Kawase T, Orikasa M, Ogata S, Burns D M
Department of Pharmacology, Niigata University School of Dentistry, Japan.
Arch Oral Biol. 1995 Oct;40(10):921-9. doi: 10.1016/0003-9969(95)00061-s.
Both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) produce a dose-dependent stimulation in the rate of cell division in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the initial mitogen-induced cellular events that may mediate mitogenic action, the effects of EGF and IGF-I on cellular protein tyrosine phosphorylation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) transiently stimulated tyrosine phosphorylation in four major proteins with apparent molecular weights of 220, 180, 140 and 120 kDa, and in five other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 160- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyrosine phosphorylation was sustained for more than 60 min, particularly that of the 160-kDa phosphoprotein. From the results of specific immunoprecipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sensitive pulp cell proteins, the 160-kDa protein was identified as insulin-receptor substrate-1. Both mitogenic treatments stimulated the tyrosine phosphorylation of a weak, 44-kDa protein, which we have identified as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (IGF-I-R) nor the phospholipase C gamma-isoform could be identified as tyrosine kinase substrates in either treatment. Pretreatment with the tyrosine kinase inhibitor genistein (20 micrograms/ml) significantly inhibited EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM) significantly prolonged the duration of the mitogen-stimulated tyrosine phosphorylation in both intact or permeabilized cells. Phorbol 12-myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth factor. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing external Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 microM) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on RDP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intracellular signalling systems that are sensitive to a PKC-dependent mechanism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell proliferation and ultimately may affect dentine formation.
表皮生长因子（EGF）和胰岛素样生长因子-I（IGF-I）均可在大鼠克隆牙髓细胞系（RDP 4-1）中产生剂量依赖性的细胞分裂速率刺激作用。为阐明可能介导有丝分裂作用的初始有丝分裂原诱导的细胞事件，研究了EGF和IGF-I对细胞蛋白酪氨酸磷酸化的影响。EGF（1 - 100 ng/ml）以剂量依赖性方式短暂刺激了四种表观分子量分别为220、180、140和120 kDa的主要蛋白以及另外五种较小蛋白（90、80、65、55和44 kDa）的酪氨酸磷酸化。IGF-I（1 - 100 ng/ml）剂量依赖性地刺激了160 kDa和140 kDa蛋白的酪氨酸磷酸化，对80 kDa、65 kDa和44 kDa蛋白的影响较小。与EGF的作用相反，IGF-I诱导的酪氨酸磷酸化持续超过60分钟，尤其是160 kDa磷蛋白的磷酸化。通过特异性免疫沉淀/蛋白质印迹分析结果，可将180 kDa的EGF敏感蛋白鉴定为EGF受体（EGF-R）。在IGF-I敏感的牙髓细胞蛋白中，160 kDa蛋白被鉴定为胰岛素受体底物-1。两种有丝分裂处理均刺激了一种弱的44 kDa蛋白的酪氨酸磷酸化，我们已将其鉴定为细胞外信号调节激酶-1。尽管存在正确大小的磷蛋白，但在两种处理中均未将IGF-I受体（IGF-I-R）或磷脂酶Cγ同工型鉴定为酪氨酸激酶底物。用酪氨酸激酶抑制剂染料木黄酮（20微克/毫升）预处理可显著抑制通透的RDP 4-1细胞中EGF和IGF-I诱导的酪氨酸磷酸化，而酪氨酸磷酸酶抑制剂原钒酸钠（1 mM）可显著延长有丝分裂原刺激的酪氨酸磷酸化在完整或通透细胞中的持续时间。佛波醇12-肉豆蔻酸酯13-乙酸酯（100 nM）可激活蛋白激酶C（PKC），抑制两种生长因子诱导的酪氨酸磷酸化。该作用可被用选择性PKC抑制剂星形孢菌素（200 nM，15分钟）预处理所阻断。然而，用EGTA（1 mM）去除细胞外Ca2+或用A23187离子载体（2 microM）诱导Ca2+内流均未显著改变EGF或IGF-I诱导的磷酸化。这些发现强烈表明，RDP 4-1细胞上的真正EGF-R和IGF-I-R与复杂的、酪氨酸激酶介导的细胞内信号系统偶联，该信号系统对PKC依赖性机制敏感。EGF和IGF-I诱导的酪氨酸磷酸化级联反应在体内牙髓细胞增殖的调节中可能具有重要作用，并最终可能影响牙本质形成。
