Suppression of hepatic stellate cell activation through downregulation of gremlin1 expression by the miR-23b/27b cluster

Abstract

The imbalance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways is a critical step in promoting hepatic stellate cell activation during hepatic fibrogenesis. Gremlin1 may impair the balance. Something remains unclear about the regulatory mechanisms of gremlin1 action on hepatic stellate cell activation and hepatic fibrosis. In the current study, gremlin1 overexpression promotes activation of hepatic stellate cells. Knockdown of gremlin1 with siRNAs suppresses hepatic stellate cell activation and attenuates hepatic fibrosis in rat model. Our results also show that miR-23b/27b cluster members bind to 3'-untranslated region of gremlin1 resulting in reduction of transforming growth factor β, α-smooth muscle actin and collagenI α1/2 gene expression. Our findings suggest that gremlin1 promotes hepatic stellate cell activation and hepatic fibrogenesis through impairment of the balance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways. The miR-23b/27b cluster suppresses activation of hepatic stellate cells through binding gremlin1 to rectify the imbalance.

Keywords: BMP-7; TGF-β; hepatic fibrosis; miR-23b/27b cluster; siRNA.

Figure 1. Overexpression of gremlin1 enhances HSC-T6 cell activation

(A) Western blot shows increased gremlin1 and α-SMA expression in HSC-T6 cells stimulated with TGF-β1. (B) In gremlin1-HSC cells, expression of α-SMA, collagen Iα1 and TGF-β are increased, while MMP-2 is decreased. (C) Western blot shows upregulation and increased phosphorylation of Smad2/3 in Gremlin1-HSC cells. (D) Western blot shows that expression of Smad6, but not Smad7, is obviously increased in Gremlin1-HSC cells.

Figure 2. Knockdown of gremlin1 expression modulates BMP-7/TGF-β signaling leading to suppression of HSC activation

(A) Transfection of HSC-T6 cells with gremlin1-specific shRNA-expressing vector, pLKO.1-si-Gremlin1-2# or 4#, led to partial downregulation of gremlin1 as well as TGF-β and α-SMA, observed via western blot. (B) Infection of HSC-T6 cells with gremlin1-specific shRNA lentivirus, Lenti-si-gremlin1 (1:1 mixture of pLKO.1-si-Gremlin1-2# and 4# packed virus particles) significantly suppressed gremlin1, α-SMA and collagen Iα1 expression. (C) Western blot shows that lenti-si-gremlin1 infection of HSC-T6 cells dramatically increases BMP-7, but not BMPR (BMP receptor) expression, and inhibits TGF-β expression, compared with Lenti-si-NC infection. (D) Western blots shows that phosphorylation of Smad1 is remarkably increased whereas that of Smad2/3 is significantly decreased in HSC-T6 cells infected with lenti-si-Gremlin1 virus.

Figure 3. siRNA-mediated downregulation of gremlin1 expression alleviates hepatic fibrosis in a SD rat model

(A) Histopathological changes in liver tissue from a CCl₄-induced hepatic fibrosis SD rat model analyzed with H&E staining and deposition of collagens visualized with Masson staining. Compared with corn-oil or Lenti-si-NC virus infection, histopathological changes and collagen deposition were almost alleviated in model SD rats. (B) Quantification of the collagen fiber area revealed a significant decrease in lenti-si-Gremlin1-infected SD rats, compared with that of corn-oil-treated or lenti-si-NC-infected model SD rats. (C) Determination of collagen content with the QuickZyme total collagen kit revealed a significant decrease following lenti-si-Gremlin1 infection, compared to corn-oil-treated or Lenti-si-NC-infected model SD rats. (D) Immunofluorescence analysis revealed increased Gremlin1 expression (green fluorescence indicated by the white arrowhead) in liver tissue in hepatic fibrosis model SD rats or those treated with corn-oil or Lenti-si-NC virus infection, and significant downregulation in model SD rats with Lenti-si-Gremlin1 virus infection. BMP-7 expression (green fluorescence indicated by the white arrowhead) in liver tissue from hepatic fibrosis model SD rats or those treated with corn-oil or Lenti-si-NC virus infection was remarkably decreased, and restored in liver tissue from model SD rats infected with Lenti-si-Gremlin1 virus (n = 6 per group, model SD rats infected with Lenti-si-Gremlin1 versus Lenti-si-NC, *p < 0.05; **p < 0.01).

Figure 4. The miR-23b/27b cluster downregulates gremlin1 expression

(A) Prediction of conserved miRNAs targeting the 3′-UTR of rat gremlin1 mRNA using Targetscan online service. Conserved binding sites for miR-23a/b, miR-27a/b and miR-181a/b/c/d were predicted in 3107–3442 nt region (G3-23-1) and for miR-182 in the 3590–3809 nt region (G3-23-3). (B) Nine conserved miRNAs, including miR-23a/b, miR-27a/b, miR-181a/b/c/d and miRNA-182, could recognize and bind the 3′-UTR of gremlin1 mRNA. (C–D) Verification of interactions between rat miR-23a/27a or miR-23b/27b and 3′-UTR of rat gremlin1 mRNA via determination of luciferase activities of HSC-T6 cells co-transfected with pMIR-Luc-3′-UTR and miRNA mimics or inhibitors. **p < 0.01 for comparing between three groups data: miRNA mimic comparing with mimic control; miRNA mimic comparing with its inhibitor; miRNA mimic inhibitor comparing with inhibitor control. (E–F) Western blot assay confirmed that gremlin1 is a cognate target of post-transcriptional repression by miR-23b/27b. Downregulation of gremlin1 expression led to inhibition of collagen Iα1 synthesis. Data in histogram figures represent means ± s.d (n = 3; *P < 0.05).

Figure 5. Downregulation of gremlin1 expression by the miR-23b/27b cluster leads to decreased expression of hepatic fibrosis-related factors

(A–C) Expression levels of hepatic fibrosis-related factors, including α-SMA, TGF-β, collagen Iα1 and collagen Iα2, were critically assessed via western blot of the proteins from HSC-T6 cells transfected with miR-23b/27b mimics, inhibitors, negative control or inhibitor control, respectively. Relative protein level of Gremlin1, TGF-β, collagen Iα1, collagen Iα2 and α-SMA vs their inhibitors, respectively and all results were expressed as means ± SD (n = 3; *P < 0.05, **P < 0.01). Relative protein level of Gremlin1, TGF-β, collagen Iα1, collagen Iα2 and α-SMA vs their NC (Negative control), respectively and all results were expressed as means ± SD (n = 3; ^#P < 0.05, ^##P < 0.01).

Figure 6. Schematic representation of suppression of HSC activation through downregulation of gremlin1 expression by the miR-23b/27b