Hypoxia stimulates hepatocyte epithelial to mesenchymal transition by hypoxia-inducible factor and transforming growth factor-beta-dependent mechanisms

Abstract

Background/aims: During development of liver fibrosis, an important source of myofibroblasts is hepatocytes, which differentiate into myofibroblasts by epithelial to mesenchymal transition (EMT). In epithelial tumours and kidney fibrosis, hypoxia, through activation of hypoxia-inducible factors (HIFs), is an important stimulus of EMT. Our recent studies demonstrated that HIF-1alpha is important for the development of liver fibrosis. Accordingly, the hypothesis was tested that hypoxia stimulates hepatocyte EMT by a HIF-dependent mechanism.

Methods: Primary mouse hepatocytes were exposed to room air or 1% oxygen and EMT evaluated. In addition, bile duct ligations (BDLs) were performed in control and HIF-1alpha-deficient mice and EMT quantified.

Results: Exposure of hepatocytes to 1% oxygen increased expression of alpha-smooth muscle actin, vimentin, Snail and fibroblast-specific protein-1 (FSP-1). Levels of E-cadherin and zona occludens-1 were decreased. Upregulation of FSP-1 and Snail by hypoxia was completely prevented in HIF-1beta-deficient hepatocytes and by pretreatment with SB431542, a transforming growth factor-beta (TGF-beta) receptor inhibitor. HIFs promoted TGF-beta-dependent EMT by stimulating activation of latent TGF-beta1. To determine whether HIF-1alpha contributes to EMT in the liver during the development of fibrosis, control and HIF-1alpha-deficient mice were subjected to BDL. FSP-1 was increased to a greater extent in the livers of control mice when compared with HIF-1alpha-deficient mice.

Conclusions: Results from these studies demonstrate that hypoxia stimulates hepatocyte EMT by a HIF and TGF-beta-dependent mechanism. Furthermore, these studies suggest that HIF-1alpha is important for EMT in the liver during the development of fibrosis.

Fig. 1

Hepatocytes were isolated from mice and exposed to room air or 1% oxygen for the indicated time. (A) FSP-1, (B) α-SMA, and (C) vimentin mRNA levels were measured by real-time PCR. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 2

Hepatocytes were isolated from mice and exposed to room air or 1% oxygen for the indicated time (A, E, and F) or for 72 hours (B–D). (A) Snail mRNA levels were measured by real-time PCR. (B) Snail protein was measured in nuclear extracts by western blot. Snail protein was detected (red fluorescence) in hepatocytes exposed to (C) room air or (D) 1% oxygen by immunocytochemistry. Arrows indicate nuclear staining of Snail protein in hepatocytes exposed to 1% oxygen. (E) TWIST and (F) SLUG mRNA levels were measured by real-time PCR. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 3

Hepatic stellate cells were isolated from mice and exposed to room air or 1% oxygen for 72 hours. (A) α-SMA, (B) FSP-1, and (C) Snail mRNA levels were measured by real-time PCR. mRNA levels are reported relative to room air. Data are expressed as means ± SEM; n = 3.

Fig. 4

Hepatocytes were isolated from mice and exposed to room air (A, C, and E) or 1% oxygen (B, D, and F). (A and B) Three days later, E-cadherin was detected by immunocytochemistry (red fluorescence). (C and D) ZO-1 was detected by immunocytochemistry (red fluorescence). Higher power image is shown in the inset. In A–D the same cells were counterstained with DAPI (blue fluorescence) to identify the nuclei. (E and F) F-actin was detected by incubating with fluorescently-labeled phalloidin (red fluorescence).

Fig. 5

Hepatocytes were isolated from mice and exposed to room air (A, C, and E) or 1% oxygen (B, D, and F). (A and B) Light microscopic images of hepatocytes. Vimentin (C and D) and FSP-1 (E and F) were detected by immunocytochemistry (red fluorescence). In C–F the same cells were counterstained with DAPI (blue fluorescence) to identify the nuclei.

Fig. 6

Hepatocytes were isolated from HIF-1β-Control and HIF-1β-Deficient mice, and exposed to room air or 1% oxygen. Seventy two hours later, (A) Glut-1, (B) FSP-1, (C) Snail, and (D) α-SMA mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). ^bSignificantly different from HIF-1β-Control hepatocytes exposed to the same concentration of oxygen (p<0.05).

Fig. 7

Hepatocytes were isolated from mice and treated with vehicle or SB-431542 for 30 minutes. The cells were then exposed to room air, 1% oxygen, or treated with TGF-β1. After 72 hours, (A) FSP-1, (B) Snail, and (C) Glut-1 mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). ^bSignificantly different from vehicle-treated hepatocytes exposed to the same concentration of oxygen (p<0.05). ^cSignificantly different from hepatocytes exposed to vehicle and room air (p<0.05). ^dSignificantly different from TGF-β1-treated hepatocytes (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 8

Hepatocytes were isolated from mice and treated with 10 μM SB-431542. Thirty minutes later, the cells were exposed to room air or 1% oxygen for 1 hour. (A) HIF-1α and (B) HIF-2α were detected by western blot. Representative of an n = 4. Hepatocytes were isolated from mice and treated with vehicle, 5 ng/ml TGF-β1, or exposed to 1% oxygen for 1 hour. (C) HIF-1α and (D) HIF-2α were detected by western blot. Representative of an n = 4.

Fig. 9

Hepatocytes were isolated from mice and exposed to room air or 1% oxygen for 24 or 48 hours. (A) TGF-β1, (B) TGF-β2, and (C) TGF-β3 mRNA levels were quantified by real-time PCR. Data are expressed as means ± SEM; n = 3.

Fig. 10

Hepatocytes were exposed to room air or 1% oxygen for 24 hours. Active and total TGF-β1 were quantified by ELISA. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). Data are expressed as means ± SEM; n = 3. Hepatocytes were treated with vehicle or 20 ng/ml latent TGF-β1 followed by exposure to room air or 1% oxygen. 72 hours later, FSP-1 and Snail mRNA levels were quantified. ^aSignificantly different from hepatocytes exposed to room air (p<0.05). ^bSignificantly different from hepatocytes exposed to vehicle and 1% oxygen (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 11

Control mice and HIF-1α-Deficient mice were subjected to bile duct ligation or sham operation. Fourteen days later, FSP-1 was detected in the liver by immunohistochemistry. Representative photomicrograph of a section of liver from a (A) Control mouse sham operation, (B) HIF-1α-Deficient mouse sham operation, (C) Control mouse bile duct ligation, and (D) HIF-1α-Deficient mouse bile duct ligation stained for FSP-1. Positive staining for collagen appears dark grey in the photomicrographs.

Fig. 12

Control mice and HIF-1α-Deficient mice were subjected to bile duct ligation or sham operation. Fourteen days later, FSP-1 was detected in the liver by immunohistochemistry. (A) Total, peribiliary, and nonperibiliary area of FSP-1 immunostaining was analyzed morphometrically. (B) FSP-1 mRNA levels were quantified in the liver by real-time PCR. ^aSignificantly different from sham-operated mice (p<0.05). ^bSignificantly different from Control mice subjected to bile duct ligation (p<0.05). Data are expressed as means ± SEM; n = 6.

Fig. 13

Wild-type mice were subjected to bile duct ligation. Ten days later, FSP-1 (A and D, red staining), CK19 (B, green staining), and albumin (E, green staining) were detected by immunohistochemistry. (C) FSP-1 immunostaining (red) in A and CK19 immunostaining (green) in B were overlaid to detect colocalization. Higher power image in A, B, and C shown in inset. (F) FSP-1 immunostaining (red) in D and albumin immunostaining (green) in E were overlaid to detect colocalization. Arrows indicate FSP-1 positive cells that also stain positive for albumin.