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Fibronectin stimulates endothelin-1 synthesis in rat hepatic myofibroblasts via a Src/ERK-regulated signaling pathway.

ABSTRACT: Liver injury serves as an excellent model of wound healing, characterized by increased synthesis of various cytokines and peptides, including the vasoactive peptide endothelin-1. In the liver, wound healing is mediated by effector cells such as hepatic stellate cells, which cause tissue contraction. Endothelin-1 has autocrine effects on stellate cells and induces their contractile activities. We explored the role of various extracellular matrix components, particularly of fibronectin, in regulating endothelin-1 production during liver injury.Hepatic stellate cells were isolated from normal and injured rats. Real-time polymerase chain reaction immunoblot and enzyme-linked immunosorbent assay analyses were used to measure specific variables, including endothelin-1 production. Preproendothelin-1 promoter activity was determined by a luciferase assay. Stellate cell contraction was measured by a gel contraction assay.Fibronectin stimulated transcription of preproendothelin-1 messenger RNA and expression of endothelin through an integrin-dependent pathway in activated hepatic stellate cells. In these cells, fibronectin induced phosphorylation/activation of extracellular signal-regulated kinase (ERK) through a Shc- and Src-dependent mechanism; ERK activation was required for fibronectin-induced endothelin-1 expression. Fibronectin stimulation by stellate cells induced expression of smooth muscle alpha-actin and endothelin-1-mediated autocrine stellate cell contraction. Stellate cells isolated from injured livers of rats exhibited increased basal phosphorylation levels of Src, Shc, and ERK, as well as increased endothelin-1 synthesis.Fibronectin stimulates activated stellate cells to produce endothelin-1 and contract, via an ERK-dependent signaling pathway. The resulting autocrine functional effects of endothelin-1 are likely to be important in the wound-healing process in injured liver.

SUBMITTER: Zhan S

PROVIDER: S-EPMC4122293 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Fibronectin stimulates endothelin-1 synthesis in rat hepatic myofibroblasts via a Src/ERK-regulated signaling pathway.

Zhan Shuxin S Chan Che-Chang CC Serdar Berrin B Rockey Don C DC

Gastroenterology 20090601 7

<h4>Background & aims</h4>Liver injury serves as an excellent model of wound healing, characterized by increased synthesis of various cytokines and peptides, including the vasoactive peptide endothelin-1. In the liver, wound healing is mediated by effector cells such as hepatic stellate cells, which cause tissue contraction. Endothelin-1 has autocrine effects on stellate cells and induces their contractile activities. We explored the role of various extracellular matrix components, particularly ...[more]

PMID: 19505428

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Project description:BackgroundThe hepatic stellate cell is the primary cell type responsible for the excessive formation and deposition of connective tissue elements during the development of hepatic fibrosis in chronically injured liver. Culturing quiescent hepatic stellate cells on plastic causes spontaneous activation leading to a myofibroblastic phenotype similar to that seen in vivo. This provides a simple model system for studying activation and transdifferentiation of these cells. The introduction of exogenous DNA into these cells is discussed controversially mainly due to the lack of systematic analysis. Therefore, we examined comparatively five nonviral, lipid-mediated gene transfer methods and adenoviral based infection, as potential tools for efficient delivery of DNA to rat hepatic stellate cells and their transdifferentiated counterpart, i.e. myofibroblasts. Transfection conditions were determined using enhanced green fluorescent protein as a reporter expressed under the transcriptional control of the human cytomegalovirus immediate early gene 1 promoter/enhancer.ResultsWith the use of chemically enhanced transfection methods, the highest relative efficiency was obtained with FuGENE6 gene mediated DNA transfer. Quantitative evaluation of representative transfection experiments by flow cytometry revealed that approximately 6% of the rat hepatic stellate cells were transfected. None of the transfection methods tested was able to mediate gene delivery to rat myofibroblasts. To analyze if rat hepatic stellate cells and myofibroblasts are susceptible to adenoviral infection, we have inserted the transgenic expression cassette into a recombinant adenoviral type 5 genome as replacement for the E1 region. Viral particles of this replication-deficient Ad5-based reporter are able to infect 100% of rat hepatic stellate cells and myofibroblasts, respectively.ConclusionsOur results indicate that FuGENE6-based methods may be optimized sufficiently to offer a feasible approach for gene transfer into rat hepatic stellate cells. The data further demonstrate that adenoviral mediated transfer is a promising approach for gene delivery to these hepatic cells.

Project description:Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4-5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in primary culture. In conclusion, this straightforward and reproducible method of PMF culture, can be used to identify new markers of PMFs at different stages of differentiation, to compare their phenotype with those of HSC-MFs and ultimately determine their progenitors and specific functions in liver wound-healing.

Project description:RATIONALE:Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BK? and auxiliary ?1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BK? is primarily plasma membrane localized, whereas the cellular location of ?1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. OBJECTIVE:Test the hypothesis that ET-1 inhibits BK channels by altering BK? and ?1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. METHODS AND RESULTS:ET-1, through activation of PKC (protein kinase C), reduced surface ?1 abundance and the proximity of ?1 to surface BK? in myocytes. In contrast, ET-1 did not alter surface BK?, total ?1, or total BK? proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1-induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface ?1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of ?1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. CONCLUSIONS:ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of ?1 subunits. The decrease in surface ?1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.

Project description:Tumor progression has been recognized as the product of evolving crosstalk between cancer cells and the surrounding stromal cells. Cancer-associated orthotopic myofibroblasts may be linked to the progression of gastric carcinomas. To understand the significance of orthotopic myofibroblasts, we examined the effects of cancer-associated orthotopic myofibroblasts on the malignant phenotype of gastric cancer cells. Three human gastric cancer cell lines (OCUM-2MD3, OCUM-12, MKN-45) and four human gastric fibroblast cell lines (cancer-associated orthotopic fibroblast [CaF]-29, CaF-33, normal orthotopic fibroblast [NF]-29, NF-33) were used. The cancer-associated orthotopic fibroblast cell lines CaF-29 and CaF-33 were established from a tumoral gastric wall, and normal orthotopic fibroblast NF-29 and NF-33 were established from a non-tumoral gastric wall. Fibroblasts that were ?-smooth muscle actin-positive were defined as myofibroblasts. We examined the effects of cancer-associated orthotopic myofibroblasts on the aggressiveness of gastric cancer cells by wound-healing assay, invasion assay, and RT-PCR. The ratios of myofibroblasts in CaF-29 (33%) and CaF-33 (46%) were significantly (P < 0.001) greater than those in NF-29 (11%) or NF-33 (13%). Although all four orthotopic fibroblast lines increased the motility of gastric cancer cells, including migration and invasion ability, the motility-stimulating activity of cancer-associated fibroblasts (CaF-29 and CaF-33) was significantly higher than that of normal fibroblasts (NF-29 and NF-33). These motility-stimulating activities of cancer-associated orthotopic fibroblasts were downregulated by Smad2 siRNA treatment and anti-transforming growth factor-? neutralizing antibody. These findings suggest that cancer-associated orthotopic myofibroblasts may play an important role in the progression of gastric cancers and that transforming growth factor-? produced by myofibroblasts may be one of the factors associated with the aggressiveness of gastric carcinoma cells.

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Fibronectin stimulates endothelin-1 synthesis in rat hepatic myofibroblasts via a Src/ERK-regulated signaling pathway.

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Fibronectin stimulates endothelin-1 synthesis in rat hepatic myofibroblasts via a Src/ERK-regulated signaling pathway.

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