Relaxin increases ubiquitin-dependent degradation of fibronectin in vitro and am - 《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 13:38 |只看该作者 |倒序浏览 |打印

作者：Glenn A. McDonald, Pradip Sarkar, Helmut Rennke, Elaine Unemori, Raghu Kalluri,  Vikas P. Sukhatme作者单位：1 Division of Renal Diseases and Hypertension, The University of Texas Medical School at Houston, Houston, Texas 77030; Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston 02215; Department of Pathology, Brigham and Women‘s Hospital and Harvard Medic ( x  }6 v- o5 s0 E' ^7 i
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      【摘要】1 _  r0 q# \0 `9 M+ S* e
   Fibronectin, a large adhesive glycoprotein, is a prominent constituent of the extracellular matrix. Abnormalities in fibronectin homeostasis occur in numerous disease states, ranging from primary fibrosing conditions to neoplastic transformation. We demonstrate that fibronectin is a target protein substrate for ubiquitin-dependent degradation. Coimmunoprecipitation experiments and confocal microscopy demonstrated ubiquitin-fibronectin interaction. In an in vitro model of renal fibrosis, relaxin, an insulin-like growth factor, increased ubiquitin-dependent fibronectin degradation. Relaxin also was evaluated in an anti-glomerular basement membrane model of renal fibrosis. Animals treated with relaxin experienced renoprotection, manifested by decreased serum creatinine and proteinuria. Histological evaluation of kidney sections from animals treated with relaxin showed decreased glomerulosclerosis and interstitial fibrosis. We conclude that relaxin might be developed as a useful agent for the treatment of renal fibrosis. ( g  h0 K9 F8 W
      【关键词】 fibrosis protein degradation kidney
   THE EXTRACELLULAR MATRIX (ECM) is the external milieu that bathes all cells of the body. Constituents of the ECM include collagens, glycoproteins, and proteoglycans. The primary function of the ECM is to provide structural support. Additionally, the ECM is critical for normal cellular morphogenesis, differentiation, and survival ( 2, 29 ). Alterations in ECM composition occur normally as cells respond to various stimuli, as in wound healing and injury ( 42 ). Changes in ECM composition are prominent pathological features of many diseases, including arthritis, scleroderma, chronic renal failure, and neoplastic transformation ( 3, 31, 33, 38 )., k- U5 d) O( Z! x" }1 ^

Fibronectin is a large adhesive glycoprotein and a prominent constituent of the ECM. Secreted as a dimer composed of 240-kDa monomers joined by two disulfide bonds, fibronectin forms an adhesive lattice surrounding virtually every cell of the body. The presence of extracellular fibronectin is critical for numerous cellular functions, including adhesion, migration, proliferation, differentiation, and survival ( 16 ). The accumulation of extracellular fibronectin is a pathological feature of most fibrosing conditions, including systemic sclerosis, arthritis, diabetic nephrosclerosis, pulmonary fibrosis, and cirrhosis ( 9, 20, 31, 33, 38 ). Abnormalities in fibronectin assembly also are associated with neoplastic transformation ( 27, 33 ).
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Fibrosis is the excessive accumulation of ECM, a process that can lead to end-organ damage. Conceptually, fibrosis can be viewed as an excessive tissue repair response with two distinct phases: an initial injury phase, followed by the progressive accumulation of ECM and an expansion of the extracellular space ( 21 ). The process can be initiated by diverse insults, including physical trauma, physical stress, immunological injury, or metabolic abnormalities. This injury phase is commonly characterized by the accumulation of macrophages, monocytes, and eosinophils and coincides with the release of inflammatory cytokines, such as transforming growth- (TGF- ), PDGF, IL-1, and TNF from the infiltrating cells. This, in turn, leads to recruitment of additional inflammatory cells and activation of resident cells ( 22 ). These cytokines promote the secretion and accumulation of ECM. Under normal circumstances, this response is transient and sufficient to restore structural integrity to the injured tissue. In fibrotic conditions, however, the response persists, resulting in the pathological accumulation of ECM ( 5 ). Despite the extensive morbidity and mortality associated with this process, effective therapy remains limited., H% L7 }' c' g1 ]

Relaxin is a polypeptide hormone member of the insulin-like growth factor family. It has a molecular weight of 5-6 kDa ( 11 ) and is a two-chain polypeptide encoded by two nonallelic genes on chromosome 9 ( 10 ). Relaxin is produced in the prostate, ovaries, and placenta, reaching its highest serum levels during pregnancy ( 4 ). Its primary function in pregnancy is in remodeling of the cervix and the interpubic ligament in preparation for parturition. Additionally, relaxin has been shown to inhibit smooth muscle contraction ( 11 ).) f' E, @; k, V1 y4 n- h( u
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Exogenous administration of relaxin in estrogen-primed animals has resulted in a decrease in the density of collagen bundles in the interpubic ligament ( 8 ). Unemori et al. ( 37 ) evaluated the effects of relaxin in an in vitro and in vivo model of pulmonary fibrosis. Relaxin inhibited TGF- 1 -induced overexpression of secreted collagen type I/III and fibronectin in human lung fibroblasts in a dose-dependent fashion. In vivo, relaxin inhibited bleomycin-induced alveolar thickening and collagen accumulation. Furthermore, while this study was in progress, Garber et al. ( 14 ) reported that relaxin decreased interstitial fibrosis in a bromo-ethylamine model of renal interstitial fibrosis. The mechanism for relaxin's effects in fibrosis models remains unclear.

EXPERIMENTAL PROCEDURES, {& @. z! H6 V" Z  d/ n
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Cell culture. Mouse cortical tubule cells (MCTs), mouse mesangial cells (MMCs), and mouse tubular fibroblasts (TFBs) were grown in DMEM (Invitrogen, Rockville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), nonessential amino acids, and 2 mM L -glutamine. Cells were seeded at 80% confluence for experiments.

Animals/reagents. Male Wistar-Kyoto rats (Charles River Laboratory, Wilmington, MA) with initial body weights of 150-180 g were used in these studies. Animal care, treatment, and housing were carried out in accordance with institutional and National Institutes of Health guidelines. Rats had ad libitum access to standard rodent lab chow and tap water, with a 12:12-h light-dark cycle in a constant-temperature facility. Recombinant human relaxin was manufactured at Connectics (Palo Alto, CA). TGF- 1 was purchased from R&D Systems (Minneapolis, MN), and MG-132 and lactacystin were purchased from Calbiochem (La Jolla, CA).; ~) F3 E% R4 ]5 O

Western blot analysis and immunoprecipitation. These experiments were performed as described previously ( 35 ). Cells were washed twice with 10 ml of cold PBS, lysed with ice-cold lysis buffer [(in mM) 50 Tris (pH 7.5), 150 NaCl, 1 Na 3 VO 4, 2 EGTA, 1 phenylmethylsulfonyl fluoride, and 2 pepstatin A, as well as 10 µg/ml leupeptin, 0.5% aprotinin, and 1% Non-idet P-40 (NP-40)], incubated for 10 min on ice, and centrifuged at 14,000 rpm for 10 min at 4°C. Protein samples were mixed with 2 x sample buffer [125 mM Tris · HCl (pH 6.8), 20% glycerol, 10% -mercaptoethanol, 4% SDS, and 0.0025% bromphenol blue], boiled, and run on 4-15% polyacrylamide gels (Ready Gel; Bio-Rad, Hercules, CA), using Tris glycine-SDS running buffer (Bio-Rad). Size-separated proteins were transferred to nylon membranes (Immobilon-P; Millipore, Austin, TX) using a semi-dry apparatus (Trans-Blot SD; Bio-Rad). For immunodetection, membranes were blocked in washing buffer (1 x TBS and 0.1% Tween 20) with 5% milk and incubated with antibody in washing buffer without Tween 20. Antibodies included a polyclonal anti-fibronectin antibody (Invitrogen), a polyclonal anti-ubiquitin antibody, and a polyclonal anti-actin antibody (Sigma, St. Louis, MO). Secondary antibodies were donkey anti-rabbit or sheep anti-mouse IgG linked to horseradish peroxidase (Amersham, Piscatawa, NJ) and were detected by chemiluminescence (Pierce, Rockford, IL). Immunocomplexes were captured with protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). After three washes with cell lysis buffer, bead-bound proteins were subjected to Western blot analysis using the anti-ubiquitin antibody (Sigma), as detailed above.- I0 }- c8 b6 @: e! T' w$ _2 V7 G- s

RNA extraction and Northern blot analysis. These experiments were performed as described previously ( 19 ). Briefly, total RNA, isolated by the single-step acid phenol extraction method, was separated on a 1.2% formaldehyde-agarose gel, transferred to a positively charged nylon membrane (Boehringer/Roche, Indianapolis, IN) using 10 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate), and probed with random primer-labeled cDNAs (Stratagene, La Jolla, CA) in a solution containing 0.5 M sodium phosphate (pH 7.2), 7% SDS, 1% BSA, 1 mM EDTA, and sonicated herring sperm DNA (50 µg/ml) at 68°C. The probes used were a 1.2-kb GAPDH cDNA fragment. Blots were washed three times with a solution containing 40 mM sodium phosphate (pH 7.2), 0.5% SDS, 0.5% BSA, and 1 mM EDTA at 68°C and autoradiographed. Fold-activation was calculated by densitometry using GAPDH expression as a normalization control.* e3 z" e* Y4 i/ A. b

Confocal microscopy. Immunofluorescent staining was performed as described previously ( 17 ). Briefly, selected cells were seeded on coverslips and grown to 50% confluence. Cells were rinsed with PBS (GIBCO BRL, Gaithersburg, MD) twice for 2 min, fixed with 3.5% paraformaldehyde (Sigma) for 30 min at 4°C, quenched with 4 M NH 4 Cl (Sigma) for 5 min, and then permeablized with PBS, 0.3% Triton X-100 (Sigma) for 40 min at room temperature. Cells were then blocked with 10% bovine serum albumin (Sigma) or 10% normal goat serum (Jackson Immunoresearch, West Grove, PA) for 1 h at 37°C. Cells were incubated with primary anti-fibronectin antibody (Sigma) at 1:100 dilution for 1 h at 37°C. FITC-conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) was at the same dilution for 1 h at 37°C. Cells were mounted using 90% glycerol (Sigma), with 0.1% p -phenylenediamine (Sigma) as an antiphoto-bleaching agent. Images were acquired at thicknesses of 0.1-0.25 µm through the samples, deconvoluted, presented as stacked-section images, and then visualized with a Molecular Dynamics scanning laser confocal microscope at a wavelength of 488 nm (Sunnyvale, CA) or a DeltaVision fluorescence scanning deconvolution microscope. The areas and volumes of the cells were determined with ImageSpace software (release 3.10; Molecular Dynamics) after optical stack-sectioning of the cells (sections were made at 180 nm).
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Biosynthetic labeling with [ 35 S]methionine. Metabolic labeling was carried out as described by Lamande et al. ( 23 ). Briefly, MMCs were grown to 80% confluence in the presence and absence of relaxin (100 ng/ml) for 36 h or in the presence of relaxin (100 ng/ml) for 36 h and lactacystin (10 µM) for 8 h. Cells were rinsed with PBS and then incubated with methionine-deficient medium with [ 35 S]methionine (200 µCi/ml medium) for 1 h. Cells were rinsed with PBS and then incubated with DMEM with 10% FBS, as described above. Cells were harvested in 1.0 ml protein lysis buffer [(in mM) 1% NP-40, 150 NaCl, 50 Tris, pH 7.5, 1 phenylmethanesulfonyl fluoride, and 11 glucose, as well as 1% NP-40, 800 units/ml] at 1, 2, 4, and 7 h. Cell lysates were centrifuged at 14,000 g after 30 min at 4°C to remove nuclei and cell debris. The supernatant was immunoprecipitated overnight with protein A/G Plus-agarose beads with polyclonal anti-rabbit fibronectin, or an isotype-equivalent control antibody. The protein A/G beads were washed three times with 1 ml lysis buffer. The beads were incubated with protein-loading buffer at 95°C for 5 min and resolved by SDS-PAGE. Fibronectin levels at different time points were quantitated by densitometry. Fibronectin half-life determinations were assessed by linear regression analysis in each condition.

Anti-glomerular basement membrane model of renal fibrosis. The protocol for preparation/isolation of NC1 hexamer/dimer of type IV collagen [ 3(IV)NC1] from seminiferous tubule basement membrane was performed as described by Kahsai et al. ( 18 ). The anti-glomerular basement membrane model was performed as described by Abbate et al. ( 1 ). Briefly, 50 µg of the antigen 3(IV)NC1 in complete Freund's adjuvant were injected subcutaneously (footpad) on day 0. A second 100-µg injection was given on day 14 (antigen  incomplete Freund's adjuvant). The correct concentration of the antigen was determined by Bradford protein assay. The animals were divided into three groups. The control group did not receive injections of 3(IV)NC1/Freund's adjuvant. A second group received daily injections of 3(IV)NC1/Freund's adjuvant as described above and 100 µl of sterile saline intraperitoneally for the duration of the experiment. The third group received 3(IV)NC1/Freund's adjuvant as described above and relaxin (100 µg/kg) intraperitoneally daily for the duration of the experiment. Blood was collected weekly and analyzed for creatinine via the picric acid columetric procedure (Sigma). Urine was collected weekly in metabolic cages and analyzed for protein content via Bradford protein assay. Histological evaluation and immunohistochemistry of the kidney were performed as described by Zhu et al. ( 44 ). A renal pathologist evaluated periodic acid-Schiff-stained sections from each group in a blinded fashion and scored them for interstitial fibrosis and glomerular sclerosis.. Z$ m. l# l2 Q1 i
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Relaxin decreases fibronectin protein in an in vitro model of renal fibrosis. TGF- is one of the most fibrogenic cytokines known and has been shown to increase fibronectin protein levels in numerous cell types ( 6 ). Three cell types implicated in renal fibrosis are MMCs, MCTs, and TFBs ( 12 ). MMCs and MCTs contribute to glomerulosclerosis and interstitial fibrosis, respectively. TFBs are nonresident cells that infiltrate the kidney in injury states and secrete ECM and fibrotic cytokines, promoting the accumulation of ECM. To establish an in vitro model of renal fibrosis, we added increasing doses of TGF- (0-1 ng/ml) to MMCs, MCTs, and TFBs. We and others have found that TGF- results in a dose-dependent increase in fibronectin levels at 36 h in all three cell types (data not shown) ( 32 ). To evaluate the effect of relaxin in an in vitro model of renal fibrosis, we added increasing doses of relaxin (0-100 ng/ml) to each of the three cell types in the presence of TGF- 1 (1 ng/ml). Whole cell lysates were isolated, subjected to SDS-PAGE, and immunoblotted with a fibronectin antibody. The 240-kDa band detected was diminished in a dose-dependent manner on the addition of relaxin in each cell type ( Fig. 1, A - C ). Variation in protein loading and transfer was controlled by monitoring the expression of actin.
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Fig. 1. Relaxin inhibition of transforming growth factor (TGF)- -induced fibronectin in renal tubular epithelial cells, renal tubular fibroblasts, and mesangial cells. A : Western blot analysis of mouse mesangial cells incubated for 36 h in the presence of TGF- with increasing concentration of human relaxin. Whole cell lysates were subjected to SDS-PAGE, blotted, and probed with a polyclonal anti-fibronectin antibody. The blot was reprobed with an anti-actin antibody to compare loading and transfer. B : optical densitometry of fibronectin protein content normalized to actin of mouse tubular fibroblast cells prepared as above. C : optical densitometry of mouse cortical epithelial cell fibronectin protein content normalized to actin prepared as above./ I4 J+ H6 G0 s! L, z

Relaxin does not decrease fibronectin RNA. To determine whether relaxin's effect on fibronectin correlated with alterations in RNA levels, we examined the effect of relaxin on TGF- -induced fibronectin RNA in MMCs, MCTs, and TFBs. Duplicate samples of MMCs, MCTs, and TFBs described in Fig. 1 were processed for RNA via the TRIzol method. RNA was resolved on a formaldehyde gel and transferred to a nitrocellulose membrane. In all three cell types, although there is some scatter in the data, RNA levels were not decreased with increasing doses of relaxin ( Fig. 2, A - C ). Variation in RNA loading and transfer was controlled by monitoring the RNA expression of GAPDH., d6 d/ u4 x: d/ {: S5 y

Fig. 2. Relaxin does not inhibit fibronectin RNA in renal tubular epithelial cells, renal tubular fibroblasts, and mesangial cells. A : Northern blot analysis of mouse mesangial cells incubated for 36 h in the presence of TGF- with increasing concentration of human relaxin. Total RNA was size separated, blotted, and probed with 32 P-labeled fibronectin cDNA. The blot was reprobed with GAPDH to compare loading and transfer. B : optical densitometry of fibronectin RNA content normalized to GAPDH in mouse tubular fibroblast cells prepared as above. C : optical densitometry of mouse cortical epithelial cell fibronectin RNA content normalized to GAPDH in cells prepared as above.2 I" I0 Z" t: x2 C& u" }

Ubiquitin associates with fibronectin. The dissociation between RNA and protein levels prompted the evaluation of a protein-degradation pathway. Ohh et al. ( 27 ) have shown that fibronectin forms a complex with von Hippel-Lindau protein, elongin B, elongin C, and cullin 2, components of the VCB E3 ligase complex. E3 ligase complexes bind to proteins and facilitate the covalent addition of ubiquitin to the target protein, thus allowing protein degradation via the ubiquitin-proteasome pathway. To determine whether ubiquitin physically associates with fibronectin, we performed coimmunoprecipitation assays on MMC whole cell lysates. Whole cell lysates from 100-mm plates were immunoprecipitated with an anti-fibronectin antibody, resolved on a 4-15% SDS-PAGE gel, transferred to a nitrocellulose membrane, and immunoblotted with anti-ubiquitin and anti-fibronectin antibodies ( Fig. 3 A ). Figure 3 A shows data from whole cell lysates immunoprecipitated with the anti-fibronectin antibody and immunoblotted with an anti-ubiquitin antibody. Multiple bands in a ladder-like electrophoresis pattern extending to the top of the immunoblot were present, starting at 240 kDa ( lane 1 ). These bands were not present in identical cell lysates immunoprecipitated with a nonrelated isotype equivalent antibody control ( lane 2 ). To confirm that these bands corresponded to fibronectin, we stripped the membrane and subsequently immunoblotted it with the anti-fibronectin antibody, indicating the presence of fibronectin ( Fig. 3 B, lane 1 ). To verify that the ubiquitinated fibronectin is degraded by the proteasome, we performed coimmunoprecipitation for fibronectin as above in the presence of the proteasomeal inhibitor lactacystin. We did find that lactacystin resulted in the accumulation of ubiquitinated intermediates (data not shown). This further supports a role for the ubiquitin-proteasome pathway in fibronectin degradation. We also evaluated the association between fibronectin and ubiquitin by confocal microscopy ( Fig. 3 B ). Fibronectin and ubiquitin colocalized primarily in a perinuclear vesicular pattern. These data demonstrate that ubiquitin physically associates with fibronectin, targeting fibronectin for ubiquitin-mediated protein degradation.
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Fig. 3. Fibronectin physically associates with ubiquitin. A : mouse cortical epithelial cell whole cell lysates were immunoprecipitated (IP) with anti-fibronectin (FN; lane 1 ) and an isotype-identical antibody (C; lane 2 ), and immunocomplexes were analyzed by immunoblotting (IB) with anti-ubiquitin (Ub; a ) and anti-fibronectin ( b ). B : confocal microscopy of mouse mesangial cells stained for fibronectin ( left ), ubiquitin ( middle ), and colocalization ( right ).

Relaxin increases ubiquitin-dependent degradation of fibronectin. Next, we hypothesized that relaxin increases ubiquitin-mediated degradation of fibronectin. To evaluate this hypothesis, we preincubated MMCs for 36 h with or without relaxin (100 ng/ml). Both groups of cells were then placed in the presence of increasing concentrations of the proteasomal inhibitor MG-132 for 6 h ( Fig. 4, A and B ). Whole cell lysates were isolated, and 20 µg of total protein were subjected to SDS-PAGE and immunoblotted with an anti-fibronectin antibody. Fibronectin levels increased in a dose-dependent manner in the cells that were pretreated with relaxin and did not change in the group without relaxin. To confirm these findings, we evaluated MMCs in the presence of relaxin with increasing doses of the proteasomal inhibitor lactacystin ( Fig. 4 C ). To demonstrate that this response was not unique to mesangial cells, we also studied MCT cells and found identical results (data not shown).% p8 J; o' s/ `: P) k
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Fig. 4. MG-132 and lactacystin increase fibronectin in the presence of relaxin. A : Western blot analysis of mouse mesangial cells with increasing concentration of MG-132. Whole cell lysates were subjected to SDS-PAGE, blotted, and probed with a polyclonal anti-fibronectin antibody. The blot was reprobed with an anti-actin antibody to compare loading and transfer. B : Western blot analysis of mouse mesangial cells incubated for 36 h in the presence of human relaxin with increasing concentration of MG-132. C : Western blot analysis of mouse mesangial cells incubated for 36 h in the presence of human relaxin with increasing concentrations of lactacystin.

Relaxin and lactacystin alter fibronectin half-life in MMCs. To confirm that the change in fibronectin levels by relaxin and lactacystin was due to protein degradation, we performed pulse-chase analysis for fibronectin in MMCs ( Fig. 5 ). MMCs were cultured for 36 h alone, with relaxin, or with relaxin and lactactacystin, metabolically labeled, and processed to determine the half-life of fibronectin. The half-life of fibronectin in MMCs was 3.47 h compared with 2.09 and 4.09 h in the presence of relaxin (100 ng/ml) or relaxin (100 ng/ml) and lactacystin (10 µM), respectively. These data support the observation that relaxin and lactacystin alter fibronectin levels via protein degradation.

Fig. 5. Relaxin decreases fibronectin half-life in mouse mesangial cells. Pulse-chase analysis for fibronectin was performed in mouse mesangial cells in the presence and absence of relaxin (Rlxn; 100 ng/ml) and relaxin (100 ng/ml) plus lactacystin (Lac; 10 µM). Fibronectin half-life was determined by linear regression analysis. The half-life of fibronectin in mouse mesangial cells was 3.47 vs. 2.09 and 4.09 h in the presence of relaxin and relaxin lactacystin, respectively.
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Relaxin has renoprotective properties in an in vivo model of renal fibrosis. To determine whether relaxin's ability to decrease fibronectin levels in vitro would translate into renoprotective properties in an in vivo model of renal fibrosis, we evaluated the effects of relaxin in a murine model of Goodpasture's syndrome. In this model, Wistar-Kyoto rats immunized with the 3 chain of type IV collagen develop immune-mediated glomerulonephritis and lung hemorrhage. The immune-mediated injury to the kidney results in both glomerulosclerosis and interstitial fibrosis. Fibronectin has been shown to be elevated in the Goodpasture's model of renal fibrosis ( 34 ). The animals' renal function was monitored by serial evaluation of serum creatinine, proteinuria, and, at the conclusion of the study, renal histology. Animals treated with relaxin showed a significant decrease in proteinuria ( Fig. 6 A ) and serum creatinine ( Fig. 6 B ) on day 40 compared with collagen-treated controls. A renal pathologist performed the histological evaluation of the kidney in a blinded fashion. The degree of focal and segmental glomerulosclerosis and interstitial fibrosis was scored on a scale of 0-3. Animals treated with relaxin revealed a decrease in both glomerulosclerosis and interstitial fibrosis on periodic aicd-Schiff staining compared with nontreated animals ( Table 1 ).

Fig. 6. Effects of relaxin on renal functional parameters of rats with anti-glomerular basement membrane model of renal fibrosis. A : urinary protein excretion. Intraperitoneal administration of relaxin to Wistar-Kyoto rats with collagen-induced anti-glomerular basement membrane renal insufficiency decreased urinary protein excretion compared with that on animals that did not receive relaxin ( * P B : serum creatinine concentration. Serum creatinine levels in the relaxin-treated group were suppressed compared with those of animals that did not receive relaxin ( ** P
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Table 1. Relaxin decreases focal glomerulosclerosis and interstitial fibrosis in an anti-GBM model of renal fibrosis& I4 c  C2 F/ _

DISCUSSION& S" D( j6 g4 [" J
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The novel findings described in this study are 1 ) fibronectin, an ECM protein, is regulated by the ubiquitin protein-degradation pathway; 2 ) relaxin increases ubiquitin-dependent fibronectin degradation; and 3 ) relaxin is effective in an in vitro model of renal fibrosis and has renoprotective properties in an in vivo anti-glomerular basement membrane model of renal fibrosis.5 h4 P' M/ q! f; l, G# D: [

The majority of the proteins targeted for degradation by the ubiquitin-proteasome pathway are either short-lived intracellular proteins and misfolded conformers of secreted and membrane-associated proteins in the endoplasmic reticulum. Proteins regulated by this pathway play an essential role in a number of key biological processes, including cell cycle regulation, signal transduction, and transcription elongation ( 30 ). Additionally, the ubiquitin-proteasome pathway degrades mutant proteins. A prominent example is the CFTR ( 40 ). Ubiquitin-dependent degradation of mutant proteins serves as a quality control measure to eliminate abnormal proteins as they traverse the endoplasmic reticulum and Golgi apparatus. To date, evidence for the involvement of the ubiquitin-proteasome pathway with secretory proteins has been limited. Fisher et al. ( 13 ) demonstrated that the lipoprotein apolipoprotein B-100 is a target for the ubiquitin-proteasome pathway. Additionally, Meerovitch et al. ( 24 ) have demonstrated that preproparathyroid hormone-related protein is degraded by the ubiquitin-proteasome pathway. Finally, Yuehong et al. ( 43 ) have demonstrated that the proteasomal inhibitor MG-132 inhibits the secretion of fetal fibronectin in placental cytotrophoblasts. This suggests that the ubiquitin proteasome pathway is involved in fetal fibronectin secretion in cytotrophoblasts. In placental mesenchymal cells, MG-132 had no effect on fibronectin secretion. In this study, we demonstrate that fibronectin is targeted for degradation by the ubiquitin-proteasome pathway. Ours is the first description of an ECM protein that is ubiquitinated and degraded by the ubiquitin-proteasome pathway. The significance of this finding is underscored by the observation that relaxin increases ubiquitin-dependent fibronectin degradation in vitro. These findings, along with those in our in vivo model of renal fibrosis, which shows decreased ECM deposition with relaxin therapy, suggest that altering the ubiquitin-proteasome pathway may have therapeutic applications in fibrotic diseases.* v9 T; {4 J% |& s

Protein degradation by the ubiquitin-proteasome pathway involves a series of complex enzymatic pathways, resulting in highly selective protein degradation ( 15 ). First, E1, an ubiquitin-activating enzyme, forms an ATP-dependent ubiquitin-thiol ester bond. This is then transferred to E2, a ubiquitin carrier protein, forming an E2 ubiquitin thiol ester. E3, an ubiquitin protein ligase, then covalently links ubiquitin to the protein target. Ubiquitin-tagged proteins are then degraded by the ubiquitin-proteasome pathway. In coimmunoprecipitation experiments we demonstrate that ubiquitin physically binds to fibronectin. Ideally, we would have expected to see identical colocalization of fibronectin and ubiquitin rather that the ubiquitin ladder extending above fibronectin. One possible reason for this observation is that stripping the membrane removed some of the fibronectin bands extending to the top of the gel. A simpler explanation would be that the fibronectin antibody is less sensitive than the antibody to ubiquitin. Finally, there are numerous examples of other polyubiquitinated proteins that demonstrate a similar ladder-like electrophoresis pattern extending to the top of the immunoblot ( 7, 25, 39 ). The association between fibronectin and ubiquitin was confirmed in fixed cells by confocal microscopy. These findings further support a role for the ubiquitin-proteasome pathway in fibronectin homeostasis, another indication that the ubiquitin-proteasome pathway is involved in the regulation of nascent secreted proteins in general and of the ECM protein fibronectin specifically.
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Regulation of fibronectin by the ubiquitin-proteasome pathway was first suggested by Ohh et al. ( 27 ). They described a physical association between fibronectin and the von Hippel-Lindau protein, cullen 2, and elonginB/C (VCB complex). Homology between Skp1 and Cdc53 of the Skp1-cullin/Cdc53-F-box protein-ubiquitin ligase complex and elongin C and cullin 2 family of the VCB complex, respectively, prompted speculation that the VCB complex targets proteins to the ubiquitin-proteasome pathway ( 28 ). How the ubiquitin-proteasome pathway affects either nascent or mutant proteins is the subject of intense investigation. Proteins targeted for either the plasma membrane or secretion are deposited in the endoplasmic reticulum. These proteins interact with molecular chaperones to ensure appropriate assembly and conformation as they proceed to their intended destination ( 26 ). Abnormalities in protein structure and conformation are monitored in the endoplasmic reticulum-Golgi and are removed as an elaborate quality control measure ( 41 ). Abnormal proteins are retrotranslocated to the cytoplasm and subsequently degraded by the ubiquitin-proteasome pathway. The mechanism by which fibronectin, a wild-type secretory protein, is affected by the ubiquitin-proteasome pathway is unclear. We speculate that fibronectin is retrotranslocated from the endoplasmic reticulum-Golgi to the cytoplasm for ubiquitination and subsequent degradation by the proteasome. This process is presently under intense investigation.  Y& w/ K0 x" M* I( e1 R. w
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The primary therapeutic strategies in fibrotic diseases range from blocking the initiating events of fibrosis, such as the treatment of hypertension; antagonizing the effects of various cytokines, including TGF-, PDGF, and angiotensin; inhibiting ECM biosynthesis; or enhancing of matrix degradation by enhancing ECM-degrading proteases, such as matrix metalloproteases (MMPs). Previously, Unemori et al. ( 37 ) demonstrated that relaxin decreases fibronectin in lung fibroblasts in a dose-dependent fashion. In their study, relaxin resulted in a dose-dependent decrease in secreted fibronectin and a dose-dependent increase in MMP-1/2. Relaxin's effects on MMPs occurred primarily at concentrations between 0 and 1 ng/ml compared with 0-100 ng/ml for fibronectin and procollagens type I/III. This disparity suggests an alternate explanation for relaxin's effects on fibronectin, collagen I, and collagen III. We demonstrate that relaxin enhances ubiquitin-dependent degradation of fibronectin. In addition to the physical association between fibronectin and ubiquitin, the specific proteasome inhibitors MG-132 and lactacystin had little effect on fibronectin in the absence of relaxin. However, fibronectin levels increased in a dose-dependent manner in the presence of relaxin. These findings confirm that relaxin increases ubiquitin-dependent degradation of fibronectin. Taken together, these data demonstrate that the ubiquitin-proteasome pathway is a potential target for therapeutic intervention.

Progressive accumulation of ECM, which results in end-organ damage, is a common pathological response and is associated with significant morbidity and mortality. Prominent disease processes that culminate in fibrosis include cirrhosis of the liver, glomerulosclerosis or interstitial fibrosis of the kidney, intimal hyperplasia of the vasculature, pulmonary fibrosis, and fibrosis of the skin in scleroderma. Relaxin has been shown to have an ECM-degrading phenotype in human dermal and lung fibroblasts ( 36, 37 ). We now extend these observations by demonstrating the effects of relaxin in three diverse renal cell types, including TFBs, an epithelial cell (MCT), and vascular smooth muscle-like mesangial cell (MCT) line. These in vitro observations translated into renal protection in vivo in an anti-glomerular basement membrane model of renal fibrosis. The recent paper by Garber et al. ( 14 ) showed similar data in a drug-induced model of severe renal interstitial fibrosis. This observation is consistent with relaxin's affecting a system, the ubiquitin-proteasome pathway, that is present in virtually every cell type/organ of the body. This attribute may have significant therapeutic implications in the treatment of other systemic diseases characterized by diffuse fibrosis.( m9 r+ E* I: j- P* Z/ `% f) y: K

ACKNOWLEDGMENTS

We thank Ryan Zabriski and Roger Bick for help with confocal microscopy.
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02546 (to G. A. McDonald), DK-55001 and DK-51711 (to R. Kalluri), and in part by a grant from Gambro (to V. P. Sukhatme).8 O' \! d: ^* G% v! o
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Present address of R. Kalluri: Div. of Gastroenterology, Beth Israel Deaconess Medical Ctr. and Harvard Medical School, Boston, MA 02215.
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