BAG3 regulates ECM accumulation in renal proximal tubular cells induced by TGF-β1

Abstract

Previously we have demonstrated that Bcl-2-associated athanogene 3 (BAG3) is increased in renal fibrosis using a rat unilateral ureteral obstruction model. The current study investigated the role of BAG3 in renal fibrosis using transforming growth factor (TGF)-β1-treated human proximal tubular epithelial (HK-2) cells. An upregulation of BAG3 in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of BAG3 induction by shorting hairpin RNA suppressed the expression of ECM proteins but had no effect on PAI-1 expression induced by TGF-β1. Forced overexpression of BAG3 selectively increased collagens. TGF-β1-induced BAG3 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. In addition, forced BAG3 overexpression blocked attenuation of collagens expression by ERK1/2 and JNK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of BAG3, which would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.

Keywords: BAG3; ECM; MAPK; tubular epithelial cell.

Figure 1

Induction of BAG3, Col I, Col IV, FN, and PAI-1 by TGF-β1 in HK2 cells. A. HK2 cells were treated with indicated dose of TGF-β1 for 24 h, mRNA levels of BAG3, Col I, Col IV, FN, and PAI-1 were measured by real time RT-PCR. B. HK2 cells were treated with 10 ng/ml TGF-β1 for indicated time, mRNA levels of BAG3, Col I, Col IV, FN, and PAI-1 were measured by real time RT-PCR. C. HK2 cells were treated with indicated dose of TGF-β1 for 24 h, western blot analysis was performed using the indicated antibodies. D. HK2 cells were treated with 10 ng/ml TGF-β1 for indicated time, western blot analysis was performed using the indicated antibodies. E. HK2 cells were treated with indicated dose of TGF-β1 for 24 h, culture supernatants were collected and dot blot was performed using the indicated antibodies. F. HK2 cells were treated with 10 ng/ml TGF-β1 for indicated time. After that, supernatant was collected and dot blot was performed using the indicated antibodies. *, P<0.01.

Figure 2

Attenuation of TGF-β1 mediated accumulation of ECM proteins by BAG3 knockdown. A. HK2 cells were infected with shBAG3 or scramble lentiviral vectors for 12 h and cultured for additional 48 h. Cells were then treated with 10 ng/ml of TGF-β1 for additional 24 h. BAG3 and ECM proteins mRNA expression was analyzed using real-time RT-PCR. B. HK2 cells were transduced with shBAG3 or scramble lentiviral vectors for 12 h and cultured for additional 48 h. Cells were then treated with 10 ng/ml of TGF-β1 for additional 24 h and Western blot was performed using the indicated antibodies. C. HK2 cells were infected with shBAG3 or scramble lentiviral vectors for 12 h and cultured for additional 48 h. Cells were then treated with 10 ng/ml of TGF-β1 for additional 24 h. Culture supernatants were collected and dot blot was performed using the indicated antibodies.

Figure 3

Selective induction of collagens generation by overexpression of BAG3. A. HK2 cells were infected with lentiviral vectors containing empty or BAG3 construct for 12 h and cultured for additional 48 h. mRNA levels of BAG3 and ECM proteins were analyzed using real-time RT-PCR. B. HK2 cells were infected with lentiviral vectors containing empty or BAG3 construct for 12 h and cultured for additional 48 h, Western blot was performed using the indicated antibodies. C. HK2 cells were infected with lentiviral vectors containing empty or BAG3 construct for 12 h and cultured for additional 48 h. Culture supernatants were collected and dot blot was performed using the indicated antibodies. D. HK2 cells were infected with lentiviral vectors containing empty or BAG3 construct, viable cells were counted daily.

Figure 4

Implication of JNK and ERK1/2 signaling pathways in induction of BAG3 expression by TGF-β1. A. HK2 cells were individually pretreated with the JNK inhibitor SP600125, p38 inhibitor SB203580, or the ERK1/2 inhibitor PD98059 for 60 min before treatment with TGF-β1. mRNA levels of BAG3 was measured by real time RT-PCR. B. HK2 cells were individually pretreated with the JNK inhibitor SP600125, p38 inhibitor SB203580, or the ERK1/2 inhibitor PD98059 for 60 min before treatment with TGF-β1. Western blot was performed using the indicated antibodies. C. HK2 cells were individually pretreated with the JNK inhibitor SP600125, p38 inhibitor SB203580, or the ERK1/2 inhibitor PD98059 for 60 min before treatment with TGF-β1. Culture supernatants were collected and dot blot was performed using the indicated antibodies.

Figure 5

Attenuation of suppressive effects of ERK1/2 and JNK inhibition on ECM accumulation mediated by TGF-β1. A. HK2 cells infected with lentivirus containing empty or BAG3 constructed vectors were pretreated with SP600125 or PD98059 for 60 min individually before treatment with TGF-β1. mRNA levels of BAG3 and ECM proteins were evaluated using real time RT-PCR. B. HK2 cells infected with lentivirus containing empty or BAG3 constructed vectors were pretreated with SP600125 or PD98059 for 60 min individually before treatment with TGF-β1. Western blot was performed using the indicated antibodies. C. HK2 cells infected with lentivirus containing empty or BAG3 constructed vectors were pretreated with SP600125 or PD98059 for 60 min individually before treatment with TGF-β1. Supernatants were collected and dot blot was performed using the indicated antibodies.