Smad3 and PKC mediate TGF-1-induced collagen I expression in human mesangial cel - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:43 |只看该作者 |倒序浏览 |打印

作者：Constance E. Runyan, H. William Schnaper,  Anne-Christine Poncelet作者单位：Department of Pediatrics, Northwestern University, Chicago, Illinois60611

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      【摘要】2 j& g" m- L* H% S
   Transforming growth factor (TGF)- has been associated with fibrogenesis in clinical studies and animal models. We previously showed thatSmad3 promotes COL1A2 gene activation by TGF- 1 inhuman mesangial cells. In addition to the Smad pathway, it has been suggestedthat TGF- 1 could also activate more classical growth factorsignaling. Here, we report that protein kinase C (PKC) plays a role inTGF- 1 -stimulated collagen I production. In an in vitro kinaseassay, TGF- 1 treatment specifically increased mesangial cellPKC activity in a time-dependent manner. Translocation to the membranewas detected by immunocytochemistry and immunoblot, suggesting activation ofPKC by TGF- 1. Inhibition of PKC by rottlerindecreased basal and TGF- 1 -stimulated collagen I production, mRNA expression, and COL1A2 promoter activity, whereas blockade ofconventional PKCs by Gö 6976 had little or no effect. In a Gal4-LUC assaysystem, inhibition of PKC abolished TGF- 1 -induced transcriptional activity of Gal4-Smad3 and Gal4-Smad4(266-552). Overexpressionof Smad3 or Smad3D, in which the three COOH-terminal serine phosphoacceptorresidues have been mutated, increased activity of the SBE-LUC construct,containing four DNA binding sites for Smad3 and Smad4. This induction wasblocked by PKC inhibition, suggesting that rottlerin decreased Smad3transcriptional activity independently of COOH-terminal serinephosphorylation. Blockade of PKC abolished ligand-independent andligand-dependent stimulation of COL1A2 promoter activity by Smad3. These data indicate that PKC is activated by TGF- 1 in humanmesangial cells. TGF- 1 -stimulated PKC activitypositively regulates Smad transcriptional activity and is required for COL1A2 gene transcription. Thus cross talk among multiple signalingpathways likely contributes to the pathogenesis of glomerular matrixaccumulation.
      【关键词】 transforming growth factor signal transduction cross talk gene regulation extracellular matrix accumulation glomerulosclerosis& I) a$ f* G6 Q+ v5 E
   CLINICAL STUDIES HAVE associated transforming growth factor (TGF)- production with glomerular matrix accumulation in diabetic nephropathy, focal segmental glomerulosclerosis, lupus nephritis, and IgAnephropathy ( 5 ). Transgenicmice with increased levels of TGF- 1 develop progressive renaldisease ( 26 ). In addition, TGF- has been shown to mediate fibrogenesis in experimental models ofglomerulonephritis and diabetic nephropathy( 5, 45 ). Extracellular matrix(ECM) accumulation in experimental glomerulonephritis induced byanti-thymocyte serum is suppressed by administration of anti-TGF- antibody ( 7 ); by the natural inhibitor of TGF-, decorin( 6 ); or by TGF- antisenseoligonucleotides ( 1 ). In vitro,we and others showed that TGF- induces type I and type IV collagen andfibronectin synthesis by human mesangial cells( 16, 40 ). Because many fibrogenicstimuli, including stretch, high glucose, platelet-activating factor, andangiotensin II, may induce TGF- 1 expression or activation( 14, 24, 42, 43 ), TGF- 1 action could represent a common pathway mediating glomerulosclerosis.) g1 n- V. {0 _- u" E/ P
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Members of the TGF- superfamily transmit their signal via heteromeric complexes of transmembrane serine/threonine kinases, the type I and type IIreceptors (T RI and T RII). The Smads are a series of proteins thatfunction downstream from the TGF- family receptors to transduce signalto the nucleus ( 2, 34, 38 ). The receptor-regulated orpathway-restricted Smads (R-Smads), Smad2 and Smad3, contain a SSXSphosphorylation site in their COOH-terminal end that is a direct target ofT RI. Upon ligand binding, the R-Smads are phosphorylated and associatewith the common partner Smad, Smad4. The resulting heteromultimer translocatesto the nucleus where it regulates expression of TGF- target genes bydirect binding to DNA and/or interaction with other transcription factors ( 2, 34, 38 ). The inhibitory Smads,Smad6 and Smad7, may participate in a negative feedback loop to controlTGF- responses by competitive interaction with T RI( 18, 21, 34, 36 ). R-Smad and Smad4 arecomposed of Mad-homology (MH)1 and MH2 domains separated by a variable linkerregion. Smad3 and Smad4 can bind directly to DNA through their MH-1 domain( 2, 34, 38 ).
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Although most studies of TGF- signal transduction have focused onSmad activity, the data also suggest a role for more classical growth factorsignaling, such as protein kinase C (PKC). The PKC family is composed of atleast 11 serine/threonine kinases. These are grouped according to thebiochemical requirement for their activation. The conventional PKCs (cPKCs),including PKC, PKC I, PKC II, and PKC, depend oncalcium and phospholipids. The novel PKCs, PKC, PKC, PKC,and PKC, do not require Ca 2 but are phospholipiddependent. The atypical enzymes, PKC and PKC, require neitherCa 2 nor phospholipid. PKC isoenzymes are expressed in atissue-specific fashion and their subcellular localization varies depending onthe cell type ( 22 ).6 p7 @, g& b# S
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Several studies have implicated TGF- 1 and PKC as mediators of ECM accumulation in diabetic animal models and in mesangial cells culturedin high glucose. In streptozotocin-induced diabetic rats, a model for type 1diabetes, administration of LY333531, a PKC inhibitor, preventedincreased expression of mRNA for TGF- 1, fibronectin, and typeIV collagen ( 28 ). In db / db mice, a model for type 2 diabetes, the same inhibitorprevented ECM expansion ( 27 ).PKC is involved in hyperglycemia-stimulated TGF- 1 promoteractivity in mesangial cells( 51 ). This could be themechanism leading to increased TGF- 1 mRNA expression andprotein synthesis that have been observed in murine mesangial cells culturedin high glucose. Thus high glucose could mediate its effect throughPKC-induced TGF- 1 activation leading to increased ECMproduction ( 56 ).# v! _( ?3 r% @9 c/ ]

Conversely, even without high concentration of glucose,TGF- 1 could exert its effect on expression of some of the ECMcomponents through PKC activation. Halstead et al.( 13 ) showed that treatment ofa human carcinoma cell line with the PKC inhibitor calphostin C blockedTGF- 1 -induced increases in plasminogen activator inhibitor-1(PAI-1) and fibronectin mRNA expression. More recently, it has been suggestedthat stabilization of elastin mRNA in lung fibroblasts by TGF- requiresSmads, PKC, and the MAP kinase p38. These data suggest potentialsynergy between classical TGF- and PKC signaling cascades. In support ofthis notion, Yakymovych et al.( 53 ) recently showed thatSmad2 and Smad3, the targets of TGF- receptors, can be phosphorylated intheir MH1 domain by PKC.- X: \8 ~8 K  r, ]4 P* |$ V5 H
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We previously showed that TGF- 1 -induced collagen I geneexpression is Smad3 dependent in human mesangial cells. Here, we investigated whether PKC might be involved in collagen I accumulation in response toTGF- 1. With the use of specific PKC isozyme inhibitors, weshowed that PKC, but not PKC or PKC I, mediates collagen Iproduction by TGF- 1 in human mesangial cells. We demonstrated that TGF- 1 activates PKC in these cells and that thisactivation plays a role in TGF- 1 -stimulated Smadtranscriptional activity and collagen I gene transcription.

MATERIALS AND METHODS

Materials. Reagents were purchased from the following vendors: active human recombinant TGF- 1 from R&D Systems(Minneapolis, MN); rabbit anti-type I collagen from Biodesign (Saco, ME); rabbit anti-PKC, rabbit anti-PKC, mouse monoclonal anti-Smad4 IgG(B-8), mouse monoclonal anti-Smad1/2/3 (H-2), and anti-mouse IgG-horseradishperoxidase (HRP) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-rabbitIgG-HRP, luciferase, and -galactosidase assay systems from Promega(Madison, WI); PMA from Sigma (St. Louis, MO); and calphostin C, rottlerin,and Gö 6976 from Calbiochem (San Diego, CA). Stock solutions were made asfollows: TGF- 1 in 4 mM HCl containing 1 mg/ml BSA; PKCinhibitors in DMSO.

Cell culture. Human mesangial cells were isolated from glomeruli by differential sieving of minced normal human renal cortex obtained fromanonymous surgery or autopsy specimens. The cells were grown in DMEM/Ham'sF-12 medium, supplemented with 20% heat-inactivated FBS, glutamine,penicillin/streptomycin, sodium pyruvate, HEPES buffer, and 8 µg/ml insulin(Invitrogen Life Technologies, Carlsbad, CA) as previously described( 44 ), and were used between passages 5 and 8.+ U4 T5 y5 K! v
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Protein kinase assay. Cells were switched to medium containing 1鸖 and then treated with 1 ng/ml TGF- 1 for various time periods leading up to simultaneous harvest in RIPA buffer (50 mMTris·HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholate; 0.1%SDS) containing protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 µg/mlleupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin). After clarification bycentrifugation, the protein content was determined by Bradford protein assay (BioRad, Hercules, CA). Immunoprecipitation was performed with 2 µganti-PKC or anti-PKC antibody and 30 µl protein G-sepharosefor 1 h at 4°C. Immunocomplexes were incubated for 10 min at roomtemperature with a PKC substrate peptide (which can be phosphorylated byboth PKC and PKC ) (Upstate, Waltham, MA) and[ 32 P] ATP in 10 mM HEPES, pH 7.0; 10 mM DTT; and 10 mMMgCl 2. The reactions were then spotted onto P81 phosphocellulose paper and washed four times with 1% phosphoric acid and once with acetone. Theamount of incorporated radioactivity into the substrate was determined byscintillation counting.
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Immunocytochemistry. Mesangial cells were grown to 60% confluence on eight-well culture slides coated with 1 mg/ml gelatin. The cells wereswitched to medium containing 1% FBS and then treated with 1 ng/mlTGF- 1 for different durations before simultaneous formalinfixation and permeabilization with Triton X-100. The cells were then stainedwith 1 µg/ml anti-PKC antibody according to the manufacturer'sinstructions. The staining was detected with Oregon Green 514-conjugatedsecondary antibodies from Molecular Probes (Eugene, OR) and evaluated under afluorescent microscope.
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Preparation of cell lysates and Western blot analysis. Cells wereswitched to medium containing 1% FBS and pretreated for 1 h with calphostin C(100 nM), rottlerin (5 µM), Gö 6976 (10 nM), or DMSO as vehiclecontrol. The cells were then incubated for 24 h with 1 ng/mlTGF- 1, followed by lysis at 4°C in RIPA or lysis buffer(10 mM Tris·HCl, pH 8.0; 150 mM NaCl; 1% Nonidet P-40) containingprotease and phosphatase inhibitors (1 mM sodium orthovanadate, 50 mM sodium fluoride, 40 mM -glycerophosphate). Lysates were clarified by centrifugation at 18,000 g for 10 min. Proteins were separated bySDS-PAGE (6 or 10% acrylamide gels), transferred onto a PVDF membrane(Millipore, Bedford, MA), and immunoblotted with anti-type I collagen,anti-Smad1/2/3, or anti-Smad4 antibody (0.2 µg/ml). The blots weredeveloped with chemiluminescence reagents according to the manufacturer'sprotocol (Santa Cruz Biotechnology). Autoradiograms were scanned with an ArcusII Scanner (AGFA) in transparency mode and densitometric analysis wasperformed using the National Institutes of Health Image 1.61 program forMacintosh.: D% J# b- y; y" G% ?0 G

Cell fractionation. Cells were scraped into a detergent-free buffer (20 mM Tris·HCl, pH 7.5; 0.5 mM EDTA; 0.5 mM EGTA; 10 mM -mercaptoethanol) containing protease and phosphatase inhibitors. Thecells were then disrupted by 15 strokes of a Dounce homogenizer. Aftercentrifugation at 100,000 g, the supernatant, representing thecytosolic fraction, was saved; the pellet, representing the particulatefraction, was resuspended in buffer; and Triton X-100 was added to be 0.5%final concentration. After 30-min incubation on ice, the pellet wascentrifuged at 18,000 g for 10 min to remove insoluble material. Thesupernatant was saved as the soluble membrane fraction. After determination ofthe protein content, each fraction was analyzed by immunoblotting withanti-PKC antibody (0.2 µg/ml) as described above.
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RNA isolation and Northern blot. Cells were plated in 100-mm culture dishes. Three days later, the cells were switched to medium containing1% FBS. They were preincubated with PKC inhibitors for 1 h before addition of1 ng/ml TGF- 1 or control vehicle for 24 h. Total RNA washarvested using Trizol (Invitrogen Life Technologies) and analyzed by Northernblot as described previously( 40 ). The same blots weresuccessively rehybridized with additional probes after confirmation ofcomplete stripping. cDNAs for human 1 (I) [clone Hf677( 8 )] and 2 (I)collagen [clone Hf1131 ( 4 )]chains were obtained from Dr. Y. Yamada. Quantification of the bands onautoradiograms was performed using densitometric analysis. The signalsobtained by hybridization with these probes were corrected for loading usingthe signal obtained with a bovine cDNA for 28S ribosomal RNA provided by Dr.H. Sage.

Transient transfection and luciferase assay. The day before thetransfection, 6.5 to 8 x 10 4 cells were seeded in six-well plates. Eighteen hours later, cells were switched to 1% FBS medium andtransfected with the indicated constructs along with 0.5 µg ofCMV-SPORT- -galactosidase (Gibco BRL) as a control of transfectionefficiency. Transfection was performed with the Fugene6 transfection reagent(Roche Applied Science, Indianapolis, IN) as previously described( 39 ). After 3 h, 1 ng/mlTGF- 1 or control vehicle was added to the cells. In someexperiments, the transfected cells were pretreated for 1 h with PKC inhibitorsbefore addition of TGF- 1. Twenty-four hours later, the cellswere harvested in 300 µl reporter lysis buffer (Promega). Luciferase and -galactosidase activities were measured as previously described( 39 ). Luciferase assay resultswere normalized for -galactosidase activity. Experimental points wereperformed in triplicates in several independent experiments. One arbitraryunit was set up as the ratio between luciferase and -galactosidase forcells cotransfected with the promoter-reporter construct and the emptyexpression vector and incubated with control vehicles (forTGF- 1 and PKC inhibitors).

Plasmid constructs. The 376COL1A2-LUC construct containing the sequence 376 bp of the 2 (I) collagen (COL1A2) promoter and 58 bp of the transcribed sequence fused to the luciferase (LUC) reporter genewas previously described ( 39 ).The SBE-LUC ( 54 ) reporterconstruct was kindly provided by Dr. B. Vogelstein. The vectors expressing theindicated Smad3 variants ( 32 )were kindly provided by Drs. H. F. Lodish and X. Liu. The Gal4-Smad constructs( 10 ) were kindly provided byDr. M. P. de Caestecker.

Statistical analysis. Statistical differences between experimental groups were determined by analysis of variance using StatView 4.02 softwareprogram for Macintosh. Values of P by Fisher's protectedleast significant difference (PLSD) were considered significant. Differencebetween two comparative groups was further analyzed by unpaired Student's t -test.
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RESULTS7 [1 O: J1 t2 o% L

Role of PKC in collagen I expression. To determine whether PKCs play a role in TGF- 1 -stimulated collagen I expression,mesangial cells were pretreated for 1 h with PKC inhibitors before addition of1 ng/ml TGF- 1 for 24 h. The PKC inhibitors examined were calphostin C, a general inhibitor of PKCs that competes at the binding sitefor diacylglycerol and phorbol esters( 47 ); rottlerin, an inhibitorof PKC ( 12 ); andGö 6976, an inhibitor of Ca 2 -dependent PKC and PKC I isozymes ( 33 ).Cell lysates were harvested and examined by immunoblotting with anti-type Icollagen and anti-Smad4 antibodies. Calphostin C and rottlerin decreased TGF- 1 -induced collagen I expression, without affecting Smad4 or Sp1 expression levels ( Fig.1 and data not shown). The inhibitory effect of rottlerin onTGF- 1 induction was dose dependent ( Fig. 2 ). In contrast, specificinhibition of cPKCs with Gö 6976 did not affectTGF- 1 -increased collagen I production. Rottlerin has beenreported to inhibit PKC activity in vitro ( 50 ); however, a concentrationover 30 µM was necessary to achieve 50% inhibition, six times higher thanthe concentration used to inhibit PKC. Moreover, while PKC isexpressed in human mesangial cells, we were not able to detect PKC inthese cells by immunoblot ( Table1 ). Thus together, these data support a role for PKC inTGF- 1 -stimulated collagen I production.

Fig. 1. Inhibition of protein kinase C (PKC), but not conventional PKCs,blocks transforming growth factor (TGF)- 1 -increased collagenI production. Human mesangial cells in 1% FBS-containing medium werepretreated for 1 h with the indicated PKC inhibitors (calphC, calphostin C;rottl, rottlerin; Gö, Gö 6976) or DMSO as a control vehicle (-).TGF- 1 (1 ng/ml) or control vehicle (1 mg/ml BSA in 4 mM HCl)was then added for 24 h. Cell lysates were harvested and analyzed by Westernblotting with anti-collagen I (Col I) or anti-Smad4 antibody. A :representative blot. B : densitometric analysis of several independentexperiments. * P 1 -treated cells in the absence of PKC inhibitor (unpairedStudent's t -test, n 3).8 N  z1 k, Z! [) O: m

Fig. 2. Rottlerin inhibition of TGF- 1 -induced collagen Iexpression is dose dependent. Mesangial cells were pretreated for 1 h with theindicated concentration of rottlerin before incubation with 1 ng/mlTGF- 1 for 24 h. Immunoblotting with anti-collagen I antibodywas then performed on cell lysates as described in MATERIALS AND METHODS. Results are shown as the ratio betweenTGF- 1 -treated cells and control cells for each concentrationof rottlerin. * P n = 3).' D% x+ e& ]% S

Table 1. Analysis of human mesangial cells for PKC isozyme expression

Activation of PKC byTGF- 1. Inhibition ofTGF- 1 -stimulated collagen I expression by blockade ofPKC suggested that TGF- 1 could activate PKC.Thus we examined the timing of PKC activation by TGF- 1 in human mesangial cells. Cells were treated with 1 ng/mlTGF- 1 for different time periods leading up to simultaneous harvest. Lysates were immunoprecipitated with an anti-PKC or -PKC antibody. Immunocomplexes were used for an in vitro kinase assay.TGF- 1 stimulates PKC in a time-dependent manner( Fig. 3 ). PKC activitybegan to increase 5 min after adding TGF- 1, although notsignificantly. Maximal activity was detected at 60 min and increased activitywas sustained for up to 24 h. In contrast, PKC activity was not affectedby TGF- 1 treatment. Incubation for 15 min with 100 nM PMA wasused as a positive control for PKC activation (not shown).+ o7 z3 ]; O* T; h1 E% y

Fig. 3. Activity of PKC, but not PKC, increases in a time-dependentmanner following TGF- 1 treatment. Cells were treated with 1ng/ml TGF- 1 for the indicated time periods, leading tosimultaneous harvest. Cell lysates were immunoprecipitated withanti-PKC or anti-PKC antibody. Immunocomplexes were used in an invitro kinase assay with the PKC pseudosubstrate. Results are presentedas fold induction over untreated cells. * P t -test, n = 3).
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Next, we examined whether increased in vitro PKC kinase activity correlates with changes in cellular localization. PKC translocates tothe plasma membrane with TGF- 1 treatment in a time-dependent manner ( Fig. 4 ). At 5 min,staining at the cell periphery slightly increased, whereas staining decreasedin the nucleus. Staining at the plasma membrane was more apparent at 60 min,when PKC activity was more robust (see Fig. 3 ). Membrane localization remained elevated for up to 24 h of treatment. Nuclear staining beganincreasing at 15 min and remained elevated for up to 24 h. Translocation tothe cell periphery in response to TGF- 1 was also demonstratedby immunocytochemistry studies on mesangial cells transfected with a greenfluorescent protein-PKC construct (data not shown). In contrast toPKC, PKC did not translocate to the plasma membrane in responseto TGF- 1, whereas translocation of both isozymes was detectedfollowing 15 min of treatment with PMA( Fig. 4 ). Because increasedPKC activity and membrane association between 5 and 30 min were subtle,we sought to further analyze early PKC activation. We performed Western blot analysis of cells separated into membrane and cytosolic fractions. Anincrease in membrane-associated PKC was detectable as early as 5 minafter adding TGF- 1 ( Fig.5 ), corresponding to the low levels of increased activity shown in Fig. 3. Together, these datasuggest that PKC, but not PKC, is activated in human mesangialcells in response to TGF- 1.

Fig. 4. TGF- 1 induces changes in the cellular localization ofPKC. Cells were treated with 1 ng/ml TGF- 1 or 100 nMPMA for the indicated time periods before fixing and immunostaining withanti-PKC or anti-PKC antibody. Arrows show membrane staining.
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Fig. 5. Timing of increased PKC activity correlates with membranetranslocation. Cells treated with TGF- 1 for different timeperiods were fractionated into cytosolic and membrane fractions beforeanalysis by Western blot using anti-PKC antibody. A representative blotfrom 3 separate experiments is shown.

TGF- 1 -induced PKC activity modulates collagen I gene expression. Because PKC isactivated by TGF- 1, and inhibition of PKC blocked TGF- 1 -stimulated collagen I production, we investigatedwhether PKC modulates collagen I gene expression. Cells were pretreated for 1 h with PKC inhibitors before addition of 1 ng/ml TGF- 1 for 24 h. Steady-state mRNA levels for 1 (I) collagen(COL1A1) and 2 (I) collagen (COL1A2) were measured byNorthern blot. As shown in Fig.6, both calphostin C and rottlerin inhibited TGF- 1 -induced COL1A1 and COL1A2 mRNA expression. Rottlerin also decreased basal collagen I mRNA levels in agreement with its effect onbasal protein synthesis (see Fig.1 ). In contrast, the cPKC inhibitor Gö 6976 slightlyincreased basal mRNA expression with minimal effect on TGF- 1 fold induction. To further define a role for PKC inTGF- 1 -induced collagen I gene expression, we performedtransient transfection experiments with 376COL1A2-LUC, a construct containingthe sequences from -376 to  58 of the human COL1A2 promoter in front of theluciferase reporter gene ( 39 ).The transfected cells were pretreated with PKC inhibitors for 1 h.TGF- 1 was then added for 24 h and luciferase activity wasdetermined. Similar to the results with protein and mRNA, calphostin C androttlerin blocked TGF- 1 -induced COL1A2 promoter activity,whereas Gö 6976 did not affect the response( Fig. 7 ). Together, theseresults suggest that PKC plays a role in basal collagen I expressionand is necessary for the transcriptional response toTGF- 1.) F/ Z0 R8 a7 m' q- q
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Fig. 6. TGF- 1 -increased 1 (I) and 2 (I) collagen mRNA expression is significantly reduced bycalphostin C and rottlerin. Mesangial cells were treated as described in Fig. 1. Total RNA was collectedafter 24 h of treatment and COL1A1 and COL1A2 mRNA levels were assessed byNorthern blotting. The same blots were stripped and reprobed with 28S rRNA asa loading control. A : representative blot of at least 2 separateexperiments. B : densitometric analysis. Data are expressed as theratio between COL1A2 and 28S, with data for untreated cells arbitrarily set upat 1. * P ** P+ k# J" j0 m- v! {$ I2 I; \

Fig. 7. Inhibition of PKC blocks COL1A2 promoter activity. Mesangial cellswere transfected with 0.5 µg of 376COL1A2-LUC construct and 0.5 µg ofCMV- -galactosidase as a control for transfection efficiency. After 3 h,the cells were pretreated with 100 nM calphostin C, 5 µM rottlerin, 10 nMGö 6976, or DMSO as vehicle control (-). One hour later,TGF- 1 was added at a final concentration of 1 ng/ml. Thecells were harvested after 24 h, and luciferase and -galactosidaseactivities were measured. Luciferase activity was normalized to -galactosidase activity. The results are means ± SE of at least 3independent experiments. * P ** P n 3).; X# p! T$ V1 \: o7 H/ A+ o3 a
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Regulation of Smad by PKC. We previously showed that Smad3 is required for TGF- 1 -stimulated COL1A2 genetranscription. Therefore, we investigated whether the inhibitory effect of PKC blockade on collagen I expression is due to modulation of Smad3expression and/or activity. Rottlerin decreased basal expression of Smad3,although not consistently, suggesting the inhibitory effect of rottlerin oncollagen I production could, at least in part, be due to decreased Smad3expression (data not shown). To determine whether PKC inhibitionimpaired TGF- 1 -induced collagen I expression by decreasingSmad3 levels, cells were pretreated with 1 µM PMA for 24 h to deplete cellular PKCs. The cells were then transfected with 376COL1A-LUC and aconstruct expressing wild-type Smad3 (Flag-N-Smad3) ( 32 ) or an empty vector (pEXL)before treatment with TGF- 1. Similar to the effect ofincubation with rottlerin, prolonged exposure to PMA not only decreased basalCOL1A2 promoter activity but also completely blocked the promoter induction byTGF- 1 ( Fig.8 A ). Overexpression of Smad3 restored the response to TGF- 1. Figure8 B shows a Western blot analysis indicating that chronicPMA treatment led to PKC downregulation and decreased Smad3 proteinlevels. Together, these data suggest that the inhibitory effect of PKC blockade or depletion on COL1A2 transcription could be partially dueto the downregulation of Smad3 expression.
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Fig. 8. Inhibitory effect of PKC downregulation on TGF- 1 -inducedCOL1A2 promoter activity can be overcome by overexpressing Smad3. To depletefrom PKCs, mesangial cells were treated for 18 to 24 h with 1 µM PMA orethanol as control vehicle. A : cells were then cotransfected with 0.5µg of 376COL1A2-LUC construct and either 0.5 µg of the vector encodingwild-type Smad3 or the empty expression vector (pEXL), along with 0.5 µg ofCMV- -galactosidase vector. After 3 h, the transfected cells were treatedwith 1 ng/ml TGF- 1 for 24 h. Luciferase activity wasnormalized to -galactosidase activity. Experimental points wereperformed in triplicate in 2 independent experiments. Values are means± SE of triplicate wells from a representative experiment. * P B : cells were treated withTGF- 1 for 30 min and lysates were harvested forimmunoblotting. Chronic treatment with PMA decreases Smad3 levels. Blottingwith anti-PKC antibody confirms downregulation of PKC byPMA.

Because it has been shown that PMA-activated PKC modulates Smad3 DNAbinding activity ( 53 ), weinvestigated whether rottlerin decreases Smad protein transcriptional activityas well as decreasing Smad3 levels. Cells were cotransfected with a reporter construct containing five Gal4 binding sites in front of the luciferase geneand a construct expressing the Gal4 DNA binding domain fused to eitherfull-length Smad3 (Gal4-Smad3) or Smad4 (266-552) [Gal4-Smad4( N)].These constructs enable us to study the transcriptional activity of Smadsindependently of their DNA binding activity. We previously showed that bothconstructs are responsive to TGF- 1 in mesangial cells( 41 ). The cells werepretreated for 1 h with the PKC inhibitor and then incubated withTGF- 1 for 24 h. Rottlerin blockedTGF- 1 -stimulated Smad3 transcriptional activity( Fig. 9 ). Smad4 transcriptionalactivity was also decreased by rottlerin pretreatment. These data suggest thatSmad activity is modulated by PKC signaling in response toTGF- 1. To further support these data, we evaluated the effect of PKC inhibition on the activity of the SBE-LUC reporter construct containing four copies of the CTCTAGAC sequence that has been shown to bindrecombinant Smad3 and Smad4( 54 ). Cells were cotransfectedwith SBE-LUC and an empty vector (pEXL) or a construct expressing wild-typeSmad3. The cells were pretreated with rottlerin for 1 h and then incubatedwith TGF- 1 for 24 h. As expected, TGF- 1 stimulated the activity of the SBE-LUC reporter indicating activation of Smadproteins ( Fig. 10, pEXL histograms). TGF- 1 -induced activity of endogenous Smads was decreased by rottlerin pretreatment. Moreover, blockade of PKC almostcompletely inhibited transcriptional activity of overexpressed Smad3. Theseresults further confirm that blockade of PKC inhibits Smad3 activity.To investigate whether PKC modulation of Smad3 activity is dependent onphosphorylation at the T RI-specific target site of Smad3, mesangialcells were cotransfected with a mutated Smad3 construct (Smad3D) in which thethree COOH-terminal serine residues are replaced by three aspartic acidresidues ( 32 ). Inhibition ofPKC drastically decreased luciferase activity induced by Smad3D. Thesedata suggest that the inhibitory effect of rottlerin on Smad3 transcriptionalactivity is not due to blocking of phosphorylation at the COOH-terminalT RI target site. Of note, Smad3D can function as a transcriptionalactivator in the absence of TGF- 1 as previously demonstrated( 32 ).

Fig. 9. Rottlerin decreases Smad3 and Smad4 transcriptional activity. Cells weretransfected with 0.5 µg of the pFR-LUC reporter construct (containing 5Gal4 binding sites in front of the luciferase gene), 0.5 µg of theindicated Gal4 DNA binding domain fusion proteins, and 0.5 µg ofCMV- -galactosidase as a control for transfection efficiency. After 3 h,the cells were incubated with 5 µM rottlerin or DMSO as vehicle control for1 h before adding 1 ng/ml TGF- 1. Twenty-four hours later, thecells were harvested, and luciferase and -galactosidase activities weremeasured. Luciferase activity was normalized to -galactosidase activity.The results are means ± SE of at least 2 independent experiments.

Fig. 10. PKC inhibition decreases Smad3 activity independently ofphosphorylation of Smad3 at the COOH-terminal T RI target site. Cellswere cotransfected with 0.5 µg of the SBE-LUC reporter construct(containing 4 DNA binding sites for Smad3/Smad4) and either 0.5 µg of thevector-encoding wild-type Smad3, mutated Smad3 (Smad3D), or the emptyexpression vector pEXL, along with 0.5 µgof CMV- -galactosidase. Thetransfected cells were incubated with 5 µM rottlerin and then treated with1 ng/ml TGF- 1 for 24 h. Luciferase activity was normalized to -galactosidase activity. Values are means ± SE of at least 3independent experiments. * P P ** P 1 -treated cells transfected with the same expressionvector in the absence of rottlerin.

Finally, we investigated whether the inhibitory effect of rottlerin on Smadactivity modulates COL1A2 promoter activity. Mesangial cells werecotransfected with the 376COL1A2-LUC and an expression vector for Smad3,Smad3D, or the corresponding empty vector. One hour after adding rottlerin orcontrol vehicle, the cells were incubated with TGF- 1 for 24h. Similar to the results obtained with the SBE-LUC reporter construct,inhibition of PKC partially blocked ligand-independent andligand-dependent, Smad3-mediated COL1A2 promoter activity( Fig. 11 ). This inhibition didnot require phosphorylation at the COOH-terminal SSXS phosphorylation site ofSmad3. Taken together, these results suggest that TGF- 1 -induced PKC activity contributes to increased COL1A2 gene transcription by modulating Smad3 expression andtranscriptional activity.! d1 L! y% N3 a* O
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Fig. 11. Inhibition of PKC abolishes ligand-dependent and -independentstimulatory effect of Smad3. Cells were cotransfected with 0.5 µg of the376COL1A2-LUC reporter construct and either 0.5 µg of the vector-encodingwild-type Smad3, Smad3D, or pEXL, along with 0.5 µg ofCMV- -galactosidase vector. The transfected cells were incubated with 5µM rottlerin and then treated with 1 ng/ml TGF- 1 for 24 h.Luciferase activity was normalized to -galactosidase activity. Valuesare means ± SE of at least 4 independent experiments. * P P ** P 1 -treated cells transfected with the same expressionvector in the absence of rottlerin.

DISCUSSION
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In the present paper, we showed that TGF- 1 stimulatesPKC activity in human mesangial cells and that PKC mediatesTGF- 1 -induced collagen I expression, probably by modulatingSmad transcriptional activity.% I  L- Y1 n; z

With the use of an in vitro kinase assay, immunocytochemistry, andimmunoblotting on cytosol/membrane fractions, we demonstrated that, in humanmesangial cells, PKC is activated by TGF- 1 in atime-dependent manner, beginning at 5 min with maximal activation at 60 min.Similarly, Perillan and colleagues( 37 ) showed thatTGF- 1 causes PKC translocation to the membrane inrat-reactive astrocytes. In contrast, Studer et al.( 46 ) suggested that TGF- does not stimulate PKC(s) in rat mesangial cells, whereas Uchiyama-Tanaka etal. ( 48 ) showed a rapid andtransient stimulation in murine mesangial cells. However, it has been reportedthat TGF- variably affects vascular smooth muscle cell PKC translocation depending on the embryonic lineage( 52 ). Thus discrepancies amongthese studies could be due to cell- or species-specific responses as well asto culture conditions.

PKC translocation to specific intracellular compartments is variabledepending on the isoform, stimulus, and/or cell type ( 22, 35 ). In ourimmunocytochemistry experiments, PKC staining increases at the membraneas well as at the nuclear area after 60 min of treatment withTGF- 1.1 _- K9 l* q/ E& f* R" p; B
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Inhibition of PKC by rottlerin blockedTGF- 1 -stimulated collagen I production. This is not due to anonspecific effect of rottlerin on cell activity because levels of Sp1 andSmad4 [2 proteins whose expression is not modulated by TGF- 1 but that are involved in TGF- 1 -induced collagen I expression( 41 )] are not affected by thePKC inhibitor. The inhibitory effect of rottlerin on collagen Iproduction is due, at least in part, to inhibition of COL1A1 and COL1A2 gene transcription because rottlerin also inhibited mRNAexpression and promoter activity. Taken together with the data showing changesof PKC, but not PKC, translocation and kinase activity, ourfindings suggest that PKC stimulates collagen I expression in responseto TGF- 1 in human mesangial cells. Recently, Rosenbloom andcolleagues ( 29, 30 ) suggested a role forPKC in mediating increased fibronectin transcription and elastin mRNAstabilization by TGF- in human lung fibroblasts. Thus TGF- couldstimulate ECM accumulation in several cell types, in part, by modulatingPKC activity.
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In contrast to rottlerin and low concentration of the general PKC inhibitorcalphostin C, Gö 6976, an inhibitor of cPKCs, did not affect collagen Iproduction or COL1A2 promoter activity in response to TGF- 1.However, in some of our experiments, Gö 6976 increased basal collagen Iprotein and 2 (I) mRNA expression. This result suggests thatcPKC isoenzymes might inhibit collagen I expression in unstimulated cells.
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In our experiments, rottlerin not only blocked TGF- 1 stimulation of COL1A2 transcription but also decreased promoteractivity in unstimulated cells. Similarly, a recent report showed blockade of COL1A1 gene transcription by inhibition of PKC in scleroderma fibroblasts ( 23 ). Thesefindings suggest that PKC is involved in maintaining basal COL1A1 and COL1A2 gene expression as well as playing a rolein gene activation by TGF- 1. Because mesangial cells havebeen shown to produce TGF- 1 ( 25 ), it is also possible thatthe inhibitory effect of rottlerin on basal collagen I expression might be dueto blockade of the autocrine/paracrine stimulation byTGF- 1.
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To determine the mechanism by which a TGF- 1 -induced PKCpathway modulates collagen I expression, we investigated whether PKC modulates Smad expression and/or activity. Both depletion of PKC by chronicPMA treatment and inhibition of PKC by rottlerin slightly decreasedSmad3 protein levels. These data suggest that PKC could play a role inmaintaining basal Smad3 expression. Downregulation of endogenous Smad3 byrottlerin or chronic PMA correlates with decreased COL1A2 promoter activityand inhibition of the TGF- 1 response. Because the inhibitionfollowing PKC depletion by PMA is overcome by ectopic expression of Smad3, this observation suggests that blockade of PKC might inhibit TGF- 1 -induced collagen I gene transcription, at least in part by decreasing Smad3 expression.! C" ], s. o. S9 @7 v

Although the effect of rottlerin on COL1A2 could be partly due to thedownregulation of Smad3 expression, our data with the Gal4 assay system andthe transfection experiments with overexpressed Smad3 demonstrate thatblockade of PKC also inhibits Smad3 transcriptional activity. ThusTGF- 1 -induced PKC activity could stimulate Smadactivity, leading to increased COL1A2 gene expression. Becauseblockade of PKC similarly decreased transcriptional activity of Smad3and Smad3D, PKC likely modulates Smad3 activity independently ofphosphorylation of the specific T RI COOH-terminal target site.' Z* j9 H7 R1 `2 ?! E
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With the use of different cell lines (Mv1Lu and NIH-3T3 cells) stablyexpressing tagged Smad proteins, Yakymovych et al. ( 53 ) showed that celltreatment with the PKC activator PMA resulted in phosphorylation of Smad2 andSmad3. The phosphorylation did not affect nuclear translocation but itabrogated direct DNA binding by Smad3. This phosphorylation-dependentmechanism involving PKC could selectively downregulate certainTGF- 1 signals. In contrast, our data indicate that blockingthe PKC pathway decreases TGF- 1 -induced collagen Iproduction and promoter activity as well as Smad activity, suggesting apositive effect of PKC on the TGF- 1 signal leading tocollagen I accumulation. Thus the same signaling pathway could inhibit oractivate the Smad pathway depending on the cell type and/or stimuli.1 D$ j; s* Q* ^) Q( y( Q- Y

In this paper, we showed modulation of the Smad pathway by PKC in responseto TGF- 1. Previously, we demonstrated that inhibition ofTGF- 1 -activated ERK1/2 decreased Smad transcriptionalactivity ( 19 ). In several celllines, PKC has been linked to ERK activation in response to some stimuli( 3, 9, 15, 17, 49, 55 ). For example, Axmann etal. ( 3 ) demonstrated thatcalcium-dependent PKCs are required for ERK1/2 phosphorylation byTGF- 1 in rat lung fibroblasts. Our laboratory showed thatTGF- 1 stimulation of Smad3 phosphorylation outside theCOOH-terminus serines is dependent on ERK activation( 20 ). However, thisobservation may not explain the role of PKC in our system because therequirement of PKC for ERK activation is dependent on the cell types and stimuli. For example, in neuronal cells, PKC mediates activation of ERKby fibroblast-derived growth factor and nerve growth factor but not byepidermal growth factor ( 9 ).Thus TGF- 1 -induced PKC in human mesangial cells couldmodulate Smad activity directly and/or be dependent on ERK activation. On theother hand, activation of PKC by TGF- 1 might itselfrequire activation of other signaling pathways. It has been shown that thephosphatidylinositol 3-kinase (PI3K) associates with PKC in theerythroleukemia cell line TF-1 stimulated with cytokines( 11 ) and that phosphorylation of PKC in response to serum in the human embryonic kidney 293 cells wasPI3K dependent ( 31 ).

In summary, we showed that, in human mesangial cells,TGF- 1 stimulates PKC translocation and activity.PKC positively regulates Smad3 and Smad4 transcriptional activity andis required for increased collagen I production, suggesting that activation and interaction of multiple signaling pathways contribute to the pathogenesisof glomerular matrix accumulation.

DISCLOSURES

This study was supported by a Young Investigator Research Grant from theNational Kidney Foundation of Illinois to A.-C. Poncelet and Grant DK-49362from the National Institute of Diabetes and Digestive and Kidney Diseases.
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ACKNOWLEDGMENTS1 C- W2 ]  W8 N, H

We thank Drs. M. P. de Caestecker, H. F. Lodish, X. Liu, H. Sage, B.Vogelstein, and Y. Yamada for the constructs described under MATERIALS AND METHODS. We express gratitude to the members of the Schnaper lab forhelpful discussion.
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