Bankey P, Carlson A, Ortiz M, Singh R, Cerra F
Department of Surgery, University of Minnesota, Minneapolis 55455.
J Surg Res. 1990 Sep;49(3):256-61. doi: 10.1016/0022-4804(90)90130-t.
Tumor necrosis factor (TNF) has been proposed as a primary inflammatory mediator of septic shock. In vitro and in vivo studies indicate that endotoxin- or lipopolysaccharide (LPS)-activated macrophages are a principle source of TNF; however, membrane signal transduction and intracellular pathways by which LPS triggers TNF production in macrophages are unclear. Recent evidence indicates that specific protein phosphorylation via activation of protein kinase C (PKC) is an early, critical step in the signaling of macrophage TNF production by phorbol esters. We hypothesize that PKC activation is also required in LPS-signaled Kupffer cell (KC) TNF production. Murine KCs were obtained by liver perfusion and digestion and then stimulated with LPS (Escherichia coli O111:B4) or LPS in the presence of H-7, a selective PKC inhibitor. Conditioned media was collected at 3 hr for assay of TNF utilizing the L929 cytolysis bioassay standardized to murine-rTNF-alpha. We found that H-7 inhibited significantly LPS signaled TNF release at a concentration of 10 microM, while H-8 (a cyclic nucleotide specific inhibitor) had no effect. The effect of H-7 was dose dependent and present at varying concentrations of LPS. Down regulation of PKC activity by preincubation of KCs with phorbol myristate acetate (PMA, a direct activator of PKC) also resulted in significantly reduced TNF release after LPS stimulation. The inhibitor H-7 (10 microM) also significantly inhibited LPS signaled prostaglandin E2 release in Kupffer cells. Total and specific intracellular protein phosphorylation was determined by trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis after labeling stimulated Kupffer cells with 32Pi. Total protein phosphorylation was not significantly altered by LPS stimulation; however, autoradiograms from PMA- and LPS-stimulated KCs demonstrate enhanced phosphorylation of a 40-kDa protein (2.7 +/- 0.9-fold) and a 33-kDa protein (3.1 +/- 1.0-fold) which were inhibited by H-7. We conclude that activation of PKC and protein phosphorylation are required steps in the signal transduction pathway of LPS-stimulated TNF production in Kupffer cells.
肿瘤坏死因子（TNF）被认为是脓毒症休克的主要炎症介质。体外和体内研究表明，内毒素或脂多糖（LPS）激活的巨噬细胞是TNF的主要来源；然而，LPS触发巨噬细胞产生TNF的膜信号转导和细胞内途径尚不清楚。最近的证据表明，通过激活蛋白激酶C（PKC）进行的特定蛋白磷酸化是佛波酯诱导巨噬细胞产生TNF信号传导的早期关键步骤。我们假设PKC激活在LPS信号传导的库普弗细胞（KC）产生TNF过程中也是必需的。通过肝脏灌注和消化获得小鼠KC，然后用LPS（大肠杆菌O111：B4）或在选择性PKC抑制剂H-7存在下的LPS进行刺激。在3小时时收集条件培养基，利用标准化为鼠rTNF-α的L929细胞溶解生物测定法测定TNF。我们发现，H-7在浓度为10μM时显著抑制LPS信号传导的TNF释放，而H-8（一种环核苷酸特异性抑制剂）则无作用。H-7的作用呈剂量依赖性，在不同浓度的LPS下均存在。用佛波醇肉豆蔻酸酯乙酸酯（PMA，PKC的直接激活剂）预孵育KC来下调PKC活性，也导致LPS刺激后TNF释放显著减少。抑制剂H-7（10μM）也显著抑制LPS信号传导的库普弗细胞中前列腺素E2的释放。在用32Pi标记受刺激的库普弗细胞后，通过三氯乙酸沉淀和SDS-聚丙烯酰胺凝胶电泳测定总蛋白和特异性细胞内蛋白磷酸化。LPS刺激未显著改变总蛋白磷酸化；然而，来自PMA和LPS刺激的KC的放射自显影片显示，一种40 kDa蛋白（2.7±0.9倍）和一种33 kDa蛋白（3.1±1.0倍）的磷酸化增强，而这被H-7抑制。我们得出结论，PKC激活和蛋白磷酸化是LPS刺激的库普弗细胞产生TNF信号转导途径中的必需步骤。
