Thrombospondin-1 Is a Major Activator of TGF-Β in Fibrotic Renal Disease In

Thrombospondin-1 Is a Major Activator of TGF-Β in Fibrotic Renal Disease In

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 65 (2004), pp. 459–468 Thrombospondin-1 is a major activator of TGF-b in fibrotic renal disease in the rat in vivo CHRISTOPH DANIEL,JULIA WIEDE,HENRY C. KRUTZSCH,SOLANGE M.F. RIBEIRO, DAVID D. ROBERTS,JOANNE E. MURPHY-ULLRICH, and CHRISTIAN HUGO From the Division of Nephrology, Universitat¨ Erlangen-Nurnberg,¨ Erlangen, Germany; Biochemical Pathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States and Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States Thrombospondin-1 is a major activator of TGF-b in fibrotic mesangial cell (MC) proliferation and extracellular ma- renal disease in the rat in vivo. trix expansion [2]. In up to 50% of the patients with Background. Transforminggrowth factor-b (TGF-b), a profi- mesangial proliferative glomerulonephritis, the disease brotic cytokine involved in many scarring processes, has to be activated extracellularly before it can bind to its receptors. process eventually progresses to end-stage renal disease Thrombospondin 1 (TSP1), a multifunctional matricellular gly- because specific treatment is still lacking [3]. Typical fea- coprotein, has been identified as an activator of TGF-b in in tures of human mesangial proliferative glomerulonephri- vitro systems and during mouse postnatal development in vivo. tis are mimicked by an experimental model in the rat, TSP1 is expressed de novo in many inflammatory disease pro- induced by an antibody against the Thy1-antigen on MC cesses, including glomerular disease. Methods. In this study we investigated whether peptides [2]. specifically interfering with the activation process of TGF-b by The role of transforming growth factor-b (TGF-b)as TSP1 may be able to block activation of TGF-b in an in vivo a major profibrotic cytokine in the anti-Thy1 model has model of mesangial proliferative glomerulonephritis. been well established [4]. It has been demonstrated that Results. Continuous intravenous infusion of blocking peptide TGF-b1 mRNA and protein are increased in the anti- by minipumps significantly reduced expression of active TGF-b in glomeruli on day 7 of disease as indicated by immunohisto- Thy1 model [5], and that blocking TGF-b1 by injections chemistry, bioassay, and activation of the TGF-b signal trans- with a polyclonal anti-TGF-b1 antibody or the proteo- duction pathway, while total TGF-b expression was unchanged. glycan decorin, a TGF-b1, -2, and -3 binding protein [7], Inhibition of glomerular TGF-b activation was accompanied by markedly reduced extracellular matrix accumulation [6]. a decrease of glomerular extracellular matrix accumulation and The results of these studies were confirmed by studies us- proteinuria, but was without effect on mesangial cell prolifera- tion or influx of monocytes/macrophages. ing equivalent gene transfer techniques against TGF-b in Conclusion. TSP1 is a major endogenous activator of TGF-b the anti-Thy1 model [8, 9]. In contrast, mice transgenic for in experimental inflammatory glomerular disease. Drugs inter- an active form of TGF-b1 exhibit elevated plasma levels fering with the activation of TGF-b by locally produced TSP1 of TGF-b1 and develop progressive renal disease charac- may be considered as a future specific treatment of scarring terized by MC matrix accumulation, interstitial fibrosis, kidney disease. and proteinuria [10]. Transfer of the TGF-b1 gene into glomeruli of normal rats caused an increase in glomerular Extracellular matrix accumulation is one of the hall- TGF-b1 protein that was linked to extracellular matrix marks of inflammatory diseases in many organ systems, formation [11]. The potential importance of TGF-b in including the kidney, and is the major cause of end- mediating fibrosis also in human kidney disease has been stage renal disease in humans. Mesangial proliferative supported by the widespread link of TGF-b up-regulation glomerulonephritis, the most common type of glomeru- and extracellular matrix excess in many different types lonephritis in the Western world [1], is characterized by of human kidney disease [4]. While these studies suggest great benefit from suppression of TGF-b function in fi- Key words: TGF-b activation, thrombospondin-1, glomerulonephritis, extracellular matrix. brotic kidney disease, it has to be considered that TGF-b is a multifunctional cytokine that exhibits other essential Received for publication May 10, 2003 functions in mammals. Mice lacking either the TGF-b1, and in revised form July 15, 2003 Accepted for publication September 9, 2003 -2, or -3 gene do not survive beyond a few weeks after birth [12–14]. Therefore, accurate regulation of TGF-b C 2004 by the International Society of Nephrology function seems to be critical for the health of mammals 459 460 Daniel et al: Thrombospondin-1 activates TGF-b in renal fibrosis and any anti-TGFb1 therapeutic approach should try to TSP 1 peptide infusion by minipump target the local overproduction (-function) of TGF-b as Group 1: SLLK (control) specifically as possible. Group 2: LSKL (LAP-peptid) One possiblilty to approach this goal could be to con- Group 3: AAWSHW (TSP-1-peptid) trol the activation process of locally produced TGF-b. 0 2 5 7 days TGF-b is secreted by most cell types as a latent, inactive procytokine complex [15]. The mature TGF-b protein has to be extracellularly released from this procytokine com- plex to be able to interact with its receptors. While various players/mechanisms such as pH changes, gamma irradia- Anti- 1. Survival 2. Survival Sacrifical tion, reactive oxygen species, plasmin, calpain, cathepsin, Thy1 biopsy biopsy biopsy or TSP1 have been identified as activating TGF-b under antibody in vitro conditions, it is still unknown how TGF-b is acti- Fig. 1. Shown is the schematic outline of the experimental design. vated in an inflammatory process in vivo [15]. Recent data have suggested the homeotrimeric extra- cellular matrix protein TSP1 as an activator of TGF-b1 b in vitro in different cell systems including MC [16–18], as press activation and thereby function of TGF- in exper- well as in cell-free systems. It has been demonstrated that imental mesangial proliferative nephritis in the rat. TSP1 forms a trimolecular complex with the TGF-b pro- cytokine complex by interacting with the mature TGF-b METHODS protein as well as the so-called latency-associated pep- Experimental design tide (LAP). Hereby, the hexapeptide AAWSHW from the type I repeat of the TSP1 molecule is required for A scheme of the experimental protocol is shown in TSP1 binding to the mature TGF-b protein, allowing in- Figure 1. An identical pilot study in four to six rats per teraction of the KRFK amino acid sequence of the TSP1 group using half of the peptide dose was done without molecule with the N-terminal LSKL sequence of the LAP glomerular preparations. Experimental mesangial prolif- [19, 20]. This complex interaction leads to a confirma- erative glomerulonephritis was induced using the mon- tional change, probably within the LAP, that allows the oclonal mouse anti-Thy1 antibody OX-7. Peptides were mature TGF-b protein to bind to its receptors. It has continuously infused intravenously via jugular vein using been shown that both the hexapeptide AAWSHW and osmotic minipumps starting 16 hours after disease induc- the LSKL peptide are able to block activation of TGF- tion. The effects of the hexapeptide AAWSHW (which b b by TSP1. In addition, comparing TSP1 null mice with interfere with the TSP1-mature TGF- interaction) or TGF-b1 null mice, Crawford et al [21] identified TSP1 as the LSKL peptide (that blocks the TSP1-LAP interac- a major activator of TGF-b1 in pancreas and lung home- tion) were compared with a control peptide (SLLK) in ostasis in mice pups in vivo. Organ pathology of TGF-b1 the anti-Thy1 model. Tissues from different time points null pups and TSP1 null pups were strikingly similar and (days 3, 5, 7) of this experiment were analyzed in regard could be induced in wild-type pups by intraperitoneal to mesangiolysis, macrophage influx, MC proliferation, treatment with the LSKL peptide that specifically blocks microaneurysm, and matrix formation, GEN prolifera- b b activation of TGF-b1 by TSP1. Loss of TSP1 expression tion, TSP1, active TGF- , total TGF- 1 and -2, as well b b in TSP1-null mice spontaneously produced inflammatory as TGF- RI and RII, and phosphorylation of the TGF- lung disease [22], and histologic changes in TSP1 null mice signaling molecule Smad2/3. Functional parameters such reverted toward wild-type by treatment with the TGF-b as blood pressure, proteinuria, and creatinine clearance activating peptide KRFK. were also determined. On day 7, glomerular secretion of b Interestingly, TSP1 expression in vitro is regulated by active TGF- was determined in individual animals using various cytokines such as platelet-derived growth factor the NRK bioassay. (PDGF), fibroblast growth factor-2 (FGF-2), or TGFb, and is frequently expressed de novo at sites of inflamma- tion and wound healing [23]. The involvement of TSP1 Animal model in the anti-Thy1 model has been currently demonstrated Experimental mesangial proliferative glomeru- [24]. lonephritis (anti-Thy1 model) was induced in Sprague Therefore, we hypothesized that TSP1 is an endoge- Dawley rats (180–200 g; Charles River, Sulzfeld, nous activator of TGF-b in inflammatory kidney disease Germany) by a single injection of 1 mg/kg of the and investigated whether systemic treatment with either mouse monoclonal anti-Thy1 antibody OX-7 (European one of two blocking peptides that interfere with TSP1- Collection of Animal Cell Culture, Salisbury, UK). In TGF-b1 binding (LSKL or AAWSHW) is able to sup- this animal model, complete anti-Thy1 antibody binding Daniel et al: Thrombospondin-1 activates TGF-b in renal fibrosis 461 occurs within an hour [26].

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