Chathuranga Kiramage, Rathnapala Pramodya, Weerawardhana Asela, Kim Tae-Hwan, Seong Yebin, Gayan Chathuranga W A, Subasinghe Ashan, Haluwana D K, Gamage Nuwan, Choi Youn Jung, Jung Jae U, Lee Jong-Soo
Laboratory of Microbiology, College of Veterinary Medicine, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea.
Kao Autoimmunity Institute and Division of Rheumatology, Department of Medicine, Ce dars-Sinai Medical Center, Los Angeles, CA, USA.
Cell Commun Signal. 2025 May 31;23(1):257. doi: 10.1186/s12964-025-02260-6.
Regulation of the nuclear factor-kappa B (NF-kB) signaling pathway is a major host homeostatic mechanism for controlling hyper-inflammation or chronic inflammation. Despite extensive research, the regulatory factors of NF-kB signaling required to preserve homeostasis and control inflammatory disorders are not fully understood. Moreover, the role of MARCH2 in chronic inflammation models and the regulation of MARCH2 activation remain to be elucidated.
We monitored disease severity and mortality in MARCH2 or MARCH2 mice induced experimental colitis. Susceptibility to DSS-induced experimental colitis was determined by various methods, including Swiss roll assay and fluorescein isothiocyanate (FITC)-dextran treatment, respectively. RNA-sequencing was conducted to recognize the inflammatory response-related genes in the distal colon of colitis-induced mice. Enzyme-linked immunosorbent assay (ELISA) was used to measure the cytokines and chemokines with in vitro and in vivo samples. Affinity purification and LC-MS/MS analysis were used to identify the MARCH2 interacting proteins and posttranslational modifications. The underlying mechanism was elucidated using immunoblotting, co-immunoprecipitation, ubiquitination assay, and confocal microscopy.
Here, we report that MARCH2 mice were more susceptible to experimental inflammatory bowel disease (IBD) due to the massive production of cytokines. Stimulation by inflammatory cytokines such as TNF induces dimerization of MARCH2 at a later stage and dimerized MARCH2 undergoes K63-linked autoubiquitination at lysine 127 and 238, which promotes NEMO recognition, ubiquitination and proteasomal degradation. We also show an interaction between MARCH2 and MARCH8 in resting cells that inhibits MARCH2 activation. Taken together, these findings provide new insights into the molecular mechanism of MARCH2 and suggest a crucial role of MARCH2 in the modulation of inflammation and cellular homeostasis.
Our results indicate that MARCH2 plays a critical role in regulating NEMO/IKKγ under the inflammatory and resting conditions, thereby suppressing excessive or unexpected inflammatory responses. Our findings here not only demonstrate a biological role of MARCH2 in inflammatory signaling pathways but also provide a novel insight in the underlying mechanism.
核因子-κB（NF-κB）信号通路的调节是控制过度炎症或慢性炎症的主要宿主稳态机制。尽管进行了广泛研究，但维持稳态和控制炎症性疾病所需的NF-κB信号调节因子仍未完全了解。此外，MARCH2在慢性炎症模型中的作用以及MARCH2激活的调节仍有待阐明。
我们监测了MARCH2基因敲除或过表达小鼠诱导实验性结肠炎后的疾病严重程度和死亡率。分别通过多种方法，包括瑞士卷试验和异硫氰酸荧光素（FITC）-葡聚糖处理，确定对葡聚糖硫酸钠（DSS）诱导的实验性结肠炎的易感性。进行RNA测序以识别结肠炎诱导小鼠远端结肠中与炎症反应相关的基因。酶联免疫吸附测定（ELISA）用于测量体外和体内样品中的细胞因子和趋化因子。亲和纯化和液相色谱-串联质谱（LC-MS/MS）分析用于鉴定MARCH2相互作用蛋白和翻译后修饰。使用免疫印迹、免疫共沉淀、泛素化测定和共聚焦显微镜阐明潜在机制。
在此，我们报告MARCH2基因敲除小鼠由于细胞因子的大量产生而对实验性炎症性肠病（IBD）更易感。肿瘤坏死因子等炎性细胞因子刺激在后期诱导MARCH2二聚化，二聚化的MARCH2在赖氨酸127和238处发生K63连接的自身泛素化，这促进了NEMO识别、泛素化和蛋白酶体降解。我们还显示在静息细胞中MARCH2和MARCH8之间存在相互作用，抑制MARCH2激活。综上所述，这些发现为MARCH2的分子机制提供了新见解，并表明MARCH2在炎症调节和细胞稳态中起关键作用。
我们的结果表明，MARCH2在炎症和静息条件下对NEMO/IKKγ的调节中起关键作用，从而抑制过度或意外的炎症反应。我们在此的发现不仅证明了MARCH2在炎症信号通路中的生物学作用，还为潜在机制提供了新见解。
