Rodriguez Marisela, Xu Haifei, Hernandez Annie, Ingraham Julia, Canizales Jason, Arce Fernando Teran, Camp Sara M, Briggs Skyler, Ooi Aikseng, Burke James M, Song Jin H, Garcia Joe G N
Center for Inflammation Science and Systems Medicine, University of Florida Scripps Research Institute, Jupiter, FL, 33458, USA.
Department of Molecular Medicine, University of Florida Scripps Research Institute, Jupiter, FL, USA.
Cell Commun Signal. 2025 Mar 30;23(1):157. doi: 10.1186/s12964-025-02164-5.
NAMPT is an important intracellular metabolic enzyme (iNAMPT) regulating the NAD salvage pathway. However, increased cellular stress (infection, inflammation, hypoxia) promotes the secretion of extracellular NAMPT (eNAMPT), a TLR4 ligand and damage-associated molecular pattern protein (DAMP) that directly drives amplification of innate immune-mediated inflammatory, fibrotic, and neoplastic responses to influence disease severity. We sought to examine the mechanisms underlying pyroptotic eNAMPT release from human monocytic THP-1 cells, evoked by Nigericin, and non-pyroptotic eNAMPT secretion elicited by lipopolysaccharide (LPS). Our data indicate eNAMPT secretion/release requires NLRP3 inflammasome activation with substantial attenuation by either NLRP3 inhibition (MCC-950) or targeted genetic deletion of key inflammasome components, including NLRP3, caspase-1, or gasdermin D (GSDMD). Pyroptosis-associated eNAMPT release involved cleavage of the pore-forming GSDMD protein resulting in plasma membrane rupture (PMR) whereas non-pyroptotic LPS-induced eNAMPT secretion involved neither GSDMD cleavage nor PMR, verified utilizing non-cleavable GSDMD mutant constructs. LPS-induced eNAMPT secretion, however, was highly dependent upon NAMPT ubiquitination catalyzed by a complex containing the NEDD4 E3 ligase, Hsp90 (a selective chaperone), and intact GSDMD verified by enzymatic inhibition or silencing of NEDD4, GSDMD, or Hsp90. NAMPT ubiquitination and secretion involves autophagy activation as super-resolution microscopy analyses demonstrate NAMPT co-localization with autophagosome marker LC3B and eNAMPT secretion was significantly reduced by targeted ATG5 and ATG7 inhibition, critical components of the autophagy E3-like complex. These studies provide key insights into eNAMPT secretion that may accelerate the development of therapeutic strategies that address unmet therapeutic needs in inflammatory, fibrotic and neoplastic disorders.
烟酰胺磷酸核糖转移酶（NAMPT）是一种调节烟酰胺腺嘌呤二核苷酸（NAD）补救途径的重要细胞内代谢酶（iNAMPT）。然而，细胞应激增加（感染、炎症、缺氧）会促进细胞外NAMPT（eNAMPT）的分泌，eNAMPT是一种Toll样受体4（TLR4）配体和损伤相关分子模式蛋白（DAMP），可直接驱动先天免疫介导的炎症、纤维化和肿瘤反应的放大，从而影响疾病严重程度。我们试图研究尼日利亚菌素诱导的人单核细胞THP-1细胞焦亡性eNAMPT释放以及脂多糖（LPS）诱导的非焦亡性eNAMPT分泌的潜在机制。我们的数据表明，eNAMPT的分泌/释放需要NLRP3炎性小体激活，通过NLRP3抑制（MCC-950）或关键炎性小体成分（包括NLRP3、半胱天冬酶-1或gasdermin D（GSDMD））的靶向基因缺失可使其显著减弱。与焦亡相关的eNAMPT释放涉及形成孔道的GSDMD蛋白的切割，导致质膜破裂（PMR），而非焦亡性LPS诱导的eNAMPT分泌既不涉及GSDMD切割也不涉及PMR，这一点通过使用不可切割的GSDMD突变体构建体得到证实。然而，LPS诱导的eNAMPT分泌高度依赖于由包含NEDD4 E3连接酶、热休克蛋白90（一种选择性伴侣蛋白）和完整GSDMD的复合物催化的NAMPT泛素化，通过对NEDD4、GSDMD或热休克蛋白90进行酶抑制或沉默来验证。NAMPT泛素化和分泌涉及自噬激活，因为超分辨率显微镜分析表明NAMPT与自噬体标志物LC3B共定位，并且通过靶向抑制自噬E3样复合物的关键成分ATG5和ATG7，eNAMPT分泌显著减少。这些研究为eNAMPT分泌提供了关键见解，可能会加速治疗策略的开发，以满足炎症、纤维化和肿瘤性疾病中未满足的治疗需求。
