Hypoxia differentially enhances the effects of transforming growth factor-β isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts
Hypoxia differentially enhances the effects of transforming growth factor-β isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts
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DOI：
10.1124/jpet.301.3.830
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发表时间：
2002-06-01
影响因子：
3.5
通讯作者：
Karakiulakis, G
中科院分区：
文献类型：
--
作者：
Papakonstantinou, E;Roth, M;Karakiulakis, G
与缺氧相关的间质性肺疾病，如肺纤维化，其特征在于转化生长因子-β（TGF-β）的产生增强和细胞外基质（ECM）分子（包括糖胺聚糖（GAG））的沉积增加。在这项研究中，我们研究了缺氧（3%O-2）对TGF-β诱导的GAG合成的原代人肺成纤维细胞，建立从肺活检。通过将[H-3]葡糖胺掺入与细胞层（细胞和ECM）相关或在培养基中分泌的GAG中，评估总GAG合成。通过凝胶过滤分离和纯化GAG，通过在醋酸纤维素膜上电泳进行分级，并使用GAG降解酶进行表征。在细胞层和培养基中鉴定的GAG分子为：透明质酸、软骨素、皮肤素和硫酸乙酰肝素。所有TGF-β同种型均时间依赖性地诱导[H-3]葡糖胺掺入细胞层或培养基的GAG中。单个GAG分子的表征表明，这归因于细胞层中的硫酸皮肤素和硫酸乙酰肝素以及培养基中的透明质酸、软骨素和硫酸皮肤素。缺氧增强了所有TGF-β亚型，特别是TGF-β 3对透明质酸和软骨素和硫酸皮肤素分泌的影响。在细胞层中，缺氧仅刺激TGF-β 2诱导的[H-3]葡糖胺掺入GAG的作用。我们的数据表明，缺氧差异增强TGF-β亚型对GAG分泌和沉积的影响，并可能加速与肺纤维化发病机制相关的ECM重塑。
Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor-beta (TGF-beta) and increased deposition of extracellular matrix (ECM) molecules, including glycosamino-glycans (GAGs). In this study, we investigated the effect of hypoxia (3% O-2) on TGF-beta-induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [H-3] glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF-beta isoforms time dependently induced [H-3] glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF-beta isoforms, particularly that of TGF-beta3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-beta2-induced [H-3] glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF-beta isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.