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. 2014 Jul 11;9(7):e101179.
doi: 10.1371/journal.pone.0101179. eCollection 2014.

TRPV4 channel inhibits TGF-β1-induced proliferation of hepatic stellate cells

Yang Song  1 , Lei Zhan  2 , Mingzhe Yu  2 , Cheng Huang  2 , Xiaoming Meng  2 , Taotao Ma  2 , Lei Zhang  2 , Jun Li  2
Affiliations

TRPV4 channel inhibits TGF-β1-induced proliferation of hepatic stellate cells

Yang Song et al. PLoS One. .

Abstract

TRPV4, one of the TRP channels, is implicated in diverse physiological and pathological processes including cell proliferation. However, the role of TRPV4 in liver fibrosis is largely unknown. Here, we characterized the role of TRPV4 in regulating HSC-T6 cell proliferation. TRPV4 mRNA and protein were measured by RT-PCR and Western blot in patients and rat model of liver fibrosis in vivo and TGF-β1-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPV4 were dramatically increased in liver fibrotic tissues of both patients and CCl4-treated rats. Stimulation of HSC-T6 cells with TGF-β1 resulted in increase of TRPV4 mRNA and protein. However, TGF-β1-induced HSC-T6 cell proliferation was inhibited by Ruthenium Red (Ru) or synthetic siRNA targeting TRPV4, and this was accompanied by downregulation of myofibroblast markers including α-SMA and Col1α1. Moreover, our study revealed that miR-203 was downregulated in liver fibrotic tissues and TGF-β1-treated HSC-T6 cell. Bioinformatics analyses predict that TRPV4 is the potential target of miR-203. In addition, overexpression of miR-203 in TGF-β1-induced HSC significantly reduced TRPV4 expression, indicating TRPV4, which was regulated by miR-203, may function as a novel regulator to modulate TGF-β1-induced HSC-T6 proliferation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Upregulation of TRPV4 mRNA and protein in the liver tissues from liver fibrosis patients and CCl4-treated rats.
A. The level of the TRPV4 was analyzed by immunohistochemistry in human normal liver and liver fibrosis patients. Representative views from each group are presented (original magnification, ×50). B. Liver tissues from liver fibrosis patients tissue were double stained with TRPV4 and α-SMA antibodies. Representative photomicrographs are shown (original magnification, ×40) in B. C. Pathology observation of the experimental rat liver sections stained with hematoxylin and eosin (H&E) staining and massion staining (×200). D. The level of the α-SMA was analyzed by immunohistochemistry in vehicle control livers and liver fibrotic tissue. Representative views from each group are presented (original magnification, ×50). E-F. Total RNAs were isolated from the livers of CCl4-treated rats or vehicle-treated groups. The expression of TRPV4 mRNA was assessed by RT-PCR(E) and realtime PCR(F). Representative images of three independent experiments are shown. *p<0.05, **p<0.01 vs. vehicle control. G. Whole-cell extracts were isolated from the liver tissues of CCl4-treated rats or vehicle-treated groups, and subjected to immunoblot for TRPV4 and β-actin control. Representative images of three independent experiments are shown. *p<0.05, **p<0.01 vs. vehicle control.
Figure 2
Figure 2. Increasing expression of TRPV4 mRNA and protein during HSC activation.
A. Total RNAs were isolated from TGF-β1-treated HSC-T6 cells, and subjected to qRT-PCR analyses. Representative images of three independent experiments are shown. *p<0.05 vs. non-treated cells. B. Whole-cell extracts were isolated from TGF-β1-treated HSC-T6 cells, and subjected to Western blot analyses with TRPV4 and β-actin antibodies. Representative blots of three independent experiments are shown. **p<0.01 vs. non-treated cells. C. Total RNAs were isolated from TGF-β1 treated HSC-T6 cells at different time points. The expression of α-SMA and Col1a1 mRNA was assessed by RT-PCR. Representative images of three independent experiments are shown. **p<0.01 vs. non-treated cells.
Figure 3
Figure 3. Blockade of TRPV4 inhibited the proliferation and decreased α-SMA expression in activated HSC-T6 cells.
A. Total RNA extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to qRT-PCR analyses of TRPV4. Representative images of three independent experiments are shown. #p<0.05 vs. TGF-β1-treated cells. B. HSC-T6 cells were seeded in triplicate on day 0 and incubated in DMEM containing 10% fetal bovine serum or same media supplemented with Ru for further 24 h. Proliferation was measured by adding 5 mg/ml MTT reagent per well and incubating it for 4 h. #p<0.05 vs. TGF-β1-treated cells. C. Total RNA extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to qRT-PCR analyses of α-SMA. Representative images of three independent experiments are shown. #p<0.05 vs. TGF-β1-treated cells. D. Whole-cell protein extracts were made from HSC-T6 cells treated with or without TGF-β1 and Ru, and subjected to Western blot analyses of TRPV4. Representative images of three independent experiments are shown. ##p<0.01 vs. TGF-β1-treated cells. E. HSC-T6 cells were treated with TGF-β1 for 48 h, followed by transfection with TRPV4-siRNA for an additional 48 h, and cell viability was determined by MTT assay. Mean±SE of two HSC preparations in quadruplets is shown; *p<0.05 vs. non-treated cells, #p<0.05 vs. TGF-β1-treated cells. F. Whole cell extracts were isolated from TGF-β1-treated HSC-T6 cells with RNAi transfection, and subjected to Western blot analyses. Representative images of three independent experiments are shown. **p<0.01 vs. non-treated cells, ##p<0.01 vs. TGF-β1-treated cells.
Figure 4
Figure 4. Overexpression of miR-203 inhibited of TGF-β1-induced HSC proliferation.
A. Downregulation of miR-203 expression in liver fibrotic tissues compared with vehicle-treated groups. The miR-203 expression was analyzed by one-step quantitative real-time PCR. *P<0.05 vs vehicle control. B. Downregulation of miR-203expression in response to TGF-β1 (0, 5, 10 ng/ml) for 24 h. The miR-203 expression was analyzed by one-step quantitative real-time PCR. **P<0.01 vs non-treated cells. C. Upregulation of miR-203 expression in transfected HSC. The miR-203 expression of HSC was analyzed by one-step quantitative real-time PCR. **P<0.01 vs NS-miRNA. D. MiR-203 overexpression significantly inhibited proliferation of TGF-β1-induced HSC. The role of miR-203 in regulating TGF-β1-treated HSC proliferation was tested by MTT assay. The data represent the mean±SD of three different experiments. *P<0.05 vs control(non-treated cells), #P<0.05 vs NS-miRNA.
Figure 5
Figure 5. TRPV4 is a target of miR-203 in HSC.
A. The region of the rat TRPV4 mRNA 3′UTR predicted to be targeted by miR-203. Three different bioinformatics approaches (miRbase, miRANDA, and Targetscan 5.1) were used for the target prediction. B. The TRPV4 mRNA expression was analyzed by real-time PCR. The results are expressed as relative expression against control expression. *P<0.05 vs control(non-treated cells), #P<0.05 vs model(TGF-β1-treated cells).C. Dual-luciferase reporter assays of TRPV4 in HSC-T6 cells. Reporter construct containing wt TRPV4 3′-UTR was cotransfected with miR-203 mimics or NC-miRNA. Relative luciferase activity was normalized to firefly luciferase. Data are representative of at least three separate experiments. *P<0.05, **P<0.01vs. NC-miRNA.
Figure 6
Figure 6. Overview of TRPV4 on liver fibrosis.
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Grants and funding

This project was supported by the National Science Foundation of China (NO 81072686, 81273526, 81202978) and publication fees were covered by NSFC (NO 81273526). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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