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

Abstract

The endothelins are a family of endothelium-derived peptides that possess a variety of biological activities, including potent vasoconstriction. Endothelin-1 (ET-1) is up-regulated during tissue repair and pulmonary fibrosis. Here, we use genome-wide expression array analysis to show that the addition of ET-1 (100 nM, 4 h) to normal lung fibroblasts directly induces expression of matrix and matrix-associated genes, including the profibrotic protein CCN2 (connective tissue growth factor, or CTGF). ET-1 induces the MEK/ERK MAP kinase pathway in fibroblasts. Blockade of the MEK/ERK kinase pathway with U0126 abrogates the ability of ET-1 to induce expression of matrix and matrix-associated mRNAs and the CCN2 protein. The CCN2 promoter possesses an ET-1 response element, which maps to the previously identified basal control element-1 (BCE-1) site. Our results suggest that ET-1 induces a program of matrix synthesis in lung fibroblasts and that ET-1 may play a key role in connective tissue deposition during wound repair and in pulmonary fibrosis.As a response to environmental insults or a consequence of local inflammatory processes, structural damage to tissue can occur, triggering a wound-healing response. This response consists of an integrated series of biochemical, immunological, and structural changes that result in the de novo synthesis of a new epithelium, blood vessels, and connective tissue (1). The proper repair of connective tissue requires the synthesis and organization of new ECM 1 components, such as collagen and fibronectin (2).A growing body of evidence implicates the vasoconstrictive peptide endothelin-1 (ET-1) as a key mediator of tissue repair (3). Each of the three known endothelin isoforms (-1, -2, and -3) arise by proteolytic processing of large precursors (ϳ200 amino acid residues). Intermediates, termed big ET-1, -2, and -3 (38 -41 amino acids), are excised from pre-propeptides at sites containing paired basic amino acids. Big endothelins, which have little or no biological activity (4), are cleaved at Trp-21-Val/Ile-22 to produce mature, 21-residue, biologically active peptides (5, 6). 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).ET-1 demonstrates a wide range of biological properties, including significant mitogenic activity toward a number of cell types such as smooth muscle cells and fibroblasts (15). ET-1 also promotes the contractile ability of normal fibroblasts (16), which is essential for wound closure and reconstitution of the dermis (17). In addition, ET-1 modifies extracellular matrix metabolism (15, 18 -20). For example, ET-1 enhances collagen types I and III and decreases...

The gene expression profile induced by Wnt 3a in NIH 3T3 fibroblasts

“…However, Wnt 3a did not cause an as potent induction of other profibrotic/ matrix remodeling genes as, say, application of TGFβ or ET-1 to fibroblasts (Shi-wen et al 2004Leask and Abraham 2004). For example, α-smooth muscle actin (α-SMA) was not detected in our studies.…”

“…Alternatively, conditioned media obtained from cell lines overexpressing Wnt 1, 4, 5 or 10 or containing empty expression vector (courtesy Cun-Yu Wang, University of Michigan) was added to DMEM/0.5% calf serum in a 1:1 ratio. RNA was then harvested using Trizol (Invitrogen) and quantified as previously described (Shi-wen et al 2004(Shi-wen et al , 2006Kennedy et al 2007). …”

“…RNA quality assessment, probe preparation and gene chip hybridization and analysis Microarrays and analysis were performed essentially as previously described (Shi-wen et al 2004;Liu et al 2007;Kennedy et al 2007). All Gene Chips were processed at the London Regional Genomics Centre (Robarts Research Institute, London, ON; http://www.lrgc.ca).…”

Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK

“…These results strongly support the notion that TGFβ is not required under situations when CCN2 is constitutively expressed. Previously, we have shown that the Ras/MEK/ERK pathway regulates CCN2 levels in response to TGF-β and ET-1 (Chen et al 2002;Leask et al 2003;Shi-wen et al 2004). Indeed, it is interesting to note that antagonizing receptors for either TGFβ or ET-1 was not effective at blocking CCN2 expression in PANC-1 cells (data not shown), in spite of the fact that ET-1 induces the CCN2 promoter via BCE-1 and ras/MEK/ERK in lung fibroblasts .…”

All in the CCN family: essential matricellular signaling modulators emerge from the bunker

“…Similarly, faithful expression of a reporter gene in transgenic mice, including in development and in response to wound healing, can be achieved if a 2 kb fragment of the CCN1 promoter is used, which indicates that this gene is also regulated at the level of transcription (Latinkic et al, 2001). Synthesis of CCN2 protein and mRNA is stimulated by specific growth factors, such as endothelin 1 and TGF␤, in addition to environment changes such as hypoxia and biomechanical stimuli (Grotendorst et al, 1996;Holmes et al, 2001;Ott et al, 2003;Shi-wen et al, 2004;Higgins et al, 2004) (Fig. 2).…”