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Overexpression of TGF-ß 1 gene induces cell surface localized glucose-regulated protein 78-associated latency-associated peptide/TGF-ß.

ABSTRACT: TGF-beta plays a crucial role in immune regulation. It has been reported that pro-TGF-beta, latency-associated peptide (LAP), latent TGF-beta and/or active TGF-beta (LAP/TGF-beta) is localized on the cell surface of Foxp3(+) regulatory T cells. However, the molecular mechanism(s) of how LAP/TGF-beta is anchored on the cell membrane is unknown. In this study, we show that forced expression of human TGF-beta(1) gene by retrovirus transduction into P3U1 mouse myeloma cells, and other cell types including murine CD4(+)CD25(-) T cells, makes these cells surface LAP/TGF-beta-positive. The surface LAP/TGF-beta contains high-glycosylated, furin-processed latent TGF-beta, which is different from the low-glycosylated, furin-unprocessed intracellular form or the high-glycosylated, furin-unprocessed secreted form. Furthermore, surface LAP/TGF-beta forms a complex with the molecular chaperone glucose-regulated protein 78 (GRP78, also known as BiP), and knockdown of GRP78 reduced the expression levels of surface LAP/TGF-beta. GRP78, however, is not involved in GARP-mediated surface LAP/TGF-beta. Our results suggest that GRP78 provides an additional surface localization mechanism for LAP/TGF-beta, which may play an important role in controlling TGF-beta activity.

SUBMITTER: Oida T

PROVIDER: S-EPMC2997468 | biostudies-other | 2010 Sep

REPOSITORIES: biostudies-other

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Overexpression of TGF-ß 1 gene induces cell surface localized glucose-regulated protein 78-associated latency-associated peptide/TGF-ß.

Oida Takatoku T Weiner Howard L HL

Journal of immunology (Baltimore, Md. : 1950) 20100818 6

TGF-beta plays a crucial role in immune regulation. It has been reported that pro-TGF-beta, latency-associated peptide (LAP), latent TGF-beta and/or active TGF-beta (LAP/TGF-beta) is localized on the cell surface of Foxp3(+) regulatory T cells. However, the molecular mechanism(s) of how LAP/TGF-beta is anchored on the cell membrane is unknown. In this study, we show that forced expression of human TGF-beta(1) gene by retrovirus transduction into P3U1 mouse myeloma cells, and other cell types inc ...[more]

PMID: 20720212

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Project description:Background Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix in the heart, which leads to the formation of cardiac scars. It causes systolic and diastolic dysfunction, and ultimately leads to cardiac dysfunction and arrhythmia. TGF-β1 is an important regulatory factor involved in cardiac fibrosis. Studies have shown that the N-terminal latency associated peptide (LAP) must be removed before TGF-β1 is activated. We hypothesize that recombinant LAP may inhibit cardiac fibrosis induced by TGF-β1. To evaluate anti-cardiac fibrosis activity of recombinant LAP, an experimental study was carried out and is reported here. Methods The pET28a-LAP plasmid was constructed and transformed into E. coli C43 (DE3) competent cells. The recombinant LAP protein was purified by Ni affinity chromatography. The cells were treated with TGF-β1 at different concentrations for 24 h. The expression of α-SMA was detected by Western blot. RTCA was used to detect the effect of recombinant LAP on the proliferation of H9C2 cells induced by 10 ng/mL TGF-β1. To detect the effect of LAP on the expression of fibrosis-related proteins, H9C2 cells were treated with 10 ng/mL TGF-β1 for 24 h, then added 60 μg/mL recombinant LAP for 48 h. The LAP group was treated with 60 μg/mL recombinant LAP alone. The LAP pre-protection group was treated with 10 ng/mL TGF-β1 and 60 μg/mL recombinant LAP at the same time. Western blot and immunofluorescence were used to detect the expression of α-SMA, collagen I and fibronectin and p-Smad2. Results The recombinant LAP was prokaryotic expressed and purified. 10 ng/mL was determined as the optimal working concentration of TGF-β1 to induce H9C2 cells fibrosis. RTCA results showed that 60 μg/mL LAP could effectively inhibit the proliferation of H9C2 cells induced by TGF-β1. Immunofluorescence results showed that compared with the control group, the fluorescence intensities of α-SMA, collagen I and FN increased significantly after TGF-β1 treatment. The fluorescence intensities in the TGF-β1+LAP group decreased significantly. Western blot results showed that 60 μg/mL LAP could inhibit the increase of α-SMA, collagen I and FN expression in H9C2 cells induced by TGF-β1. Compared with the control, the LAP alone group has no significant difference in α-SMA and p-Smad2 expression level. The expression of α-SMA and p-Smad2 in the TGF-β1 model group was significantly increased compared with the control group. Compared with the TGF-β1 group, both TGF-β1+LAP group and LAP pre-protection group significantly reduced the increase in α-SMA and p-Smad2 levels. Conclusions Recombinant LAP was prokaryotic expressed and purified. The results showed that recombinant LAP can inhibit the cell proliferation and expression increase of α-SMA, collagen I, fibronectin and p-Smad2 in H9C2 cells induced by TGF-β1. These results suggested that recombinant LAP might inhibit TGF-β1-induced fibrosis of H9C2 cells through the TGF-β/Smad pathway.

Project description:Rho GTPases mediate stromal-epithelial interactions that are important for mammary epithelial cell (MEC) morphogenesis. Increased extracellular matrix (ECM) deposition and reorganization affect MEC morphogenesis in a Rho GTPase-dependent manner. Although the effects of altered ECM on MEC morphogenesis have been described, how MECs regulate stromal deposition is not well understood. Previously, we showed that p190B RhoGAP overexpression disrupts mammary gland morphogenesis by inducing hyperbranching in association with stromal alterations. We therefore hypothesized that MEC overexpression of p190B regulates paracrine interactions to impact fibroblast activation. Using a combination of in vivo morphometric and immunohistochemical analyses and primary cell culture assays, we found that p190B overexpression in MECs activates fibroblasts leading to increased collagen, fibronectin, and laminin production and elevated expression of the collagen crosslinking enzyme lysyl oxidase. Phosphorylation of the TGF-? effector SMAD2 and expression of the TGF-? target gene ?Sma were increased in p190B-associated fibroblasts, suggesting that elevated TGF-? signaling promoted fibroblast activation. Mechanical tension and TGF-? cooperate to activate fibroblasts. Interestingly, active TGF-? was elevated in conditioned medium from p190B overexpressing MECs compared to control MECs, and p190B overexpressing MECs exhibited increased contractility in a collagen gel contraction assay. These data suggest that paracrine signaling from the p190B overexpressing MECs may activate TGF-? signaling in adjacent fibroblasts. In support of this, transfer of conditioned medium from p190B overexpressing MECs onto wildtype fibroblasts or co-culture of p190B overexpressing MECs with wildtype fibroblasts increased SMAD2 phosphorylation and mRNA expression of ECM genes in the fibroblasts when compared to fibroblasts treated with control CM or co-cultured with control MECs. The increased ECM gene expression and SMAD2 phosphorylation were blocked by treatment with a TGF-? receptor inhibitor. Taken together, these data suggest that p190B overexpression in the mammary epithelium induces fibroblast activation via elevated TGF-? paracrine signaling.

Dataset Information

Overexpression of TGF-ß 1 gene induces cell surface localized glucose-regulated protein 78-associated latency-associated peptide/TGF-ß.

Publications

Overexpression of TGF-ß 1 gene induces cell surface localized glucose-regulated protein 78-associated latency-associated peptide/TGF-ß.

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