tPA Protects Renal Interstitial Fibroblasts and Myofibroblasts from Apoptosis

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.

tPA Is a Potent Mitogen for Renal Interstitial Fibroblasts

“…In summary, this study, together with previous reports, 25,28 has established that tPA, a serine protease whose expression is induced in fibrotic kidney, is a fibrogenic mediator that promotes fibroblast proliferation, survival, and myofibroblast activation through ␤1 integrinmediated signaling cascades. The challenges ahead are how to translate this knowledge into new therapeutic strategies, thereby eventually benefitting the patients with fibrotic kidney diseases.…”

“…It is worthwhile to point out that downstream signaling of FAK, namely the ERK1/2/p90RSK/Bad cascade, is also important in promoting tPA-mediated fibroblast survival, as previously reported. 28 Collectively, these observations indicate that FAK and ILK, two different branches of ␤1 integrin signaling, are selectively responsible for the tPA-mediated cell proliferation/survival and myofibroblast activation/matrix production, respectively. Of note, tPA-triggered ERK1/2 activation apparently mediates both interstitial fibroblast proliferation ( Figure 8E) and matrix metalloproteinase-9 induction in NRK-49F cells, 22 whether these two events are functionally linked remains to be determined.…”

“…The role and relevant pathways of tPA in regulating fibroblast survival and myofibroblastic activation (gray arrows) are as elucidated previously. 25,28 Open circle denotes tPA; asterisk indicates LRP-1 tyrosine phosphorylation.…”

“…28 Briefly, NRK-49F cells were cultured in Dulbecco's modified Eagle's medium/Ham's F12 (1:1) supplemented with 5% fetal bovine serum, while PEA-13 and MEF-1 were incubated in Dulbecco's modified Eagle's medium with supplements specified by the American Type Culture Collection. Cells were seeded onto six-well plates in complete medium and incubated overnight, and then changed to serum-free medium for serum-starvation for 24 hours.…”

“…26 Increased expression of tPA appears detrimental, as genetic ablation of tPA protects kidney against development of renal fibrosis in obstructive nephropathy. 27 tPA has been shown to prolong the lifespan of interstitial fibroblasts by protecting them from apoptosis and to potentiate myofibroblastic activation, 25,28 which likely leads to an exaggerated fibroblast pool in diseased kidney. However, whether tPA also directly regulates fibroblast proliferation remains completely unknown.…”