Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas‐4 null mice leads to embryonic arrest around E9. Centriole loss in Sas‐4 ^−/− embryos causes prolonged mitosis and p53‐dependent cell death. Studies in vitro discovered a similar USP28‐, 53BP1‐, and p53‐dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas‐4 ^−/− embryos. The data indicate that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.     

Acetylshikonin induces autophagy‐dependent apoptosis through the key LKB1‐AMPK and PI3K/Akt‐regulated mTOR signalling pathways in HL‐60 cells

Acetylshikonin (ASK) is a natural naphthoquinone derivative of traditional Chinese medicine Lithospermum erythrorhyzon. It has been reported that ASK has bactericidal, anti‐inflammatory and antitumour effects. However, whether ASK induces apoptosis and autophagy in acute myeloid leukaemia (AML) cells and the underlying mechanism are still unclear. Here, we explored the roles of apoptosis and autophagy in ASK‐induced cell death and the potential molecular mechanisms in human AML HL‐60 cells. The results demonstrated that ASK remarkably inhibited the cell proliferation, viability and induced apoptosis in HL‐60 cells through the mitochondrial pathway, and ASK promoted cell cycle arrest in the S‐phase. In addition, the increased formation of autophagosomes, the turnover from light chain 3B (LC3B) I to LC3B II and decrease of P62 suggested the induction of autophagy by ASK. Furthermore, ASK significantly decreased PI3K, phospho‐Akt and p‐p70S6K expression, while enhanced phospho‐AMP‐activated protein kinase (AMPK) and phospho‐liver kinase B1(LKB1) expression. The suppression of ASK‐induced the conversion from LC3B I to LC3B II caused by the application of inhibitors of AMPK (compound C) demonstrated that ASK‐induced autophagy depends on the LKB1/AMPK pathway. These data suggested that the autophagy induced by ASK were dependent on the activation of LKB1/AMPK signalling and suppression of PI3K/Akt/mTOR pathways. The cleavage of the apoptosis‐related markers caspase‐3 and caspase‐9 and the activity of caspase‐3 induced by ASK were markedly reduced by inhibitor of AMPK (compound C), an autophagy inhibitor 3‐methyladenine (3‐MA) and another autophagy inhibitor chloroquine (CQ). Taken together, our data reveal that ASK‐induced HL‐60 cell apoptosis is dependent on the activation of autophagy via the LKB1/AMPK and PI3K/Akt‐regulated mTOR signalling pathways.     

Curcumin analog HO‐3867 triggers apoptotic pathways through activating JNK1/2 signalling in human oral squamous cell carcinoma cells

Human oral squamous cell carcinoma (OSCC) is the common head and neck malignancy in the world. While surgery, radiotherapy and chemotherapy are emerging as the standard treatment for OSCC patients, the outcome is limited to the recurrence and side effects. Therefore, patients with OSCC require alternative strategies for treatment. In this study, we aimed to explore the therapeutic effect and the mode of action of the novel curcumin analog, HO‐3867, against human OSCC cells. We analysed the cytotoxicity of HO‐3867 using MTT assay. In vitro mechanic studies were performed to determine whether MAPK pathway is involved in HO‐3867 induced cell apoptosis. As the results, we found HO‐3867 suppressed OSCC cells growth effectively. The flow cytometry data indicate that HO‐3867 induce the sub‐G1 phase. Moreover, we found that HO‐3867 induced cell apoptosis by triggering formation of activated caspase 3, caspase 8, caspase 9 and PARP. After dissecting MAPK pathway, we found HO‐3867 induced cell apoptosis via the c‐Jun N‐terminal kinase (JNK)1/2 pathway. Our results suggest that HO‐3867 is an effective anticancer agent as its induction of cell apoptosis through JNK1/2 pathway in human oral cancer cells.     

Nucleolar c‐Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood‐borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpA^L10A) did not alter its translocation to the nucleus but abolished the effector’s capacity to interact with EBP2. VgpA‐EBP2 interaction led to the re‐localization of c‐Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA‐EBP2 interaction elevated EBP2’s affinity for c‐Myc and prolonged the oncoprotein’s half‐life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re‐localization of c‐Myc. Moreover, the in vivo VgpA‐EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus’ colonization and virulence. These observations suggest that direct effector stimulation of a c‐Myc controlled host cell growth program can contribute to pathogenesis.     

ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

Acute myeloid leukaemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. 4‐Amino‐2‐trifluoromethyl‐phenyl retinate (ATPR), a novel all‐trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show superior anticancer effect compared with ATRA on various cancers. However, its potential effect on AML remains largely unknown. Lactate dehydrogenase B (LDHB) is the key glycolytic enzyme that catalyses the interconversion between pyruvate and lactate. Currently, little is known about the role of LDHB in AML. In this study, we found that ATPR showed antileukaemic effects with RARα dependent in AML cells. LDHB was aberrantly overexpressed in human AML peripheral blood mononuclear cell (PBMC) and AML cell lines. A lentiviral vector expressing LDHB‐targeting shRNA was constructed to generate a stable AML cells with low expression of LDHB. The effect of LDHB knockdown on differentiation and cycle arrest of AML cells was assessed in vitro and vivo, including involvement of Raf/MEK/ERK signalling. Finally, these data suggested that ATPR showed antileukaemic effects by RARα/LDHB/ ERK‐glycolysis signalling axis. Further studies should focus on the underlying leukaemia‐promoting mechanisms and investigate LDHB as a therapeutic target.     

Non‐apoptotic caspase activation preserves Drosophila intestinal progenitor cells in quiescence

Caspase malfunction in stem cells often precedes the appearance and progression of multiple types of cancer, including human colorectal cancer. However, the caspase‐dependent regulation of intestinal stem cell properties remains poorly understood. Here, we demonstrate that Dronc, the Drosophila ortholog of caspase‐9/2 in mammals, limits the number of intestinal progenitor cells and their entry into the enterocyte differentiation programme. Strikingly, these unexpected roles for Dronc are non‐apoptotic and have been uncovered under experimental conditions without epithelial replenishment. Supporting the non‐apoptotic nature of these functions, we show that they require the enzymatic activity of Dronc, but are largely independent of the apoptotic pathway. Alternatively, our genetic and functional data suggest that they are linked to the caspase‐mediated regulation of Notch signalling. Our findings provide novel insights into the non‐apoptotic, caspase‐dependent modulation of stem cell properties that could improve our understanding of the origin of intestinal malignancies.     

Identification of SRY‐box 30 as an age‐related essential gatekeeper for male germ‐cell meiosis and differentiation

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY‐box 30 (Sox30) is an age‐related and essential regulator of meiosis in the postnatal testis. Sox30‐null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ‐cell differentiation and irregular Leydig cell proliferation. In aged Sox30‐null mice, the observed testicular impairments were more severe. Furthermore, the germ‐cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a “fail‐safe” mechanism for Sox30 to facilitate germ‐cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30‐null testes. Re‐expression of Sox30 in Sox30‐null mice successfully restored germ‐cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age‐associated for germ‐cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ‐cell meiosis and differentiation in the postnatal testis.     

Tumour necrosis factor‐α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen‐deficient osteoporosis

The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis.     

MUC3A promotes non-small cell lung cancer progression via activating the NFκB pathway and attenuates radiosensitivity

Mucin 3A (MUC3A) is highly expressed in non-small cell lung cancer (NSCLC), but its functions and effects on clinical outcomes are not well understood. Tissue microarray of 92 NSCLC samples indicated that high levels of MUC3A were associated with poor prognosis, advanced staging, and low differentiation. MUC3A knockdown significantly suppressed NSCLC cell proliferation and induced G1/S accumulation via downregulating cell cycle checkpoints. MUC3A knockdown also inhibited tumor growth in vivo and had synergistic effects with radiation. MUC3A knockdown increased radiation-induced DNA double strain breaks and γ-H2AX phosphorylation in NSCLC cells. MUC3A downregulation inhibited the BRCA-1/RAD51 pathway and nucleus translocation of P53 and XCRR6, suggesting that MUC3A promoted DNA damage repair and attenuated radiation sensitivity. MUC3A knockdown also resulted in less nucleus translocation of RELA and P53 in vivo. Immunoprecipitation revealed that MUC3A interacted with RELA and activated the NFκB pathway via promoting RELA phosphorylation and interfering the binding of RELA to IκB. Our studies indicated that MUC3A was a potential oncogene and associated with unfavorable clinical outcomes. NSCLC patients with a high MUC3A level, who should be more frequent follow-up and might benefit less from radiotherapy.     

Circular RNA circEIF4G2 aggravates renal fibrosis in diabetic nephropathy by sponging miR‐218

Circular RNAs play essential roles in the development of various human diseases. However, how circRNAs are involved in diabetic nephropathy (DN) are not fully understood. Our study aimed to investigate the effects of circRNA circEIF4G2 on DN. Experiments were performed in the db/db mouse model of type 2 diabetes and NRK‐52E cells. We found that circEIF4G2 was significantly up‐regulated in the kidneys of db/db mice and NRK‐52E cells stimulated by high glucose. circEIF4G2 knockdown inhibited the expressions of TGF‐β1, Collagen I and Fibronectin in high glucose‐stimulated NRK‐52E cells, which could be rescued by miR‐218 inhibitor. Knockdown of SERBP1 reduced the expression of TGF‐β1, Collagen I and Fibronectin in HG‐stimulated NRK‐52E cells. In summary, our findings suggested that circEIF4G2 promotes renal tubular epithelial cell fibrosis via the miR‐218/SERBP1 pathway, presenting a novel insight for DN treatment.     

Nbs1‐mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.     

MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.