GM-CSF TRIGGERS A RAPID, GLUCOSE DEPENDENT EXTRACELLULAR ACIDIFICATION BY TF-1 CELLS - EVIDENCE FOR SODIUM PROTON ANTIPORTER AND PKC MEDIATED ACTIVATION OF ACID PRODUCTION
GM-CSF TRIGGERS A RAPID, GLUCOSE DEPENDENT EXTRACELLULAR ACIDIFICATION BY TF-1 CELLS - EVIDENCE FOR SODIUM PROTON ANTIPORTER AND PKC MEDIATED ACTIVATION OF ACID PRODUCTION
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DOI：
10.1002/jcp.1041540116
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发表时间：
1993-01-01
影响因子：
5.6
通讯作者：
PARCE, JW
中科院分区：
文献类型：
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作者：
WADA, HG;INDELICATO, SR;PARCE, JW
人骨髓细胞系TF-1的细胞外酸化率在重组粒细胞-巨噬细胞集落刺激因子（GM-CSF）的推注下迅速增加。使用硅微生理计测量细胞外酸化速率。该仪器包含配备电位传感器的微流室，用于监测pH值。细胞固定在夹在两个多孔聚碳酸酯膜之间的纤维蛋白凝块中。这些膜是一次性塑料“细胞胶囊”的一部分，可以装入微生理计的流动室。GM-CSF激活的酸化爆发是剂量依赖性的，并且可以通过用抗GM-CSF抗体预处理细胞因子来中和。酸化爆发可以在动力学上分解成至少两个组分。爆发的快速成分是由于钠/质子反向转运蛋白的激活，如其在无钠介质中和阿米洛利存在下的消除所证明的。GM-CSF反应的较慢组分是糖酵解代谢增加的结果，如通过其对作为培养基营养物的D-葡萄糖的依赖性所证明的。冈田酸（一种磷酸丝氨酸/苏氨酸磷酸酶抑制剂）、佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA，一种蛋白激酶C（PKC）激活剂）和离子霉素（一种钙离子载体）都能在TF-1细胞中产生类似于GM-CSF反应的代谢爆发。用PMA预处理TF-1细胞18 h导致GM-CSF酸化反应丧失。虽然这种治疗被报道破坏蛋白激酶活性，我们在这里证明，它也下调TF-1细胞表面上的高亲和力GM-CSF受体的表达。此外，PMA处理18 h后，GM-CSF驱动的TF-1细胞增殖减少。PMA短期处理（1 -2小时）再次导致GM-CSF酸化反应的丧失，但高亲和力GM-CSF受体的表达没有降低。PKC参与GM-CSF信号转导的证据是使用calphostin C获得的，calphostin C是PKC的特异性抑制剂，其在亚毒性浓度下抑制GM-CSF代谢爆发。酪氨酸激酶抑制剂Genistein和herbimycin A均能抑制TF-1细胞的GM-CSF反应，但仅在足以抑制PMA刺激的水平。这些结果表明，GM-CSF激活的TF-1细胞的细胞外酸化是由钠/质子逆向转运蛋白活性和糖酵解的增加引起的，通过蛋白激酶信号通路，其可以被PMA激活和下调。
The extracellular acidification rate of the human bone marrow cell line, TF-1, increases rapidly in response to a bolus of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF). Extracellular acidification rates were measured using a silicon microphysiometer. This instrument contains micro-flow chambers equipped with potentiometric sensors to monitor pH. The cells are immobilized in a fibrin clot sandwiched between two porous polycarbonate membranes. The membranes are part of a disposable plastic ''cell capsule'' that fits into the microphysiometer flow chamber. The GM-CSF activated acidification burst is dose dependent and can be neutralized by pretreating the cytokine with anti-GM-CSF antibody. The acidification burst can be resolved kinetically into at least two components. A rapid component of the burst is due to activation of the sodium/proton antiporter as evidenced by its elimination in sodium-free medium and in the presence of amiloride. A slower component of the GM-CSF response is a consequence of increased glycolytic metabolism as demonstrated by its dependence on D-glucose as a medium nutrient. Okadaic acid (a phospho-serine/threonine phosphatase inhibitor), phorbol 12-myristate 13-acetate (PMA, a protein kinase C (PKC) activator), and ionomycin (a calcium ionophore) all produce metabolic bursts in TF-1 cells similar to the GM-CSF response. Pretreatment of TF-1 cells with PMA for 18 h resulted in loss of the GM-CSF acidification response. Although this treatment is reported to destroy protein kinase activity, we demonstrate here that it also down-regulates expression of high-affinity GM-CSF receptors on the surface of TF-1 cells. In addition, GM-CSF driven TF-1 cell proliferation was decreased after the 1 8 h PMA treatment. Short-term treatment with PMA (I-2 h) again resulted in loss of the GM-CSF acidification response, but without a decrease in expression of high-affinity GM-CSF receptors. Evidence for involvement of PKC in GM-CSF signal transduction was obtained using calphostin C, a specific inhibitor of PKC, which inhibited the GM-CSF metabolic burst at a subtoxic concentration. Genistein and herbimycin A, tyrosine kinase inhibitors, both inhibited the GM-CSF response of TF-1 cel ls, but only at levels high enough to also inhibit stimulation by PMA. These results indicate that GM-CSF activated extracellular acidification of TF-1 cells is caused by increases in sodium/proton antiporter activity and glycolysis, through protein kinase signalling pathways which can be both activated and down-regulated by PMA.