Nimah Marianne, Zhao Bin, Denenberg Alvin G, Bueno Orlando, Molkentin Jeffery, Wong Hector R, Shanley Thomas P
Division of Critical Care Medicine, Cincinnati Children's Hospital Research Foundation, University of Cincinnati Medical School, Cincinnati, Ohio 45229-3039, USA.
Shock. 2005 Jan;23(1):80-7. doi: 10.1097/01.shk.0000145206.28812.60.
Endotoxin tolerance has been characterized as diminished TNF-alpha expression after a second LPS stimulus and is dependent on new protein synthesis. LPS-induced expression of TNF-alpha is partly regulated by the p38 mitogen-activated protein (MAP) kinase, which post-transcriptionally stabilizes TNF-alpha mRNA. The dual-specific phosphatase, MKP-1, has been shown to negatively regulate p38 via dephosphorylation. We hypothesized that MKP-1 expression induced during tolerance regulates TNF-alpha expression by inhibiting p38 activity. To test this hypothesis, tolerance was induced in THP-1 cells, and naive or tolerized cells were rechallenged 18 h later with LPS (1 microg/mL) and TNF-alpha production was measured. Under similar conditions, nuclear proteins were isolated after LPS stimulation and were analyzed for phospho-p38 and MKP-1 by Western blot. Transient overexpression of MKP-1 was achieved using an adenoviral expression strategy and infected cells subsequently treated with LPS for TNF-alpha production and p38 activation. Results showed that LPS tolerance was induced as reflected by decreased TNF-alpha. Induction of LPS hyporesponsiveness could be mimicked by overexpression of MKP-1 but not beta-gal. MKP-1 expression was noted only in LPS-tolerized or Ad-MKP-1 infected cells. In the canonical and Ad-MKP-1-mediated tolerance models, decreased phospho-p38 activity was observed. MKP-1s role in mediating endotoxin tolerance was further confirmed by demonstrating the inability to fully tolerize peritoneal macrophages isolated from MKP-1 null mutant (vs. wild type) mice (24% vs. 72% reductions, respectively). These data demonstrate that the dual specific phosphatase MKP-1 is an important mediator of endotoxin tolerance via p38 regulation.
内毒素耐受的特征是在第二次脂多糖（LPS）刺激后肿瘤坏死因子-α（TNF-α）表达减少，且依赖于新的蛋白质合成。LPS诱导的TNF-α表达部分受p38丝裂原活化蛋白（MAP）激酶调节，该激酶在转录后使TNF-α mRNA稳定。双特异性磷酸酶MKP-1已被证明可通过去磷酸化对p38进行负调控。我们推测，耐受过程中诱导产生的MKP-1通过抑制p38活性来调节TNF-α表达。为验证这一假设，在THP-1细胞中诱导耐受，18小时后用LPS（1微克/毫升）再次刺激未处理或已耐受的细胞，并检测TNF-α的产生。在相似条件下，LPS刺激后分离核蛋白，通过蛋白质免疫印迹法分析磷酸化p38和MKP-1。采用腺病毒表达策略实现MKP-1的瞬时过表达，随后用LPS处理感染细胞以检测TNF-α产生和p38激活情况。结果显示，LPS耐受诱导成功，表现为TNF-α降低。过表达MKP-1而非β-半乳糖苷酶可模拟LPS低反应性的诱导。仅在LPS耐受或Ad-MKP-1感染的细胞中观察到MKP-1表达。在经典和Ad-MKP-1介导的耐受模型中，均观察到磷酸化p38活性降低。通过证明无法使从MKP-1基因敲除突变体（与野生型相比）小鼠分离的腹膜巨噬细胞完全耐受（分别降低24%和72%），进一步证实了MKP-1在介导内毒素耐受中的作用。这些数据表明，双特异性磷酸酶MKP-1是通过p38调节内毒素耐受的重要介质。
