Increased TG2 Expression Can Result in Induction of Transforming Growth Factor β1, Causing Increased Synthesis and Deposition of Matrix Proteins, Which Can Be Regulated by Nitric Oxide

Dilek Telci,Russell John Collighan,Huveyda Basaga,Martin Griffin

doi:10.1074/jbc.m109.041806

Dilek Telci, Russell John Collighan + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109.041806

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Abstract

In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-κB activation, resulting in the increased expression of transforming growth factor (TGF) β1. In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFβ1 in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-κB and TGFβ1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-κB and TGFβ1 production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.

Highlights

Tissue transglutaminase (TG)2 2 is a member of a family of enzymes that in mammals mediate the Ca2ϩ-dependent formation of ⑀-(␥-glutamyl)-lysine protein cross-links or incorporation of polyamines into proteins to form (␥-glutamyl)-polyamine bonds [1]
We demonstrate for the first time that TG2 induction stimulates NF-␬B activity, which is paralleled by an increase in the expression and bioactivation of transforming growth factor (TGF)␤1, with a subsequent elevation in the synthesis and deposition of collagen I, III, and IV and fibronectin into the ECM in a transglutaminase activity-dependent manner
Induction of TG2 Expression and Activity Increases the Synthesis and Deposition of Matrix Proteins—In agreement with our previous report [12] TG2 expression was induced upon removal of tetracycline from the culture medium of transfected Swiss 3T3 cells for 72 h, which was evident in whole cell lysates

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Cell culture medium, growth supplements, and other chemicals of cell culture grade were purchased from Sigma-Aldrich. For detection of cell surface TG2 antigen via flow cytometry, the detached cells were resuspended in Dulbecco’s modified Eagle’s medium containing 3 ␮g of anti-TG2 monoclonal antibody Cub7402 (Lab Vision) followed by an incubation with secondary FITC-labeled mouse IgG (3 ␮g/ml) for 1 h at 4 °C as previously described [7]. TG2-mediated fluorescein cadaverin incorporation in ECM/WCL fractions was detected using mouse anti-fluorescein antibody (Roche Applied Science) by Western blots, whereas analysis of incorporation in cell monolayers was performed on cells seeded into Permanox eight-well chamber slides by methanol fixation, mounting (Vector Laboratories), and laser confocal microscopy (Zeiss LSM510) using the Zeiss LSM image browser as described previously [10]. Col 1A1, collagen type 1 ␣1 chain; Col 1A2, collagen type 1 ␣2 chain; Col 4A1, collagen type 4 ␣1 chain; Col 4A2, collagen type 4 ␣2 chain; Col 3A1, collagen type 3 ␣1 chain; TGF␤1, transforming growth factor ␤1; 18 S RNA, 18 S ribosomal RNA

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RESULTS

DISCUSSION