The Role of P38 MAPK and PKC in BLP Induced TNF-α Release, Apoptosis, and NFκB Activation in THP-1 Monocyte Cells
The Role of P38 MAPK and PKC in BLP Induced TNF-α Release, Apoptosis, and NFκB Activation in THP-1 Monocyte Cells
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DOI：
10.1016/j.jss.2008.02.031
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发表时间：
2009-01-01
影响因子：
2.2
通讯作者：
Redmond, Henry P.
中科院分区：
文献类型：
--
作者：
O'Sullivan, Adrian W.;Wang, Jiang H.;Redmond, Henry P.
背景P38丝裂原活化蛋白激酶（p38 MAPK）是炎症反应的关键介质，这使其成为一种合适的候选者作为一种新的治疗炎症的策略。在这项研究中，我们着手研究蛋白激酶C（PKC）和P38 MAPK信号激酶在细菌脂蛋白（BLP）诱导的核因子-κ B（NF-κ B B）激活和肿瘤坏死因子-α（TNF-α）释放THP-1单核细胞系中的确切作用。将THP-1细胞与BLP（0-1000 ng/mL）、佛波醇肉豆蔻酸酯乙酸酯（PMA，0-100 μ g/mL）或两者的组合一起孵育6和24小时，用或不用SB 202190（p38 MAPK的特异性抑制剂）和双吲哚基马来酰亚胺1（PKC的特异性抑制剂）（0-200 μ m）预处理。分析细胞上清液的TNF-α释放和细胞凋亡。采用电迁移率超移法检测NF κ B B活性。用所有浓度的SB 202190预处理显著降低了BLP诱导的TNF-α释放（428.7 +/-5.9对51 +/-0.8 rho g/mL，P < 0.05）。用bis I预处理在较高浓度（200 μ M）下显著抑制TNF-α释放（429.7 +/- 5.9对194.9 +/- 42.68 rho g（mL，P < 0.05），但这远不如SB 202190有效。PMA诱导的TNF-α释放在6 h时不被SB 202190或his 1抑制，但在24 h时显著抑制（148.5 +/- 9.8 vs 24 +/- 1.7和25.1 +/- 4.4 rho g/mL，P < 0.05）。BLP和脂多糖（LPS）均不能诱导THP-1细胞凋亡（P > 0.05），PMA诱导THP-1细胞凋亡呈时间和剂量依赖性。与BLP（1000 ng/mL）而非LPS（1000 ng/mL）组合时，低剂量PMA导致细胞凋亡显著增加，分别为6% +/-0.5%（对照）对9.2% +/-0.3%（P < 0.05）和7% +/-2.2%（对照）对7.7% +/-0.3%（P > 0.05）。这种协同作用被双吲哚基马来酰亚胺100 nm抑制，8.9% +/- 0.9%（对照）对9.8% +/- 0.2%（P > 0.05）。PMA和BLP可诱导NF κ B B快速核转位，SB-202190和His I预处理可抑制PMA和BLP诱导的NF κ B快速核转位，SB-202190预处理可抑制PMA和BLP诱导的NF κ B快速核转位。P38是BLP诱导的TNF-α释放和NF κ B B活化的关键介质，而PKC仅部分负责其应答。P38和PKC都是PMA诱导TNF-α释放和NF κ B B活化的关键介质。(C)2009 Elsevier Inc. All rights reserved.
Background. P38 mitogen activated protein kinase (p38 MAPK) is a critical mediator of the inflammatory response, which makes it a suitable candidate as a novel therapeutic strategy for inflammatory conditions. In this study, we set out to examine the precise role of both protein kinase C (PKC) and P38 MAPK signaling kinases in bacterial lipoprotein (BLP) induced nuclear factor-kappa B (NF kappa B) activation and tumor necrosis factor-alpha (TNF alpha) release in THP-1 monocytic cell line.Materials and methods. THP-1 cells were incubated with BLP(0-1000 ng/mL), phorbol myristate acetate (PMA, 0-100 mu g/mL) or a combination of both for 6 and 24 h, with or without pretreatment with SB202190, a specific inhibitor of p38 MAPK and bisindolylmaleimide 1, a specific inhibitor of PKC (0-200 mu m). Cell supernatants were analyzed for TNF-alpha release and apoptosis. NF kappa B activity was analyzed by electromobility supershift assay.Results. BLP induced TNF-alpha release was significantly reduced by pretreatment with SB202190 at all concentrations (428.7 +/- 5.9 versus 51 +/- 0.8 rho g/mL, P < 0.05). Pretreatment with bis I significantly inhibited TNF-alpha release at higher concentrations (200 mu M) (429.7 +/- 5.9 versus 194.9 +/- 42.68 rho g(mL, P < 0.05) but this was much less effective than SB202190. PMA induced TNF-alpha release was not inhibited at 6 h by either SB202190 or his 1, but was significantly so at 24 h (148.5 +/- 9.8 versus 24 +/- 1.7 and 25.1 +/- 4.4 rho g/mL, P < 0.05). BLP or lipopolysaccharide (LPS) did not result in apoptosis in THP-1 cells (P > 0.05) with PMA inducing apoptosis in a time- and dose-dependent manner. In combination with BLP (1000 ngmL) but not LPS (1000 ng/mL), low dose PMA resulted in a significant increase in apoptosis, 6% +/- 0.5% (Control) versus 9.2% +/- 0.3% (P < 0.05) and 7% +/- 2.2% (Control) versus 7.7% +/- 0.3% (P > 0.05), respectively. This synergistic effect was inhibited by bisindolylmaleimide 100 nm, 8.9% +/- 0.9% (Control) versus 9.8% +/- 0.2% (P > 0.05). PMA and BLP induced rapid nuclear translocation of NF kappa B, which was inhibited by pretreatment with both SB-202190 and his I, and SB202190 but not his I, respectively.Conclusions. P38 is a critical mediator of BLP induced TNF-alpha release and NF kappa B activation, whereas PKC is only partially responsible for its response. P38 and PKC are both critical mediators of PMA induced TNF-alpha release and NF kappa B activation. (C) 2009 Elsevier Inc. All rights reserved.