LPS mediates cuproptosis and inflammation in THP-1 macrophages through HKDC1

Abstract

Cuproptosis is a recently identified form of copper-driven cell death characterized by the aggregation of acylated proteins and proteotoxic stress in the mitochondrial tricarboxylic acid cycle, which plays a role in inflammation. Recent studies suggest that hexokinase structural domain protein 1 (HKDC1), a fifth hexokinase, is involved in regulating mitochondrial function. However, the role of HKDC1 in cuproptosis and LPS-induced macrophage inflammation remains unclear. Here, we assess macrophage plasticity using CCK8 viability assays and phagocytosis activity experiments in an in vitro inflammatory model of THP-1 cells. We measure the levels of inflammatory factors and cuproptosis-related proteins using western blot analysis and RT-qPCR. Additionally, we examine the expression and localization of the HKDC1 protein using ChIP-qPCR and immunofluorescence staining. We find that LPS promotes the expressions of inflammatory factors and decreases cuproptosis levels in THP-1-derived macrophages while also activating glycolysis and inducing the expression of HKDC1 via the Toll-like receptor 4 (TLR4) receptor. We further demonstrate that HKDC1 knockdown inhibits glycolysis and induces cuproptosis. Mechanistically, we provide the first evidence that LPS promotes the binding of Yin Yang 1 (YY1) to the HKDC1 promoter, thereby regulating HKDC1 transcription. HKDC1 interacts with heat shock cognate B (HSCB) and ferredoxin 1 (FDX1), leading to increased intracellular copper levels and subsequent cuproptosis. HKDC1 knockdown in vivo alleviates acute sepsis by activating copper-dependent cell death pathways. Collectively, our findings suggest that LPS mitigates cuproptosis and promotes inflammation via HKDC1, suggesting a new cuproptosis-dependent anti-inflammatory strategy.

1. Introduction

Inflammation is a vital component of a series of reactions, such as cell dysfunction and immune abnormalities triggered by various factors, including ischemia and infection [1]. The role of inflammation in promoting the occurrence and progression of diseases, such as pulmonary vascular inflammation caused by hypoxia, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS), has been demonstrated [2–4]. Long-term inflammation of the liver and rectum increases the risk of hepatocellular carcinoma and colorectal cancer, respectively [5]. Additionally, inflammation is a key pathogenic factor of cardiovascular and cerebrovascular diseases [6]. Macrophages play a central role in the development of chronic inflammation and excessive scarring by driving and resolving inflammation and restoring tissue homeostasis [7]. The THP-1 cell line, which was treated with phorbol myristate acetate (PMA; 100 ng/mL) and stimulated with lipopolysaccharide (LPS), is widely used to polarize M1 macrophages and serves as a typical inflammatory model in research on aging, pulmonary hypertension, and cancer [8–10]. In this study, THP-1-derived macrophages stimulated with LPS were used to further explore the diversity of macrophages and their contributions to inflammatory responses.

Glycolysis, the fundamental process of glucose metabolism, is catalyzed by hexokinase and pyruvate kinase, which decompose glucose into pyruvate and produce ATP for the body [11]. Hexokinase domain containing 1 (HKDC1) is a novel hexokinase that is widely expressed in various tissues in humans and mice [12,13]. Previous studies have demonstrated that aberrantly expressed HKDC1 plays an essential role in the progression of malignant tumors, such as lymphoma and liver, breast, and colorectal cancers [13–16]. Moreover, Cui et al. [17] reported that HKDC1 is critical for maintaining mitochondrial homeostasis by regulating the PINK1/Parkin-dependent pathway and is important for maintaining mitochondria-lysosome contact. However, the effects of HKDC1 on inflammation in macrophages and the underlying mechanisms remain unclear.

Cuproptosis is a recently discovered type of copper-dependent cell death [17,18]. Copper dyshomeostasis leads to Cu2+ overload. Ferredoxin 1 (FDX1), a reducing protein, converts Cu2+ to the more toxic Cu+ [18]. Cu+ subsequently causes abnormal lipoylation and aggregation of lipoyl synthase (LIAS), a lipoylation-related enzyme and a key component of pyruvate dehydrogenase. This process also results in the loss of iron-sulfur (Fe-S) clusters.