Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.
Endogenous protein kinase-C activation in osteoblast-like cells modulates responsiveness to estrogen and estrogen receptor levels.
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成骨细胞样细胞中的内源性蛋白激酶-C 激活调节对雌激素和雌激素受体水平的反应。
作者：
Silvia Migliaccio;W. C. Wetsel;William M. Fox;T. Washburn;K. S. Korach
为了更容易地评价雌激素在骨细胞动态平衡中的生理作用，将雌激素受体(ER)基因稳定地转染成骨样骨肉瘤细胞系ROS17/2.8。这些转基因的ROS.SMER 14细胞在融合后对雌激素刺激高度敏感，但在融合后变得对雌激素刺激不敏感。这些研究的目的是要确定这些ER稳定转导的细胞在融合后失去反应性的机制。用溴脱氧尿嘧啶核苷免疫细胞化学检测细胞增殖能力时，约70%的融合细胞处于活跃分裂状态，而融合后细胞均未分裂。亚汇合体细胞含有2500-3000个ER结合位点/细胞，而融合后细胞的ER含量很低，常常检测不到。融合后，稳态ER基因表达水平没有明显改变。ER蛋白水平也不受汇合状态的影响。由于蛋白激酶C(PKC)已被报道影响细胞增殖和类固醇激素受体结合，因此我们测定了融合后和亚融合细胞中的PKC活性。融合后细胞中钙依赖的PKC活性约为融合后细胞的2倍，而不依赖钙的PKC活性则无明显差异。为了更详细地研究PKC的作用，用PKC抑制剂(H-7或星形孢子素)或肿瘤促进剂TPA(12-O-十四酰佛波醇-13-醋酸酯)处理融合后的细胞以下调PKC的活性，并评估ER的变化。抑制或下调融合后细胞中的PKC活性以剂量依赖的方式增强ER结合能力和外源报告基因和内源性碱性磷酸酶的雌激素反应性，这是一种内源性雌激素刺激基因。这些数据表明，在骨细胞中存在PKC和ER信号系统之间的相互作用，这种相互作用可能受到细胞的增殖和/或分化状态的影响，从而导致激素反应性的调节。
The osteoblast-like osteosarcoma cell line ROS 17/2.8, which expresses very low levels of estrogen receptor (ER), was stably transfected with the mouse ER in order to more easily evaluate the physiological role of estrogens in bone cell homeostasis. These transfected ROS.SMER 14 cells are highly responsive to estrogenic stimulation at subconfluence, but become refractory to estrogenic stimulation when postconfluency is reached. The purpose of these studies was to determine the mechanisms underlying this loss of responsiveness in these ER stably transfected cells at postconfluence. When proliferative capacity was evaluated by bromodeoxyuridine immunocytochemistry, approximately 70% of the subconfluent cells were actively dividing, whereas none of the postconfluent cells underwent division. Subconfluent cells were found to contain 2500-3000 ER-binding sites/cell, whereas the ER in postconfluent cells was low and often undetectable. Steady state ER mRNA levels were not significantly modified by postconfluency. ER protein levels were also unaffected by confluency status. Since protein kinase-C (PKC) has been reported to influence cell proliferation and steroid hormone receptor binding, PKC activity was measured in sub- and postconfluent cells. Calcium-dependent PKC activity was approximately about 2-fold higher in postconfluent compared to subconfluent cells, whereas no differences were discerned in calcium-independent PKC activity. In an effort to examine the role of PKC in greater detail, postconfluent cells were treated with PKC inhibitors (H-7 or staurosporine) or with the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) to down-regulate PKC activity, and changes in ER were evaluated. Inhibition or down-regulation of the PKC activity in postconfluent cells enhanced ER-binding capacity in a dose-dependent manner and estrogen responsiveness of an exogenous reporter gene and of the endogenous alkaline phosphatase, representing an endogenous estrogen-stimulated gene. These data indicate that there is an interaction between the PKC and ER signaling systems in bone cells and that this interaction may be influenced by the proliferative and/or differentiative state of the cells, resulting in modulation of hormone responsiveness.
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