u-0126 and Fibrosis

To study the possible mechanism of ghrelin in heart failure and how it works.. In vitro results demonstrated that ghrelin alleviates cardiac function and reduces myocardial fibrosis in rats with heart failure. Moreover, ghrelin intervention increased PTEN expression level and reduced ERK, c-jun, and c-Fos expression level; in vivo experiments demonstrated that ghrelin intervention reduces mast memory expression and increases cardiomyocyte surface area, PTEN expression level, ERK, c-jun, c-Fos expression level, and cell surface area, while ERK blockade suppresses mast gene expression and reduces cell surface area.. In vitro experimental results prove that we have successfully constructed a rat model related to heart failure, and ghrelin can alleviate the heart function of heart failure rats and reduce myocardial fibrosis. In addition, ghrelin is closely related to the decrease of the expression levels of ERK, c-jun, and c-Fos, but it can also increase the expression of PTEN in the rat model; in vivo experiments proved that we successfully constructed an in vitro cardiac hypertrophy model, and the intervention of ghrelin would reduce the expression of hypertrophic memory and increase the surface area of cardiomyocytes, increase the expression level of PTEN, and reduce the expression levels of ERK, c-jun, and c-Fos, while the blockade of PTEN will increase the expression of hypertrophy genes and increase the cell surface area, while the blockade of ERK will increase the expression of hypertrophic genes, which in turn will make the cell surface area reducing.. Ghrelin inhibits the phosphorylation and nuclear entry of ERK by activating PTEN, thereby controlling the transcription of hypertrophic genes, improving myocardial hypertrophy, and enhancing cardiac function.

Topics: Animals; Butadienes; Cell Enlargement; Cell Line; Computational Biology; Disease Models, Animal; Female; Fibrosis; Gene Expression; Ghrelin; Heart Failure; MAP Kinase Signaling System; Mast Cells; Myocytes, Cardiac; Nitriles; Phenanthrenes; PTEN Phosphohydrolase; Rats; Rats, Sprague-Dawley

Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation.. Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI.. Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft.. Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.

Topics: Adenosine; Allopurinol; Animals; Apoptosis; Butadienes; Cell Hypoxia; Cell Line; Cold Ischemia; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Glutathione; Heart Transplantation; Insulin; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitriles; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Phosphorylation; Protein Kinase Inhibitors; Raffinose; Rats; Signal Transduction; Ventricular Function, Left

Interleukin (IL)-17A is upregulated in several renal diseases and plays a crucial role in renal inflammation. However, it remains unclear how IL-17A contributes to renal fibrosis. Our result demonstrated that IL-17A expression was upregulated in the obstructed kidney of unilateral ureter obstruction (UUO) mice when compared to the contralateral control kidney. Inhibition of IL-17A functions by the intravenous administration of an anti-IL-17A receptor antibody (100 μg) 2 h prior to UUO and on post-UUO day 1 and 3 significantly reduced fibronectin expression in the UUO kidney. The addition of IL-17A (25-100 μg) to human renal proximal tubular cells or renal fibroblasts caused an increase in fibronectin production and extracellular signal-regulated kinase (ERK)1/2 activation, which were reduced upon pretreatment with the ERK inhibitor U0126. The level of phosphorylated (p)-ERK1/2 was increased in the UUO kidney, but reduced by the administration of the anti-IL-17A receptor antibody, verifying the importance of the ERK pathway in vivo. TGF-β1 mRNA expression and protein were increased in the UUO kidney and in IL-17A-stimulated cultured cells. The administration of an anti-TGF-β1 neutralizing antibody or TGF-β1 receptor I inhibitor (SB431542) to cells abrogated the IL-17A-mediated increase of fibronectin production. IL-17A induced an increase in p-Smad2 and p-Smad3 expression at 7.5 min and 24 h and pretreatment with the anti-TGF-β1 neutralizing antibody, and SB431542 reduced the IL-17A-stimulated increase of p-Smad2. Knockdown of Smad2 or Smad3 expression inhibited the IL-17A-enhanced production of fibronectin. These results suggest an essential role for the TGF-β/Smad pathway in the IL-17A-mediated increase of fibronectin production. This study demonstrates that IL-17A contributes to the production of extracellular matrix, and targeting its associated signaling pathways could provide a therapeutic target for preventing renal fibrosis.

Topics: Animals; Benzamides; Butadienes; Cell Line; Cytokines; Dioxoles; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Fibronectins; Fibrosis; Humans; Interleukin-17; Kidney; Mice; Mice, Inbred BALB C; Models, Animal; Nitriles; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Ureteral Obstruction

Endoplasmic reticulum stress (ERS) is involved in a variety of diseases. Recently, it was found that ERS induces not only apoptosis but also autophagy. Previous studies showed that inhibition of autophagy alleviates cell injury. The purpose of our study was to investigate the involvement of the R-like ER kinase (PERK) in ERS-induced autophagy in H9c2 cardiomyoblasts. To address this aim, therefore, H9c2 cells were treated with PERK agonist and inhibitor after establishment of rapamycin-induced ERS models in H9c2 cardiomyoblasts. Transmission electron microscopy and immunofluorescence staining were used to detect degrees of ERS-induced autophagy, apoptosis and myocardial fibrosis. Western blotting was employed to detect the levels of total and phosphorylated PERK, light chain 3 (LC3), P62, Caspase3, Bcl2 and Bax. Immunofluorescence staining was used to assess α-SMA density. TGF-β induced H9c2 cardiomyoblasts time-dependently upregulated col I, col III, FN, and LC3 expressions, PERK phosphorylation and α-SMA density, and downregulated P62 level compared with control cells. Treatment with PERK agonist and inhibitor respectively increased and decreased LC3 expression, conversely in P62 level, which is consistent with effect of ERS agonists and inhibitors. And a PERK inhibitor upregulated the expressions of Caspase3 and Bax, and downregulated Bcl2 level, which developed H9c2 cardiomyoblasts. Moreover, siRNA-mediated knockdown of PERK reduced ERS mediated autophagy activity and increased cells apoptosis. On the other hand, elevated autophagy activity could downregulated PERK level. Our finding showed that PERK activity mediates upregulation of ERS-induced autophagy and regulation of cardiomyocyte apoptosis in H9c2 cardiomyoblasts.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Butadienes; Cell Line; eIF-2 Kinase; Endoplasmic Reticulum Stress; Fibrosis; Humans; Myocytes, Cardiac; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Rats; Signal Transduction; Sirolimus

Subconjunctival fibrosis represents the primary cause of postoperative failure of trabeculectomy, and at present there is a lack of effective intervention strategies. The present study aimed to investigate the effect of the mitogen‑activated protein kinase kinase (MEK) inhibitor U0126 on human tenon fibroblast (HTF) myofibrosis transdifferentiation, and to illuminate the underlying molecular mechanisms involved. It was demonstrated that U0126 significantly inhibited the proliferation, migration and collagen contraction of HTFs stimulated with TGF‑β1. In addition, U0126 largely attenuated the TGF‑β1‑induced conversion of HTFs into myofibroblasts, as indicated by a downregulation of the mRNA and protein expression of α‑smooth muscle actin and zinc finger protein SNAI1, and by ameliorating the 3D‑collagen contraction response. Mechanistically, U0126 suppressed the TGF‑β1‑stimulated phosphorylation of mothers against decapentaplegic homolog 2/3, P38 mitogen‑activated protein kinase and extracellular signal‑regulated kinase 1/2, indicating that U0126 may inhibit HTF activation through the canonical and non‑canonical signaling pathways of TGF‑β1. Therefore, U0126 exhibits a potent anti‑fibrotic effect among HTFs, and the inhibition of MEK signaling may serve as an alternative intervention strategy for the treatment of trabeculectomy‑associated fibrosis.

Topics: Actins; Butadienes; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Transdifferentiation; Cells, Cultured; Collagen; Down-Regulation; Fibroblasts; Fibrosis; Humans; Mitogen-Activated Protein Kinases; Myofibroblasts; Nitriles; Phosphorylation; RNA, Messenger; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta1

Topics: Animals; Apoptosis; Butadienes; Cell Line; Cell-Derived Microparticles; Enzyme Inhibitors; Fibrosis; G2 Phase Cell Cycle Checkpoints; Hepatocyte Growth Factor; Humans; Kidney; MAP Kinase Signaling System; Mesenchymal Stem Cells; Nitriles; Rats; Reperfusion Injury; Wharton Jelly

In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFβ1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFβ/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFβ1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFβ1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFβ1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFβ1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.

Topics: 3T3 Cells; Animals; Basic Helix-Loop-Helix Transcription Factors; Butadienes; Cardiomegaly; Chlorocebus aethiops; COS Cells; Fibrosis; Male; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Myocytes, Cardiac; Nitriles; Primary Cell Culture; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1

Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.

Topics: Animals; Blotting, Western; Butadienes; Cilia; Creatinine; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Fluorescent Antibody Technique; Immunoenzyme Techniques; Kidney Tubules; Male; MAP Kinase Kinase 1; Mice; Mice, Inbred C57BL; NF-kappa B; Nitriles; Phosphorylation; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta1

A stable 40-kDa fragment is produced from cardiac myosin-binding protein C when the heart is stressed using a stimulus, such as ischemia-reperfusion injury. Elevated levels of the fragment can be detected in the diseased mouse and human heart, but its ability to interfere with normal cardiac function in the intact animal is unexplored.. To understand the potential pathogenicity of the 40-kDa fragment in vivo and to investigate the molecular pathways that could be targeted for potential therapeutic intervention.. We generated cardiac myocyte-specific transgenic mice using a Tet-Off inducible system to permit controlled expression of the 40-kDa fragment in cardiomyocytes. When expression of the 40-kDa protein is induced by crossing the responder animals with tetracycline transactivator mice under conditions in which substantial quantities approximating those observed in diseased hearts are reached, the double-transgenic mice subsequently experience development of sarcomere dysgenesis and altered cardiac geometry, and the heart fails between 12 and 17 weeks of age. The induced double-transgenic mice had development of cardiac hypertrophy with myofibrillar disarray and fibrosis, in addition to activation of pathogenic MEK-ERK pathways. Inhibition of MEK-ERK signaling was achieved by injection of the mitogen-activated protein kinase (MAPK)/ERK inhibitor U0126. The drug effectively improved cardiac function, normalized heart size, and increased probability of survival.. These results suggest that the 40-kDa cardiac myosin-binding protein C fragment, which is produced at elevated levels during human cardiac disease, is a pathogenic fragment that is sufficient to cause hypertrophic cardiomyopathy and heart failure.

Topics: Animals; Butadienes; Carrier Proteins; Female; Fibrosis; Gene Expression Regulation; Heart Failure; Heart Ventricles; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Myosin Heavy Chains; Nitriles; Peptide Fragments; Phosphorylation; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Sarcomeres

To explore the effects of valsartan and MEK1/2 inhibitor U0126 on atrial fibrosis and connexin40 (Cx40) remodeling in rats treated with isoproterenol (ISO).. 32 male SD rats were randomly divided into control group (A), ISO (5 mg · kg(-1) · d(-1) for 7 days) + DMSO group (B), ISO + U0126 (0.5 mg · kg(-1) · d(-1) for 28 days) group (C, U0126 was dissolved in DMSO), ISO + valsartan (30 mg · kg(-1) · d(-1) for 28 days) + DMSO group (D). Rats were sacrificed after 28 days. The AngIIcontent in myocardial tissue was measured by radioimmunoassay, P-MEK1/2, P-ERK1/2 and Cx40 was detected by immunohistochemistry, atrial fibrosis was determined on HE and Masson stained heart sections.. The content of AngII was significantly higher in group B, C and D compared with group A [(368.243 ± 6.283) ng/L, (357.175 ± 5.944) ng/L, (359.908 ± 2.496) ng/L vs (250.380 ± 4.261) ng/L, P < 0.01]; the degree of atrial fibrosis was significantly lower in group C and D compared with group B [CVF(10.260 ± 0.525)%, (10.238 ± 0.524)% vs (78.710 ± 1.587)%, P < 0.01] while there was no atrial fibrosis in group A [CVF(9.025 ± 0.456)%]; the expression of P-MEK1/2 and P-ERK1/2 was significantly upregulated in group B compared with group A (P-MEK1/2: 0.311 ± 0.007 vs 0.203 ± 0.009, P < 0.01; P-ERK1/2: 0.259 ± 0.003 vs 0.173 ± 0.006, P < 0.01) and significantly lower in group C and D compared with group B (P-MEK1/2: 0.212 ± 0.004, 0.213 ± 0.005 vs 0.311 ± 0.007, P < 0.01, P-ERK1/2: 0.178 ± 0.004, 0.175 ± 0.007 vs 0.259 ± 0.003, P < 0.01). The content of Cx40 was obviously reduced and the distribution of Cx40 was disordered in group B compared with A (0.199 ± 0.007 vs 0.241 ± 0.004, P < 0.01) which could be partly reversed in group C and D (0.239 ± 0.037, 0.235 ± 0.006 vs 0.199 ± 0.007, P < 0.01). All parameters in group C and D were similar (P > 0.05).. The chronically elevated AngII content in myocardium may be related to atrial fibrosis and Cx40 remodeling in this model, valsartan and U0126 were equivalent on attenuating atrial fibrosis and Cx40 remodeling by inhibiting ERK pathways at different levels.

Topics: Animals; Butadienes; Connexins; Fibrosis; Gap Junction alpha-5 Protein; Heart Atria; Male; Myocardium; Nitriles; Rats; Rats, Sprague-Dawley; Tetrazoles; Valine; Valsartan

Aldosterone plays a crucial role in renal fibrosis by inducing mesangial cell proliferation and promoting collagen synthesis in renal fibroblasts. However, renal proximal tubule involvement in aldosterone-induced collagen synthesis has not yet been identified. The aim of this study was to examine the potential role of aldosterone in collagen expression and its possible mineralocorticoid receptor (MR)-dependent pathway, mediated by activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured human renal proximal tubular epithelial (HKC) cells.. After HKC cells were stimulated by aldosterone with different concentrations for various time and periods, the gene expression and protein synthesis of collagen I, II, III and IV were measured by real-time polymerase chain reaction and western blot, respectively. ERK1/2 activation, alpha-smooth muscle actin (alpha-SMA), and E-cadherin were also detected by western blot.. Aldosterone can increase ERK1/2 phosphorylation of human renal proximal tubular epithelial cells in a time- and dose-dependent manner. Although aldosterone had no effect on collagen I and II expression, it increased expression of alpha-SMA and collagen III and IV and decreased that of E-cadherin in HKC cells after 48 h. These effects could be prevented by a ERK pathway inhibitor, U0126, or by a selective MR antagonist, spironolactone.. The results suggest that aldosterone plays a pivotal role in tubulointerstitial fibrosis by promoting tubular epithelial-mesenchymal transition and collagen synthesis in proximal tubular cells. The process is MR-dependent, and mediated by ERK1/2 mitogen-activated protein kinase pathway.

Topics: Actins; Aldosterone; Butadienes; Cadherins; Cell Transdifferentiation; Cells, Cultured; Collagen; Enzyme Activation; Epithelial Cells; Fibrosis; Humans; Kidney Tubules, Proximal; Mineralocorticoid Receptor Antagonists; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Receptors, Mineralocorticoid; Signal Transduction; Spironolactone; Time Factors

Cyclosporine A (CsA) significantly improves the success of organ transplantation, however renal fibrosis, characterised by severe tubulointerstitial fibrosis is a complication of CsA therapy. Previously we have reported the involvement of PKC-beta isoforms in a model of CsA-induced tubulointerstitial fibrosis and we have now further elucidated this role. Treatment of human proximal tubular epithelial cells with CsA resulted in increased fibronectin production which coincided with increased PKC activity. To delineate the respective contributions of the two PKC-beta isoforms in fibrotic events, we overexpressed PKC-betaI, -betaII, or both in combination. Overexpression of the two PKC-beta isoforms induced morphological alterations, secretion of the profibrotic cytokine TGF-beta1, and fibronectin release from proximal tubular cells however PKC-betaII induced more significant effects in all parameters examined. Inhibition of PKC-beta completely abrogated the CsA-induced increase in fibronectin secretion demonstrating a direct antifibrotic effect of PKC-beta inhibition. Further studies also identified a role for the p44/42 mitogen activated kinase signalling pathway in CsA-induced fibrotic effects downstream of PKC-beta. Overall, these findings demonstrate a central role for PKC-beta, and particularly PKC-betaII in the development of tubulointerstitial fibrosis and suggest that PKC-beta may be a viable therapeutic target in CsA nephropathy.

Topics: Butadienes; Cell Line; Cell Shape; Cyclosporine; Enzyme Activation; Epithelial Cells; Fibronectins; Fibrosis; Green Fluorescent Proteins; Humans; Imidazoles; Isoenzymes; Kidney Tubules, Proximal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Phenotype; Protein Kinase C; Protein Kinase C beta; Pyridines; Pyrones; Recombinant Fusion Proteins; Signal Transduction; Transforming Growth Factor beta1

Pancreatic islet fibrosis observed in Type 2 diabetes is one of the major factors leading to progressive beta-cell loss and dysfunction. Despite its importance, the mechanism of islet-restricted fibrogenesis associated with pancreatic stellate cell (PSC) activation and proliferation remains to be defined. Therefore, we studied whether the islet-specific environment represented by hyperglycemia and hyperinsulinemia had additive effects on the activation and proliferation of cultured rat PSCs. Cells were stimulated to activate and proliferate with glucose and insulin, either individually or concomitantly. Both stimuli promoted PSC proliferation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation independently, but an additive effect was also demonstrated. Blockade of ERK signaling by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, suppressed both glucose- and insulin-induced ERK 1/2 phosphorylation and PSC proliferation. Glucose and insulin-induced ERK 1/2 phosphorylation also stimulated connective tissue growth factor gene expression. Thus, hyperglycemia and hyperinsulinemia are two crucial mitogenic factors that activate and proliferate PSCs, and the presence of both states will amplify this response.

Topics: Animals; Blood Glucose; Blotting, Western; Butadienes; Cell Proliferation; Cells, Cultured; Connective Tissue Growth Factor; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Gene Expression; Glucose; Hyperglycemia; Hyperinsulinism; Immediate-Early Proteins; Insulin; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Male; MAP Kinase Signaling System; Nitriles; Pancreas; Phosphorylation; Rats; Rats, Sprague-Dawley

Plasminogen (Plg) activator inhibitor-1 (PAI-1) is an important fibrosis-promoting molecule. Whether this effect can be attributed to PAI-1's activity as an inhibitor of plasmin generation is debated. This study was designed to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. Plg-deficient (Plg-/-) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n = 8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 d of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35% in the Plg-/- mice. Epithelial-to-mesenchymal transition (EMT), as typified by tubular loss of E-cadherin and acquisition of alpha-smooth muscle actin, was also significantly reduced in Plg-/- mice, 76% and 50%, respectively. Attenuation of EMT and fibrosis severity in the Plg-/- mice was associated with significantly lower levels of phosphorylated extracellular signal-regulated kinase (ERK) and active TGF-beta. In vitro, addition of plasmin (20 microg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, alpha-smooth muscle actin+, fibronectin-producing fibroblast-like cells. Both plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti-PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1-overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1-dependent ERK signaling and EMT induction.

Topics: Actins; Animals; Butadienes; Cadherins; Cell Movement; Collagen; Disease Models, Animal; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Fibrinolysin; Fibrosis; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitriles; Phosphorylation; Plasminogen Activator Inhibitor 1; Receptor, PAR-1; Signal Transduction; Transforming Growth Factor beta; Ureteral Obstruction

The roles of MEK, ERK, the epsilon and alpha isoforms of protein kinase C (PKC), and caveolin-1 in regulating collagen expression were studied in normal lung fibroblasts. Knocking down caveolin-1 gave particularly striking results. A 70% decrease caused a 5-fold increase in MEK/ERK activation and collagen expression. The combined data reveal a branched signaling pathway. In its central portion MEK activates ERK, leading to increased collagen expression. Two branches converge on MEK/ERK. In one, increased PKCepsilon leads to MEK/ERK activation. In another, increased PKCalpha induces caveolin-1 expression, which in turn inhibits MEK/ERK activation and collagen expression. Lung fibroblasts from scleroderma patients with pulmonary fibrosis showed altered signaling. Consistent with their overexpression of collagen, scleroderma lung fibroblasts contain more activated MEK/ERK and less caveolin-1 than normal lung fibroblasts. Because cutaneous fibrosis is the hallmark of scleroderma, we also studied dermal fibroblasts. As in lung, there was more activated MEK/ERK in cells from scleroderma patients than in control cells, and MEK inhibition decreased collagen expression. However, the distinctive levels of PKCepsilon, PKCalpha, and caveolin-1 in lung and dermal fibroblasts from scleroderma patients and control subjects indicate that the links between these signaling proteins and MEK/ERK must function differently in the four cell types. Finally, we confirmed the relevance of these signaling cascades in vivo. The combined results demonstrate that a branched signaling pathway involving MEK, ERK, PKCepsilon, PKCalpha, and caveolin-1 regulates collagen expression in normal lung tissue and is perturbed during fibrosis.

Topics: Animals; Butadienes; Caveolin 1; Caveolins; Cells, Cultured; Collagen; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Fibrosis; Humans; Isoenzymes; Lung; Male; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase Kinases; Nitriles; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-epsilon; Scleroderma, Systemic

The natriuretic peptides, including human B-type natriuretic peptide (BNP), have been implicated in the regulation of cardiac remodeling. Because transforming growth factor-beta (TGF-beta) is associated with profibrotic processes in heart failure, we tested whether BNP could inhibit TGF-beta-induced effects on primary human cardiac fibroblasts. BNP inhibited TGF-beta-induced cell proliferation as well as the production of collagen 1 and fibronectin proteins as measured by Western blot analysis. cDNA microarray analysis was performed on RNA from cardiac fibroblasts incubated in the presence or absence of TGF-beta and BNP for 24 and 48 hours. TGF-beta, but not BNP, treatment resulted in a significant change in the RNA profile. BNP treatment resulted in a remarkable reduction in TGF-beta effects; 88% and 85% of all TGF-beta-regulated mRNAs were affected at 24 and 48 hours, respectively. BNP opposed TGF-beta-regulated genes related to fibrosis (collagen 1, fibronectin, CTGF, PAI-1, and TIMP3), myofibroblast conversion (alpha-smooth muscle actin 2 and nonmuscle myosin heavy chain), proliferation (PDGFA, IGF1, FGF18, and IGFBP10), and inflammation (COX2, IL6, TNFalpha-induced protein 6, and TNF superfamily, member 4). Lastly, BNP stimulated the extracellular signal-related kinase pathway via cyclic guanosine monophosphate-dependent protein kinase signaling, and two mitogen-activated protein kinase kinase inhibitors, U0126 and PD98059, reversed BNP inhibition of TGF-beta-induced collagen-1 expression. These findings demonstrate that BNP has a direct effect on cardiac fibroblasts to inhibit fibrotic responses via extracellular signal-related kinase signaling, suggesting that BNP functions as an antifibrotic factor in the heart to prevent cardiac remodeling in pathological conditions.

Topics: Adolescent; Blotting, Western; Butadienes; Cell Division; Cells, Cultured; Cyclic GMP; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Humans; Inflammation; Male; MAP Kinase Signaling System; Middle Aged; Muscle Proteins; Natriuretic Peptide, Brain; Nitriles; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Ventricular Remodeling