Manumycin Regulates Cytokine-induced NF-kappaB Activatio

NF-kappaB/Rel家族转录因子在炎症反应中发挥重要作用。在未受刺激的细胞中，异二聚体NF-kappaB复合物位于细胞质中，在那里它与抑制分子IkappaBalpha相关。在促炎细胞因子肿瘤坏死因子（TNF）和白细胞介素-1的刺激下，IkappaBalpha经历磷酸化和随后的蛋白水解降解，使p50/p65 NF-kappaB异源二聚体迁移到细胞核并反激活各种诱导靶基因。IkappaB激酶（IKK）复合物在ikappabα的磷酸化中起关键作用，以响应细胞因子刺激。该复合物由至少两个催化亚基IKKalpha和IKKbeta以及一个紧密相关的调控亚基NEMO组成。许多研究已经证实，小gtp结合蛋白Ras超家族在信号转导、增殖和恶性转化中发挥重要作用，但也在炎症中发挥重要作用。Ras和一些Ras样蛋白（如RhoB， Rheb, TC10）需要通过在c端偶联法尼基（15碳异戊二烯基）片段进行翻译后修饰。经过法尼化后，这些Ras蛋白定位于质膜的内表面，并发挥功能。蛋白法尼化抑制剂已被证明可以阻断致癌Ras对NF-kappaB的核靶向；然而，蛋白法尼化是否在NF-kappaB的细胞因子依赖性调节中发挥作用尚不清楚。在这项研究中，我们检测了马霉素A（一种有效的选择性法尼基转移酶抑制剂）对CHO细胞和人肝源性HepG2细胞中细胞因子诱导的NF-kappaB活化的影响。马霉素A在体外具有抗肿瘤活性，并且在裸鼠异种移植模型的体内研究表明几乎没有毒副作用。为了评估蛋白法尼化是否可能在细胞因子诱导NK-kappaB中发挥作用，将细胞暴露于10 μ m马霉素a长达6小时。这种抑制剂明显阻断NF-kappaB的反激活和p65/RelA对TNF的核易位反应。马霉素A也阻断了ikk依赖性的IkappaB α由TNF或白细胞介素-1 β引起的磷酸化。通过固相激酶实验，我们确定TNF对IKK复合物和上游激酶NF-kappaB诱导激酶（NIK）的激活在马霉素A处理细胞30-60分钟后迅速减弱。此外，在HepG2细胞中，NIK或ikkβ的异位表达并未赋予对马霉素a的保护作用。这些结果表明，半衰期短的法酰化蛋白通过控制IKK通路的信号机制参与了NF-kappaB的上调。使用蓝色天然凝胶电泳，我们发现~750 kDa的IKK复合物在马霉素A存在下是稳定的，但IKK对脂筏的tnf依赖性募集丢失，这一事件与肌动蛋白解聚相关。因此，在没有K-ras突变的细胞中，当细胞因子刺激时，马霉素A似乎会干扰质膜上信号传导复合物的募集和/或形成。我们的研究结果表明，在免疫霉素敏感靶点和TNF诱导的IKK激活之间存在一种新的联系。

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.