Abstract
Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.
Highlights
Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis
To clarify the significance of Endoplasmic reticulum oxidase 1 (ERO1)␣ in HSCs, we investigated the proliferation of HSCs by silencing ERO1␣ expression
Because the knockdown efficiency of ERO1␣ expression in LX-2 cells transfected with ERO1␣-A siRNA was the highest among the three batches of ERO1␣ siRNAs, and transfection with ERO1␣-A siRNA inhibited the expression of ERO1␣ protein in LX-2 cells, LI90, cells and primary HSCs (Fig. 2B), we used ERO1␣-A siRNA for the cell proliferation assay
Summary
We first confirmed the expression of ERO1␣ in human hepatic stellate cell lines (LX-2 and LI90) and primary human. There was no significant difference in the expression levels of collagen 1␣1 mRNA and collagen 1␣2 mRNA between cells stably expressing control shRNA with the Akt inhibitor and cells stably expressing ERO1␣ shRNA with the Akt inhibitor, immunoblotting revealed that excessive accumulation of intracellular collagen type 1 in cells (LX-2 and LI90) stably expressing ERO1␣ shRNA was markedly ameliorated by treatment with the Akt inhibitor (Fig. 6G) These results indicate that silencing of ERO1␣ inhibits autophagy through activation of the Akt signaling pathway, causing excessive accumulation of collagens in HSCs. It has been shown that the proliferation of HSCs progresses through the modification of collagens with MMP2 and MT1MMP, which are expressed by HSCs themselves [8, 10]. A cell proliferation assay showed that the proliferation of two cell lines (LX-2 and LI90) stably expressing ERO1␣ shRNA was significantly enhanced by addition of MT1-MMP– cleaved collagen type 1, as was observed in two cell lines (LX-2 and LI90) stably expressing control shRNA (Fig. 8, A and B)
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