Kebin Hu

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression.

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the β subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.

Hepatocyte Growth Factor Is a Downstream Effector that Mediates the Antifibrotic Action of Peroxisome Proliferator–Activated Receptor-γ Agonists

Peroxisome proliferator–activated receptor-γ (PPAR-γ) is a ligand-dependent transcription factor that plays an important role in the regulation of insulin sensitivity and lipid metabolism. Evidence shows that PPAR-γ agonists also ameliorate renal fibrotic lesions in both diabetic nephropathy and nondiabetic chronic kidney disease. However, little is known about the mechanism underlying their antifibrotic action. This study demonstrated that PPAR-γ agonists could exert their actions by inducing antifibrotic hepatocyte growth factor (HGF) expression. Incubation of mesangial cells with natural or synthetic PPAR-γ agonists 15-deoxy-Δ 12,14 -prostaglandin J2 (15d-PGJ2) or troglitazone and ciglitazone suppressed TGF-β1–mediated α-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression. PPAR-γ agonists also induced HGF mRNA expression and protein secretion. Transfection studies revealed that 15d-PGJ2 stimulated HGF gene promoter activity, which was dependent on the presence of a novel peroxisome proliferator response element. Treatment of mesangial cells with 15d-PGJ2 induced the binding of PPAR-γ to the peroxisome proliferator response element in the HGF promoter region. PPAR-γ agonists also activated c-met receptor tyrosine phosphorylation, induced Smad transcriptional co-repressor TG-interacting factor expression, and blocked TGF-β/Smad-mediated gene transcription in mesangial cells. Furthermore, ablation of c-met receptor through the LoxP-Cre system in mesangial cells abolished the antifibrotic effect of 15d-PGJ2. PPAR-γ activation also induced HGF expression in renal interstitial fibroblasts and repressed TGF-β1–mediated myofibroblast activation. Both HGF and 15d-PGJ2 attenuated Smad nuclear translocation in response to TGF-β1 stimulation in renal fibroblasts. Together, these findings suggest that HGF may act as a downstream effector that mediates the antifibrotic action of PPAR-γ agonists.

Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling

The activation of interstitial fibroblasts to become alpha-SMA-positive myofibroblasts is an essential step in the evolution of chronic kidney fibrosis, as myofibroblasts are responsible for the production and deposition of the ECM components that are a hallmark of the disease. Here we describe a signaling pathway that leads to this activation. Tissue-type plasminogen activator (tPA) promoted TGF-beta1-mediated alpha-SMA and type I collagen expression in rat kidney interstitial fibroblasts. This fibrogenic effect was independent of its protease activity but required its membrane receptor, the LDL receptor-related protein 1 (LRP-1). In rat kidney fibroblasts, tPA induced rapid LRP-1 tyrosine phosphorylation and enhanced beta1 integrin recruitment by facilitating the LRP-1/beta1 integrin complex formation. Blockade or knockdown of beta1 integrin abolished type I collagen and alpha-SMA expression. Furthermore, inhibition of the integrin-linked kinase (ILK), a downstream effector of beta1 integrin, or disruption of beta1 integrin/ILK engagement, abrogated the tPA action, whereas ectopic expression of ILK mimicked tPA in promoting myofibroblast activation. In murine renal interstitium after obstructive injury, tPA and alpha-SMA colocalized with LRP-1, and tPA deficiency reduced LRP-1/beta1 integrin interaction and myofibroblast activation. These findings show that tPA induces LRP-1 tyrosine phosphorylation, which in turn facilitates the LRP-1-mediated recruitment of beta1 integrin and downstream ILK signaling, thereby leading to myofibroblast activation. This study implicates tPA as a fibrogenic cytokine that promotes the progression of kidney fibrosis.

tPA Protects Renal Interstitial Fibroblasts and Myofibroblasts from Apoptosis

Activation and expansion of interstitial fibroblasts and myofibroblasts play an essential role in the evolution of renal fibrosis. After obstructive injury, mice lacking tissue-type plasminogen activator (tPA) have fewer myofibroblasts and less interstitial fibrosis than wild-type controls. This suggests that tPA controls the size of the fibroblast/myofibroblast population in vivo, and this study sought to determine the underlying mechanism. In vitro, tPA inhibited staurosporine or H(2)O(2)-induced caspase-3 activation, prevented cellular DNA fragmentation, and suppressed the release of cytochrome C from mitochondria into the cytosol in a rat interstitial fibroblast cell line (NRK-49F). tPA also protected TGF-beta1-activated myofibroblasts from apoptosis. This antiapoptotic effect of tPA was independent of its protease activity but required its membrane receptor, the LDL receptor-related protein 1 (LRP-1). Deletion or knockdown of LRP-1 abolished tPA-mediated cell survival, whereas re-introduction of an LRP-1 minigene in a mouse LRP-1-deficient fibroblast cell line (PEA-13) restored the cytoprotective ability of tPA. tPA triggered a cascade of survival signaling involving extracellular signal-regulated kinase 1/2 (Erk1/2), p90RSK, and phosphorylation of Bad. Blockade of Erk1/2 activation abrogated the antiapoptotic effect of tPA, whereas expression of constitutively active MEK1 promoted cell survival similar to tPA. In vivo, compared with wild-type controls, apoptosis of interstitial myofibroblasts was increased in tPA(-/-) mice after obstructive injury, and myofibroblasts were completely depleted 4 wk after relief of the obstruction. Together, these findings illustrate that tPA is a survival factor that prevents apoptosis of renal interstitial fibroblasts and myofibroblasts through an LRP-1-, Erk1/2-, p90RSK-, and Bad-dependent mechanism.

Hepatocyte growth factor receptor signaling mediates the anti-fibrotic action of 9-cis-retinoic acid in glomerular mesangial cells

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the regulation of cell proliferation, survival, and differentiation. RA action is primarily mediated through its receptors, ligand-dependent transcription factors of the steroid/thyroid/vitamin D nuclear receptor superfamily. Recent studies indicate that administration of RA mitigates progressive kidney disease, underscoring its renoprotective potential. In this study, we investigated the effects of 9-cis-RA on glomerular mesangial cell activation induced by transforming growth factor (TGF)-beta1 using an in vitro cell culture system. In human mesangial cells 9-cis-RA suppressed TGF-beta1-induced alpha-smooth muscle actin, fibronectin, and plasminogen activator inhibitor-1 expression, but it did not significantly affect cell proliferation and survival. Interestingly, 9-cis-RA induced hepatocyte growth factor (HGF) mRNA expression and protein secretion, stimulated HGF promoter activity, and activated c-met receptor phosphorylation. Similar to HGF, 9-cis-RA induced expression of the Smad transcriptional co-repressor TGIF in mesangial cells. Overexpression of exogenous TGIF by transfection or 9-cis-RA treatment suppressed trans-activation of the TGF-beta-responsive promoter. Moreover, conditional ablation of the c-met receptor completely abolished the anti-fibrotic effect of 9-cis-RA and abrogated TGIF induction. Collectively, these results indicate that 9-cis-RA possesses anti-fibrotic ability by antagonizing TGF-beta1 in mesangial cells and that 9-cis-RA activity is likely mediated through a mechanism dependent on HGF/c-met receptor signaling.

Novel actions of tissue-type plasminogen activator in chronic kidney disease.

Tissue-type plasminogen activator (tPA) is traditionally viewed as a simple serine protease whose main function is to convert plasminogen into biologically active plasmin. As a protease, tPA plays a crucial role in regulating blood fibrinolysis, in maintaining the homeostasis of extracellular matrix and in modulating the post-translational activation of growth factors. However, emerging evidence indicates that tPA also functions as a cytokine that transmits its signal across the cell membrane, initiates a diverse array of intracellular signaling, and dictates gene expression in the nuclei. tPA binds to the cell membrane LDL receptor-related protein 1 (LRP-1), triggers its tyrosine phosphorylation. As a cytokine, tPA plays a pivotal role in the pathogenesis of renal interstitial fibrosis through diverse mechanisms. It facilitates tubular epithelial to mesenchymal transition, potentiates myofibroblast activation, and protects renal interstitial fibroblasts/myofibroblasts from apoptosis. Together, growing evidence has implicated tPA as a fibrogenic cytokine that promotes the progression of kidney diseases. These new findings have radically changed our conception of tPA in renal fibrogenesis and represent a paradigm shift towards uncovering its cytokine function.

tPA Activates LDL Receptor-Related Protein 1-Mediated Mitogenic Signaling Involving the p90RSK and GSK3β Pathway

In renal fibrosis, interstitial fibroblasts have an increased proliferative phenotype, and the numbers of interstitial fibroblasts closely correlate with the extent of kidney damage. The mechanisms underlying proliferation and resulting expansion of the interstitium remain largely unknown. Here we define the intracellular signaling events by which tissue plasminogen activator (tPA) promotes renal interstitial fibroblast proliferation. tPA promoted the proliferation of renal interstitial fibroblasts independent of its protease activity. The mitogenic effect of tPA required Tyr(4507) phosphorylation of the cytoplasmic tail of its receptor LDL receptor-related protein 1. tPA triggered sequential proliferative signaling events involving Erk1/2, p90RSK, GSK3β phosphorylation, and cyclin D1 induction. Blockade of Erk1/2 activation or knockdown of p90RSK suppressed tPA-induced GSK3β phosphorylation, cyclin D1 expression, and fibroblast proliferation. In contrast, expression of constitutively active Mek1 mimicked tPA in inducing GSK3β phosphorylation and cyclin D1 expression. Ectopic overexpression of an uninhibitable GSK3β mutant eliminated tPA-induced cyclin D1 expression. In the murine obstruction model, tPA deficiency reduced renal GSK3β phosphorylation and induction of PCNA and FSP-1. These findings show that tPA induces Tyr(4507) phosphorylation of LDL receptor-related protein 1, which in turn leads to the downstream phosphorylation of Erk1/2, p90RSK, and GSK3β, followed by the induction of cyclin D1 in murine interstitial fibroblasts. This study implicates tPA as a mitogen that promotes interstitial fibroblast proliferation, leading to expansion of these cells.

LRP-1: Functions, Signaling and Implications in Kidney and Other Diseases

Low-density lipoprotein (LDL)-related protein-1 (LRP-1) is a member of LDL receptor family that is implicated in lipoprotein metabolism and in the homeostasis of proteases and protease inhibitors. Expression of LRP-1 is ubiquitous. Up-regulation of LRP-1 has been reported in numerous human diseases. In addition to its function as a scavenger receptor for various ligands, LRP-1 has been shown to transduce multiple intracellular signal pathways including mitogen-activated protein kinase (MAPK), Akt, Rho, and the integrin signaling. LRP-1 signaling plays an important role in the regulation of diverse cellular process, such as cell proliferation, survival, motility, differentiation, and transdifferentiation, and thus participates in the pathogenesis of organ dysfunction and injury. In this review, we focus on the current understanding of LRP-1 signaling and its roles in the development and progression of kidney disease. The role and signaling of LRP-1 in the nervous and cardiovascular systems, as well as in carcinogenesis, are also briefly discussed.

Inhibition of Wnt/β-catenin signaling suppresses bleomycin-induced pulmonary fibrosis by attenuating the expression of TGF-β1 and FGF-2

Pulmonary fibrosis is a progressive lung disorder of unknown etiology, which is characterized by alterations in alveolar epithelium function, fibroblast activation, and increased extracellular matrix deposition. Recent studies have demonstrated that PF is associated with uncontrolled production of cytokines after lung injury. In the present study, we found that transforming growth factor-β1 (TGF-β1) and fibroblast growth factor 2 (FGF-2) were both upregulated in bleomycin-induced fibrotic lung tissue and primary murine alveolar epithelial Type II (ATII) cells treated with bleomycin. Furthermore, we discovered that TGF-β1 could induce the differentiation of lung resident mesenchymal stem cells (LR-MSCs) into fibroblasts, which may play an essential role in PF. LR-MSCs incubated with FGF-2 showed modest alterations in the expression of α-SMA and Vimentin. Moreover, in our study, we found that Wnt/β-catenin signaling was activated both in vitro and in vivo as a result of bleomycin treatment. Interestingly, we also found that suppression of the Wnt/β-catenin signaling could significantly attenuate bleomycin-induced PF accompanied with decreased expression of TGF-β1 and FGF-2 in vitro and in vivo. These results support that controlling the aberrant expression of TGF-β1 and FGF-2 via inhibition of Wnt/β-catenin signaling could serve as a potential therapeutic strategy for PF.

miR-877-3p targets Smad7 and is associated with myofibroblast differentiation and bleomycin-induced lung fibrosis.

Myofibroblast differentiation of lung resident mesenchymal stem cells (LR-MSC) plays an important role in idiopathic pulmonary fibrosis. By comparing the expression profiles of miRNAs before and after myofibroblast differentiation of LR-MSC, we identified miR-877-3p as a fibrosis-related miRNA. We found that miR-877-3p sequestration inhibited the myofibroblast differentiation of LR-MSC and attenuates bleomycin-induced lung fibrosis by targeting Smad7. Smad7, as an inhibitory smad in the TGF-β1 signaling pathway, was decreased in the myofibroblast differentiation of LR-MSC and up-regulation of Smad7 could inhibit the differentiation process. Our data implicates a potential application of miR-877-3p as a fibrosis suppressor for pulmonary fibrosis therapy and also as a fibrosis marker for predicting prognosis.