Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.
Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma.
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DOI：
10.1371/journal.pone.0047312
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Cohen-Solal KA
中科院分区：
文献类型：
--
作者：
Abushahba W;Olabisi OO;Jeong BS;Boregowda RK;Wen Y;Liu F;Goydos JS;Lasfar A;Cohen-Solal KA
利鲁唑是一种谷氨酸释放的抑制剂，已经显示出抑制黑色素瘤细胞异种移植生长的能力。作为单一药物的利鲁唑在黑色素瘤患者中的0期临床试验导致34%的患者肿瘤消退，并同时抑制了丝裂原激活蛋白激酶(MAPK)和磷脂酰肌醇-3-激酶/AKT(PI3K/AKT)通路。在本研究中，我们证明了利鲁唑抑制AKT介导的黑色素瘤细胞株中糖原合成酶激酶3(GSK3)的磷酸化。由于我们已经证明Gsk3参与了转化生长因子β的两个下游效应分子Smad2和Smad3在其连接域的磷酸化，我们的目的是确定利鲁唑是否能诱导Gsk3β介导的连接物Smad2和Smad3的磷酸化。我们提供的证据表明，利鲁唑分别增加了丝氨酸245/250/255和丝氨酸204簇上的Smad2和Smad3连接蛋白的磷酸化。使用Gsk3抑制剂和siRNA敲除，我们证明了利鲁唑诱导Smad磷酸化的机制涉及Gsk3β。此外，GSK3β在体外也能使相同的连接位点磷酸化。利鲁唑诱导的Smad连接子磷酸化与转化生长因子β诱导的Smad连接子磷酸化在机制上是不同的。我们还证明了利鲁唑诱导的Smad连接蛋白的磷酸化不依赖于代谢性谷氨酸受体1(GRM1)的表达，GRM1是参与人类黑色素瘤的谷氨酸受体之一。我们进一步证明利鲁唑上调INHBB和PLAU的表达，这两个基因与转化生长因子β信号通路有关。在晚期黑色素瘤中，利鲁唑诱导的Smad连接蛋白磷酸化的非典型性增加可能有助于调节Smads的致癌功能。
Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.