Research ArticleLIVER FIBROSIS

Hyaluronan synthase 2–mediated hyaluronan production mediates Notch1 activation and liver fibrosis

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Science Translational Medicine  12 Jun 2019:
Vol. 11, Issue 496, eaat9284
DOI: 10.1126/scitranslmed.aat9284
  • Fig. 1 Expression of HAS2 and HA is increased in human liver fibrosis.

    (A) Hepatic HAS2 mRNA expression in patients with fibrosis and chronic hepatitis B virus (HBV). (B) Representative immunohistochemical (IHC) staining images of HAS2 in liver sections of patients with HBV with F0/1 or F4 stages. Boxed regions are shown at higher magnification in the bottom row. Scale bar, 100 μm. (C) Colocalization of α-SMA and HAS2 in the liver of patients with HBV. Representative immunofluorescence staining images are shown (F0/1, n = 10; F4, n = 10). Boxed regions are shown at higher magnification. Blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. (D) Pearson correlation coefficient analysis of HAS2 mRNA expression and serum HA concentration of patients with HBV. (E and F) Representative images and quantification of HA staining in liver sections of patients with NASH-mediated liver fibrosis. HABP, HA-binding protein. Scale bar, 100 μm. (G) Hepatic HAS2 mRNA expression in patients with NAFLD. F0, no fibrosis; F1 to F4, fibrosis. In (A), (F), and (G), data are means ± SD. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis (A and F) and two-tailed Student’s t test (G). *P < 0.05 and **P < 0.01.

  • Fig. 2 HAS2 and HA are overexpressed in murine liver fibrosis.

    (A) Representative images of HA staining in mouse liver fibrosis. ND, normal diet; CSAA, choline-sufficient amino acid–defined. Scale bar, 100 μm. (B) Representative double immunofluorescence staining images of desmin and HA. Boxed regions are shown at higher magnification. (C) Hepatic Has mRNA expression in mouse liver fibrosis (sham, n = 10 or BDL, n = 12; ND, n = 5 or DDC, n = 9; ND, n = 5 or CDHFD diet, n = 7; corn oil, n = 5 or CCl4, n = 8). Mice underwent BDL for 3 weeks. *P < 0.05 and **P < 0.01 versus relative control. (D) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for Has2 mRNA in primary HSCs, Kupffer cells (KC), and hepatocytes (HEP) isolated from sham- or BDL-operated mice 5 days after surgery (n = 3 per group). **P < 0.01; N.S., not significant. (E) qRT-PCR assays for Has2 mRNA in mouse liver cells (n = 3 per group). Activated HSC: 7-day culture-activated HSC. **P < 0.01. Data are means ± SEM. Two-tailed Student’s t test (C and D) and one-way ANOVA with Tukey’s post hoc analysis (E).

  • Fig. 3 HSC-derived HAS2 promotes liver fibrosis.

    (A) Representative images of HA and Sirius red staining of sections of mouse liver. Mice underwent BDL for 2 weeks (Sham-WT, n = 5; Sham-Tg, n = 3; BDL-WT, n = 10; BDL-Tg, n = 7). (B) Liver HA content by enzyme-linked immunosorbent assay (ELISA). (C) Sirius red–positive area (collagen) from (A). (D) HA and Sirius red staining of sections of mouse liver. Mice were injected with corn oil (Con) or CCl4 intraperitoneally twice a week for 5 weeks (Con, n = 3 per group; CCl4-WT, n = 7; CCl4ASMA-HAS2 Tg, n = 8). (E) Liver HA content by ELISA. (F) Sirius red–positive area from (D). (G) Representative images of HA and Sirius red staining of sections of mouse liver (Sham-WT, n = 8; Sham-Has2ΔHSC, n = 3; BDL-WT, n = 11; BDL-Has2ΔHSC, n = 9). (H) Liver HA content by ELISA. (I) Quantification of Sirius red–positive area from (G) (Sham groups, n = 3, each). (J) HA and Sirius red staining of sections of mouse liver. Mice were fed with ND or CDHFD for 12 weeks. (K) Liver HA content by ELISA (ND, n = 5 per group; CDHFD, n = 8 per group). (L) Sirius red–positive area from (J) (ND, n = 5 per group; CDHFD, n = 7 per group). Data are means ± SEM. *P < 0.05 and **P < 0.01. One-way ANOVA with Tukey’s post hoc analysis. Scale bars, 100 μm.

  • Fig. 4 HSC-derived HAS2 regulates HSC function.

    (A) A heat map of genes related to ECM-receptor interaction. RNA-seq was performed using quiescent Has2ΔHSC HSCs or HAS2 Tg HSCs (n = 3 per group). RPKM, reads per kilobase million. (B) qRT-PCR assays for Itga1, Fn1, Col4a1, Col1a1, and Acta2 mRNA expression in quiescent WT, Has2ΔHSC, or HAS2 Tg HSCs (n = 3 to 6 per group). (C) BrdU (5-bromo-2′-deoxyuridine) incorporation assay. Proliferation of WT, Has2ΔHSC, or HAS2 Tg HSCs was assessed by BrdU incorporation for 24 hours (n = 3 per group). (D) Microscopic images of the Matrigel-invading capacity of HSCs isolated from WT, Has2ΔHSC, or HAS2 Tg mice. Representative images are shown. Scale bar, 100 μm (n = 4 per group). Data are means ± SEM. **P < 0.01 versus WT HSC. One-way ANOVA with Tukey’s post hoc analysis.

  • Fig. 5 CD44 and TLR4 mediate HA-induced HSC profibrogenic phenotypic change.

    (A) Total and fractionated serum HA content in patients with fibrosis and NAFLD (F0/1, F2, and F3, n = 5 per group; F4, n = 9). *P < 0.05 and **P < 0.01 versus F0/1; #P < 0.05 versus F2; δP < 0.05 versus F3. (B) Fractionated HA content in murine liver (sham-operated, n = 7; BDL-operated, n = 10). (C) Profibrogenic and proinflammatory gene expression in mouse HSCs and (D) in mouse Kupffer cells (n = 3 to 5 per group). Cells were treated with vehicle (Con), LMW-HA, or HMW-HA. *P < 0.05 and **P < 0.01 versus Con. (E) Representative images of immunofluorescence staining of sections of mouse liver. Blue, DAPI. Scale bar, 100 μm. (F) A heat map of genes related to ECM-receptor interaction. Each lane represents pooled WT, Cd44−/−, or Tlr4−/− HSCs from three mice. (G) qRT-PCR assay (n = 3 per group). Mouse HSCs were treated with or without LMW-HA for 12 hours. (H) BrdU incorporation assay. HSCs were treated with conditioned medium (CM, days 4 to 7) (n = 6 per group). (I) Matrigel invasion assay (n = 4 per group). WT, Cd44−/−, or Tlr4−/− HSCs were plated onto the upper chamber, and conditioned medium was placed in the lower chamber. (J) Migration of WT, Cd44−/−, or Tlr4−/− Kupffer cells toward HSC-conditioned medium (n = 3 to 6 per group). In (G) to (J), **P < 0.01 versus vehicle (or WT CM)–treated WT; #P < 0.05 and ##P < 0.01 versus LMW-HA (or HAS2 Tg CM)–treated WT. Data are means ± SEM. One-way ANOVA with Tukey’s post hoc analysis.

  • Fig. 6 Notch1, as a downstream molecule of HAS2-HA-CD44 signaling, promotes liver fibrosis.

    (A and B) Heat maps of genes related to Notch pathway. (A) RNA-seq for HSCs from Has2ΔHSC or HAS2 Tg mice. (B) RNA-seq for HSCs from WT, Cd44−/−, or Tlr4−/− mice. (C to F) Effect of Has2 or CD44 modulation on Notch1. (C) Liver (n = 8 per group), (C and D) HSCs (n = 3 to 4 per group), and (E and F) human HSC line LX-2 (n = 5 to 6). (G) ChIP assay (n = 4). IgG, immunoglobulin G. (H) NOTCH1 promoter activity in human embryonic kidney 293 A (HEK293A) (three replicates in each of four experiments). **P < 0.01 versus −1239/+1684 (WT) and mock-transfected; ##P < 0.01 versus −1239/+1684 (WT) and CD44-transfected. (I) qRT-PCR for Timp1 and Ccl2 (n = 3). Mouse HSCs were treated with vehicle (Con), LMW-HA, or LPS for 12 hours. **P < 0.01 versus WT-Con; ##P < 0.01 versus WT-LMW; N.S. versus WT-LPS. (J) BrdU incorporation assay (n = 9). (K) Matrigel invasion assay (n = 3). (L) Effect of Notch1 depletion on expression of fibrogenic mRNAs in HSCs (n = 3). (M) Sirius red staining and quantification of collagen deposition in liver sections from mice with HSC-specific Notch1 deletion 3 weeks after BDL (sham-operated, n = 3 per group; BDL-operated, n = 7 per group). Scale bar, 100 μm. (N) Sirius red staining in liver sections 2 weeks after BDL (WT, n = 10; Notch1ΔHSC, n = 9; ASMA-HAS2 Tg, n = 6; ASMA-HAS2 Tg/Notch1ΔHSC, n = 7). (C to N) Data are means ± SEM. (O) qRT-PCR (means ± SD) and (P) Pearson correlation coefficient analysis in liver tissue from patients with fibrosis and chronic hepatitis B. Two-tailed Student’s t test (C to F and L) and one-way ANOVA with Tukey’s post hoc analysis (G to K and M to O). *P < 0.05 and **P < 0.01.

  • Fig. 7 WT1 transcriptionally regulates HAS2 in HSCs.

    (A) qRT-PCR assay for Has2 after TGF-β treatment in mouse HSCs (n = 5). (B) Site-directed mutagenesis analysis of WT1-binding sites in the Has2 promoter (three replicates in each of five experiments). **P < 0.01 versus pGL3-Has2 (WT) and mock-transfected cells; ##P < 0.01 versus pGL3-Has2 (WT) and WT1-transfected cells. (C) Effect of WT1 overexpression on Has2 mRNA expression (n = 3). (D) qRT-PCR assays for Wt1 after TGF-β treatment (n = 5). (E and F) Effect of WT1 knockdown on TGF-β–induced Has2 and Col1a1 mRNA (n = 3 to 7). (G) Col1a1 mRNA expression (n = 7 to 8). Mouse HSCs were treated with or without TGF-β. (H) Wt1 mRNA expression in quiescent (day 1) or activated (day 7) HSCs (n = 3 per group) and (I) in the liver (sham, n = 10; BDL, n = 12; Con, n = 8; CCl4, n = 24) and in vivo–activated HSCs (n = 3 per group). (J) WT1 mRNA expression in patients with fibrosis and chronic hepatitis B. (K) Pearson correlation coefficient analysis of WT1 and HAS2. (A to I) Data are means ± SEM and (J) SD. *P < 0.05 and **P < 0.01 versus relative control; #P < 0.05 and ##P < 0.01 versus WT–TGF-β. Two-tailed Student’s t test (A, C and D, and H and I) and one-way ANOVA with Tukey’s post hoc analysis (B, E to G, and J).

  • Fig. 8 4-MU treatment prevents liver fibrosis progression.

    (A) Representative immunofluorescence staining images. HSCs were isolated from Col-GFP mice. Two days after seeding, cells were treated with either vehicle (Veh) or 0.5 mM 4-MU for 6 days. (B) Quantification of GFP-positive area in (A) by ImageJ (n = 3). (C) Col1a1 mRNA expression (n = 3). (D) Representative images of HA and Sirius red staining in mouse liver. One week after BDL, mice were orally gavaged with either Veh or 4-MU (450 mg/kg) once daily for 2 weeks. (E) Liver HA content after 4-MU treatment (Sham-Veh, n = 4; Sham–4-MU, n = 5; BDL-Veh, n = 11; BDL–4-MU, n = 10). (F) Quantification of Sirius red staining in (D) (Sham-Veh, n = 4; Sham–4-MU, n = 5; BDL-Veh, n = 5; BDL–4-MU, n = 10). (G) Sirius red staining in mouse liver. At 11 weeks of CDHFD feeding, mice were orally gavaged daily with Veh or 4-MU (450 mg/kg) for 4 weeks (ND, n = 6 per group; CDHFD-Veh, n = 9; CDHFD–4-MU, n = 10). Data are means ± SEM. **P < 0.01. Two-tailed Student’s t test (B and C) and one-way ANOVA with Tukey’s post hoc analysis (E to G). Scale bars, 100 μm.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/496/eaat9284/DC1

    Materials and Methods

    Fig. S1. Expression of HAS in patients with fibrosis.

    Fig. S2. Expression of HA and Has expression in mouse liver cells.

    Fig. S3. Increased susceptibility to BDL- or CCl4-induced liver fibrosis in ASMA-HAS2 Tg mice.

    Fig. S4. Generation of HSC-specific Has2 knockout mice.

    Fig. S5. Effect of HSC-specific Has2 deficiency on DDC-induced liver fibrosis.

    Fig. S6. No effect of HSC-specific Has2 deletion on CDHFD-mediated fat accumulation or glucose intolerance.

    Fig. S7. Cd44 and Tlr4 mRNA expression in in vivo–activated HSCs.

    Fig. S8. HA concentration in the supernatant of WT HSCs or HAS2 Tg HSCs.

    Fig. S9. Notch1 and its target gene Hes1 mRNA expression.

    Fig. S10. Jagged-1 expression.

    Fig. S11. Inhibition of BDL-induced HSC activation and macrophage infiltration by 4-MU treatment.

    Table S1. Sequence of primers used for qPCR.

    Data file S1. Individual subject-level data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of HAS in patients with fibrosis.
    • Fig. S2. Expression of HA and Has expression in mouse liver cells.
    • Fig. S3. Increased susceptibility to BDL- or CCl4-induced liver fibrosis in ASMA-HAS2 Tg mice.
    • Fig. S4. Generation of HSC-specific Has2 knockout mice.
    • Fig. S5. Effect of HSC-specific Has2 deficiency on DDC-induced liver fibrosis.
    • Fig. S6. No effect of HSC-specific Has2 deletion on CDHFD-mediated fat accumulation or glucose intolerance.
    • Fig. S7. Cd44 and Tlr4 mRNA expression in in vivo–activated HSCs.
    • Fig. S8. HA concentration in the supernatant of WT HSCs or HAS2 Tg HSCs.
    • Fig. S9. Notch1 and its target gene Hes1 mRNA expression.
    • Fig. S10. Jagged-1 expression.
    • Fig. S11. Inhibition of BDL-induced HSC activation and macrophage infiltration by 4-MU treatment.
    • Table S1. Sequence of primers used for qPCR.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data.

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