Inhibition of Transforming Growth Factor (TGF)- 1–Induced Extracellular Matrix with a Novel Inhibitor of the TGF- Type I Receptor Kinase Activity: SB-431542-猫眼课题宝

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2002

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N. Laping;E. Grygielko;A. Mathur;S. Butter;J. Bomberger;C. Tweed;W. Martin;J. Fornwald;R. Leh

转化生长因子 1 (TGF1) 是一种有效的纤维化因子，负责细胞外基质的合成。 TGF1 通过 TGF I 型和 II 型受体发挥作用，激活细胞内介质，例如 Smad 蛋白、p38 丝裂原激活蛋白激酶 (MAPK) 和细胞外信号调节激酶途径。我们表达了 TGF I 型受体 [激活素受体样激酶 (ALK)5] 的激酶结构域和底物 Smad3，并确定 SB431542 是 Smad3 磷酸化的选择性抑制剂，IC50 为 94 nM。它抑制 TGF1 诱导的 Smad3 核定位。 p38 丝裂原激活蛋白激酶抑制剂 SB-203580 和 SB-202190 还可抑制 ALK5 对 Smad3 的磷酸化，IC50 值分别为 6 和 3 M。这表明这些 p38 MAPK 抑制剂必须以低于 10 M 的浓度使用才能选择性地解决 p38 MAPK 机制。然而，p38 MAPK 抑制剂 SB242235 不抑制 ALK5。为了评估 Smad 信号传导和 p38 MAPK 信号传导在 TGF1 诱导的基质产生中的相对贡献，将 SB-431542 与 SB-242235 在肾上皮癌 A498 细胞中的效果进行了比较。所有化合物均抑制 TGF1 诱导的纤连蛋白 (FN) mRNA，表明 FN 合成部分通过 p38 MAPK 途径介导。相比之下，SB-431542（而非选择性 p38 MAPK 抑制剂 SB-242235）抑制 TGF1 诱导的胶原蛋白 I 1 (col I 1)。这些数据表明，一些受 TGF1 刺激的基质标记物是通过 p38 MAPK 途径（即 FN）介导的，而其他标记物似乎是通过独立于 p38 MAPK 途径的 ALK5 信号传导激活的（即 col I 1）。许多报告将 TGF1 描述为细胞外基质的有效调节剂。因此，外源TGF1可以诱导细胞产生细胞外基质蛋白并抑制蛋白酶活性（Nakamura等人，1992；Ziyadeh等人，1994）。过度表达 TGF1 的转基因小鼠会出现严重的肾小球硬化症和肝纤维化（Kopp 等人，1996；Kanzler 等人，1999）。最后，针对 TGF1 的中和抗体可以防止肾病和肺纤维化模型中细胞外基质的积累（Border 等人，1990；Sharma 等人，1996；Ziyadeh 等人，2000）。一些信号通路与介导 TGF 诱导的细胞外基质产生和纤维化有关。通过两个具有细胞内丝氨酸/苏氨酸激酶结构域的高度保守的单跨膜受体转化生长因子信号。具体而言，II 型受体磷酸化配体占据的 I 型受体 (ALK5) 富含甘氨酸丝氨酸 (GS) 结构域中的苏氨酸残基，从而导致 ALK5 的激活 (Wrana, 1998)。 ALK5 反过来又磷酸化 Smad 蛋白（Abdollah 等，1997），从而介导细胞内信号传导至细胞核。 TGF 还通过 TGF 激活激酶 TAK1 激活 p38 丝裂原激活蛋白激酶 (MAPK)（Hanafusa 等，1999）。因此，TGF受体可以通过激活Smad转录因子直接调节基因转录，通过p38 MAPK间接调节基因转录，或通过这两种途径的组合。其他激酶，例如 JNK1 和 MKK4，似乎也参与纤连蛋白合成的 TGF 调节（Hocevar 等，1999）。此外，TGF1可以通过蛋白质合成激活细胞外信号调节激酶。缩写：TGF1，转化生长因子1； GS，甘氨酸-丝氨酸； ALK，激活素受体样激酶； MAPK，丝裂原激活蛋白激酶； SB-203580, 4-[4-氟苯基]-2-[4-甲基亚磺酰基苯基]-5-[4-吡啶基]咪唑； SB-431542, 4-(5-苯并[1,3]二氧杂环己烷-5-基-4-吡啶-2-基1H-咪唑-2-基)-苯甲酰胺； SB-242235, 4-[4-(4-氟苯基)-1-(4-哌啶基)-1H-咪唑-5-基]-2-甲氧基嘧啶； GST，谷胱甘肽S-转移酶； RPTEC，肾近曲小管上皮细胞； XTT，2,3-双[2-甲氧基-4-硝基-5-磺基苯基]-2H-四唑鎓-5-羧基苯胺； BMP-- 骨形态发生蛋白； PBS，磷酸盐缓冲盐水； PAI-1，纤溶酶原激活剂抑制剂-1； TSP-1、血小板反应蛋白-1； FN，纤连蛋白； rpL32，核糖体蛋白L32； RT-PCR、逆转录聚合酶链式反应； SB-202190，4-(4-氟苯基)-2-(4-羟基苯基)-5-(4-吡啶基)-1H-咪唑。 0026-895X/02/6201-58–64$7.00 分子药理学卷。 62, No. 1 版权所有 © 2002 美国药理学和实验治疗学会 1514/989396 Mol Pharmacol 62:58–64, 2002 2017 年 8 月 8 日美国印刷 58 at A PE T Journals m oharm .aspeurnals.org涉及碱性成纤维细胞生长因子（Finlay 等，2000）。很明显，TGF 可以与多种激酶途径相互作用，从而影响细胞外基质的许多成分。尽管已表明p38 MAPK抑制剂SB-203580可以抑制ALK5自磷酸化，IC50为20 M（Eyers等人，1998），但更具选择性的抑制剂尚未应用于细胞外基质的TGF调节。我们表征了几种化合物选择性抑制 ALK5 或 p38 MAPK 的能力，以描述 Smad 和 p38 MAPK 激活在细胞外基质 TGF 调节中的相对贡献。因此，在本研究中，我们研究了选择性 ALK5 抑制剂 SB-431542（Callahan 等人，2002）和选择性 p38 MAPK 抑制剂 SB-242235（Badger 等人，2000）对 TGF1 诱导的纤连蛋白、胶原蛋白 I 1、血小板反应蛋白-1 和纤溶酶原激活剂抑制剂-1 mRNA 的影响。材料和方法重组蛋白的表达。 ALK5的激酶结构域、C端第200个氨基酸（缺少GS区）和全长Smad3蛋白在杆状病毒表达系统中表达为N端谷胱甘肽S-转移酶（GST）融合蛋白。用谷胱甘肽琼脂糖珠 4B（Amersham Biosciences，乌普萨拉，瑞典）纯化蛋白质（Roshak 等人，

Transforming growth factor 1 (TGF1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF1 acts through the TGFtype I and type II receptors to activate intracellular mediators, such as Smad proteins, the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGFtype I receptor [activin receptor-like kinase (ALK)5] and the substrate, Smad3, and determined that SB431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF1–induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 M, respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 M to selectively address p38 MAPK mechanisms. However, the p38 MAPK inhibitor SB242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF1– induced matrix production, the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF1–induced fibronectin (FN) mRNA, indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast, SB-431542, but not the selective p38 MAPK inhibitor SB-242235, inhibited TGF1– induced collagen I 1 (col I 1). These data indicate that some matrix markers that are stimulated by TGF1 are mediated via the p38 MAPK pathway (i.e., FN), whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e., col I 1). Numerous reports describe TGF1 as a potent regulator of extracellular matrix. Thus, cells can be induced to produce extracellular matrix protein and inhibit protease activity by exogenous TGF1 (Nakamura et al., 1992; Ziyadeh et al., 1994). Transgenic mice that overexpress TGF1 develop severe glomerulosclerosis and liver fibrosis (Kopp et al., 1996; Kanzler et al., 1999). Finally, neutralizing antibodies against TGF1 can prevent the accumulation of extracellular matrix in models of renal disease and lung fibrosis (Border et al., 1990; Sharma et al., 1996; Ziyadeh et al., 2000). Several signaling pathways have been implicated in mediating TGF–induced extracellular matrix production and fibrosis. Transforming growth factorsignals through two highly conserved single transmembrane receptors with intracellular serine/threonine kinase domains. Specifically, the type II receptor phosphorylates threonine residues in the glycine-serine (GS)-rich domain of the ligand-occupied type I receptor (ALK5), which results in the activation of ALK5 (Wrana, 1998). ALK5, in turn, phosphorylates Smad proteins (Abdollah et al., 1997), which mediate intracellular signaling to the nucleus. TGFalso activates the p38 mitogen-activated protein kinase (MAPK) through the TGFactivated kinase, TAK1 (Hanafusa et al., 1999). Thus, the TGFreceptors can regulate gene transcription directly by activating the Smad transcription factors, indirectly via p38 MAPK, or by a combination of both pathways. Other kinases, such as JNK1 and MKK4, also seem to be involved in TGFregulation of fibronectin synthesis (Hocevar et al., 1999). Furthermore, TGF1 can activate the extracellular signal-regulated kinase through a protein synthesis ABBREVIATIONS: TGF1, transforming growth factor 1; GS, glycine-serine; ALK, activin receptor-like kinase; MAPK, mitogen-activated protein kinase; SB-203580, 4-[4 -fluorophenyl]-2-[4 -methylsulfinylphenyl]-5-[4 -pyridyl] imidazole; SB-431542, 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl1H-imidazol-2-yl)-benzamide; SB-242235, 4-[4-(4-flurophenyl)-1-(4-piperidinyl)-1H-imidazol-5-yl]-2-methoxypyrimidine; GST, glutathione S-transferase; RPTEC, renal proximal tubule epithelial cells; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline; BMP, bone morphogenetic protein; PBS, phosphate-buffered saline; PAI-1, plasminogen activator inhibitor-1; TSP-1, thrombospondin-1; FN, fibronectin; rpL32, ribosomal protein L32; RT-PCR, reverse transcription-polymerase chain reaction; SB-202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole. 0026-895X/02/6201-58–64$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 1 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1514/989396 Mol Pharmacol 62:58–64, 2002 Printed in U.S.A. 58 at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from requiring a mechanism that may involve basic fibroblast growth factor (Finlay et al., 2000). It is clear that TGFcan interact with several kinase pathways to influence many components of the extracellular matrix. Although it has been shown that the p38 MAPK inhibitor SB-203580 can inhibit ALK5 autophosphorylation with an IC50 of 20 M (Eyers et al., 1998), more selective inhibitors have not been applied to TGFregulation of extracellular matrix. We characterized several compounds for their ability to selectively inhibit ALK5 or p38 MAPK to delineate the relative contribution of Smad and p38 MAPK activation in TGFregulation of extracellular matrix. In this study, therefore, we examine the effects of the selective ALK5 inhibitor SB-431542 (Callahan et al., 2002) and the selective p38 MAPK inhibitor SB-242235 (Badger et al., 2000) on TGF1–induced fibronectin, collagen I 1, thrombospondin-1, and plasminogen activator inhibitor-1 mRNA. Materials and Methods Expression of Recombinant Protein. The kinase domain of ALK5, amino acids 200 to the C terminus, which lacks the GS region, and the full-length Smad3 protein were expressed as N-terminal glutathione S-transferase (GST) fusion proteins in baculovirus expression system. Proteins were purified with glutathione Sepharose beads 4B (Amersham Biosciences, Uppsala, Sweden) (Roshak et al.,

Inhibition of Transforming Growth Factor (TGF)- 1–Induced Extracellular Matrix with a Novel Inhibitor of the TGF- Type I Receptor Kinase Activity: SB-431542

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