Concentration-dependent stimulation by tissue inhibitor of metalloproteinases(TIMP)-2 of two signaling pathways in human osteosarcoma (MG-63) Cells

Ting Wang, Chihiro Kondo, K. Yamashita, M. Oguchi, K. Iwata, T. Noguchi, T. Hayakawa
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引用次数: 1

Abstract

We reported previously that tyrosine kinase (TYK) and mitogen-activated protein kinase (MAPK) play a role in TIMP-dependent growth signaling. Here we demonstrate that the activation of MAPK stimulated by TIMP-2 was sharply inhibited in MG-63 cells by PP1, a selective inhibitor of Src-family tyrosine kinases, suggesting that Src activation is possibly required for the MAPK activity in response to 1 ng/mL TIMP-2. A specific cell-permeable inhibitor of MAPK kinase (MEK), PD 98058 had an inhibitory effect on MAPK activity, indicating that MEK is an upstream effector of MAPK. By transfecting MG-63 cells with dominant-negative Ras, RasN17, and furthermore, by using a selective farnesyltransferase inhibitor, FTI-277, we found that MAPK activation stimulated by TIMP-2 was independent of Ras. We also demonstrated that the activation of MAPK in response to 1 ng/mL TIMP-2 was inhibited by a cAMP agonist, 8-bromo-cAMP. On the contrary, [3H]thymidine incorporation and the activation of MAPK, all of which were heavily suppressed by increasing the TIMP-2 concentration up to 100 ng/mL, were recovered by the addition of a cell-permeable specific PKA inhibitor, H-89. These results strongly suggest the presence of a negative crosstalk from the cAMP/PKA pathway to the TYK/MAPK one.
组织金属蛋白酶抑制剂(TIMP)-2对人骨肉瘤(MG-63)细胞两种信号通路的浓度依赖性刺激
我们之前报道过酪氨酸激酶(TYK)和丝裂原活化蛋白激酶(MAPK)在timp依赖性生长信号传导中发挥作用。在MG-63细胞中,由TIMP-2刺激的MAPK激活被PP1(一种Src家族酪氨酸激酶的选择性抑制剂)急剧抑制,这表明MAPK在1 ng/mL TIMP-2下的活性可能需要Src激活。PD 98058是一种特异性的细胞渗透性MAPK激酶(MEK)抑制剂,对MAPK活性有抑制作用,表明MEK是MAPK的上游效应物。通过用显性阴性Ras、RasN17以及选择性法尼基转移酶抑制剂FTI-277转染MG-63细胞,我们发现TIMP-2刺激的MAPK活化与Ras无关。我们还证明,MAPK对1 ng/mL TIMP-2的反应被cAMP激动剂8-溴cAMP抑制。相反,通过将TIMP-2浓度增加至100 ng/mL而严重抑制的[3H]胸腺嘧啶掺入和MAPK的激活,通过添加具有细胞渗透性的特异性PKA抑制剂H-89得以恢复。这些结果强烈表明cAMP/PKA通路与TYK/MAPK通路之间存在负串扰。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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