Manumycin Regulates Cytokine-induced NF-kappaB Activatio

Manumycin 调节细胞因子诱导的 NF-kappaB 激活

• 批准号: 6815464
• 负责人: MICHEL BERNIER
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6815464
• 关键词: I kappa B beta; biological signal transduction; enzyme activity; enzyme inhibitors; farnesyl compound; membrane structure; nuclear factor kappa beta; posttranslational modifications; tissue /cell culture; tumor necrosis factor alpha

The NF-kappaB/Rel family of transcription factors plays an important role in inflammatory responses. In nonstimulated cells, the heterodimeric NF-kappaB complex is located in the cytoplasm, where it is associated with the inhibitory molecule, IkappaBalpha. In response to stimulation by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1, IkappaBalpha undergoes phosphorylation and subsequent proteolytic degradation, allowing the p50/p65 NF-kappaB heterodimer to migrate to the nucleus and transactivate various inducible target genes. The IkappaB kinase (IKK) complex plays a pivotal role in the phosphorylation of IkappaBalpha in response to cytokine stimulation. This complex is composed of at least two catalytic subunits, IKKalpha and IKKbeta, and a tightly associated regulatory subunit known as NEMO. A number of studies have established that the Ras superfamily of small GTP-binding proteins plays important roles in signal transduction, proliferation, and malignant transformation but also inflammation. Ras and some Ras-like proteins (e.g., RhoB, Rheb, TC10) require posttranslational modification by conjugation of a farnesyl (15-carbon isoprenyl group) moiety to the C-terminal. After farnesylation, these Ras proteins are localized to the inner surface of the plasma membrane, and become functional. Inhibitors of protein farnesylation have been shown to block nuclear targeting of NF-kappaB by oncogenic Ras; however, whether protein farnesylation plays a role in cytokine-dependent regulation of NF-kappaB is unknown. In this study, we examined the effect of manumycin A, a potent and selective farnesyltransferase inhibitor, on cytokine-induced NF-kappaB activation in CHO cells and human liver-derived HepG2 cells. Manumycin A has antitumor activity in vitro, and in vivo studies in nude mouse xenograft models has demonstrated little toxic side effects. To assess whether protein farnesylation may play a role in the induction of NK-kappaB by cytokines, cells were exposed to 10 uM manumycin A for periods up to 6 h. This inhibitor clearly blocked NF-kappaB transactivation and nuclear translocation of p65/RelA in response to TNF. Manumycin A also blocked IKK-dependent phosphorylation of IkappaB alpha elicited by TNF or interleukin-1 beta. Using solid-phase kinase assays, it was determined that activation by TNF of the IKK complex and an upstream kinase, NF-kappaB inducing kinase (NIK), was rapidly attenuated after cell treatment with manumycin A for 30-60 min. Moreover, ectopic expression of NIK or IKKbeta in HepG2 cells did not confer protection against manumycin A. These results indicate that farnesylated proteins with a short half-life are involved in the up-regulation of NF-kappaB through signaling mechanisms that control the IKK pathway. Using blue native gel electrophoresis, we found the ~750 kDa IKK complex to be stable in the presence of manumycin A, but the TNF-dependent recruitment of IKK to lipid rafts was lost, an event that was correlated with actin depolymerization. Therefore, it would appear that manumycin A interferes with the recruitment and/or formation of signaling-competent complexes at the plasma membrane upon cytokine stimulation in cells devoid of K-ras mutation. Our results indicate a novel link between manumycin-sensitive targets and IKK activation elicited by TNF. As proinflammatory cytokines play an important role in the pathogenesis of insulin resistance, our findings suggest that inhibition of cytokine signaling at a converging step at or upstream of IKK may represent a potential target for new strategies to improve insulin-resistant states.

NF-κ B/Rel转录因子家族在炎症反应中起重要作用。在未受刺激的细胞中，异源二聚体NF-κ B复合物位于细胞质中，在那里它与抑制性分子IkappaB α结合。响应于促炎细胞因子肿瘤坏死因子（TNF）和白细胞介素-1的刺激，IkappaB α经历磷酸化和随后的蛋白水解降解，允许p50/p65 NF-kappaB异源二聚体迁移到细胞核并反式激活各种可诱导的靶基因。IkappaB激酶（IKK）复合物在响应于细胞因子刺激的IkappaB α磷酸化中起关键作用。该复合物由至少两个催化亚基（IKK α和IKK β）和一个紧密相关的调节亚基（称为NEMO）组成。许多研究已经确定小GTP结合蛋白的Ras超家族在信号转导、增殖和恶性转化以及炎症中起重要作用。Ras和一些Ras样蛋白（例如，RhoB，Rheb，TC 10）需要通过将法尼基（15-碳异戊二烯基）部分缀合至C-末端进行翻译后修饰。法尼基化后，这些Ras蛋白定位于质膜的内表面，并具有功能。蛋白质法尼基化的抑制剂已被证明可以阻断致癌Ras对NF-κ B的核靶向作用;然而，蛋白质法尼基化是否在NF-kappaB的尼古丁依赖性调节中起作用尚不清楚。在这项研究中，我们研究了manumycin A，一种有效的和选择性的法尼基转移酶抑制剂，在CHO细胞和人肝源性HepG 2细胞中对奎宁诱导的NF-κ B活化的影响。Manumycin A在体外具有抗肿瘤活性，在裸鼠异种移植模型中的体内研究表明几乎没有毒副作用。为了评估蛋白法尼基化是否在细胞因子诱导NK-κ B中起作用，将细胞暴露于10 μ M manumycin A长达6小时。该抑制剂明显阻断了TNF-α诱导的NF-κ B反式激活和p65/RelA核转位。Manumycin A还阻断了TNF或白细胞介素-1 β引起的IKK依赖性IkappaB α磷酸化。使用固相激酶试验，确定了TNF对IKK复合物和上游激酶NF-κ B诱导激酶（NIK）的激活在用manumycin A处理细胞30-60 min后迅速减弱。此外，HepG 2细胞中NIK或IKK β的异位表达并不能提供针对manumycin A的保护。这些结果表明，具有短半衰期的法尼基化蛋白通过控制IKK通路的信号传导机制参与NF-κ B的上调。使用蓝色天然凝胶电泳，我们发现约750 kDa的IKK复合物在manumycin A的存在下是稳定的，但TNF依赖的IKK向脂筏的募集丢失，这与肌动蛋白解聚相关。因此，似乎manumycin A干扰了缺乏K-ras突变的细胞中细胞因子刺激后质膜上信号活性复合物的募集和/或形成。我们的研究结果表明，一个新的链接之间的manumycin敏感的目标和IKK激活引起的TNF。 由于促炎细胞因子在胰岛素抵抗的发病机制中起着重要作用，我们的研究结果表明，在IKK或上游的会聚步骤抑制细胞因子信号传导可能是改善胰岛素抵抗状态的新策略的潜在靶点。