Shames B D, Selzman C H, Pulido E J, Meng X, Meldrum D R, McIntyre R C, Harken A H, Banerjee A
Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
J Surg Res. 1999 May 1;83(1):69-74. doi: 10.1006/jsre.1998.5564.
Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes.
Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay.
LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes.
These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.
肿瘤坏死因子α（TNF-α）是脓毒症休克的重要介质。内毒素（LPS）在人单核细胞中的信号转导导致核因子κB（NF-κB）激活和TNF-α释放。先前的研究表明蛋白激酶C（PKC）和蛋白酪氨酸激酶（PTK）的激活均参与LPS诱导的NF-κB激活和TNF-α产生。我们推测抑制PKC或PTK会减少LPS诱导的人单核细胞中NF-κB与DNA的结合及TNF-α释放。
用人单核细胞分别单独用佛波酯（PMA，50 ng/ml）刺激，或用LPS（100 ng/ml）刺激，同时加入或不加入非特异性丝氨酸/苏氨酸蛋白激酶抑制剂星形孢菌素（Stauro）、特异性泛PKC抑制剂双吲哚马来酰胺（Bis）或PTK抑制剂染料木黄酮（Gen）。通过酶联免疫吸附测定法（ELISA）检测培养上清液中TNF-α的释放。通过电泳迁移率变动分析评估NF-κB与DNA的结合。
LPS增加人单核细胞中NF-κB与DNA的结合及TNF-α释放。非特异性蛋白激酶抑制可抑制NF-κB激活和TNF-α释放，而用Bis特异性抑制PKC对LPS诱导的NF-κB与DNA的结合或TNF-α释放无影响。用Gen抑制PTK可减弱LPS诱导的人单核细胞中NF-κB与DNA的结合及TNF-α产生。用PMA直接激活PKC可诱导人单核细胞的NF-κB激活和TNF-α产生。
这些结果表明LPS诱导的人单核细胞中NF-κB激活和TNF-α释放与PKC活性无关。此外，我们的结果证明PTK在LPS诱导的人单核细胞中NF-κB激活和TNF-α释放中起作用，因此可能是炎症状态下的潜在治疗靶点。
