Gremlin activates the Smad pathway linked to epithelial mesenchymal transdifferentiation in cultured tubular epithelial cells

Abstract

Gremlin is a developmental gene upregulated in human chronic kidney disease and in renal cells in response to transforming growth factor-β (TGF-β). Epithelial mesenchymal transition (EMT) is one process involved in renal fibrosis. In tubular epithelial cells we have recently described that Gremlin induces EMT and acts as a downstream TGF-β mediator. Our aim was to investigate whether Gremlin participates in EMT by the regulation of the Smad pathway. Stimulation of human tubular epithelial cells (HK2) with Gremlin caused an early activation of the Smad signaling pathway (Smad 2/3 phosphorylation, nuclear translocation, and Smad-dependent gene transcription). The blockade of TGF-β, by a neutralizing antibody against active TGF-β, did not modify Gremlin-induced early Smad activation. These data show that Gremlin directly, by a TGF-β independent process, activates the Smad pathway. In tubular epithelial cells long-term incubation with Gremlin increased TGF-β production and caused a sustained Smad activation and a phenotype conversion into myofibroblasts-like cells. Smad 7 overexpression, which blocks Smad 2/3 activation, diminished EMT changes observed in Gremlin-transfected tubuloepithelial cells. TGF-β neutralization also diminished Gremlin-induced EMT changes. In conclusion, we propose that Gremlin could participate in renal fibrosis by inducing EMT in tubular epithelial cells through activation of Smad pathway and induction of TGF-β.

Figure 1

(a) Stimulation with Gremlin rapidly increased Smad 3 phosphorylation in cultured human tubuloepithelial cells. HK2 cells were stimulated with Gremlin (50 ng/mL) for different times. (b) Early Smad 3 phosphorylation induced by stimulation with Gremlin was not mediated by TGF-β. TGF-β was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-β neutralizing antibody and then treated with Gremlin for 10 minutes. (c) In some points, HK2 cells were preincubated with BMP-2 or BMP-4 and then treated with Gremlin for 10 minutes. Total proteins were isolated and protein levels were evaluated by western blot. GAPDH or Smad 3 were used as loading controls. Figures show a representative western blot of phosphorylated levels of Smad 3 and data are expressed as n-fold over control (considered as 1), as the mean ± SEM of 3-4 independent experiments. *P < 0.05 versus control.

Figure 2

Stimulation with Gremlin induces a rapid activation of the Smad pathway in cultured human tubuloepithelial cells. HK2 cells were stimulated with Gremlin (50 ng/mL) for 15 minutes. The localization of R-Smad 3 (a) and 2 (b) and Smad 4 (c) was evaluated by confocal microscopy with FITC-secondary antibodies (green staining). Nuclei were stained with propidium iodide (I.P.) (red). In the merge, the yellow staining indicates the nuclear localization of Smad proteins. The results are representative of 3 independent confocal microscopy experiments.

Figure 3

Gremlin overexpression causes a sustained Smad activation in cultured human tubuloepithelial cells. HK2 cells were transiently transfected with a Gremlin expression vector (GREM-GFP; green) or empty vector for 24 hours. The levels and localization of R-Smad 3 (a) and R-Smad 2 (b) were evaluated by confocal microscopy with Alexa-633 secondary IgG (red). Nuclei were stained using 4′,6-diamino-2-phenylindole dihydrochloride (DAPI; blue). In Gremlin-transfected cells (green staining by GFP), the Smad 2 and Smad 3 were found in the nuclei (white staining in the merge). Figures show representative images out of 3 independent observations.

Figure 4

Gremlin overexpression induces Smad-dependent gene transcription. HK2 cells were transfected with GREM-GFP or empty vector, Smad/luc promoter, and TK-renilla for 24 hours. In some points, cells were cotransfected with Smad 7. Then, luciferase/renilla activity was measured. Data are expressed as increase in Smad binding element (SBE) promoter-luciferase dependent expression. Data are expressed as n-fold over control (considered as 1), as the mean ± SEM of 5 experiments. *P < 0.05 versus control. ^# P < 0.05 versus Gremlin.

Figure 5

Gremlin-induced EMT via the Smad pathway. HK2 cells were transiently transfected with empty, Gremlin (GREM-GFP) alone or cotransfected with Smad 7 expression vectors. EMT markers were evaluated after 48 hours. Gremlin transfected cells express GFP (green staining). Confocal microscopy analysis of cytokeratin and α-SMA immunofluorescence was performed using specific primary antibodies and Alexa-633 secondary IgG (red staining). Representative image out of 3 experiments.

Figure 6

(a) Gremlin increased TGF-β production. HK2 cells were stimulated with Gremlin (50 ng/mL) for 24 and 48 hours in serum-free medium. TGF-β1 protein levels were measured in the cell-conditioned medium using a specific ELISA. Data are expressed as mean ± SEM of 6 independent experiments. *P < 0.05 versus control. (b) The late increase in gene expression of profibrotic factors caused by Gremlin is mediated by endogenous TGF-β production. HK2 cells were stimulated with Gremlin (50 ng/mL) for 24 hours in serum-free medium. TGF-β was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-β neutralizing antibody. Total cell RNA was isolated to assess mRNA levels by real-time PCR. Data are expressed as n-fold over control (considered as 1), as the mean ± SEM of 3 experiments. *P < 0.05 versus control. ^# P < 0.05 versus Gremlin.

Figure 7

TGF-β is a mediator of EMT-related changes following stimulation with Gremlin. HK2 cells were stimulated with Gremlin (50 ng/mL) for 48 hours in serum-free medium. TGF-β was blocked or not (control) by pretreatment of cells for 1 hour with an anti-TGF-β neutralizing antibody. (a) EMT changes were evaluated by confocal microscopy. E-cadherin, pan-Cytokeratin, and Vimentin were studied by indirect immunofluorescence using FITC-secondary IgG (green) and confocal microscopy. Nuclei are shown in blue. Figure shows a representative image out of 3 independent observations. (b)Total proteins were isolated and Vimentin, E-cadherin, and Slug levels were analyzed by western blot. Data are expressed as n-fold over control (considered as 1), as the mean ± SEM of 3 experiments. *P < 0.05 versus control. ^# P < 0.05 versus Gremlin.

Figure 8

Dual effects of Gremlin on Smad activation. Gremlin induces an early (minutes) and direct, TGF-β-independent, Smad pathway activation. After 24 hours Gremlin increased several profibrotic genes, including TGF-β, and after 48 hours increased TGF-β production and induced EMT features. These long-term Gremlin-induced profibrotic events require autocrine TGF-β.