Tamura Akiyoshi, Kitayama Koji, Adachi Mutsumi, Hashimoto Kentaro, Oguro Ami, Imaoka Susumu
Department of Biomedical Sciences, School of Biological and Environmental Sciences, Kwansei Gakuin University.
Graduate School of Biomedical and Health Sciences, Hiroshima University.
J Toxicol Sci. 2025;50(3):105-116. doi: 10.2131/jts.50.105.
Hypoxia induces the expression of nuclear factor kappa B (NF-kappa-B). NF-kappa-B functions by forming dimers from five main subunits: p65 (RelA), RelB, p52, p50, and c-Rel. In the classical pathway, NF-kappa-B activity is regulated by the degradation-inducing factor I kappa B kinase (IKK). IKK is composed of an α/β isomer and essential modulator NEMO (γ) subunits in the classical pathway, which may be the major pathway for NF-kappa-B signaling. In the present study, we focused on factor-inhibiting HIF-1 (FIH-1) and Prolyl hydroxylase domain enzyme (PHD), which have been identified as oxygen concentration-dependent regulators of HIF-1α. PHD has three isoforms: PHD1, PHD2, and PHD3, which have different affinities towards HIF-1α. We examined the interactions between IKKα/β and PHD1-3 by immunoprecipitation. PHDs efficiently interacted with IKKα/β. Furthermore, the overexpression of PHDs decreased the mRNA level of IL-1β, a downstream factor of NF-kappa-B activated by LPS. The overexpression of PHD1 and PHD2 markedly reduced IKKα/β protein levels; however, the effects of PHD3 were weaker than those of PHD1 and PHD2. Mutants of the active sites of PHD1 and PHD2 did not decrease IKKα/β protein levels, and a mutation in the active site of PHD3 did not affect IKKα/β protein levels. We also attempted to investigate the interactions of FIH-1 with IKKα/β and IκBα by immunoprecipitation, but found none. Moreover, IKKα/β and p65 protein levels were not affected by the overexpression of FIH-1. Collectively, these results suggest that PHDs directly regulated IKK protein levels, while FIH-1 did not affect the NF-kappa-B classical pathway.
缺氧诱导核因子κB（NF-κB）的表达。NF-κB通过由五个主要亚基形成二聚体来发挥作用：p65（RelA）、RelB、p52、p50和c-Rel。在经典途径中，NF-κB的活性由降解诱导因子IκB激酶（IKK）调节。在经典途径中，IKK由α/β异构体和必需调节因子NEMO（γ）亚基组成，这可能是NF-κB信号传导的主要途径。在本研究中，我们重点关注了抑制因子HIF-1（FIH-1）和脯氨酰羟化酶结构域酶（PHD），它们已被确定为HIF-1α的氧浓度依赖性调节因子。PHD有三种同工型：PHD1、PHD2和PHD3，它们对HIF-1α具有不同的亲和力。我们通过免疫沉淀检测了IKKα/β与PHD1-3之间的相互作用。PHD与IKKα/β有效相互作用。此外，PHD的过表达降低了IL-1β的mRNA水平，IL-1β是由LPS激活的NF-κB的下游因子。PHD1和PHD2的过表达显著降低了IKKα/β蛋白水平；然而，PHD3的作用比PHD1和PHD2弱。PHD1和PHD2活性位点的突变体没有降低IKKα/β蛋白水平，PHD3活性位点的突变也不影响IKKα/β蛋白水平。我们还试图通过免疫沉淀研究FIH-1与IKKα/β和IκBα的相互作用，但未发现相互作用。此外，FIH-1的过表达不影响IKKα/β和p65蛋白水平。总体而言，这些结果表明PHD直接调节IKK蛋白水平，而FIH-1不影响NF-κB经典途径。
