Figure 7 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Mitochondrial STAT3 driven CPT1a-mediated FAO via USP50. (A) CPT1a complexes were purified from 293T cells overexpressing either empty (Ctrl) or CPT1a tagged at the C-terminus with a flag epitope (CPT1a). The complexes were analyzed by mass spectrometry and a Western blot analysis. (B) USP50 complexes were purified from 293T cells overexpressing either empty (Ctrl) or USP50-flag (USP50). The complexes were analyzed by a Western blot analysis. (C) BMDMs of the indicated
Source publication
Rationale: We found that a subset of signal transducer and activator of transcription 3 (STAT3) translocated into mitochondria in phagocytes, including macrophages isolated from individuals with sepsis. However, the role of mitochondrial STAT3 in macrophages remains unclear.
Method: To investigate the function of mitochondrial STAT3 in vivo, we gen...
Contexts in source publication
Context 1
... identify the proteins involved in blocking CPT1a proteasomal degradation mediated by mitochondrial STAT3, we first performed IP coupled with LC-MS and revealed that USP50, a deubiquitinating enzyme, could interact with CPT1a ( Figure 7A). Additionally, CPT1a interacted with USP50 in 293T cells transfected with flag-USP50 ( Figure 7B). ...
Context 2
... identify the proteins involved in blocking CPT1a proteasomal degradation mediated by mitochondrial STAT3, we first performed IP coupled with LC-MS and revealed that USP50, a deubiquitinating enzyme, could interact with CPT1a ( Figure 7A). Additionally, CPT1a interacted with USP50 in 293T cells transfected with flag-USP50 ( Figure 7B). Importantly, the IP of CPT1a pulled down a significant amount of USP50 in the LPS-treated ...
Context 3
... LSL-MLS-mSTAT3 ; Ubc ERT2Cre/+ macrophage lysates (Figure 7C, IP Lane 2, Figure S5A). ...
Context 4
... comparison, in the LPS-treated Rosa26 LSL-MLS-mSTAT3 macrophage lysates, CPT1a pulled down much less USP50 ( Figure 7C, IP Lane 1, Figure S5A), suggesting that mitochondrial STAT3 could contribute to CPT1a stabilization by influencing CPT1a-USP50 binding, at least partially. ...
Context 5
... determine whether UPS50 is a key mediator of mitochondrial STAT3-triggered FAO, we knocked down USP50 by siRNAs and found that the levels of UPS50 were decreased along with CPT1a, especially in #3 siRNA (Figure 7D, Figure S5B). To explore whether USP50 is critical for mediating CPT1a polyubiquitination, we performed IP and found that polyubiquitinated CPT1a protein species were observed in 293T cells transfected with flag-CPT1a and HA-ubiquitin ( Figure 7E). However, the levels of polyubiquitinated CPT1a protein were decreased in the 293T cells transfected with flag-CPT1a, myc-USP50 and HA-ubiquitin ( Figure 7E). ...
Context 6
... explore whether USP50 is critical for mediating CPT1a polyubiquitination, we performed IP and found that polyubiquitinated CPT1a protein species were observed in 293T cells transfected with flag-CPT1a and HA-ubiquitin ( Figure 7E). However, the levels of polyubiquitinated CPT1a protein were decreased in the 293T cells transfected with flag-CPT1a, myc-USP50 and HA-ubiquitin ( Figure 7E). Furthermore, silencing USP50 suppressed the OCR (Figure 7F). ...
Context 7
... the levels of polyubiquitinated CPT1a protein were decreased in the 293T cells transfected with flag-CPT1a, myc-USP50 and HA-ubiquitin ( Figure 7E). Furthermore, silencing USP50 suppressed the OCR (Figure 7F). These results suggest that USP50 is a key factor contributing to mitochondrial STAT3-triggered FAO. ...
Context 8
... uncover the mechanisms by which mitochondrial STAT3 influenced the expression of USP50, we examined the mRNA levels of USP50 and found that the levels of USP50 mRNA were dramatically increased in the LPS-treated Rosa26 LSL-MLS-mSTAT3 ; Ubc ERT2Cre/+ macrophages ( Figure 7G). Next, we predicted the binding sites of transcription factors on the USP50 promoter. ...
Context 9
... we predicted the binding sites of transcription factors on the USP50 promoter. As shown in Figure 7H, NF-κB bound the UPS50 promoter. ...
Context 10
... the presence of ATP, the NF-κB levels in the nucleus were higher ( Figure 7I, Figure S5C). In addition, ATP alone induced NF-κB nuclear localization ( Figure 7J). These observations suggest that mitochondrial STAT3 facilitated NF-κB nuclear localization by elevating ATP, leading to USP50 expression. ...
Context 11
... our study demonstrates that in the presence of LPS, mitochondrial STAT3 drives FAO by inducing CPT1a stabilization mediated by USP50 in macrophages, several questions emerge from our study. Regarding nuclear NF-κB expression in the presence of ATP or LPS (Figures 7I and 7J), it seems that the effect of ATP and LPS on NF-κB nuclear expression is stronger than that of mitochondrial STAT3 alone in vitro, leading us to consider whether sepsis or metabolic disorder patients can benefit from mitochondrial STAT3 inhibitors, although we cannot exclude the possibility that the difference between ATP/LPS-induced NF-κB expression and mitochondrial STAT3-mediated nuclear NF-κB expression is due to differences in the treatment conditions. Unfortunately, we could not test this hypothesis because of the unavailability of specific mitochondrial STAT3 inhibitors. ...
Citations
... The mechanisms by which CPT1A plays a role in promoting tumor progression are diverse, but largely dependent on fatty acid oxidation [19] , in pancreatic adenocarcinoma [5] and clear cell renal carcinoma [20]. Excessive fatty acid oxidation is induced by the overexpression of CPT1A to provide more energy for tumor cells [11], and also aggravates the radioresistance of tumor cells [21]. ...
... In sepsis, bacterial infection induces the excessive release of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1, and IL-18, causing cellular injury and multiple-organ dysfunction syndrome [7,8]. Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria, which is an important substance in the inflammatory response of sepsis [9]. It has been reported that cardiomyocytes has the ability to secrete IL-1β, IL-18, and TNF-a after LPS treatment [10][11][12]. ...
Cardiomyocyte pyroptosis is a primary contributor to sepsis-induced cardiac dysfunction (SICD). Recombinant phospholipid transfer protein (PLTP) have been demonstrated to possess anti-inflammatory and antiseptic properties. However, the effect of PLTP on SICD remains unknown. In this study, we established the in vivo and in vitro sepsis model with the recombinant PLTP treatment. The survival rates of mice, mouse cardiac function, cell viability, the protein level of proinflammatory cytokine, and lactate dehydrogenase level were evaluated. The cardiomyocyte pyroptotic changes were observed. The distribution of PLTP and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in mouse myocardial tissue and expression of PLTP, apoptosis associated speck like protein containing a CARD (ASC), NLRP3, caspase-1, interleukin (IL)-1β, and Gasdermin D (GSDMD) were detected. PLTP ameliorated the cecal ligation and puncture-induced mouse survival rate decrease and cardiac dysfunction, inhibited the IL-1β, IL-18, and tumor necrosis factor (TNF)-α release, and blocked the NLRP3 inflammasome/GSDMD signaling pathway in septic mice. In vitro, PLTP reversed the lipopolysaccharide-induced cardiomyocyte pyroptosis, expression of IL-1β, IL-6, TNF-α, and activation of the NLRP3 inflammasome/GSDMD signal pathway. Moreover, PLTP could bind to NLRP3 and negatively regulate the activity of the NLRP3 inflammasome/GSDMD signal pathway. This study demonstrated that PLTP can ameliorate SICD by inhibiting inflammatory responses and cardiomyocyte pyroptosis by blocking the activation of the NLRP3 inflammasome/GSDMD signaling pathway.
... Curcumin is a traditional Chinese medicine with hypolipidemic, anti-tumor, anti-inflammatory, choleretic, and antioxidant effects and has been widely used to treat cardiovascular diseases [37,38]. Prior studies have explored the cellular protective mechanisms of curcumin, including its ability to reduce the release of pro-inflammatory mediators, such as IL-6, IL-1, and TNF-α, and inhibit the anti-inflammatory effect of NF-κB [23,39,40]. Curcumin has high antioxidant activity, which can protect cell membranes by inhibiting lipid peroxidation [23]. ...
Septic cardiomyopathy (SCM) is associated with an imbalance in mitochondrial quality and high mortality rates, with no effective treatment developed to date. Curcumin provides antioxidant, anti-inflammatory, cardiovascular, and mitochondrial protection. However, curcumin has not been confirmed to improve cardiac dysfunction in sepsis. We hypothesized that curcumin can reduce abnormal inflammatory responses by improving mitochondrial function as a novel mechanism to improve SCM. To explore this hypothesis, we used an in vivo male C57BL/6 mouse sepsis model and an in vitro model of lipopolysaccharide-stimulated HL-1 cells. The effects of curcumin on sepsis-induced cardiac dysfunction, inflammatory responses, and mitochondrial quality of cardiac cells were observed using quantitative polymerase chain reaction, western blotting, echocardiography, and transmission electron microscopy. Curcumin activated sirtuin 1 (SIRT1); increased expression of the mitochondrial biogenesis-related genes Pgc1α, Tfam, and Nrf2; reduced dynamin-related protein 1 translocation from the cytoplasm to mitochondria; and restored the mitochondrial morphology and function in cardiac cells. Accordingly, curcumin protected heart function after septic shock and alleviated the effects of SCM. SIRT1 knockdown reversed the protective effects of curcumin on mitochondria. Therefore, curcumin promotes mitochondrial biogenesis and inhibits mitochondrial fragmentation by activating SIRT1, thereby improving the mitochondrial quality and reducing oxidative stress in cardiomyocytes and sepsis-induced cardiac dysfunction. These findings provide new evidence supporting the use of curcumin to treat SCM.
... Bone marrow-derived macrophages (BMDMs), 293 T, and RAW264.7 cells were cultured as described [51]. Murine intestinal cells were isolated as described [49]. ...
... #2, 5′-CCAACGAUAUCGAGUUUGUTT-3′; 5′-ACAAACUCGAUAUCGUUGGTT-3′) was purchased from Jima (Shanghai, China). BMDMs were transfected with the indicated siRNAs as described [51]. After 24 h, cells were harvested and subjected to the indicated experiments. ...
Targeting C5aR1 modulates the function of infiltrated immune cells including tumor-associated macrophages (TAMs). The gut microbiome plays a pivotal role in colorectal cancer (CRC) tumorigenesis and development through TAM education. However, whether and how the gut flora is involved in C5aR1 inhibition-mediated TAMs remains unclear. Therefore, in this study, genetic deletion of C5ar1 or pharmacological inhibition of C5aR1 with anti-C5aR1 Ab or PMX-53 in the presence or absence of deletion Abs were utilized to verify if and how C5aR1 inhibition regulated TAMs polarization via affecting gut microbiota composition. We found that the therapeutic effects of C5aR1 inhibition on CRC benefited from programming of TAMs toward M1 polarization via driving AKT2-mediated 6-phosphofructokinase muscle type (PFKM) stabilization in a TLR5-dependent manner. Of note, in the further study, we found that C5aR1 inhibition elevated the concentration of serum IL-22 and the mRNA levels of its downstream target genes encoded antimicrobial peptides (AMPs), leading to gut microbiota modulation and flagellin releasement, which contributed to M1 polarization. Our data revealed that high levels of C5aR1 in TAMs predicted poor prognosis. In summary, our study suggested that C5aR1 inhibition reduced CRC growth via resetting M1 by AKT2 activation-mediated PFKM stabilization in a TLR5-dependent manner, which relied on IL-22-regulated gut flora.
... Chandra reported that when FAO is inhibited in macrophages, the bacteria cannot survive, and the tissue bacterial load of mice decreases (55). Enhancing FAO exacerbates LPS-induced sepsis (56). ...
Macrophages polarized into distinct phenotypes play vital roles in inflammatory diseases by clearing pathogens, promoting tissue repair, and maintaining homeostasis. Metabolism serves as a fundamental driver in regulating macrophage polarization, and understanding the interplay between macrophage metabolism and polarization is crucial for unraveling the mechanisms underlying inflammatory diseases. The intricate network of cellular signaling pathway plays a pivotal role in modulating macrophage metabolism, and growing evidence indicates that the Hippo pathway emerges as a central player in network of cellular metabolism signaling. This review aims to explore the impact of macrophage metabolism on polarization and summarize the cell signaling pathways that regulate macrophage metabolism in diseases. Specifically, we highlight the pivotal role of the Hippo pathway as a key regulator of cellular metabolism and reveal its potential relationship with metabolism in macrophage polarization.
... Inflammatory responses caused by sepsis can be mimicked with lipopolysaccharide (Lps), which is responsible for the virulence of gram-negative bacteria, as shown in animal studies [10]. Lps endotoxins activate Toll-like receptor 4 (TLR4) in human cells [11] stimulating pathways that trigger each other and initiate damage mechanisms, such as oxidative stress, inflammation, and apoptosis [12]. ...
Introduction: Lipopolysaccharide (Lps) is an essential component responsible for the virulence of
gram-negative bacteria. Lps can cause damage to many organs, including the heart, kidneys, and
lungs. Dexpanthenol (Dex) is an agent that exhibits anti-oxidative and anti-inflammatory effects
and stimulates epithelialization. In this study, we aimed to investigate the effects of Dex on Lpsinduced
cardiovascular toxicity.
Methods: Rats were divided into four groups: control, Lps (5 mg/kg, intraperitoneal), Dex (500
mg/kg, intraperitoneal), and Lps + Dex. The control group received saline intraperitoneally (i.p.)
once daily for three days. The Lps group received saline i.p. once daily for three days and a single
dose of Lps i.p. was administered on the third day. The Dex group received Dex i.p. once daily for
three days and saline on the third day. The Lps + Dex group received Dex i.p. once daily for three
days and a single dose of Lps i.p. on the third day. Heart and aortic tissues were taken for
biochemical, histopathological, immunohistochemical, and genetic analysis.
Results: Lps injection caused histopathological changes in both heart and aortic tissues and
significantly increased total oxidant status and oxidative stress index levels. Interleukin-6, and
Tumor necrosis factor-α mRNA expressions were significantly altered in heart and aorta, likely do
to the anti-inflammatory and antioxidative effects of Dex. Furthermore, Dex affected Caspase-3
and Hypoxia-inducible factor 1-α staining patterns.
Conclusions: Our results show that Dex treatment has a protective effect on Lps-induced cardiac
and endothelial damage in rats by reducing inflammation, oxidative stress, and apoptosis.
... In the high-fat diet mice, a high-fat feed comprising 60% fat calories (High Fat Diet60, Oriental Yeast Co., Ltd., Tokyo, Japan) was administered for two months [56][57][58]. To simulate the state of sepsis, lipopolysaccharide LPS (Sigma-Aldrich, Co., St. Louis, MO, USA) adjusted to various concentrations using Phosphate-Buffered Saline (PBS, Nacalai Tesque, Kyoto, Japan) was directly administered into the mice's abdominal cavity [59,60]. ...
In recent years, attention has increasingly focused on various infectious diseases. Although some fatalities are directly attributed to the causative virus, many result from complications and reactive inflammation. Patients with comorbidities are at a higher risk of mortality. Refractory skin conditions such as atopic dermatitis, psoriasis, and epidermolysis bullosa, known for an elevated risk of sepsis, partly owe this to compromised surface barrier function. However, the detailed mechanisms underlying this phenomenon remain elusive. Conversely, although the detrimental effects of a high-fat diet on health, including the onset of metabolic syndrome, are widely recognized, the association between diet and susceptibility to sepsis has not been extensively explored. In this study, we examined the potential causes and pathogenesis of increased sepsis susceptibility in inflammatory skin diseases using a mouse dermatitis model: keratin 14-driven caspase-1 is overexpressed (KCASP1Tg) in mice on a high-fat diet. Our findings reveal that heightened mortality in the dermatitis mouse model is caused by the inflamed immune system due to the chronic inflammatory state of the local skin, and administration of LPS causes a rapid increase in inflammatory cytokine levels in the spleen. Intake of a high-fat diet exacerbates these cytokine levels. Interestingly, we also observed a reduced expression of Toll-like receptor 4 (TLR4) in monocytes from KCASP1Tg mice, potentially predisposing these animals to heightened infection risks and associated complications. Histological analysis showed a clear decrease in T and B cells in the spleen of KCASP1Tg mice fed a high-fat diet. Thickening of the alveolar wall, inflammatory cell infiltration, and alveolar hemorrhage were more prominent in the lungs of KCASP1Tg and KCASP1Tg with fat mice. We postulate that the chronic, non-infectious inflammation induces a negative feedback loop within the inflammatory cascade, and the suppressed expression of TLR4 renders the mice more susceptible to infections. Therefore, it is imperative for individuals with chronic skin inflammation to closely monitor disease progression upon infection and seek timely and appropriate treatment. Additionally, chronic inflammation of adipose tissue, induced by high-fat food intake, combined with dermatitis inflammation, may exacerbate infections, necessitating a review of dietary habits.
... Ubiquitin C-terminal hydrolase L3 (UCHL3) is an important member of the ubiquitin C-terminal hydrolase family, exercising DNA damage repairing and homeostasis maintaining functions under heat stress [70]. Additionally, it has been reported that the downregulation of the ubiquitination-related gene Usp50 can suppress the expression of mitochondrial Carnitine Palmitoyl transferase 1A (CPT1A), leading to the decrease of fatty acid beta-oxidation and energy supply, which play an important role in energy supply during heat stress [71]. Based on the expression results, genes related to the ubiquitination process were highly expressed in relatively heat-tolerant C. angulata after heat stress. ...
As the world’s largest farmed marine animal, oysters have enormous economic and ecological value. However, mass summer mortality caused by high temperature poses a significant threat to the oyster industry. To investigate the molecular mechanisms underlying heat adaptation and improve the heat tolerance ability in the oyster, we conducted genome-wide association analysis (GWAS) analysis on the F2 generation derived from the hybridization of relatively heat-tolerant Crassostrea angulata ♀ and heat-sensitive Crassostrea gigas ♂, which are the dominant cultured species in southern and northern China, respectively. Acute heat stress experiment (semi-lethal temperature 42 °C) demonstrated that the F2 population showed differentiation in heat tolerance, leading to extremely differentiated individuals (approximately 20% of individuals die within the first four days with 10% survival after 14 days). Genome resequencing and GWAS of the two divergent groups had identified 18 significant SNPs associated with heat tolerance, with 26 candidate genes located near these SNPs. Eleven candidate genes that may associate with the thermal resistance were identified, which were classified into five categories: temperature sensor (Trpm2), transcriptional factor (Gata3), protein ubiquitination (Ube2h, Usp50, Uchl3), heat shock subfamily (Dnajc17, Dnaja1), and transporters (Slc16a9, Slc16a14, Slc16a9, Slc16a2). The expressional differentiation of the above genes between C. gigas and C. angulata under sublethal temperature (37 °C) further supports their crucial role in coping with high temperature. Our results will contribute to understanding the molecular mechanisms underlying heat tolerance, and provide genetic markers for heat-resistance breeding in the oyster industry.
... Subsequently, mitochondrial STAT3 exacerbates LPS-induced sepsis [19] along with promoting mitochondrial metabolism and mediating ROS production [17,20]. Additionally, different from nuclear STAT3 functions, mitochondrial STAT3 affects the enzymatic activities of complexes I and II of the electron transport chain (ETC) [21]. ...
... STAT3 is a critical transcription factor for the proliferation, survival, and stemness maintenance of HSCs, as well as the proliferation and transformation of cancer cells [37][38][39][40][41]. In addition to its role in the nucleus, previous studies have demonstrated that under stressed conditions, such as infection, STAT3 is phosphorylated at residue 727 and translocated into mitochondria, leading to the metabolic reprogramming for biosynthetic precursors and cellular activity [17,19]. A recent study demonstrated that upon STAT3 inhibition, decreases in OXPHOS and TCA cycles elucidate succinate which are observed in leukemia stem cells (LSCs), indicating that STAT3 could indirectly regulate mitochondrial functions in LSCs [41]. ...
... A recent study demonstrated that upon STAT3 inhibition, decreases in OXPHOS and TCA cycles elucidate succinate which are observed in leukemia stem cells (LSCs), indicating that STAT3 could indirectly regulate mitochondrial functions in LSCs [41]. In contrast to a previous study in which mitochondrial STAT3 effectively in uenced hematopoiesis under physiological conditions utilizing Stat3 knockout mice [38], we found that without stimulus, mitochondrial STAT3 barely affected hematopoiesis in vivo [19]. Previous studies have indicated that within the rst weeks after allogeneic hematopoietic stem cell transplantation (aHSCT), the lower intestinal diversity of the gut microbial community could elevate the risk of aGVHD-related mortality and disease relapse [42][43][44]. ...
It has been shown that under various conditions, such as aging, the gut flora contributes to modulating the status and functions of hematopoietic stem cells (HSCs). However, which species/strains of probiotics and the underlying mechanisms involved in controlling HSC function remain obscure, especially under irradiation conditions. In this study, we found that an antibiotic cocktail caused STAT3 localization in the mitochondria of epithelial cells along with a decrease in the abundance of F. rodentium . Feeding mice with F. rodentium and its metabolite butyrate prolonged the survival of irradiated mice. In a further study, we found that the administration of butyrate sustained nuclear PKM2 expression, attenuating p53-induced apoptosis. These results suggested that in response to irradiation, a reduced abundance of F. rodentium led to decreased butyrate content, thereby promoting PKM2 nuclear translocation, which subsequently enhanced p53 transcriptional activity.
... Sepsis is commonly reported to be induced by bacterial components [24]. To avoid interference from live bacteria in the ALI model, we used inactivated Hypervirulent Klebsiella pneumoniae (iHvKp) to establish a mouse model of ALI. ...
... The invasive and pathogenic properties of hvKp pose significant challenges in the clinical management of infections and subsequent sepsis. Sepsis is an excessive immune response triggered by bacterial infections, often driven by bacterial components such as LPS rather than intact bacteria themselves [24]. Notably, approximately 70% of severe sepsis patients exhibit negative blood cultures, further supporting the notion that sepsis is triggered by the release of bacterial components during infection [38]. ...
Background
Hypervirulent Klebsiella pneumoniae (hvKp) infection-induced sepsis-associated acute lung injury (ALI) has emerged as a significant clinical challenge. Increasing evidence suggests that activated inflammatory macrophages contribute to tissue damage in sepsis. However, the underlying causes of widespread macrophage activation remain unclear.
Methods
BALB/c mice were intravenously injected with inactivated hvKp (iHvKp) to observe lung tissue damage, inflammation, and M1 macrophage polarization. In vitro, activated RAW264.7 macrophage-derived exosomes (iHvKp-exo) were isolated and their role in ALI formation was investigated. RT-PCR was conducted to identify changes in exosomal miRNA. Bioinformatics analysis and dual-luciferase reporter assays were performed to validate MSK1 as a direct target of miR-155-5p. Further in vivo and in vitro experiments were conducted to explore the specific mechanisms involved.
Results
iHvKp successfully induced ALI in vivo and upregulated the expression of miR-155-5p. In vivo, injection of iHvKp-exo induced inflammatory tissue damage and macrophage M1 polarization. In vitro, iHvKp-exo was found to promote macrophage inflammatory response and M1 polarization through the activation of the p38-MAPK pathway. RT-PCR revealed exposure time-dependent increased levels of miR-155-5p in iHvKp-exo. Dual-luciferase reporter assays confirmed the functional role of miR-155-5p in mediating iHvKp-exo effects by targeting MSK1. Additionally, inhibition of miR-155-5p reduced M1 polarization of lung macrophages in vivo, resulting in decreased lung injury and inflammation induced by iHvKp-exo or iHvKp.
Conclusions
The aforementioned results indicate that exosomal miR-155-5p drives widespread macrophage inflammation and M1 polarization in hvKp-induced ALI through the MSK1/p38-MAPK Axis.
Graphical Abstract
Supplementary Information
The online version contains supplementary material available at 10.1186/s11658-023-00505-1.
