UBD participates in neutrophilic asthma by promoting the activation of IL-17 signaling.

Neutrophilic asthma is a persistent and severe inflammatory lung disease characterized by neutrophil activation and the mechanisms of which are not completely elucidated. Ubiquitin D (UBD) is a ubiquitin-like modifier participating in infections, immune responses, and tumorigenesis, while whether UBD involves in neutrophilic asthma needs further study. In this study, we initially found that UBD expression was significantly elevated and interleukin 17 (IL-17) signaling was enriched in the endobronchial biopsies of severe asthma along with neutrophils increasing by bioinformatics analysis. We further confirmed that UBD was upregulated in the lung tissues of neutrophilic asthma mouse model. UBD overexpression promoted IL-17 signaling activation. Knockdown of UBD suppressed the activation of IL-17 signaling. UBD interacted with TRAF2 and reduced the total and the K48-linked ubiquitination of TRAF2. However, IL-17 A stimulation increased both the total and the K48-linked ubiquitination of TRAF2. Together, these findings indicated that UBD was upregulated and played a critical role in IL-17 signaling which contributed to a better understanding of the complex mechanisms in neutrophilic asthma.

REGγ Mitigates Radiation-Induced Enteritis by Preserving Mucin Secretion and Sustaining Microbiome Homeostasis.

Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.

Ferritinophagy-mediated ferroptosis facilitates methotrexate-induced hepatotoxicity by high-mobility group box 1 (HMGB1).

BACKGROUND AND AIM: Hepatotoxicity is a well-defined reaction to methotrexate (MTX), a drug commonly used for the treatment of rheumatoid arthritis and various tumours. We sought to elucidate the mechanism underlying MTX-induced hepatotoxicity and establish a potentially effective intervention strategy. METHODS: We administered MTX to liver cells and mice and assessed hepatotoxicity by cell viability assay and hepatic pathological changes. We determined ferroptosis and ferritinophagy by detecting ferroptosis-related markers and autophagic degradation of ferritin heavy chain 1 (FTH1). RESULTS: We have shown that hepatocytes treated with MTX undergo ferroptosis, and this process can be attenuated by ferroptosis inhibitors. Interestingly, NCOA4-mediated ferritinophagy was found to be involved in MTX-induced ferroptosis, which was demonstrated by the relief of ferroptosis through the inhibition of autophagy or knockdown of Ncoa4. Furthermore, MTX treatment resulted in the elevation of high-mobility group box 1 (HMGB1) expression. The depletion of Hmgb1 in hepatocytes considerably alleviated MTX-induced hepatotoxicity by limiting autophagy and the subsequent autophagy-dependent ferroptosis. It is noteworthy that glycyrrhizic acid (GA), a precise inhibitor of HMGB1, effectively suppressed autophagy, ferroptosis and hepatotoxicity caused by MTX. CONCLUSION: Our study shows the significant roles of autophagy-dependent ferroptosis and HMGB1 in MTX-induced hepatotoxicity. It emphasizes that the inhibition of ferritinophagy and HMGB1 may have potential as a therapeutic approach for preventing and treating MTX-induced liver injury.

Reciprocal REGγ-Nrf2 Regulation Promotes Long Period ROS Scavenging in Oxidative Stress-Induced Cell Aging.

Increased accumulation of reactive oxygen species (ROS) and decline of adaptive response of antioxidants to oxidative stimuli has been implicated in the aging process. Nuclear factor erythroid 2-related factor 2 (Nrf2) activation is a core event in attenuating oxidative stress-associated aging. The activity is modulated by a more complex regulatory network. In this study, we demonstrate the proteasome activator REGγ function as a new regulator of Nrf2 activity upon oxidative stress in cell aging model induced by hydrogen peroxide (H2O2). REGγ deficiency promotes cell senescence in primary MEF cells after H2O2 treatment. Accordingly, ROS scavenging is accelerated in WT cells but blunted in REGγ lacking cells during 12-hour recovery from a 1-hour H2O2 treatment, indicating long-lasting antioxidant buffering capacity of REGγ. Mechanistically, through GSK-3ß inhibition, REGγ enhances the nuclear distribution and transcriptional activity of Nrf2, which is surveyed by induction of phase II enzymes including Ho1 and Nqo1. Meanwhile, Nrf2 mediates the transcriptional activation of REGγ upon H2O2 stimulation. More interestingly, short-term exposure to H2O2 leads to transiently upregulation and gradually descent of REGγ transcription, however sustained higher REGγ protein level even in the absence of H2O2 for 24 hours. Thus, our results establish a positive feedback loop between REGγ and Nrf2 and a new layer of adaptive response after oxidative stimulation that is the REGγ-GSK-3ß-Nrf2 pathway.

Quercetin Mitigates Radiation-Induced Intestinal Injury and Promotes Intestinal Regeneration via Nrf2-Mediated Antioxidant Pathway1.

Radiation-induced intestinal injury is one the most common adverse events of radiotherapy, which can severely affect quality of life. There are currently no effective preventive and therapeutic options for this disorder. Quercetin is a natural flavonoid found in common food species, with the characteristics of antioxidative, anti-inflammatory, and anti-cancerous activity. However, the role of quercetin on radiation-induced intestinal injury and the underlying mechanism remains poorly understood. In this study, we found quercetin treatment can improve the survival rate of mice after a single-dose (10 Gy) abdominal irradiation. Quercetin-pretreated mice significantly reduced radiation-induced DNA damage and intestinal epithelium cell apoptosis. In addition, quercetin also improved the proliferation activity of intestinal stem cells and promoted intestine epithelium repair after irradiation. Further studies demonstrated that quercetin treatment curtailed radiation-induced reactive oxygen species generation via regulating Nrf2 signaling in intestinal epithelium cells. Furthermore, treatment with Nrf2 inhibitor, could reverse the above effects. Altogether, quercetin can ameliorate radiation-induced intestine injury via regulating Nrf2 signaling, scavenging free radicals, and promoting intestinal epithelium repair.

Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

TRIM56 positively regulates TNFα-induced NF-κB signaling by enhancing the ubiquitination of TAK1.

Nuclear factor-κB (NF-κB) signaling participates in many biologic processes including immunity, inflammation, and cancer. Here we reported that tripartite motif-containing protein 56 (TRIM56), an E3 ligase enzyme, participated in TNFα-induced NF-κB signaling by interacting with TAK1. Overexpression of TRIM56 potentiated the activation of TNFα-induced NF-κB signaling, whereas knockdown of TRIM56 had an opposite effect. TRIM56 enhanced the ubiquitination of TAK1, specifically enhanced the M1-linked polyubiquitin chains to TAK1, leading to the tight interactions of the TAK1-IKKα complex. Consequently, the stimulation of TNFa and TRIM56 strengthened the interaction with TAK1. Furthermore, we found that the C terminal (CT) domain was the binding region of TRIM56, and the RING domain of TRIM56 was the E3 enzyme activity region which was important to the ubiquitination of TAK1. Together, these results reveal that TRIM56 positively regulates TNFα-induced NF-κB signaling by heightening the ubiquitination of TAK1 and provide new insight into the complicated mechanisms of the inflammatory and immune response.

REGγ drives Lgr5+ stem cells to potentiate radiation induced intestinal regeneration.

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a marker of intestinal stem cells (ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5+ stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated ß-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+ stem cells in the regeneration of intestinal epithelial cells. Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

YAP/TEAD4-induced KIF4A contributes to the progression and worse prognosis of esophageal squamous cell carcinoma.

Aberrant expression of kinesin family member 4A (KIF4A), which is associated with tumor progression, has been reported in several types of cancer. However, its expression and the underlying molecular mechanisms regulating the transcription of KIF4A in esophageal squamous cell carcinoma (ESCC) remain largely unclear. Here, we found that high KIF4A expression was positively correlated with tumor stage and poor prognosis in ESCC patients. KIF4A silencing significantly inhibited the growth and migration of ESCC cells, arrested cell cycle, and induced apoptosis. Interestingly, KIF4A expression was positively related to the expression of YAP in human ESCC tissues. YAP knockdown or disrupting YAP/TEAD4 interaction by verteporfin repressed KIF4A expression. Also, KIF4A knockdown significantly inhibited the cell growth induced by YAP overexpression. Mechanistically, YAP activated KIF4A transcriptional expression by TEAD4-mediated direct binding to KIF4A promoter. Finally, KIF4A knockdown and verteporfin treatment synergistically inhibited tumor growth in xenograft models. Together, these results indicated that KIF4A, a novel target gene of YAP/TEAD4, may be a progression and prognostic biomarker of ESCC. Targeting drugs for KIF4A combined with YAP inhibitor may be a novel therapeutic strategy for ESCC.

Procyanidin B2 Promotes Intestinal Injury Repair and Attenuates Colitis-Associated Tumorigenesis via Suppression of Oxidative Stress in Mice.

Aims: Intact intestinal epithelium is essential to maintain normal intestinal physiological function. Irradiation-induced gastrointestinal syndrome or inflammatory bowel disease occurred when epithelial integrity was impaired. This study aims at exploring the mechanism of procyanidin B2 (PB2) administration to promote intestinal injury repair in mice. Results: PB2 treatment reduces reactive oxygen species (ROS) accumulation and protects the intestine damage from irradiation. Mechanistic studies reveal that PB2 could effectively slow down the degradation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and it significantly triggers Nrf2 into the nucleus, which leads to subsequent antioxidant enzyme expression. However, knockdown of Nrf2 attenuates PB2-induced protection in the intestine. More importantly, PB2 also promotes leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive intestinal stem cells (Lgr5+ ISCs) driven regeneration via enhancing Wnt/ß-catenin signaling, which depends on, at least in part, activation of the Nrf2 signal. Evidence from an injury model of intestinal organoids is similar with in vivo results. Correspondingly, results from flow cytometric analysis and luciferase reporter assay reveal that PB2 also inhibits the level of ROS and promotes Lgr5 expression in vitro. Finally, PB2 alleviates the severity of experimental colitis and colitis-associated cancer in a long-term inflammatory model via inhibiting nuclear localization of p65. Innovation: This study, for the first time, reveals a role of PB2 for intestinal regeneration and repair after radiation or dextran sulfate sodium-induced injury in mice. Conclusion: Our results indicate that PB2 can repress oxidative stress via Nrf2/ARE signaling and then promote intestinal injury repair.

(-)-Epicatechin mitigates radiation-induced intestinal injury and promotes intestinal regeneration via suppressing oxidative stress.

The high radiosensitivity of the intestinal epithelium limits the survival of victims by nuclear accidents or terrorism and limits effective radiotherapy against abdominal malignancies. Recently, we reported that (-)-epicatechin (EC) modulates oxidative stress and exerts neuroprotection. Here, we investigate the protective effects of EC against intestinal damage induced by radiation. The established model is acute moderate but reversible intestinal injury damage. We also set up the injured model of "minigut" ex vivo, which mimic the process of intestinal regeneration in vivo. We found that EC can repress oxidative stress by regulating SOD and MDA levels in serum and intestine tissue. Correspondingly, EC can decrease apoptosis of crypt cells in Lgr5-EGFP-IRES-creERT2 mice after radiation. Further studies demonstrated that EC can promote Nrf2 translocation from cytoplasm to nuclear and then activate the expression of HO1 and NQO1. Interestingly, EC can enhance the activity of intestine stem cells labelled by Lgr5 and promote intestinal epithelium regeneration determined by HE and immunofluorescence staining in vivo and in vitro. We also found that EC can activate the Wnt/ß-catenin signal pathway confirmed by TCF/LEF luciferase reporter assay. Together, EC can provide the protective effect on intestine and promote intestinal regeneration after radiation through Nrf2 and Wnt/ß-catenin signal pathway.

RING box protein-1 gene involved in flagellar disassembly of Dunaliella salina.

Ring box protein-1 (RBX1), also called Regulator of Cullins-1 (ROC1), is a key component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting protein substrates for degradation. Although RBX1 plays an important role in ubiquitination machinery of both prokaryotes and eukaryotes, studies on the RBX1 have not been involved in the unicellular green alga Dunaliella salina. In this study, a full-length RBX1 cDNA fragment of 817 bp was cloned using rapid amplification of cDNA end (RACE) technique. The full-length sequence contained an open reading frame of 411 bp encoding 136 amino acids. The predicted protein had a molecular molar mass of 14.8 kDa and pI of 5.9 with a high degree of homology to RBX1 from Chlamydomonas reinhardtii (92 %). Recombinant RBX1 was expressed in Escherichia coli BL21 and was purified and characterized. The apparent molecular mass of the recombinant protein was approximately 17 kDa, and the optimal induction time and concentration were 3 h and 0.1 mmol/L IPTG, respectively. The predicted 3D structures of RBX1 proteins contained RING-H2 finger domain including "Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115." The expression of RBX1 protein was increased by 132 % during flagellar disassembly and decreased by 76 % during flagellar assembly of D. salina. The expression of RBX1 mRNA had a similar tendency with the expression of RBX1 protein. The results indicated that RBX1 responded to flagellar disassembly of D. salina.

ß-Carotene synergistically enhances the anti-tumor effect of 5-fluorouracil on esophageal squamous cell carcinoma in vivo and in vitro.

Recently, we reported that ß-carotene exhibited anticancer activity against human esophageal squamous cell carcinoma cells in vitro. In the present study, we examined a novel therapeutic strategy by combining ß-carotene with 5-fluorouracil (5-FU) in human esophageal cancer in vitro and in vivo, and elucidated the underlying mechanisms. We found that the combination of 5-FU and ß-carotene displayed greater growth inhibitory effects than did either compound alone in esophageal squamous cell carcinoma (ESCC) cells. In addition, the combination of 5-FU and ß-carotene displayed greater tumor growth inhibition in an Eca109 xenograft mouse model than did a single agent with low systemic toxicity. ß-Carotene enhanced 5-FU-induced apoptosis. TUNEL staining revealed that the rate of TUNEL-positive cells was markedly increased in tumor tissues after treatment with 5-FU and ß-carotene. Western blotting and immunohistochemistry revealed the down-regulation of Bcl-2 and PCNA and the up-regulation of Bax and caspase-3 in tumor tissues. Further studies demonstrated that the combined administration of 5-FU and ß-carotene significantly down-regulated the protein levels of Cav-1, p-AKT, p-NF-κB, p-mTOR and p-p70S6K in Eca109 cells more effectively than did 5-FU alone. These data suggested that the combined therapy of 5-FU and ß-carotene exerted synergistic antitumor effects in vivo and in vitro and could constitute a novel therapeutic treatment for ESCC.

ß-Carotene Induces Apoptosis in Human Esophageal Squamous Cell Carcinoma Cell Lines via the Cav-1/AKT/NF-κB Signaling Pathway.

ß-carotene, a type of terpenoid, has many metabolic and physiological functions. In particular, ß-carotene has an antitumor effect. However, the efficacy of ß-carotene against esophageal squamous cell carcinoma (ESCC) remains unclear. In our study, ß-carotene inhibited the growth of ESCC cells and downregulated expression of the Caveolin-1 (Cav-1) protein. Cav-1 protein was expressed only in ESCC cells, not in Het-1A cells. Moreover, ß-carotene triggered apoptosis, induced cell cycle G0/G1 phase arrest, and inhibited cell migration. To explore the mechanism involved in these processes, we further examined the effect of ß-carotene on the Cav-1-mediated AKT/NF-κB pathway. The results showed that the level of AKT and NF-κB phosphorylation was dramatically inhibited, which led to an increase in the Bax/Bcl-2 ratio. Correspondingly, the activity of Caspase-3 was also enhanced. These data suggest that ß-carotene has an antiproliferative role in ESCC cells and may be a promising chemotherapeutic agent for use against ESCC cells.