Hes1, an important gene for activation of hepatic stellate cells, is regulated by Notch1 and TGF-β/BMP signaling

Abstract

Aim: To determine the role of Notch1 and Hes1 in regulating the activation of hepatic stellate cells (HSCs) and whether Hes1 is regulated by transforming growth factor (TGF)/bone morphogenetic protein (BMP) signaling.

Methods: Immunofluorescence staining was used to detect the expression of desmin, glial fibrillary acidic protein and the myofibroblastic marker α-smooth muscle actin (α-SMA) after freshly isolated, normal rat HSCs had been activated in culture for different numbers of days (0, 1, 3, 7 and 10 d). The expression of α-SMA, collagen1α2 (COL1α2), Notch receptors (Notch1-4), and the Notch target genes Hes1 and Hey1 were analyzed by reverse transcriptase-polymerase chain reaction. Luciferase reporter assays and Western blot were used to study the regulation of α-SMA, COL1α1, COL1α2 and Hes1 by NICD1, Hes1, CA-ALK3, and CA-ALK5 in HSC-T6 cells. Moreover, the effects of inhibiting Hes1 function in HSC-T6 cells using a Hes1 decoy were also investigated.

Results: The expression of Notch1 and Hes1 mRNAs was significantly down-regulated during the culture of freshly isolated HSCs. In HSC-T6 cells, Notch1 inhibited the promoter activities of α-SMA, COL1α1 and COL1α2. On the other hand, Hes1 enhanced the promoter activities of α-SMA and COL1α2, and this effect could be blocked by inhibiting Hes1 function with a Hes1 decoy. Furthermore, co-transfection of pcDNA3-CA-ALK3 (BMP signaling activin receptor-like kinase 3) and pcDNA3.1-NICD1 further increased the expression of Hes1 compared with transfection of either vector alone in HSC-T6 cells, while pcDNA3-CA-ALK5 (TGF-β signaling activin receptor-like kinase 5) reduced the effect of NICD1 on Hes1 expression.

Conclusion: Selective interruption of Hes1 or maintenance of Hes1 at a reasonable level decreases the promoter activities of α-SMA and COL1α2, and these conditions may provide an anti-fibrotic strategy against hepatic fibrosis.

Keywords: Hepatic fibrosis; Hepatic stellate cells; Hes1; Notch1; TGF-β/BMP.

Figure 1

Characteristics of quiescent and activated hepatic stellate cells. A: Microscopic pictures of freshly isolated hepatic stellate cells (HSCs) after 1, 3, 7 and 10 d of culture; B: The expression of α-smooth muscle actin (α-SMA), desmin and GFAP detected by immunofluorescence staining. Red fluorescence represents α-SMA in pictures a, b, c, and d; desmin in pictures e, f, g, and h; and GFAP in pictures i, j, k, and l. The cell nuclei were visualized by DAPI (4, 6-diamidino-2-phenylindole) staining (blue). C: Expression analysis of collagen1α2, α-SMA, E-cadherin, transforming growth factor-β1 (TGFβ1), bone morphogenetic protein-7 (BMP7), and β-catenin in the cells in (A) measured by RT-PCR. RT-PCR of Gapdh served as a control; D: Analysis of α-SMA, Hes1 and TGF-β (R) by Western blotting of the cells in (A). The β-actin protein served as a control; E: RT-PCR analysis of Notch receptors (Notch1-4) and the Notch target genes Hes1 and Hey1. Notably, the data for Hes1 in D and E do not agree with each other.

Figure 2

NICD1 suppresses the expression of myofibroblastic markers in hepatic stellate cell-T6 cells. A-C: Luciferase reporter assays for the promoter activities of α-SMA, COL1α1, COL1α2 in cells transfected with empty plasmid-pcDNA3.1(+) as a control or with pcDNA3.1-NICD1 (^aP < 0.05 vs the control group, n = 8). α-SMA: α-smooth muscle actin; COL1α1: Collagen1α1.

Figure 3

Over-expression of the Notch target gene Hes1 increases the expression of α-smooth muscle actin and Collagen1α2 in hepatic stellate cell-T6 cells but has no effect on the expression of Collagen1α1. A, C, D: Luciferase reporter assays for the promoter activities of α-SMA, COL1α1 and COL1α2 in cells transfected with pcDNA-Hes1 or empty plasmid-pcDNA3.1(+) as a control (^aP < 0.05, ^bP < 0.01 vs the control group, n = 8); B: Analysis of α-SMA and Hes1 by Western blot analysis of transfected cells. The β-actin protein served as a control (^cP < 0.05 vs all the control groups). α-SMA: α-smooth muscle actin; COL1α1: Collagen1α1.

Figure 4

Inhibition of Hes1 transcription factor function by a Hes1 decoy down-regulates the promoter activities of α-smooth muscle actin and Collagen1α2 in hepatic stellate cell-T6 cells. The promoter activities of α-SMA and col1α2 decreased in cells treated with a Hes1 ODN decoy compared with the control (SCR) (A, B). (^aP < 0.05 vs the control and SCR groups, n = 8). α-SMA: α-smooth muscle actin; ODN: Oligodeoxynucleotide.

Figure 5

Notch target gene Hes1 is regulated by the transforming growth factor-β/bone morphogenetic protein signaling pathway in hepatic stellate cell-T6 cells. A: Western blot analysis of Hes1 expression in cells transfected with pcDNA3.1-NICD1. The β-actin protein served as a control; B: Luciferase reporter assays for the Hes1 promoter in cells transfected with pcDNA3-NICD1 and/or pcDNA3-CA-ALK3 (^aP < 0.05, ^bP < 0.01 vs all the control groups, n = 8); C: Analysis of Hes1 and TGF-β(R) by Western blot analysis of cells transfected with pcDNA3-CA-ALK3 with the β-actin protein as a control; D: Luciferase reporter assays for the Hes1 promoter in cells transfected with pcDNA3.1-NICD1 and/or pcDNA3-CA-ALK5 together (^dP < 0.01 vs the control group, n = 8). TGF: Transforming growth factor.