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 ␤ 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. Tissue-type plasminogen activator (tPA)2 is a serine protease that plays a pivotal role in blood coagulation and fibrinolysis. In the kidney, the main function of tPA is to convert plasminogen into the biologically active plasmin, which in turn participates in regulating the catabolism of extracellular matrix and tissue fibrosis (1). In light of its proteolytic potential, tPA has generally been considered to be beneficial in the pathogenesis of fibrotic lesions, leading to increased matrix degradation and decreased matrix accumulation (2). However, previous studies from our laboratory demonstrate that genetic ablation of tPA actually protects the kidney from development of interstitial fibrosis in obstructive nephropathy (3), suggesting that increased tPA is detrimental in certain pathological situations. The deleterious effect of tPA is attributed to its ability to induce matrix metalloproteinase-9 (MMP-9) production, which leads to the destruction of the tubular basement membrane and subsequent generation of the matrix-producing myofibroblasts via tubular epithelial to mesenchymal transition (4, 5). In vitro, tPA directly induces MMP-9 gene expression and protein secretion in renal interstitial fibroblast cells (3). However, how tPA, a well known serine protease, transduces its signal into the nucleus to control specifi...

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.

No abstract

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-␤1-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 Ϫ/Ϫ 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.

NF-kB activation is central to the initiation and progression of inflammation, which contributes to the pathogenesis of CKD. Tissue plasminogen activator (tPA) modulates the NF-kB pathway, but the underlying mechanism remains unknown. We investigated the role of tPA signaling in macrophage NF-kB activation and found that tPA activated NF-kB in a time-and dose-dependent manner. tPA also induced the expression of the NF-kB-dependent chemokines IP-10 and MIP-1a. The protease-independent action of tPA required its membrane receptor, annexin A2. tPA induced the aggregation and interaction of annexin A2 with integrin CD11b, and ablation of CD11b or administration of anti-CD11b neutralizing antibody abolished the effect of tPA. Knockdown of the downstream effector of CD11b, integrin-linked kinase, or disruption of its engagement with CD11b also blocked tPA-induced NF-kB signaling. In vivo, tPA-knockout mice had reduced NF-kB signaling, fewer renal macrophages, and less collagen deposition than their counterparts. Taken together, these data suggest that tPA activates the NF-kB pathway in macrophages through a signaling pathway involving annexin A2/CD11b-mediated integrin-linked kinase.