Kawase T, Orikasa M, Oguro A, Burns D M
Department of Pharmacology, Niigata University School of Dentistry, Japan.
Arch Oral Biol. 1999 Feb;44(2):157-71. doi: 10.1016/s0003-9969(98)00105-8.
A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both Ca2+- and G protein-mediated pathways in UMR106 cells, revealing a previously unrecognized modulation of EGF signalling in osteoblast-like cells that contrasts with the simpler regulatory mechanisms found in A431 cells.
为了研究调节成骨细胞中蛋白质酪氨酸磷酸化的机制，建立了一种使用化学通透细胞的模型。利用通透的UMR106成骨细胞或A431（对照）细胞，研究了表皮生长因子（EGF）诱导的细胞酪氨酸磷酸化，以及该转导途径与Ca2 +或G蛋白依赖性信号通路之间是否存在以前未被认识到的相互作用。当两种通透细胞类型维持在未补充的细胞质替代溶液（基础CSS）中时，它们对EGF（1 - 100 ng/ml）的反应是酪氨酸磷酸化呈剂量依赖性增加。由此产生了一种复杂且随时间变化的含磷酸酪氨酸蛋白模式，但酪氨酸磷酸化蛋白的图谱比完整细胞中的明显不那么复杂。用MgATP补充基础CSS可恢复两种通透细胞系中EGF诱导的酪氨酸磷酸化蛋白图谱的正常复杂性，并在A431细胞中产生更持久的磷酸化蛋白产物积累。添加Ca2 +（≤10^(-6) M），无论有无外源性MgATP，均能剂量依赖性地减弱UMR106细胞中EGF诱导的EGF受体（EGFR）和其他底物的酪氨酸磷酸化，但在A431细胞中效果较差。在两种细胞类型中，酪氨酸激酶抑制剂染料木黄酮在减弱通透细胞中EGF诱导的受体酪氨酸磷酸化方面更有效。同样，蛋白质酪氨酸磷酸酶抑制剂原钒酸钠在通透细胞中更有效地刺激了磷酸化蛋白产物的积累。因此，通透作用保留了完整细胞的许多特征，同时便于对细胞内条件进行操作。NaF在通透细胞中可重复性地产生显著的类似原钒酸钠的作用，其作用比在完整细胞中的作用稍强。相比之下，佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）对酪氨酸磷酸化的著名抑制作用在通透细胞中比在完整细胞中效果更差；PMA的这些作用是Ca2 +依赖性的。此外，鸟苷酰亚胺二磷酸（Gpp(NH)p）减弱了UMR106细胞中的酪氨酸磷酸化，并且这种作用被5'-O-(2-硫代二磷酸)鸟苷（GDPβS）特异性阻断。这些结果强烈表明，在UMR106细胞中，EGFR激活的酪氨酸磷酸化/去磷酸化途径与Ca2 +和G蛋白介导的途径之间存在相互作用，揭示了以前未被认识到的成骨样细胞中EGF信号的调节，这与在A431细胞中发现的更简单的调节机制形成对比。
