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

Abstract

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.

Figure 1

The effects of 9-cis-RA on mesangial cell proliferation and survival. HMCs were treated with increasing amounts of 9-cis-RA as indicated (A, C, and E) or with 9-cis-RA (10⁻⁶ mol/L) or/and TGF-β1 (1 ng/ml) (B, D, and F) for 48 hours. Cell proliferation was assessed by MTT assay (A and B). Cell cytotoxicity was estimated by lactate dehydrogenase release (C and D), whereas apoptosis was detected by TUNEL staining (E and F). No statistical difference was found between the treatment groups and controls (n = 3).

Figure 2

9-cis-RA inhibits TGF-β1-mediated α-SMA expression in mesangial cells. A: HMCs were treated with or without TGF-β1 (1 ng/ml) and different amounts of 9-cis-RA as indicated for 72 hours. Whole cell lysates were immunoblotted with specific antibodies against α-SMA and actin, respectively. B: HMCs were incubated without or with TGF-β1 (1 ng/ml) and/or 9-cis-RA (10⁻⁶ mol/L) for different periods of time as indicated. 9-cis-RA inhibited α-SMA expression induced by TGF-β1 at 48 and 72 hours, respectively. C to F: Immunofluorescence staining for α-SMA in HMCs after various treatments. HMCs were treated without (C) or with TGF-β1 (1 ng/ml) (D), 9-cis-RA (10⁻⁶ mol/L) (E), or both (F) for 72 hours.

Figure 3

9-cis-RA suppresses TGF-β1-induced fibronectin expression and deposition. A: 9-cis-RA suppresses fibronectin expression at both resting and TGF-β1-stimulated conditions in a time-dependent manner. HMCs were treated with or without TGF-β1 (1 ng/ml) in the absence or presence of 9-cis-RA (10⁻⁶ mol/L) for 48 and 72 hours, respectively. Whole cell lysates were immunoblotted with specific antibodies against fibronectin and actin, respectively. B: 9-cis-RA suppresses fibronectin expression in HMCs in a dose-dependent manner. C to F: Indirect immunofluorescence staining shows fibronectin deposition after various treatments. HMCs were treated without or with TGF-β1 (1 ng/ml) and/or 9-cis-RA (10⁻⁶ mol/L) for 72 hours. C: Control; D: TGF-β1; E: 9-cis-RA; F: TGF-β1 plus 9-cis-RA.

Figure 4

9-cis-RA abolishes TGF-β1-mediated plasminogen activator inhibitor-1 (PAI-1) induction in HMCs. HMCs were treated with or without TGF-β1 (1 ng/ml) and different concentrations of 9-cis-RA as indicated for 72 hours. Whole cell lysates were immunoblotted with specific antibodies against PAI-1 and actin, respectively.

Figure 5

9-cis-RA induces HGF protein and mRNA expression, stimulates HGF promoter activity, and activates c-met receptor phosphorylation. A: HMCs were incubated with different doses of 9-cis-RA as indicated for 72 hours. The supernatants were collected and concentrated, followed by Western blot analysis with anti-HGF antibody. The authenticity of HGF was confirmed by loading the purified human recombinant HGF (10 ng) on the adjacent lane (not shown). Cell lysates were probed with actin to ensure same number of cells used. B: Graphical presentation of the relative abundance of HGF protein in the supernatants of the HMCs after treatment with different concentrations of 9-cis-RA. Data are presented as mean ± SEM of three experiments. *P < 0.05 versus control (n = 3). C: Real-time RT-PCR shows that 9-cis-RA induced HGF mRNA abundance in HMCs. Cells were treated with 9-cis-RA (10⁻⁶ mol/L) for different periods of time as indicated. *P < 0.05 versus control (n = 3). D: 9-cis-RA induced HGF expression in the presence of TGF-β1. HMCs were treated with TGF-β1 (1 ng/ml) alone or TGF-β1 plus 9-cis-RA (10⁻⁶ mol/L) for 24 hours. E: 9-cis-RA stimulated HGF promoter activity. Rat mesangial cells were transfected with HGF promoter-Luc reporter construct and internal control reporter pRL-TK, followed by incubation with 9-cis-RA (10⁻⁶ mol/L) for another 48 hours. *P < 0.01 versus control (n = 3). F: 9-cis-RA activated c-met receptor phosphorylation. HMCs were incubated without or with 10⁻⁶ mol/L 9-cis-RA for 24 hours. Cell lysates were immunoblotted with antibodies against phospho-specific and total c-met, respectively. Fold induction after quantitative analysis was also presented in the bottom of the picture.

Figure 6

9-cis-RA induces Smad transcriptional co-repressor TGIF expression, which in turn blocks TGF-β1/Smad-mediated gene transcription. A: HMCs were incubated with 10⁻⁶ mol/L 9-cis-RA for various periods of time as indicated. Whole cell lysates were immunoblotted with antibodies against TGIF and actin, respectively. B: 9-cis-RA and TGIF repress TGF-β1/Smad-mediated gene transcription. Rat mesangial cells were transfected with reporter plasmid p3TP-Lux together with internal control plasmid pRL-TK; with or without Smad2/Smad3 and TGIF expression vectors as indicated. The transfected cells were pretreated with 9-cis-RA (10⁻⁶ mol/L) for 16 hours, followed by incubation with or without TGF-β1 (1 ng/ml) for another 36 hours. *P < 0.01 versus control groups without Smad2/3 (n = 9). ^†P < 0.01 versus pSmad2/3-transfected groups (n = 9).

Figure 7

Conditional knockout of c-met receptor in mesangial cells abolishes the anti-fibrotic effect of 9-cis-RA. A: Mouse mesangial cells (MMCs) were isolated from c-met-floxed mice by differential sieving technique. MMCs were infected with adenoviral vectors containing Cre recombinase or GFP (control vector), respectively. Cre expression and c-met ablation in the MMCs were confirmed by Western blot analysis of whole cell lysates. B: Ablation of c-met receptor abolished the suppressive effect of 9-cis-RA on fibronectin expression induced by TGF-β1. MMCs infected with Ad.GFP (control) or Ad.Cre (c-met knockout) were treated with or without TGF-β1 (1 ng/ml) and/or 9-cis-RA (10⁻⁶ mol/L) for 72 hours. Whole cell lysates were immunoblotted with antibodies against fibronectin and actin, respectively. C: Ablation of c-met receptor abolished 9-cis-RA inhibition of the α-SMA expression induced by TGF-β1. MMCs were treated as indicated for 72 hours. D: Ablation of c-met receptor abrogated TGIF induction by 9-cis-RA. MMCs after infection with adenoviral vectors were incubated with 9-cis-RA for 24 hours.