Cailotto Frederic, Bianchi Arnaud, Sebillaud Sylvie, Venkatesan Narayanan, Moulin David, Jouzeau Jean-Yves, Netter Patrick
UMR 7561 CNRS-Nancy-Université, Laboratoire de Physiopathologie et Pharmacologie Articulaires, France.
Arthritis Res Ther. 2007;9(6):R122. doi: 10.1186/ar2330.
ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-beta1 on Ank mRNA level. These data show that TGF-beta1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes.
ANK是一种多次跨膜蛋白转运体，被认为在细胞内无机焦磷酸的输出中发挥作用，从而参与软骨钙质沉着症的病理生理过程。由于转化生长因子-β1（TGF-β1）已被证明有利于二水焦磷酸钙沉积，我们研究了ANK对软骨细胞产生细胞外无机焦磷酸（ePPi）的作用以及TGF-β1调节Ank表达所涉及的信号通路。将软骨细胞暴露于10 ng/mL的TGF-β1中，通过定量聚合酶链反应和蛋白质印迹法检测Ank表达。对细胞上清液中的ePPi进行定量分析。采用RNA沉默技术来确定Ank和PC-1在TGF-β1诱导的ePPi生成中的各自作用。最后，使用选择性激酶抑制剂以及显性负性/过表达质粒策略来探究几种信号通路对TGF-β1诱导Ank表达的作用。TGF-β1在mRNA和蛋白质水平上均强烈增加Ank表达以及ePPi的产生。使用小干扰RNA技术，我们发现Ank对TGF-β1诱导的ePPi生成贡献约60%，PC-1贡献近20%。TGF-β1诱导Ank需要激活细胞外信号调节激酶（ERK）通路，但不需要p38丝裂原活化蛋白激酶或蛋白激酶A的激活。与一般的蛋白激酶C（PKC）抑制剂钙泊三醇C一致，Gö6976（一种钙依赖性PKC抑制剂）使TGF-β1诱导的Ank表达降低60%，而使用rottlerin（一种PKCδ抑制剂）观察到10%的抑制作用。这些数据表明钙在TGF-β1诱导的Ank表达中具有调节作用。最后，我们证明TGF-β1对Ank表达的刺激作用在Ras/Raf-1通路受抑制时受到抑制，而在其组成性激活时增强。抑制性Smad蛋白Smad 7的瞬时过表达未能影响TGF-β1对Ank mRNA水平的诱导作用。这些数据表明，TGF-β1主要通过诱导Ank基因来增加ePPi水平，这需要在软骨细胞中激活Ras、Raf-1、ERK和钙依赖性PKC通路。
