Jessop H L, Rawlinson S C F, Pitsillides A A, Lanyon L E
Department of Veterinary Basic Sciences, The Royal Veterinary College, London, UK.
Bone. 2002 Jul;31(1):186-94. doi: 10.1016/s8756-3282(02)00797-4.
Extracellular regulated kinases (ERKs)-1 and -2 are members of the MAPK family of protein kinases involved in the proliferation, differentiation, and apoptosis of bone cells. We have shown previously that ROS 17/2.8 cells show increased activation of ERK-1 or -2, which is sustained for 24 h, when the strips onto which they are seeded are subjected to a 10 min period of cyclic four point bending that produces physiological levels of mechanical strain along with associated fluid movement of the medium. Movement of the strips through the medium without bending causes fluid movement without strain. This also increases ERK-1/2 activation, but in a biphasic manner over the same time period. Our present study investigates the role of components of signaling pathways in the activation of ERK-1/2 in ROS 17/2.8 cells in response to these stimuli. Using a range of inhibitors we show specific differences by which ERK-1 and ERK-2 are activated in response to fluid movement alone, compared with those induced in response to strain plus its associated fluid movement. ERK-1 activation induced by fluid movement was markedly reduced by nifedipine, and therefore appears to involve L-type calcium channels, but was unaffected by either L-NAME or indomethacin. This suggests independence from prostacyclin (PGI(2)) and nitric oxide (NO) production. In contrast, ERK-1 activation induced by application of strain (and its associated fluid disturbance) was abrogated by TMB-8 hydrochloride, L-NAME, and indomethacin. This suggests that strain-induced ERK-1 activation is dependent upon calcium mobilization from intracellular stores and production of NO and PGI(2). ERK-2 activation appears to be mediated by a separate mechanism in these cells. Its activation by fluid movement alone involved both PGI(2) and NO production, but its activation by strain was not affected by any of the inhibitors used. The G protein inhibitor, pertussis toxin, did not cause a reduction in the activation of ERK-1 or -2 in response to either stimulus. These results are consistent with earlier observations of ERK activation in bone cells in response to both strain (with fluid movement) and fluid movement alone, and further demonstrate that these phenomena stimulate distinct signaling pathways.
细胞外调节激酶（ERKs）-1和-2是丝裂原活化蛋白激酶（MAPK）家族的成员，参与骨细胞的增殖、分化和凋亡。我们之前已经表明，当接种了ROS 17/2.8细胞的条带经历10分钟的周期性四点弯曲时，ERK-1或-2的激活会增加，并持续24小时，这种弯曲会产生生理水平的机械应变以及培养基相关的液体流动。条带在培养基中移动但不弯曲会导致没有应变的液体流动。这也会增加ERK-1/2的激活，但在相同时间段内呈双相方式。我们目前的研究调查了信号通路成分在ROS 17/2.8细胞中响应这些刺激而激活ERK-1/2的过程中的作用。使用一系列抑制剂，我们展示了ERK-1和ERK-2在仅响应液体流动时与响应应变及其相关液体流动时被激活的特定差异。硝苯地平显著降低了液体流动诱导的ERK-1激活，因此似乎涉及L型钙通道，但不受L-NAME或吲哚美辛的影响。这表明其独立于前列环素（PGI(2)）和一氧化氮（NO）的产生。相反，施加应变（及其相关的液体扰动）诱导的ERK-1激活被盐酸TMB-8、L-NAME和吲哚美辛消除。这表明应变诱导的ERK-1激活依赖于细胞内钙库的钙动员以及NO和PGI(2)的产生。在这些细胞中，ERK-2的激活似乎由一种单独的机制介导。仅液体流动对其激活涉及PGI(2)和NO的产生，但应变对其激活不受所用任何抑制剂的影响。G蛋白抑制剂百日咳毒素在响应任何一种刺激时都不会导致ERK-1或-2的激活降低。这些结果与早期关于骨细胞中ERK在响应应变（伴有液体流动）和仅液体流动时激活的观察结果一致，并进一步证明这些现象刺激了不同的信号通路。
