Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) has widespread effects on extracellular matrix production by many cultured cell lines and appears to play a role in the pathological accumulation of extracellular matrix that accompanies inflammatory and fibrotic diseases such as glomerulonephritis. Earlier experiments have shown that mesangial cells respond to TGF-beta 1 with a marked increase in the production of two chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, but their production of other matrix components elevated in glomerulonephritis is not substantially affected by TGF-beta 1. Since the glomerular epithelial cells are also thought to contribute to matrix production in the glomerulus, we examined the ability of these cells to produce some of the nonproteoglycan matrix components in response to TGF-beta 1. Exposure of glomerular epithelial cells to TGF-beta 1 increased the production of fibronectin and type IV collagen, in addition to biglycan. Enhancement of the cell layer accumulation of laminin was also observed. These results show that TGF-beta 1 has a differential effect on extracellular matrix production by epithelial and mesangial cells from glomeruli. TGF-beta 1 released in the glomerulus secondary to injury could thus affect both cell types and lead to increased intraglomerular production of proteoglycans, whereas the increased fibronectin, type IV collagen, and laminin may primarily originate from the epithelial cells.     

High glucose decreases matrix metalloproteinase-2 activity in rat mesangial cells via transforming growth factor-beta1.

Diabetic nephropathy is characterized by accumulation of mesangial matrix. Glucose-induced inhibition of matrix-degrading enzymes such as collagenases is believed to contribute to matrix accumulation. We have previously demonstrated that 72 kDa type IV collagenase activity is decreased in the rat mesangial cells cultured in high glucose media [Diabetes 1995;44:929-935]. The present studies were designed to investigate if the cytokine transforming growth factor-beta1 (TGF-beta1) mediates this effect of glucose. Type IV collagenases degrade type IV collagen as well as gelatin (denatured collagen) and are thus also called gelatinases. They belong to the family of matrix metalloproteinases (MMPs); MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs). The activity of 72 kDa type IV collagenase, also known as matrix metalloproteinase-2 (MMP-2), was assessed using three methods: (1) fluoresceinated gelatin degradation assay to detect free enzyme activity (activity which is present in excess of TIMP-inhibited activity); (2) zymography to measure total (free + TIMP-bound) enzyme activity; (3) ELISA using specific antibodies to measure MMP-2 levels. TGF-beta1 and TIMP-2 levels were also determined by ELISA. Incubation of primary cultures of rat mesangial cells for 5 days in 30 vs. 5 mM glucose resulted in a 3-fold increase in production of total TGF-beta1, a significant decrease in MMP-2 activity and immunoreactive MMP-2 levels, and an increase in TIMP-2 levels. Addition of exogenous TGF-beta1 to mesangial cells incubated in 5 mM glucose replicated the high glucose effect by producing a significant decrease in MMP-2 levels with a concurrent increase in TIMP-2 levels. Furthermore, glucose-induced inhibition of MMP-2 activity was completely blocked by neutralization of TGF-beta1 with anti-TGF-beta1 antibody. We conclude that the decrease in MMP-2 activity induced by glucose loading is mediated via TGF-beta1.     

Lovastatin inhibits transforming growth factor-beta1 expression in diabetic rat glomeruli and cultured rat mesangial cells

Diabetic nephropathy is a leading cause of end-stage renal disease and is characterized by excessive deposition of extracellular matrix (ECM) proteins in the glomeruli. Transforming growth factor-beta (TGF-beta) is the major mediator of excessive accumulation of ECM proteins in diabetic nephropathy through upregulation of genes encoding ECM proteins as well as downregulation of genes for ECM-degrading enzymes. It has been shown that lovastatin, an inhibitor of 3-hydroxy3-methylglutaryl CoA reductase, delays the onset and progression of different models of experimental nephropathy. To evaluate the effect of lovastatin on the development and progression of diabetic nephropathy, streptozotocin-induced diabetic rats were studied for 12 mo. In untreated diabetic rats, there were significant increases in blood glucose, urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression in the glomeruli compared with normal control rats treated with citrate buffer only. Treatment with lovastatin in diabetic rats significantly suppressed the increase in urine albumin excretion, kidney weight, glomerular volume, and TGF-beta1 mRNA expression despite high blood glucose levels. To elucidate the mechanisms of the renal effects of lovastatin, rat mesangial cells were cultured under control (5.5 mM) or high (30 mM) glucose with lovastatin alone, mevalonate alone, or with both. Under high glucose, TGF-beta1 and fibronectin mRNA and proteins were upregulated. These high glucose-induced changes were suppressed by lovastatin (10 micro/M) and nearly completely restored by mevalonate (100 microM). These results suggest that lovastatin has a direct cellular effect independent of a cholesterol-lowering effect and delays the onset and progression of diabetic nephropathy, at least in part, through suppression of glomerular expression of TGF-beta1.     

Autocrine secretion of transforming growth factor-beta in cultured rat mesangial cells.

Transforming growth factor-beta (TGF-beta) recently has been shown to modulate mesangial cell growth and to stimulate mesangial matrix synthesis by mesangial cells. Here we examined whether mesangial cells expressed TGF-beta mRNA and secreted mature TGF-beta, and we investigated the role of TGF-beta in mesangial cell growth. Cultured rat mesangial cells expressed 2.5 kb TGF-beta mRNA, and removal of fetal calf serum (FCS) for two days decreased the TGF-beta mRNA level, which was then stimulated by re-addition of 17% FCS reaching the maximum at nine hours. 12-O-tetradecanoyl phorbol-13-acetate (TPA), one of the phorbol esters, markedly increased the mRNA level and reached the maximum at six or nine hours. Immunoblot analysis of the conditioned media using specific anti-TGF-beta 1 antibodies revealed single 12.5 kDa proteins, the size compatible with mature TGF-beta subunits. By means of bioassay using CCL-64 cell line, TGF-beta production rate by mesangial cells was estimated to be 22.1 +/- 6.5 (mean +/- SD) ng/10(6) cells/24 hours, 96% of which was in latent forms. Exogenously added TGF-beta inhibited mesangial cell growth at 10 pM or higher. Moreover, addition of anti-TGF-beta neutralizing antibodies augmented mesangial cell growth, indicating that the secreted TGF-beta actually exerted a growth-inhibitory action. In summary, mesangial cells produce and secrete substantial amounts of TGF-beta but mostly in latent forms, and the secreted TGF-beta may regulate mesangial cell growth and differentiation. We conclude that TGF-beta may function as an autocrine factor in mesangial cells.     

Expression of transforming growth factor-beta 1 and beta 2 in rat glomeruli

The transforming growth factors-beta are potent modulators of cell growth and extracellular matrix metabolism in most types of cultured cells. The distribution and functions of TGF-beta in vivo are less well known. We utilized several different techniques including northern blots, a CCl-64 cell growth inhibition assay, and sandwich enzyme-linked immunosorbent assays (SELISA) to examine the expression of TGF-beta 1 and TGF-beta 2 in rat glomeruli. High levels of TGF-beta 1 mRNA and protein were found in glomeruli (56 +/- 22 ng TGF-beta 1/g tissue). These levels were several-fold higher than those present in whole kidney (10 +/- 5 ng/g). TGF-beta 2 mRNA was present in glomeruli but was not detected in whole kidney. TGF-beta 2 concentrations by SELISA were 19 +/- 8 ng TGF-beta 2/g in glomeruli and less than 5 ng/g in whole kidney. Since TGF-beta has such marked effects on cell growth, we also examined whether alterations in TGF-beta expression were associated with the renal hypertrophy which follows unilateral nephrectomy. Expression of TGF-beta 1 mRNA decreased in glomeruli following nephrectomy. However, this was not associated with a significant fall in glomerular TGF-beta 1 protein concentration. Whole kidney levels of TGF-beta 1 and its mRNA were unchanged following nephrectomy. Similar results were obtained for TGF-beta 2. Our data document the presence of high concentrations of TGF-beta 1 and beta 2 and their corresponding mRNAs in normal rat glomeruli. These results suggest that TGF-beta may play important regulatory roles in the normal glomerulus.     

Thrombin stimulates production of transforming growth factor-beta by cultured human mesangial cells

Fibrin formation within the glomeruli occurs in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Transforming growth factor-beta (TGF- beta) has been shown to participate in the glomerular accumulation of extracellular matrix in glomerulonephritis. We investigated whether thrombin, an important coagulation factor, could modulate the production of TGF-beta by cultured human mesangial cells (HMC). TGF- beta levels in the culture supernatants were measured by ELISA using a specific antibody. The TGF-beta concentration was significantly increased by incubation of HMC with thrombin in a time-dependent manner. The stimulating effect of thrombin on TGF-beta was inhibited by addition of hirudin (a natural thrombin inhibitor) and argatroban (a synthetic thrombin inhibitor). In addition DFP-inactivated thrombin, which has no enzymatic activity, did not stimulate TGF-beta production. A protein kinase C inhibitor (H7) and a tyrosine kinase inhibitor (herbimycin A) also inhibited thrombin induced TGF-beta production. These findings suggested that thrombin may modulate the synthesis of TGF-beta via protein kinase C- and tyrosine kinase-dependent mechanisms in cultured HMC. Thus thrombin may participate in the accumulation of extracellular matrix in glomeruli through the augmentation of TGF-beta production.      

Mechanical stretch-induced fibronectin and transforming growth factor-beta1 production in human mesangial cells is p38 mitogen-activated protein kinase-dependent

Hemodynamic abnormalities are important in the pathogenesis of the excess mesangial matrix deposition of diabetic and other glomerulopathies. p38-Mitogen-activated protein (MAP) kinase, an important intracellular signaling molecule, is activated in the glomeruli of diabetic rats. We studied, in human mesangial cells, the effect of stretch on p38 MAP kinase activation and the role of p38 MAP kinase in stretch-induced fibronectin and transforming growth factor-beta1 (TGF-beta1) accumulation. p38 MAP kinase was activated by stretch in a rapid (11-fold increase at 30 min, P < 0.001) and sustained manner (3-fold increase at 33 h, P < 0.001); this activation was mediated by protein kinase C (PKC). Stretch-induced fibronectin and TGF-beta1 protein levels were completely abolished (100% inhibition, P < 0.001; and 92% inhibition, P < 0.01, respectively) by SB203580, a specific p38 MAP kinase inhibitor. At 33 h, TGF-beta1 blockade did not affect stretch-induced fibronectin production, but partially prevented stretch-induced p38 MAP kinase activation (59% inhibition, P < 0.05). TGF-beta1 induced fibronectin accumulation after 72 h of exposure via a p38 MAP kinase-dependent mechanism (30% increase over control, P < 0.01). In human mesangial cells, stretch activates, via a PKC-dependent mechanism, p38 MAP kinase, which independently induces TGF-beta1 and fibronectin. In turn, TGF-beta1 contributes to maintaining late p38 MAP kinase activation, which perpetuates fibronectin accumulation.     

Transforming growth factor-beta is a potent inhibitor of extracellular matrix degradation by cultured human mesangial cells

Accumulation of the glomerular extracellular matrix (ECM) is a pivotal event in the progression from acute glomerular injury to end-stage renal disease. Although enhanced ECM synthesis has been demonstrated to contribute to ECM accumulation, the role of decreased ECM degradation is largely unknown. It was previously shown that glomerular ECM degradation is mediated by a plasminogen activator (PA)/plasmin/matrix metalloproteinase 2 (MMP-2) cascade. However, little information is available regarding the factors that regulate the activity of this degradative cascade in normal or pathologic states. Transforming growth factor-beta1 (TGF-beta1) is shown here to be a potent inhibitor of ECM degradation by cultured human mesangial cells. Using human mesangial cells grown on thin films of 125I-labeled Matrigel, dose-dependent inhibition of ECM degradation in the presence of TGF-beta1 was observed, reaching >90% inhibition with 0.4 ng/ml TGF-beta1. Addition of anti-TGF-beta antibodies (4 microg/ml) in the absence of exogenous TGF-beta increased ECM degradation (1.8+/-0.2-fold versus controls, P<0.05). In contrast, platelet-derived growth factor, at concentrations up to 10 ng/ml, had no effect on ECM degradation. TGF-beta completely blocked the conversion of plasminogen to plasmin and markedly reduced the conversion of latent MMP-2 to active MMP-2. TGF-beta did not significantly alter the levels of tissue PA, total MMP-2, or tissue inhibitor of metalloproteinase-1, but did increase the levels of PA inhibitor- (1.8-fold, P<0.05), the major physiologic inhibitor of PA. These data document that TGF-beta is a potent inhibitor of ECM degradation by cultured human mesangial cells, and they suggest that decreased mesangial matrix degradation, caused by TGF-beta-mediated decreases in the activity of the PA/plasmin/MMP-2 cascade, may contribute to the glomerular matrix accumulation that occurs in progressive renal disease.     

Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms

Recent evidence suggesting a strong interplay between components of the renin-angiotensin system and key mediators of fibrosis led us to hypothesize that renin, independent of its enzymatic action to enhance angiotensin (Ang) II synthesis, directly increases production of the fibrogenic cytokine transforming growth factor (TGF)-beta. Human or rat mesangial cells (MCs) were treated with human recombinant renin (HrRenin) or rat recombinant renin (RrRenin) and the effects on TGF-beta1, plasminogen activator inhibitor-type 1 (PAI-1), fibronectin (FN) and collagen 1 mRNA and protein were investigated. Blockade of the rat MC renin receptor was achieved using siRNA. HrRenin or RrRenin, at doses shown to be physiologically relevant, induced marked dose- and time-dependent increases in TGF-beta1. These effects were not altered by adding an inhibitor of renin's enzymatic action (RO 42-5892), the Ang II receptor antagonist losartan or the Ang-converting enzyme inhibitor enalapril. RrRenin also induced PAI-1, FN and collagen 1 mRNA and PAI-1 and FN protein in a dose-dependent manner. Neutralizing antibodies to TGF-beta partially blocked these effects. Supernatant and cell lysate Ang I and Ang II levels were extremely low. MC angiotensinogen mRNA was undetectable both with and without added renin. Targeting of the rat renin receptor mRNA with siRNA blocked induction of TGF-beta1. We conclude that renin upregulates MC TGF-beta1 through a receptor-mediated mechanism, independent of Ang II generation or action. Renin-induced increases in TGF-beta1 in turn stimulate increases in PAI-1, FN and collagen I. Thus, renin may contribute to renal fibrotic disease, particularly when therapeutic Ang II blockade elevates plasma renin.     

The involvement of beta1 integrin in the modulation by collagen of chondrocyte-response to transforming growth factor-beta1.

The physiologic response of chondrocytes to maintenance of the matrix and response to injury likely involves signaling from multiple sources including soluble cytokines, mechanical stimulation, and signaling from the extracellular matrix. The signaling from the extracellular matrix may serve to effect cell differentiation and to modulate the response to cytokines. We have previously reported that type II collagen modulates the response of bovine articular chondrocytes to TGF-beta1. The molecular nature of the signaling mechanism has not been elucidated but presumably involves a similar mechanism by which the cell attaches to the surrounding matrix. An alginate bead culture system is utilized to which exogenous type II collagen is added. The inclusion of type II collagen results in an alteration of integrin expression with a down regulation of alpha2. The response of the chondrocyte to TGF-beta1 can be modulated by the inclusion of exogenous type II collagen. The modulation of DNA and proteoglycan synthesis was blocked by the treatment of anti-beta1 integrin antibody (4B4) or by cyclic RGD containing peptides. These events occur at concentrations that block cell adhesion to type II collagen. Linear RGD containing peptides and anti-anchorin antibodies had no effect on the modulation by type II collagen. These results suggest that type II collagen binding by chondrocytes at least in part occurs through the beta1 integrin. This binding results in modulation of the cell response to TGF-beta1. This modulation may serve to provide physiologic specificity to the cytokine-signaling cascade. An understanding of the regulatory milieu of the chondrocyte may permit the stimulation of an intrinsic repair of articular cartilage in the future. A near term application of this understanding can be made to tissue engineering attempts at articular cartilage repair.     

Angiotensin II-mediated collagen production is inhibited by a transforming growth factor-beta receptor kinase inhibitor

Introduction. The purpose of this experiment is to determine if the pro-fibrotic effect of angiotensin II (AII) is mediated through a transforming growth factor-beta (TGF-beta) receptor kinase. Methods. Neonatal rat heart fibroblast were cultured and incubated with SB 431542 (an inhibitor of TGF-beta receptor kinase activity) or vehicle. Recombinant human TGF-beta or AII was then added to the cardiac fibroblasts, and cells were incubated for 48 h under the experimental conditions. The cells were then fixed and stained with Sirius red collagen stain. Sirius red was extracted and quantified by absorbance at 540 nm. Five replicates were performed in each group and compared using the unpaired Student’s t-test. Results. Addition of SB 431542 to cells treated with TGF-beta showed an absorbance of 0.14 ± 0.01 (mean ± SE) compared to treatment with TGF-beta alone which showed an absorbance of 0.19 ± 0.01 representing a 26% reduction in total collagen (P = 0.03). Cells treated with SB 431542 and AII showed an absorbance of 0.105 ± 0.004, while cells treated with AII alone showed an absorbance of 0.147 ± 0.008 representing a 28% reduction in total collagen (P < 0.01). Conclusion. Angiotensin II induced collagen production is mediated through a TGF-beta receptor kinase in neonatal rat heart fibroblasts. The TGF-beta receptor kinase-mediated effect of AII may be responsible for the increased fibrosis seen in the myocardium of patients with congestive heart failure and may provide a target for therapeutic intervention.     

转化生长因子β1反义RNA对肾小球系膜细胞基质合成及分泌的影响

目的：观察转化生长因子beta1(TGF-β1）反义RNA对系膜细胞基质合成的影响。方法：构建含TGF－β1反义RNA的重组腺病毒，将重组腺病毒转染系膜细胞，用Northern blot检测转染系膜细胞TGF－β1及ColIV mRNA的含量，用免疫组化半定量分析转染的系膜细胞中TGF－β1，纤连蛋白（FN）及胶原（Col)IV蛋白水平，并与无转染的系膜细胞对照组比较其表达的变化。结果：构建了含TGF－β1反义RNA的重组腺病，重组反义TGF－β1腺病毒转染系膜细胞后，与对照组相比，在第24hTGF-β1及Col IV的mRNA无明显抑制，在48hTGF－β1及Col IV mRNA抑制率分别为22．5％，18．2％，72h TGF-β1及Col IV mRNA抑制率分别为29．5％，27．3％，免疫组织化学半定时结果显示；与对照组相比，在48h始转系膜细胞TGF－β1，FN及ColIV蛋白含量开始下降，48h TGF-β1，FN及Col IV蛋白抑制率分别为16．5％，18．2％及14．6％，72h TGF－β1，FN及ColⅣ蛋白抑制率分别为23．5％，27．3％，26．8％。结论：重组反义TGF－β1可抑制系膜细胞合成细胞外基质，在肾小球肾炎及肾小球硬化的研究及治疗中有潜在的应用价值。     

Mechanical stretching force promotes collagen synthesis by cultured cells from human ligamentum flavum via transforming growth factor-beta1.

Although mechanical stress as a result of spinal instability is known to cause hypertrophy of the ligamentum flavum resulting in degenerative spinal canal stenosis, the mechanism of the ligament hypertrophy is not well understood. In the present study, we investigated the effect of mechanical stretching force on collagen synthesis and transforming growth factor-beta1 (TGF-beta1) production using ligament cells isolated from human ligamentum flavum in vitro. Ligamentum flavum cells (LFCs) were isolated from human ligamentum flavum obtained from patients who underwent lumbar spine surgery. The LFCs were subjected to a mechanical stretching force using a commercially available stretching device that physically deformed the cells. Collagen synthesis and TGF-beta1 production levels in the LFCs were then examined. Notable increases were observed in the gene expressions of collagen types I, III, and V in LFCs subjected to mechanical stretching force. The increase in collagen gene expression of LFCs was inhibited in the presence of anti-TGF-beta1 antibodies. Production of TGF-beta1 by the LFCs also increased significantly by the mechanical stretching force. Exogenous application of TGF-beta1 was confirmed to increase collagen synthesis of the LFCs. This data indicated that mechanical stretching force can promote TGF-beta1 production by LFCs, resulting in hypertrophy of the ligament.