Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling
Kebin Hu, Chuanyue Wu, Wendy M. Mars, Youhua Liu
Kebin Hu, Chuanyue Wu, Wendy M. Mars, Youhua Liu
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Research Article Nephrology

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

Abstract

The activation of interstitial fibroblasts to become α-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-β1–mediated α-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 β1 integrin recruitment by facilitating the LRP-1/β1 integrin complex formation. Blockade or knockdown of β1 integrin abolished type I collagen and α-SMA expression. Furthermore, inhibition of the integrin-linked kinase (ILK), a downstream effector of β1 integrin, or disruption of β1 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 α-SMA colocalized with LRP-1, and tPA deficiency reduced LRP-1/β1 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 β1 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.

Authors

Kebin Hu, Chuanyue Wu, Wendy M. Mars, Youhua Liu

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Figure 5

tPA promotes LRP-1 and β1 integrin interaction and facilitates LRP-1–mediated recruitment of β1 integrin.

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tPA promotes LRP-1 and β1 integrin interaction and facilitates LRP-1–med...
(A–D) Coimmunoprecipitation shows that tPA promoted the physical interaction of LRP-1 and β1 integrin, while RAP abolished such interaction in NRK-49F cells. Cell lysates were precipitated with anti–LRP-1, followed by immunoblotting with antibodies against β1 integrin or LRP-1, respectively (A and B). In reciprocal experiments, cell lysates were first precipitated with anti–β1 integrin, followed by blotting with antibodies against LRP-1 or β1 integrin (C and D). Representative western blots (A and C) and graphic representations of the relative levels of the LRP-1/β1 integrin complexes (B and D) are presented. **P < 0.01. (E) Inhibition of LRP-1 tyrosine phosphorylation by genistein (50 μM) abolished the tPA ability to promote LRP-1/β1 integrin interaction. (F) Treatments of NRK-49F cells with tPA for 2 hours had no effect on the abundance of total (top panel) or the cell membrane surface β1 integrin (bottom panel). (G) Blockade of TGF-β signaling with neutralizing antibody (25 μg/ml) did not affect the tPA-induced LRP-1/β1 integrin complex formation. Same amount of normal mouse IgG (mIgG) was used as a negative control.