1999[Medline]
Huang WC, Chen JJ, Inoue H, Chen CC: Tyrosine phosphorylation of I-kappaB kinase alpha/beta by protein kinase C-dependent c-Src activation is involved in TNF-alpha-induced cyclooxygenase-2 expression.
J Immunol170
:4767
–4775,2003[Abstract/Free Full Text]
Gupta AK, Diaz RA, Higham S, Kone BC: Alpha-MSH inhibits induction of C/EBPbeta-DNA binding activity and NOS2 gene transcription in macrophages.
Kidney Int57
:2239
–2248,2000[CrossRef][Medline]
Zou L, Sato N, Kone BC: Alpha-melanocyte stimulating hormone protects against H2O2-induced inhibition of wound restitution in IEC-6 cells via a Syk kinase- and NF-kappaB-dependent mechanism.
Shock22
:453
–459,2004[CrossRef][Medline]
Cui QL, Zheng WH, Quirion R, Almazan G: Inhibition of Src-like kinases reveals Akt-dependent and -independent pathways in insulin-like growth factor I-mediated oligodendrocyte progenitor survival.
J Biol Chem280
:8918
–8928,2005[Abstract/Free Full Text]
Natarajan R, Fisher BJ, Jones DG, Fowler AA 3rd: Atypical mechanism of NF-kappaB activation during reoxygenation stress in microvascular endothelium: A role for tyrosine kinases.
Free Radic Biol Med33
:962
–8928,2002[CrossRef][Medline]
Xie QW, Kashiwabara Y, Nathan C: Role of transcription factor NF-kappaB/Rel in induction of nitric oxide synthase.
J Biol Chem269
:4705
–4708,1994[Abstract/Free Full Text]
Schaeffer G, Levak-Frank S, Spitaler MM, Fleischhacker E, Esenabhalu VE, Wagner AH, Hecker M, Graier WF: Intercellular signalling within vascular cells under high D-glucose involves free radical-triggered tyrosine kinase activation.
Diabetologia46
:773
–783,2003[CrossRef][Medline]
Cao Z, Xiong J, Takeuchi M, Kurama T, Goeddel DV: TRAF6 is a signal transducer for interleukin-1.
Nature383
:443
–446,1996[CrossRef][Medline]
This article has been cited by other articles:
W. S. Yang, J. W. Seo, N. J. Han, J. Choi, K.-U. Lee, H. Ahn, S. K. Lee, and S.-K. Park High glucose-induced NF-{kappa}B activation occurs via tyrosine phosphorylation of I{kappa}B{alpha} in human glomerular endothelial cells: involvement of Syk tyrosine kinase
Am J Physiol Renal Physiol,
May 1, 2008;
294(5):
F1065 - F1075.
[Abstract][Full Text][PDF]
B Augments IL-1
–Induced Nitric Oxide Production in Mesangial Cells
,
Top
Abstract
Introduction
B is a critical transcription factor that is involved in glomerulonephritis and inflammatory host responses and a critical transactivator of the inducible nitric oxide (NO) synthase gene in mesangial cells. The Src protein tyrosine kinases (SFK) are involved in several signaling pathways and have been proposed to mediate cytokine activation of NF-
induction of NO production has not been clearly established. Accordingly, pharmacologic and molecular tools were used to clarify this issue in cultured murine mesangial cells. The SFK antagonist 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo(3,4-d)pyrimidine (PP2) dramatically inhibited IL-1
and anti-phosphotyrosine antibodies revealed that PP2 also inhibited IL-1
(14), and oxidative stress (15) activate this transcription factor in mesangial cells. NF-
