Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

Abstract

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.

Keywords: Hepatic stellate cell; IL-1α; Liver fibrosis; TNF-α.

Fig. 1

Effect of inflammatory cytokines on MMP1 expression in primary HSCs. Primary HSCs were isolated from excised human livers. Following serum starvation, TGF-β, IL-1α, IL-6, TNF-α and PDGF were added to the medium at 1.0 ng/mL and the cells were incubated for a further 24 h. MMP1 mRNA expression was measured by qPCR. Untreated cells were used as a control. The results derived from two patients are shown (panel A and B). Because only a low number of HSCs can be obtained from a hepatocellular carcinoma patient liver, experiments using identical HSCs could not be repeated. However, the synergistic effect of the combined cytokines was similar among two different patients

Fig. 2

Identification of cytokines enhancing MMP1 expression in primary HSCs. Primary HSCs were isolated from excised human livers. Following serum starvation, TGF-β, IL-1α, IL-6, TNF-α and PDGF were added to the medium at 1.0 ng/mL as indicated in Figure and the cells were incubated for a further 24 h. MMP1 mRNA expression was measured by qPCR. Untreated cells were used as a control. The results derived from two patients are shown (panel A and B)

Fig. 3

Effect of TNF-α and IL-1 α on MMP1 expression in primary HSCs. TNF-α and IL-1α cytokines were simultaneously exposed to primary HSCs at 1.0 ng/mL for 24 h. MMP1 mRNA expression levels were measured after exposure to individual and combined cytokines using qPCR. Untreated HSCs were used as a control. The results derived from two patients are shown (panel A and B). Each bar represents the means and standard deviations of three independent experiments

Fig. 4

Effect of TNF-α and IL-1 α on α-SMA expression in primary HSCs. TNF-α and IL-1α cytokines were simultaneously exposed to primary HSCs at 1.0 ng/mL for 24 h. α-SMA mRNA expression levels were measured after exposure to individual and combined cytokines using qPCR. Untreated HSCs were used as a control. The results derived from two patients are shown (panel A and B). Each bar represents the means and standard deviations of three independent experiments

Fig. 5

Attenuation of α-SMA protein expression in primary HSC by TNF-α and IL-1α. Following serum starvation, IL-1α and TNF-α were added to the medium at 1.0 ng/mL and incubated for further 24 h. The cells were fixed and α-SMA protein was identified by immunohistochemistry

Fig. 6

Effect of TNF-α and IL-1 α on MMP1 and α-SMA expression in the LX2 cell line. Following serum starvation, IL-1α and TNF-α were added to the LX2 cultured medium at 1.0 ng/mL and cells were incubated for a further 24 h. MMP1 and α-SMA mRNA expression was measured by qPCR. Untreated cells were used as a control. Each bar represents the means and standard deviations of five independent experiments