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

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.