Hypoxia-inducible factor-1α regulates the expression of genes in hypoxic hepatic stellate cells important for collagen deposition and angiogenesis

Abstract

Background/aims: Several studies have shown that regions of hypoxia develop in the liver during chronic injury. Furthermore, it has been demonstrated that hypoxia stimulates the release of mediators from hepatic stellate cells (HSCs) that may affect the progression of fibrosis. The mechanism by which hypoxia modulates gene expression in HSCs is not known. Recent studies demonstrated that the hypoxia-activated transcription factor, hypoxia-inducible factor (HIF)-1α, is critical for the development of fibrosis. Accordingly, the hypothesis was tested that HIF-1α is activated in HSCs and regulates the expression of genes important for HSC activation and liver fibrosis.

Methods: Hepatic stellate cells were isolated from mice and exposed to hypoxia. HIF-1α and HIF-2α activation were measured, and gene expression was analysed by gene array analysis. To identify the genes regulated by HIF-1α, HSCs were isolated from control and HIF-1α-deficient mice.

Results: Exposure of primary mouse HSCs to 0.5% oxygen activated HIF-1α and HIF-2α. mRNA levels of numerous genes were increased in HSCs exposed to 0.5% oxygen, many of which are important for HSC function, angiogenesis and collagen synthesis. Of the mRNAs increased, chemokine receptor (Ccr) 1, Ccr5, macrophage migration inhibitory factor, interleukin-13 receptor α1 and prolyl-4-hydroxylase α2 (P4h α2) were completely HIF-1α dependent. Upregulation of the vascular endothelial growth factor and the placental growth factor was partially HIF-1α dependent and upregulation of angiopoietin-like 4 and P4h α1 was HIF-1α independent.

Conclusions: Results from these studies demonstrate that hypoxia, through activation of HIF-1α, regulates the expression of genes that may alter the sensitivity of HSCs to certain activators and chemotaxins, and regulates the expression of genes important for angiogenesis and collagen synthesis.

Fig. 1

Hepatic stellate cells were isolated from mice and exposed to room air or 0.5% oxygen for 1 hour. HIF-1α and lamin B1 (i.e., loading control) were then detected in nuclear extracts by western blot. (A) Representative western blot of an n=3. Hepatic stellate cells were exposed to room air (B and C) or 0.5% oxygen (D and E) for 1 hour. Immunohistochemistry was used to detect HIF-1α (red fluorescence; B and D) and α-smooth muscle actin (green fluorescence; BE). The same cells were counterstained with DAPI (blue fluorescence; C and E) to identify the nuclei and show colocalization of HIF-1α immunostaining with nuclear staining. Arrows indicate nuclear HIF-1α immunostaining. Representative of an n=3.

Fig. 2

Hepatic stellate cells were isolated from HIF-1α^fl/fl MxCre- mice and HIF-1α^fl/fl MxCre+ mice and exposed to 0.5% oxygen for 1 hour. HIF-1α and lamin B1 (i.e., loading control) were then detected in nuclear extracts by western blot.

Fig. 3

Hepatic stellate cells were isolated from HIF-1α-Control and HIF-1α-Deficient mice, activated in culture for 7 days, and then exposed to room air or 0.5% oxygen. Eighteen hours later, (A) VEGF, (B) PGF, (C) AngL4, (D) MIF, and (E) PDGF-B mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatic stellate cells exposed to room air (p<0.05). ^bSignificantly different from HIF-1α-Control hepatic stellate cells exposed 0.5% oxygen (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 4

Hepatic stellate cells were isolated from HIF-1α-Control and HIF-1α-Deficient mice, activated in culture for 7 days, and then exposed to room air or 0.5% oxygen. Eighteen hours later, (A) Ccr1 and (B) Ccr5 mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatic stellate cells exposed to room air (p<0.05). ^bSignificantly different from HIF-1α-Control hepatic stellate cells exposed 0.5% oxygen (p<0.05). (C) Ccr5 and β-actin (i.e., loading control) proteins were detected by western blot. Data are expressed as means ± SEM; n = 3.

Fig. 5

Hepatic stellate cells were isolated from HIF-1α-Control and HIF-1α-Deficient mice, activated in culture for 7 days, and then exposed to room air or 0.5% oxygen. Eighteen hours later, (A) Adrα2b and (B) IL-13rα1 mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatic stellate cells exposed to room air (p<0.05). ^bSignificantly different from HIF-1α-Control hepatic stellate cells exposed 0.5% oxygen (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 6

Hepatic stellate cells were isolated from HIF-1α-Control and HIF-1α-Deficient mice, activated in culture for 7 days, and then exposed to room air or 0.5% oxygen. Eighteen hours later, (A) P4hα1 and (B) P4hα2 mRNA levels were quantified by real-time PCR. ^aSignificantly different from hepatic stellate cells exposed to room air (p<0.05). ^bSignificantly different from HIF-1α-Control hepatic stellate cells exposed 0.5% oxygen (p<0.05). Data are expressed as means ± SEM; n = 3.

Fig. 7

Hepatic stellate cells were isolated from mice, activated in culture for 7 days, and then exposed to room air or 0.5% oxygen for 1 hour. HIF-2α and lamin B1 were then detected in nuclear extracts by western blot. (A) Representative western blot of an n=3. Hepatic stellate cells were exposed to room air (B and C) or 0.5% oxygen (D and E) for 1 hour. Immunohistochemistry was used to detect HIF-2α (red fluorescence; B and D) and α-smooth muscle actin (green fluorescence; B–E). The same cells were counterstained with DAPI (blue fluorescence; C and E) to identify the nuclei and show colocalization of HIF-2α immunostaining with nuclear staining. Arrows indicate nuclear HIF-2α immunostaining. Representative of an n=3.