BMP7 antagonizes TGF--dependent fibrogenesis in mesangialcells - 《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 13:37 |只看该作者 |倒序浏览 |打印

作者：ShinongWang and RaimundHirschberg作者单位：Division of Nephrology and Hypertension, Research andEducation Institute at Harbor-UCLA Medical Center, Torrance,California 90502 : I% i% ~% {9 s' a' Y

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      【摘要】* q# H; X6 `/ v  P
   Exogenous administration ofrecombinant human bone morphogenetic protein (BMP)-7 was recently shownto ameliorate renal glomerular and interstitial fibrosis in rodentswith experimental renal diseases. We tested the hypothesis that BMP7functions by antagonizing profibrogenic events that are induced bytransforming growth factor (TGF)- in cultured mesangial cells.Incubation of murine mesangial cells with TGF- (50-200 pM)increased cell-associated collagen type IV and fibronectin, solublecollagen type IV, thrombospondin, and connective tissue growth factor(CTGF). Coincubation with recombinant human BMP7 (200 pM) reduced theincrease of these ECM proteins and CTGF. The changes incollagen type IV and fibronectin proteins occurred without concomitantchanges in collagen type 1 IV and fibronectin mRNAlevels, suggesting that TGF- and BMP7 act primarily by affecting ECMprotein degradation. Indeed, TGF- decreases the levels and activityof matrix metalloprotease (MMP)-2, the major metalloprotease that issecreted by mesangial cells. Moreover, BMP7 inhibits TGF- -inducedactivation of MMP2. Because TGF- reduces the activity of MMPsthrough increasing plasminogen activator inhibitor (PAI)-1, we testedwhether BMP7 interferes with this TGF- effect. BMP7 reduces, byabout two-thirds, the activation of a PAI-1 promoter/luciferasereporter in cells stably transfected with this construct. The findingsfrom these studies indicate that BMP7 reduces TGF- -induced ECMprotein accumulation in cultured mesangial cells primarily bymaintaining levels and activity of MMP2 partially through prevention ofTGF- -dependent upregulation of PAI-1. * L+ y. ^* v8 F) m+ n
      【关键词】 bone morphogenetic protein transforming growth factor kidney fibrosis matrix metalloprotease plasminogen activatorinhibitor+ ^: V% B, y; p& j: J& u0 P& i
   INTRODUCTION

RENAL FIBROSIS, I.E., ACCUMULATION of ECM proteins in glomeruli and renalinterstitium, is the hallmark of most advanced chronic renal diseases.Progressive fibrosis is the result of an imbalance between synthesisand degradation of ECM proteins. Several cytokines contribute toincreased matrix accumulation, but transforming growth factor (TGF)- has evolved as the single most important profibrogenic mediator inrenal diseases.

Recent studies showed that another member of the TGF- superfamily ofcysteine-knot cytokines, bone morphogenetic protein (BMP)-7, canprevent or reduce the progression of renal fibrosis in animals withexperimental renal diseases ( 7, 12, 13, 16, 18, 20 ).Morrissey and associates ( 18 ) showed that exogenouslyadministered recombinant human (rh)BMP7 may even resolve, at leastpartially, early stages of established glomerular and interstitialfibrosis in experimental diabetic nephropathy.( H; e! {3 Y9 V& P* m) ]! s
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BMP7 plays major roles during embryonic development, but in adultorganisms expression of BMP7 and its receptors is retained in only fewtissues, most prominently in the kidney ( 3, 4, 22, 29 ).Its functions in adult kidney are presently unknown but may includepromotion of differentiated epithelial phenotype in tubular cells( 7, 8, 31 ). Moreover, BMP7 may counteract some of theprofibrogenic actions of TGF-. Consistent with this latterhypothesis are previous findings indicating that a decrease in renalBMP7 expression predates the onset of glomerular sclerosis andinterstitial fibrosis in experimental obstructive nephropathy anddiabetic nephropathy ( 12, 29 ).! D2 U5 y! F2 @$ C. P

The present studies were performed to examine the hypothesis that BMP7reduces ECM accumulation in mesangial cells and antagonizes (some ofthe) profibrogenic events that are induced by TGF-.7 J6 J$ u0 \9 U7 S
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MATERIALS AND METHODS$ c6 ~9 d6 M: H; \
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Mouse mesangial cells derived from a SV40-expressing mouse wereobtained from ATCC, Manassas, VA ( 17 ). Cells were grown 90% confluence in DMEM/F-12 (3:1) containing 14 mM HEPES and 5黃. Before each experiment, cells were incubated in serum-free mediumcontaining 0.1% BSA. In individual experiments, cells were incubatedfor 24 to 72 h with rhTGF- (Biosource, Camarillo, CA), 50, 100, or 200 pM, in the presence or absence of 200 pM rhBMP7 (kind gift fromDr. J. McCartney, Curis, Cambridge, MA). The concentrations ofTGF- were selected to be within the physiological range that hasbeen found in serum ( 9, 11 ) but less than 1 nM, which usually induces maximal effects in most cell types. In individual experiments, we examined the effects of TGF- and BMP7 on collagen type IV (col IV), fibronectin (FN), and thrombospondin (TSP), onconnective tissue growth factor (CTGF) and matrix metalloproteases (MMP) and their activity regulator plasminogen activator inhibitor (PAI)-1., D& `$ n6 j; P3 x$ ~% C* i. G0 ?+ H
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Effects of BMP7 on collagen 1 IV and FN mRNAlevels. Near-confluent mesangial cells in six-well plates were growth arrestedby serum starvation in medium containing 0.1% BSA. Cells were thenincubated with 200 pM rhBMP7, 200 pM rhTGF- 1, or bothfor 72 h ( n = 6 each). Total RNA was extractedwith RNA-Stat-60 reagent and the procedure recommended by themanufacturer (Tel Test, Friendswood, TX). mRNA levels encoding FN orcollagen 1 IV (col4A1) were measured by quantitativereverse-transcription polymerase chain reaction. Random-primed firststrand cDNA was synthesized from RNA with Omniscript ReverseTranscriptase and random primers (Qiagen, Valencia, CA). Aliquots ofcDNA were amplified with Taq polymerase (Qiagen) and the followingspecies- and gene-specific primers. Col4A1: 5'-TAGGTGTCAGCAATTAGG-3'(sense) and 5'-TCACTTCAAGCATAGTGGTCCG-3' (antisense); FN:5'-ATGCACCGATTGTCAACAGA-3' (sense) and 5'-TGCCGCAACTACTGTGATTC-3' (antisense). The optimal number of cycles for amplification in thelinear range was determined in pilot assays. For col4A1, 42 cycles of94°C for 45 s, 60°C for 45 s, and 72°C for 60 s;and for FN, 30 cycles of 94°C for 45 s, 58°C for 45 s,and 72°C for 60 s were found optimal followed by 72°C for 7 min to complete the last cycle. 18S rRNA was used as endogenouscoamplification standard. The optimal ratio of 18S primers to 18Scompatimers (Qiagen) for col4A1 was 1:9 and for FN it was 3:7 to derivesimilar 18S yields compared with that of col4A1 and FN, respectively. PCR products were electrophoretically resolved in 2% agarose gels containing ethidium bromide, illuminated with UV light and analyzed bydigital densitometry using Alpha DigiDoc 100 (Alpha Innotech, SanLeandro, CA).

Effects of BMP7 on TGF- -induced accumulation ofcell-associated FN and col IV. Mesangial cells were incubated without or with rhTGF- 1 (200 pM) in the presence or absence of rhBMP7 (200 pM) for 72 h, n = 4 each. Media were removed and cell layers werewashed three times with ice-cold PBS. Cells and matrix were lysed with2× reducing Laemmli buffer, scraped off the plates, sonicated withthree short 1-s bursts at 4 W, and heated at 80°C for 10 min.Proteins were resolved by SDS-PAGE in 5% gels. Resolved proteins wereelectrotransfered onto nitrocellulose. Membranes were blocked with 5%dry milk (DM) in Tris-buffered saline containing 0.05% Tween 20 (TTBS). For visualization of FN, membranes were incubated with anti-FNmonoclonal antibody (1:1,500; BD-Biosciences, Palo Alto, CA) andsubsequently conjugated with horseradish peroxidase (HRP) anti-mouseIgG. Bands were visualized by enhanced chemiluminescence and capturedon X-ray film. For col IV Western blot, blocked membranes wereincubated with biotin-conjugated goat anti-col IV antibody (1:2,000;Southern Biotechnology, Birmingham, AL). Bands werevisualized with Neutralite Avidin-HRP (Southern Biotechnology) andsubsequent chemiluminescence.  g+ ?* g  l, f5 K
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Cell-associated col IV was also measured quantitatively by ELISA asdescribed previously with some modifications ( 10 ).Briefly, cells were washed three times with ice-cold PBS, scraped into 3 M guanidine/0.05 M Tris, pH 7.5, and homogenized. Precipitates weresuspended in incubation buffer (0.1 M NaHCO 3, pH 9.8),sonicated, and cleared by centrifugation. Ninety-six-well plates werecoated with 100 µl/well of cleared supernatants, each in triplicate, for 48 h at 4°C. Then 100 µl/well of blocking buffer (0.2%BSA in PBS) were added to each well and incubated for 24 h. Wells were washed three times with washing buffer (PBS containing 0.05% Tween 20) and incubated with biotinylated anti-col IV antibody (1:1,500) in incubation buffer (0.2% BSA in PBS, 0.05% Tween 20). Wells were washed six times and incubated with Neutralite-Avidin-HRP inincubation buffer at room temperature for 1 h. Wells were washed again and 100 µl/well of 0.2% O -phenylenediamine inreaction buffer (200 mM Tris · HCl, 150 mM NaCl, pH 6.0, 0.01%H 2 O 2 ) were added to each well. Plates wereincubated for 45 min in the dark and 50 µl/well of 1.3% Na sulfitein 4 M H 2 SO 4 were added. Color intensity wasmeasured at 492 nm in an ELISA-plate reader (Molecular Devices, MenloPark, CA). Results were expressed as percent mean control (cellsincubated in the absence of TGF- and BMP7).0 h1 W0 ~4 t* R
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Effect of BMP7 on TGF- -induced soluble col IV and TSP. Near-confluent mesangial cells were incubated with protein-free mediumcontaining TGF- (0, 50, 100, or 200 pM) in the presence or absenceof BMP7 (200 pM), n = 6 each in six-well culture plates for 72 h. Conditioned media were then cleared by centrifugation and concentrated 10-fold with spin concentrators (Amicon, Bedford, MA).Col IV was examined by Western blotting and quantified by ELISA. ForWestern blot analysis, aliquots of concentrated conditioned media wereelectrophoresed in 5% SDS-PAGE minigels and transferred tonitrocellulose. Membranes were blocked and incubated with biotinylated anti-col IV (Southern Biotechnology) and then withNeutralite-Avidin-HRP (Southern Biotechnology). Bands were visualizedwith enhanced chemiluminescence.

Soluble col IV was also measured by ELISA in concentrated, conditionedmedia essentially as described above with the exception that wells werecoated with 25 µl of concentrated media in coating buffer.

TSP in conditioned media was examined by Western blotting.Heparin-binding proteins were precipitated from cleared media with 40 µl/ml of a 1:1 slurry of preswollen heparin-conjugated Sepharose CL-6B in PBS (Pharmacia, Piscataway, NJ) and rocking for 4 h at 4°C. Precipitates were washed three times with ice-cold PBS, taken upin reducing Laemmli buffer, and electrophoresed in 7.5% SDS-PAGE gels.After electroblotting of resolved proteins, membranes were blocked with5% DM/TTBS and immunoblotted with anti-TSP antibody (Santa CruzBiotechnology, Santa Cruz, CA) and subsequently HRP-anti goat-IgG.Bands were visualized with chemiluminescence.( p3 L' E# M9 n( U5 c! T
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Effects of BMP7 on TGF- -induced CTGF. CTGF is thought to mediate some of the fibrogenic actions of TGF- insome cell types including mesangial cells ( 6 ). Thus wetested whether coincubation of mesangial cells with TGF- in thepresence of BMP7 would reduce secreted CTGF levels. Arrested mesangialcells in six-well plates were incubated with rhTGF- 1 (200 pM), rhBMP7 (200 pM), both, or neither (control) for 48 h. Conditioned media were cleared by centrifugation. Heparin-binding proteins were precipitated as described above. Washed precipitates weretaken up in nonreducing sample buffer and boiled for 3 min. Proteinswere separated in 15% SDS-PAGE gels and electroblotted ontonitrocellulose. Membranes were blocked and incubated with chickenanti-CTGF-IgY (0.5 µg/ml; gift from Fibrogen, South San Francisco,CA) and HRP-anti-chicken IgY. Bands were visualized withchemiluminescence and captured on X-ray film.

MMP activity in mesangial cell-conditioned media. To examine whether BMP7 antagonizes the TGF- -induced decrease in MMPactivity that contributes to the accumulation of ECM, mesangial cellswere incubated in media containing TGF- (0-200 pM) or BMP7 (0 or 200 pM) for 24 h. MMP activity was examined by gelatinzymography in media that had been cleared by centrifugation. Briefly,10% SDS-PAGE minigels were prepared with 1 mg/ml presolved gelatin assubstrate. Samples in reducing agent-free sample buffer were separatedby electrophoresis and fixed in 2.5% Triton X-100 and then developedin 50 mM Tris, 200 mM NaCl, 5 mM CaCl 2, 0.02% Brij-35, pH7.5 for 24 h at 37°C. Gels were stained with 0.5% Coomassieblue R-250 and lightly destained. Additional zymograms were performedwith casein as substrate. However, no protease bands were visualized." m0 w  F: \! |0 {+ @
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Effect of BMP7 on TGF- -dependent PAI-1 promoter activation. MMPs can be activated by proteolysis of Pro-MMPs by plasmin. Thisenzyme, in turn, is derived from plasminogen by a proteolysis steprequiring urokinase-type plasminogen activator (uPA). The activity ofuPA and, hence, MMP2 is negatively regulated by the PAI-1. The PAI-1promoter contains a TGF- -regulated response element and TGF- upregulates PAI-1 transcription ( 25 ). We hypothesized thatBMP7 may function by antagonizing TGF- -dependent activation of MMPsby reducing PAI-1 promoter activation by TGF-. This question wasexamined in mink lung epithelial cells (MLECs) that had been stablytransfected with a construct containing the PAI-1 promoter andluciferase reporter ( 1 ) (kindly provided by D. Rifkin, NYU). Cells were grown to confluence in DMEM/F-12 containing G418 and10% FCS. Cells were washed three times with PBS and then incubated for16 h with serum/G418-free medium containing 0.1% BSA and TGF- (0, 50, 100, or 200 pM) in the presence or absence of BMP7 (200 pM), n = 6-8 each. Additional incubations were madewith excess 29 nM BMP7. Cells were washed three times with ice-cold PBSand lysed in 20 µl/well of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM1,2-diaminocyclohexane- N, N, N ', N '-tetraacetic acid, 10% glycerol, 1% Triton X-100). Luciferase activity in cell lysates was measured in a luminometer with a commercially available luciferase reagent using the manufacturer's instructions (Promega, Madison, WI).9 N% G8 J. s5 O
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Effects of TGF- and BMP7 on PAI-1 and MMP2 protein levels. To examine protein levels of secreted PAI-1 and MMP2 in media,mesangial cells were incubated with serum/BSA-free medium for 48 hwithout (control) or with TGF- or BMP7, each at 200 pM, or both( n = 4 each). Media were cleared by centrifugation. For PAI-1 Western blot assay, aliquots of cleared media were concentrated in spin concentrators (Amicon), taken up in 1× reducing sample buffer,boiled for 5 min, electrophoresed in 10% SDS-PAGE gels, andtransferred to nitrocellulose. Blocked membranes were incubated withpolyclonal anti-PAI-1 antibody (1:800; Santa Cruz Biotechnology) inTTBS/4% BSA overnight at 4°C, washed, and then incubated with HRP-conjugated second antibody (1:12,000 in TTBS/5% BSA) for 1 h.Bands were visualized with enhanced chemiluminescence and captured onX-ray film.; C+ X+ E+ ^  l5 }+ Z- h! h

For MMP2 Western blot analysis, heparin-binding proteins wereprecipitated from aliquots of cleared media as described above. Washedprecipitates were taken up in 1× reducing sample buffer, boiled for 4 min, electrophoresed, transferred, and blotted as above, except thatpolyclonal anti-MMP2 primary antibody was used at 1:500 (Santa CruzBiotechnology). For derivation of semiquantitative results, bandintensity was measured densitometrically and data were expressed aspercentage of mean control.

Statistical analysis. Results are expressed as means ± SE. Significance of differenceswas examined by analysis of variance and subsequent Newman-Keuls multicomparison test. P of difference.) v! N+ [& M. T' ?
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RESULTS3 a: `4 P# V4 m+ b% A! ^; s

BMP7 reduces TGF- -induced accumulation of cell-associated FN andcol IV. At the concentrations tested in the present studies, TGF- did notsignificantly raise steady-state FN or col 1 IV mRNAlevels in cultured murine mesangial cells. Both were also unaffected byBMP7 (Fig. 1 ). Despite the lack ofsignificantly increased mRNAs, both FN and col IV proteins accumulatedduring incubation with TGF-. Cell-associated col IV moderatelyincreased with each of the three TGF- levels by up to 2.2-fold (Fig. 2 ). BMP7 did not reduce baseline levelsof col IV but amerliorated the TGF- -induced increase incell-associated col IV by ~25%.

Fig. 1. Quantitative rtPCR of fibronectin (FN) and collagen 1 IV (C4A1) mRNAs and 18S rRNA (internal control) inmesangial cells that were incubated in the presence or absence ofrecombinant human transforming growth factor (rhTGF)- 1,bone morphogenetic protein (BMP)7, or both (T B) for 3 days. Neitherof the conditions caused a significant change in mRNA levels.3 Z/ x, X# o( A2 C6 a( D

Fig. 2. Changes in cell-associated collagen type IV in mesangialcells that were incubated with rhTGF- 1 or rhBMP7 or bothfor 3 days. A : representative Western blot. B :quantitative ELISA ( n = 6). * P in the absence of BMP7.2 w, ]1 v, j3 y) k' K

Cell-associated matrix FN was also increased by TGF-, almost2.4-fold compared with control. Coincubation with BMP7 also reduced therise in FN by almost one-half (Fig. 3 ).! M& \( |( Q( G$ J9 H  W$ p; K

Fig. 3. Changes in cell-associated FN in mesangial cells afterincubation with rhTGF- 1, rhBMP7 (200 pM), or both for 3 days. A : representative Western blot. B :quantitative densitometric units ( n = 4).* P- t3 K. }  ]! a. H6 z
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BMP7 reduces TGF- -induced soluble col IV and secreted TSP. Collagens are secreted by cells and assemble into complex matrixstructures that become cell associated. Some of the secreted collagenin cell cultures is present in soluble forms. As shown previously byother investigators, TGF- raises col IV in mesangial cell media. Thesoluble col IV that was immunodetected in concentrated, conditionedmedia increased more than threefold with the highest TGF- concentration and increased significantly by ~50%, even with thelowest TGF- concentration (Fig. 4 ).Coincubation with BMP7 reduced the levels of col IV in media by~30-40% (Fig. 4 ). The rise in col IV that is induced by thelowest concentration of TGF- (50 pM) is actually quantitativelyinhibited by BMP7. The rise in col IV that is associated with the twogreater levels of TGF- was significantly reduced but not completelyprevented by BMP7 (Fig. 4 ). Incubation of cells with BMP7 (200 pM) inthe absence of TGF- does not significantly reduce secreted orcell-associated col IV levels.- M2 r) Z+ ?" N" _/ C
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Fig. 4. Collagen type IV that accumulated in mesangial cell mediaduring incubation with rhTGF- 1 and/or BMP7 for 3 days. A : representative Western blot of concentrated media. B : quantitative ELISA ( n = 6).* P P in the absence of BMP7.
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TSP is also a secreted ECM protein and is known to be induced byTGF-, in part, through TGF- -induced CTGF ( 19, 27, 28 ). As shown in Fig. 5, TGF- (200 pM) increases the levels of TSP in conditioned media, and thiseffect of the cytokine is also ameliorated by coincubation with BMP7 atequimolar concentrations.

Fig. 5. Thrombospondin (TSP) Western blot. Mesangial cells wereincubated with rhBMP7, rhTGF- 1 (200 pM), or both for 3 days. Heparin-binding proteins were precipitated from cleared media andimmunoblotted with anti-TSP. Representative blot of 3 experiments.
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BMP7 reduces TGF- -induced, secreted CTGF. CTGF is a secreted mediator of many (but not all) effects of TGF- infibroblasts and also in mesangial cells ( 6, 15, 19 ).Consistent with previous findings from other laboratories, TGF- increases the levels of CTGF in mesangial cell media in the presentstudies about threefold (Fig. 6 ). Theaccumulation of CTGF in conditioned media was significantly, althoughnot completely, reduced by coincubation with BMP7 at equimolarconcentrations (Fig. 6 ).
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Fig. 6. Connective tissue growth factor (CTGF) Western blot ofheparin-Sepharose-precipitated proteins from conditioned mesangial cellsupernatant after incubation with rhBMP7 and/or rhTGF- 1 (200 pM). A : representative of 4 blots. B :relative densitometric units in percent of control. * P P alone.# U: B/ ]7 J0 h) }# p9 E$ A7 g7 i) ?
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BMP7 antagonizes the reduction in MMP2 activity by TGF-. TGF- is known to reduce the activity of MMPs in various cell types.Mesangial cells primarily secrete MMP2, and its activity and perhapslevels are downregulated by TGF- ( 2, 23, 24 ). To testwhether BMP7 alters the reduced activation that occurs with TGF-,mesangial cells were incubated with both proteins and zymography wasperformed on conditioned media. Zymograms with gelatin as substrateshowed a single band at 70 kDa, known to represent MMP2 (gelatinaseA) (Fig. 7 ). -Casein zymography did not visualize any band (not shown). Incubation of cells with TGF-, 50 and 100 pM, tended to reduce MMP2 activity moderately ( P = not significant), but at 200 pM, TGF- substantially reducedMMP2 activity ( P 7 ). BMP7, 200 pM, almost quantitatively prevented the reduction in MMP2 activity thatwas induced by the two lower concentrations of TGF- and maintainedMMP2 activity at about three-quarters of control even in the presenceof TGF-, 200 pM (Fig. 7 ).0 A6 O6 ?5 }0 b4 [+ }( |

Fig. 7. Gelatin zymography of cleared, conditioned mesangial cellmedia after incubation with rhTGF- 1 (50-200 pM)with or without rhBMP7 (200 pM). A single band was visualized at ~70kDa corresponding to matrix metalloprotease 2 (MMP2; gelatinase A). Arepresentative of 5 zymograms is shown, and means ± SE ofdensitometric units are depicted in the bar graph. * P& S/ g: N/ O- y7 I- c, O' j3 v- u

BMP7 blocks TGF- -induced PAI-1 promoter activation. A major mechanism through which TGF- downregulates MMP activitieshas been demonstrated previously, namely, through increasing PAI-1levels that blocks upstream steps required for MMP activation. Specifically, PAI-1 reduces the generation of active plasminogen activator (PA) from inactive pro-PA. TGF- upregulates PAI-1transcription through smad3 and a smad response element in the PAI-1promoter. We tested the question of whether BMP7 blocks this action ofTGF- in MLECs stably expressing a PAI-1 promoter/luciferase reporter construct. As expected, TGF- substantially increases PAI-1 promoter activity (Fig. 8 ). Coincubation of thecells with TGF- in the presence of BMP7, each at 200 pM, reducedPAI-1 promoter activation by ~65% compared with incubation withTGF- alone (Fig. 8 ). At excessively high levels, 29 nM, BMP7downregulates PAI-1 promoter activity by ~50% below control levelseven in the absence of TGF- (not shown).

Fig. 8. Assessment of plasminogen activator inhibitor (PAI)-1promoter activation by TGF- and response to coincubation with rhBMP7in mink lung epithelial cells that had been stably transfected with aPAI-1 promoter/luciferase reporter construct ( 1 ). At eachlevel of TGF-, BMP7 reduces PAI-1 promoter activity significantly byabout two-thirds ( P n = 6-8 each).
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BMP7 reduces TGF- -induced PAI-1 levels in mesangial cell media. Consistent with the findings in MLECs, incubation of mesangial cellswith TGF- increases accumulation of PAI-1 protein in media (Fig. 9 ). This rise in accumulated PAI-1 wassubstantially less in media from cells that were coincubated withTGF- in the presence of BMP7. These findings indicate that BMP7 alsoblocks TGF- -induced PAI-1 in mesangial cells, presumably also byantagonizing the increase in PAI-1 transcription.; d  v. B5 ?* }! E% z. [/ m
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Fig. 9. A : Western blot analysis of PAI-1 and MMP2 insupernatants from cultured mesangial cells that were incubated with orwithout TGF- and/or BMP7, both at 200 pM ( n = 4 each). B : TGF- raises the accumulation of PAI-1 andreduces levels of free heparin-binding MMP2 in media. C :isomolar BMP7 antagonizes these TGF- effects significantly, althoughnot completely. * P P alone.. K/ G* [5 G! G. J+ w
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BMP7 blocks TGF- -induced decrease in MMP2 levels in mesangialcells. Incubation of mesangial cells with TGF- also substantially reducesthe levels of MMP2 that were recovered from media by precipitation withheparin-Sepharose (Fig. 9 ). The reduction in secreted MMP2 wasprevented, in part, by coincubation with BMP7. In the MMP2 Westernblot, only a single band corresponding to ~70 kDa apparent molecularweight band in the zymograms but not higher molecular weight forms wasfound. This may suggest that perhaps only free but not complexed MMP2was detected.5 K/ ]4 _- L2 f) k+ Y8 `% E/ A4 F% p
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DISCUSSION; G% j: C2 w5 J3 E- P7 y  |

Glomerular sclerosis and renal interstitial fibrosis are featuresof most progressive renal diseases and cause renal failure. TGF- hasbeen recognized as an important profibrogenic agent in the kidney, andits antagonism such as with administration of neutralizing antibodiesor soluble receptor decoy proteins ameliorates nephron fibrosis inexperimental glomerular and renal diseases ( 14, 32 ).TGF- contributes to progressive glomerular and interstitialfibrogenesis by increasing gene expression of some ECM proteins, but amajor action of this cytokine is the reduction of degradation andthereby increasing half-lives and, hence, progressive extracellulardeposition of matrix proteins ( 2, 26, 30 ). To this end, inmesangial cells, TGF- reduces MMP2 activation but also its levels asshown in the present studies and in previous experiments by otherinvestigators ( 2 ). MMP2 contributes to the proteolyticdegradation of several different matrix proteins (including collagens,elastin, FN, and laminin) and contributes to the proteolytic activationof several other MMPs ( 5 ). Downregulation of MMP2activity, therefore, contributes to accumulation of several differentECM proteins. Moreover, maintenance of MMP2 activity by BMP7 may haveimportant antifibrogenic effects.
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BMP7 is another member of the TGF- superfamily of cysteine-knotgrowth factors. It plays major roles during renal and eye development( 8 ). In adults, BMP7 expression is very limited, andtissues with greatest levels include the kidney ( 7, 22 ). Its function in the kidney is largely unknown. During progression ofchronic renal diseases such as diabetic nephropathy and obstructive nephropathy, renal BMP7 levels decrease substantially, which may becaused, in part, by rising TGF- ( 12, 29 ).
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Exogenous administration of rhBMP7 to rats with experimentalobstructive nephropathy, streptozotocin-induced diabetic nephropathy, or other experimental renal diseases ameliorates the development ofglomerular sclerosis ( 7, 12, 13, 16, 18, 20 ). Fibrosis inthese models is, at least in part, TGF- dependent, which gives riseto the possibility that BMP7 antagonizes profibrogenic actions ofTGF- in the kidney.8 A  U' ~- M, Q$ y, Y

The present experimental studies examine this hypothesis. Mesangialcells are the pivotal cell type in the glomerulus elaborating the ECMproteins that accumulate to progressive glomerular fibrosis in manyglomerular diseases, and FN and col IV contribute to glomerular scarformation. TGF- -dependent accumulation of these ECM proteins resultsfrom an imbalance between increased production and/or reducedmetabolism of individual ECM proteins. Although the present studies usean in vitro model of SV40-transfected murine mesangial cells, previousobservations from other laboratories strongly suggest that the findingsapply to the pathogenesis of glomerular fibrosis in vivo ( 21, 26 )." i6 w9 z  j7 z* H
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The present in vitro experiments in cultured mesangial cells show thatthe TGF- -induced accumulation of FN, col IV, and TSP can be reducedby coincubation with BMP7, suggesting an antifibrogenic role of thelatter protein. A major although perhaps not exclusive mode of actionof BMP7 is to block the TGF- -induced downregulation of MMP2, i.e.,to prevent TGF- 's reduction of ECM protein metabolism. MMP activityis regulated by activators (uPA) that upregulate plasmin activity,which, in turn, activates MMPs, and by tissue inhibitors ofmetalloproteases that block MMPs. PAI-1 is a principal antagonist tothe activity of uPA, and the upregulation of its promoter activity byTGF- is a major mechanism through which TGF- downregulatesglomerular ECM degradation ( 26 ). Increased transcriptionof the PAI-1 gene by TGF- is mediated by direct interaction ofTGF- -activated smad3/4 complex with a smad response element in thePAI-1 promoter ( 25 ). In the present studies, we testedwhether BMP7 inhibits this particular step in the profibrogenic chainof actions of TGF-. Indeed, BMP7 ameliorates the activation of PAI-1transcription that normally occurs on incubation with TGF-. Thislikely causes or importantly contributes to the maintenance ofnear-normal MMP activity despite the presence of TGF-. This cytokinehas previously been shown to also downregulate MMP2 levels in mesangialcells ( 24 ). Findings in the present studies confirm areduction of (free) MMP2 in media from TGF- -conditioned cells (Fig. 9 ). Thus TGF- may reduce both levels and PAI-1-driven activation ofMMP2, and both effects are apparently blocked by BMP7.0 [7 d( I7 S$ A- W* g. |+ N

In addition to its effects on PAI-1 and MMP2, BMP7 also antagonizessecretion of TSP and CTGF that is induced by TGF-. The moderatereduction in CTGF may also mediate some of the antifibrogenic effectsBMP7. This may include TSP, which is, in part, regulated through CTGF( 27 ). Detailed mechanisms of how BMP7 may regulate CTGFare not revealed by the present studies.

In summary, the present experimental studies indicate that BMP7partially blocks TGF- -induced CTGF as well as accumulation of colIV, FN, and TSP in cultured mesangial cells. BMP7 antagonizes TGF- -induced downregulation of MMP2 and increased transcription andsecretion of PAI-1, suggesting that BMP7 opposes downregulation ofmatrix degradation by TGF-. Thus BMP7 blocks several profibrogenic activities of TGF- in mesangial cells.! ~% |, m0 f2 R9 `
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ACKNOWLEDGEMENTS

The authors appreciate the kind gift of MLECs stably overexpressingPAI-1/luciferase from Dr. D. Rifkin, New York University, New York.rhBMP7 was a generous gift from Dr. J. McCartney, Curis, Cambridge, MA.; }7 ]9 L7 o* T* g- Y3 v
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