Abstract
Transforming growth factor (TGF)-beta1 plays an important role in the development of pulmonary fibrosis. In this study we examined the relationship between TGF-beta1 stimulation and the expression of heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) in cultured normal human lung fibroblasts (NHLFs) and in murine lungs in vivo. By removing 6-O-sulfates from specific HS intrachain sites on the cell surface, Sulf1 has been shown to modulate the activities of many HS binding growth factors and morphogens including fibroblast growth factor (FGF)-2. Real time reverse transcription-PCR analysis revealed that TGF-beta1 increased Sulf1 expression in NHLFs in a dose- and time-dependent manner which was accompanied by a decrease in 6-O-sulfated disaccharides as revealed by high performance liquid chromatography analysis. Decreased ERK activation after FGF-2 stimulation was observed in TGF-beta1-treated NHLFs compared with control cells without changes in HS-dependent FGF-2 binding or FGF-2.FR1c complex formation. To study the function of Sulf1, negative control or Sulf1-specific small interference RNA (siRNA)-transfected NHLFs were stimulated with TGF-beta1. Enhanced Smad2/3 phosphorylation and elevated total Smad2 protein level were observed in Sulf1 siRNA-transfected cells and were accompanied by enhanced expression of alpha-smooth muscle actin and fibronectin. In addition, Sulf1 siRNA transfection enhanced the anti-proliferative effect of TGF-beta1. Finally Sulf1 expression was up-regulated in the lungs of mice treated with adenovirus encoding active TGF-beta1. Taken together, our data indicate that Sulf1 is a TGF-beta1-responsive gene both in vitro and in vivo and may function as a negative regulator of TGF-beta1-induced fibrogenesis.
Highlights
JULY 18, 2008 VOLUME 283 NUMBER 29 extensive accumulation of collagen and other extracellular matrix (ECM) molecules in the lung resulting in loss of pulmonary function [1]
In this study we examined the relationship between transforming growth factor (TGF)-1 stimulation and the expression of heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) in cultured normal human lung fibroblasts (NHLFs) and in murine lungs in vivo
In this study we show for the first time that TGF-1, a profibrotic cytokine, induces Sulf1 expression in NHLFs and in murine lungs in vivo and that Sulf1 may function as a negative regulator of TGF-1induced fibrogenesis
Summary
Cell Culture—Primary NHLFs and the culture reagent fibroblast growth medium-2 Bullet Kit were obtained from Cambrex Bioproducts (Walkersville, MD). The NHLFs were received at passage 3 and cultured in fibroblast growth medium-2 complete medium. Subconfluent NHLFs were rendered quiescent in fibroblast basal medium (FBM, Cambrex Bioproducts) containing 0.2% bovine serum albumin (BSA, Sigma) overnight before treatment with TGF-1 and/or FGF-2 (R&D systems Inc., Minneapolis, MN) in the presence or absence of heparin (porcine intestinal mucosal heparin, Sigma). All experiments were performed in FBM containing 0.2% BSA and with cells at passages 5– 6. Real-time Quantitative Reverse Transcription-PCR—Messenger RNA (mRNA) of the gene of interest was quantified by quantitative real-time reverse transcription (RT)-PCR. Total RNA was isolated using the RNA mini plus kit
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