HOPX is methylated and exerts tumour‐suppressive function through Ras‐induced senescence in human lung cancer

HOPX acts as a tumour suppressor in various cancers. However, the regulation of HOPX in human lung cancer as well as the mechanism underlying its tumour‐suppressive function has not yet been well elucidated. Here we investigated the epigenetic regulation and molecular mechanism by which HOPX exerts growth inhibitory effects. We found that HOPX was down‐regulated in 12 out of 13 lung cancer cell lines and in 69 out of 120 primary lung tumours at mRNA and protein levels. Patients with lung adenocarcinoma (ADC) exhibited significantly more positive staining of HOPX protein compared with lung squamous cell carcinoma (SCC) (p =0.036). Again in ADC, patients with higher HOPX expression had a significantly longer disease‐free survival (p =0.001). Methylation analysis showed that down‐regulation of HOPX was associated with DNA methylation (p =0.011). To analyse the function of HOPX in lung cancer cells, stable transfection with an expression vector of HOPX was performed. It turned out that HOPX inhibited tumour cell proliferation rate, migration, and invasion, and, more interestingly, forced expression of HOPX enhanced cellular senescence via activation of oncogenic Ras and the downstream MAPK pathway, which in turn led to decreased MDM2 and increased p21. On the contrary, knockdown of HOPX by siRNA resulted in reduced Ras activity, inactivation of the MAPK pathway, and decreased p21 levels, accompanied by reduced cellular senescence. Additionally, the HOPX‐induced senescence pathway was also active in human bronchial epithelial cells. Taken together, our data suggest that down‐regulation of HOPX was related to DNA methylation and that HOPX exerts tumour‐suppressive activity by oncogenic Ras‐induced cellular senescence in lung cancer cells. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Differential cell surface recruitment of the superoxide‐producing NADPH oxidases Nox1, Nox2 and Nox5: The role of the small GTPase Sar1

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII‐coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide‐producing NADPH oxidase, is transported to the cell surface in a manner resistant to co‐expression of Sar1 (H79G), a GTP‐fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER‐to‐Golgi transport of glycoproteins including the Nox5‐related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over‐expression of syntaxin 5 (Stx5), a t‐SNARE required for COPII ER‐to‐Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5‐dependent and ‐independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N‐glycosylated forms, one complex and one high mannose, in a Sar1/Stx5‐dependent and ‐independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation‐defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1‐dependent manner; N‐glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1‐dependently, albeit mainly as the high‐mannose form in a Sar1‐independent manner.     

Over‐expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress‐induced premature senescence

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress‐induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over‐expression of NAMPT, which is the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, delays replicative senescence in vitro. However, whether Nampt‐overexpressing cells are tolerant of stress‐induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt‐overexpressing transgenic mice (Nampt Tg‐MEF cells) possess resistance against stress‐induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg‐MEF cells compared to wild‐type cells. Moreover, we found that Nampt Tg‐MEF cells show acute expression of unfolded protein response (UPR)‐related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD⁺ axis functions to protect cells not only from replicative senescence, but also from stress‐induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD⁺ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.     

Mannan‐binding lectin attenuates acetaminophen‐induced hepatotoxicity by regulating CYP2E1 expression via ROS‐dependent JNK/SP1 pathway

Mannan‐binding lectin (MBL) acts as a soluble pattern recognition molecule in the innate immune system, which is primarily produced by the liver. MBL deficiency occurs with high frequency in the population and is reported to be associated with susceptibility to several liver diseases. In the present study, we investigated the pathophysiological role of MBL in acetaminophen (APAP)‐induced hepatotoxicity. After APAP treatment, MBL‐deficient (MBL^?/?) mice had significantly higher mortality and aggravated hepatic necrosis as well as elevated serum lactate dehydrogenase and alanine aminotransferase levels compared to control mice. The enhanced hepatotoxicity in MBL^?/? mice was associated with increased concentration of APAP toxic metabolisms. Furthermore, we demonstrated here that genetic ablation of MBL resulted in excessive reactive oxygen species (ROS) production and enhanced c‐Jun N‐terminal kinase (JNK) activation, leading to up‐regulated specificity protein 1 (SP1) nuclear expression, thus promoted CYP2E1 hepatic expression and consequently exacerbated APAP‐induced liver injury in mice. Importantly, we have validated that MBL protected against APAP toxicity in human HepaRG cells in vitro with the same mechanism. Our study revealed an unexpected function of MBL in drug metabolism, thus providing new insight into the drug‐induced liver injury in patients with MBL deficiency.     

hace1 Influences zebrafish cardiac development via ROS‐dependent mechanisms

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B‐1‐cell subpopulations contribute differently to gut immunity

In mice, B‐1 (B1a/B1b) cells are mainly located in the peritoneal cavity. B‐1 cells are well known for their role in the early stages of Ab‐mediated immune responses against pathogenic invasion as well as for the production of natural IgM antibodies. Although such B cells have been claimed to give rise to intestinal plasma cells producing IgA, a clear role of B‐1 cells in IgA production in the gut‐associated tissues is still not defined. Here, we employed the transgenic L2 mouse model characterized by the lack of B‐2 cells and presence of B‐1 cells as major B‐cell subpopulation. The oligoclonality of the Ab repertoire in this mouse allowed us to take typical B1a cell VH sequences as indicators of the presence of IgM‐producing B‐1a cells in Peyer's patches as well as in lamina propria. However, amongst the IgAVH sequences recovered from the same tissues, none of the sequences showed B1a‐cell specificity. Interestingly, all IgAVH sequences derived from the lamina propria of L2 mice displayed extensive numbers of nucleotide exchanges, indicating somatic hypermutation, and affinity maturation. This suggests that the contribution of natural unmutated IgA by B‐1a cells to intestinal immunity is negligible.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation,leading to Arf/p53-independent senescence

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.     

NADPH oxidases in the gastrointestinal tract: a potential role of Nox1 in innate immune response and carcinogenesis

The gastrointestinal epithelium functions as physical and innate immune barriers against commensal or pathogenic microbes. NADPH oxidase 1 (Nox1) and dual oxidase 2 (Duox2), highly expressed in the colon, are suggested to play a potential role in host defense. Guinea-pig gastric pit cells and human colonic epithelial cells (T84 cells) express Nox1. With regard to activation of Nox1, the gastric epithelial cells are primed with Helicobacter pylori lipopolysaccharide, whereas T84 cells preferentially use the Toll-like receptor (TLR) 5, rather than TLR4, against Salmonella enteritidis infection. Thus, gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environments and to activate Nox1 appropriately for host defense. Nox1-derived reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammation-associated tumor development. The human stomach does not express Nox1. Helicobacter pylori infection alone does not induce it, whereas Nox1 is specifically expressed in gastric adenocarcinomas. In the human colon, Nox1 is differentiation-dependently expressed, and its expression is upregulated in adenomas and well-differentiated adenocarcinomas. Although Nox1 expression may not be directly linked to mitogenic activity, Nox1-derived ROS may exert a cancer-promoting effect by increasing resistance to programmed cell death of tumor cells.     

p21WAF1/Cip1 limits senescence and acinar‐to‐ductal metaplasia formation during pancreatitis

Trans‐differentiation of pancreatic acinar cells into ductal‐like lesions, a process defined as acinar‐to‐ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre‐malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down‐regulation of p21^WAF1/Cip1, a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down‐regulation is implicated in controlling the early events of acinar cell trans‐differentiation and ADM formation. p21‐mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein‐induced pancreatitis, using wild‐type (WT) and p21‐deficient (p21^?/?) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up‐regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down‐regulation is associated with ADM formation. In support of this hypothesis, p21^?/? mice showed a significant increase in number and size of metaplasia. In addition, p21 over‐expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell‐autonomous manner. p21^?/? mice displayed increased expression and relocalization of β‐catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate‐keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

IL‐4‐induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells

Human Th17 cells have a limited proliferative capacity compared to other T‐cell subsets. We have shown that human Th17 cells display impaired IL‐2 production due to IL‐4‐induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B‐cell traslocation gene) antiproliferative protein family, which prevents cell‐cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR‐activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)‐related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR‐mediated expansion not only by blocking the molecular pathway involved in the activation of the IL‐2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.