Cell and Molecular Regulation of Endothelin-1 Production during Hepatic Wound Healing

Abstract

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelinconverting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56-and 62-kDa ECE-1 3Ј-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3Ј UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3Ј UTR mRNA (62 kDa). A functional role for the 3Ј UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-␤1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56-and 62-kDa ECE-1 3Ј UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56-and 62-kDa proteins, which in turn are regulated by transforming growth factor-␤. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised. INTRODUCTIONThe response to hepatic wounding is characterized by the activation of hepatic stellate cells (also lipocytes, fat-storing cells), critical effectors of the wound healing process (Ballardini et al., 1988;Friedman and Arthur, 1989;Kent et al., 1976;Maher and McGuire, 1990). During stellate cell activation, these cells exhibit features of myofibroblasts, producing increased quantities of extracellular matrix proteins as well as smooth muscle ␣-actin. A further characteristic of stellate cell activation is enhanced production of the vasoconstrictive peptide, endothelin-1 (ET-1), which contributes to perpetuation of the fibrogenic process (Gandhi et al., 1998;Rockey and Chung, 1996) as well as in control hepatic vascular tone (Bauer et al., 1994;Kawada et al., 1993;Rockey and Weisiger, 1996).Each of the three known endothelin isoforms (-1, -2, -3) arise by proteolytic processing of large precursors (ϳ200 amino acid residues). Intermediates, termed big ET-1, -2, and -3 (38 -41 aa) are excised from prepropeptides at sites containing paired basic amino acids. Big endothelins, which have little or no biological activity (Yanagisawa, 1994), are cleaved at Trp-21-Val/Ile-22 to produce mature 21-residue, biologically active peptides. The enzyme responsible for the specific cleavage at Trp-21 has been termed endothelinconverting enzyme (ECE); it is a neutral membrane-bound metalloprotease with M r ϭ 120 kDa, belonging to the endopeptidase-24.11 family found in brain (Ohnaka et al., 1993;...

Vascular biology and pathobiology of the liver: Report of a single-topic symposium

“…20 In addition to the well established up-regulation of ET-1 production by TGF-␤, overproduction of endothelin-1 in cirrhotic liver is also due to dysregulation of the converting enzyme that cleaves precursor ET-1 to the mature peptide. 21 Interestingly, endothelin converting enzyme-1 is up-regulated in HSCs, rather than in SECs, indicating that the major cellular source of endothelin in the injured liver are HSCs rather than SECs, thereby highlighting an important autocrine activation loop in HSCs. In addition to alterations in ET-1, eNOS-derived NO is reduced after liver injury, independent of changes in eNOS expression and through defects in posttranslational regulation of the enzyme within ECs.…”

Endothelin-1 Promotes Myofibroblast Induction through the ETA Receptor via a rac/Phosphoinositide 3-Kinase/Akt-dependent Pathway and Is Essential for the Enhanced Contractile Phenotype of Fibrotic Fibroblasts

“…The enzyme responsible for the specific cleavage at Trp-21 has been termed endothelin-converting enzyme; it is a neutral membranebound metalloproteinase with M r ϭ 120 kDa, belonging to the endo-peptidase-24.11 family found in brain (Ohnaka et al, 1993;Turner and Murphy, 1996). Injury and the wound healing response lead to stabilization of endothelin-converting enzyme-1 mRNA and to the generation of bioactive endothelin (Shao et al, 2003).…”

“…The enzyme responsible for the specific cleavage at Trp-21 has been termed endothelin-converting enzyme; it is a neutral membranebound metalloproteinase with M r ϭ 120 kDa, belonging to the endo-peptidase-24.11 family found in brain (Ohnaka et al, 1993;Turner and Murphy, 1996). Injury and the wound healing response lead to stabilization of endothelin-converting enzyme-1 mRNA and to the generation of bioactive endothelin (Shao et al, 2003).ET-1 demonstrates a wide range of biological properties on normal cells (Rubanyi and Botelho, 1991;Levin, 1995), including significant mitogenic activity toward a number of cell types, such as smooth muscle cells and fibroblasts, and the modification of extracellular matrix metabolism (Levin, 1995;Saita et al, 1998;Xu et al, 1998;Shi-Wen et al, 2001). In addition, ET-1 has been reported to promote the contractile ability of normal dermal fibroblasts (Guidry and Hook, 1991;Appleton et al, 1992), which is essential for wound closure and reconstitution of the dermis (Grinnell, 1994), but also contributes to scar formation (Schmitt-Graff et al, 1994).…”

Endothelin-1 Induces Expression of Matrix-associated Genes in Lung Fibroblasts through MEK/ERK

“…The enzyme responsible for the specific cleavage at Trp-21 has been termed the endothelin-converting enzyme (7,8). Injury and the wound-healing response lead to stabilization of endothelin-converting enzyme-1 mRNA and to the generation of bioactive endothelin (9). Elevated levels of ET-1 have been shown in patients with fibrotic disease, suggesting that ET-1 may play a key role not only in normal wound repair but also in the pathogenesis of fibrosis (10 -14).…”