Inhibitor-κB kinase-β regulates LPS-induced TNF-α production in cardiac myocytes through modulation of NF-κB p65 subunit phosphorylation
Inhibitor-κB kinase-β regulates LPS-induced TNF-α production in cardiac myocytes through modulation of NF-κB p65 subunit phosphorylation
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DOI：
10.1152/ajpheart.00393.2005
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发表时间：
2005-11-01
影响因子：
4.8
通讯作者：
Rogers, TB
中科院分区：
文献类型：
--
作者：
Hall, G;Singh, IS;Rogers, TB
肿瘤坏死因子-α被认为是心肌功能不全的重要因素。尽管一些研究表明核因子-kappa B家族的成员是心肌肿瘤坏死因子-α基因表达的重要调节因子，但最近我们对核因子-kappa B亚基翻译后修饰对核因子-kappaB活性的调控的了解表明，目前关于心脏核因子-kappaB依赖的细胞因子表达的观点是不完整的。因此，本研究的目的是研究p65亚单位磷酸化在调节培养的新生大鼠心室肌细胞产生肿瘤坏死因子-kappa中的作用。细菌内毒素诱导的肿瘤坏死因子-α的产生伴随着p65在Ser(536)的磷酸化增加12倍，这一修饰与p65反式激活潜能的增强有关。药物抑制IKK-β可使脂多糖诱导的肿瘤坏死因子-α的产生减少38倍，肿瘤坏死因子-α的mRNA水平降低6倍，使I-kappa B-α的磷酸化水平降低5倍，使I-kappa B-α的磷酸化水平降低2倍，使p65的磷酸化水平降低6倍。显性负性p65的过度表达使肿瘤坏死因子-α的产生减少3.5倍，而显性负性IKK-β的过度表达使脂多糖诱导的肿瘤坏死因子-α的产生减少2倍，p65的磷酸化减少2倍。显性负性IKK-α的过表达对p65的磷酸化或TNF-α的产生没有影响，表明在心脏Ser536处p65的磷酸化和TNF-α的产生中起核心作用的是IKK-β，而不是IKK-α。最后，我们用染色质免疫沉淀法证明，在心肌细胞中，内毒素刺激Ser(536)磷酸化的p65向肿瘤坏死因子-α基因启动子募集。综上所述，这些数据为核因子-kappa B信号在心脏中肿瘤坏死因子-α基因表达中的作用提供了令人信服的证据，并强调了这一促炎症基因调控途径在管理细胞因子诱导的心肌功能障碍中作为潜在治疗靶点的重要性。
TNF-alpha\ is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappa B family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappa B activity through posttranslational modification of NF-kappa B subunits suggest that the present view of NF-kappa B-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-kappa production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser(536), a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and I kappa B-alpha phosphorylation 5-fold and degraded I kappa B-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser(536)-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappa B signaling in TNF-alpha gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.