Activating p53 function by targeting RLIP

Aberrations in RLIP, p53, and PKCα represent essentially the entire spectrum of all human neoplasms. Elevated PKCα expression, failure of the cell cycle checkpoint (p53 dysfunction), and abnormal glutathione (GSH) metabolism are fundamental hallmarks of carcinogenesis and drug/radiation resistance. However, a lack of investigations into the interactions between these important regulatory nodes has fundamentally limited our understanding of carcinogenesis and the development of effective interventions for cancer prevention and therapy. Loss of p53, perhaps the most powerful tumor suppressor gene, predisposes rodents to spontaneous cancer and humans to familial, as well as acquired, cancers. Until recently, no genetic manipulation of any oncogene had been reported to abrogate spontaneous carcinogenesis in p53^?/? rodent models. However, the overexpression of RLIP, a GSH-electrophile conjugate (GS-E) transporter, has been found to enhance cancer cell proliferation and confer drug/radiation resistance, whereas its depletion causes tumor regression, suggesting its importance in cancer and drug/radiation resistance. Indeed, RLIP is an essential effector of p53 that is necessary for broad cancer-promoting epigenetic remodeling. Interestingly, through a haploinsufficiency mechanism, the partial depletion of RLIP in p53^?/? mice provides complete protection from neoplasia. Furthermore, RLIP^?/? mice exhibit altered p53 and PKCα function, marked deficiency in clathrin-dependent endocytosis (CDE), and almost total resistance to chemical carcinogenesis. Based on these findings, in this review, we present a novel and radical hypothesis that expands our understanding of the highly significant cross-talk between p53, PKCα, and GSH signaling by RLIP in multiple tumor models.     

RLIP controls receptor-ligand signaling by regulating clathrin-dependent endocytosis

RLIP (Ral-interacting protein) is a multifunctional protein that couples ATP hydrolysis with the movement of substances. Its primary function appears to be in the plasma membrane, where it catalyzes the ATP-dependent efflux of glutathione-conjugates (GS-Es), as well as un-metabolized drugs and toxins. In the plasma membrane, its interaction with the clathrin adaptor protein AP2 localizes it to endocytic vesicle, where its GS-E-stimulated ATPase and transport activity are required for clathrin-dependent endocytosis (CDE). CDE is an essential mechanism for internalizing ligand-receptor complexes that signal proliferation (EGF, insulin, IGF1), apoptosis (TNFα, TRAIL, Fas-L), and differentiation and morphogenesis (TGFβ, WNT, Notch, SHH). Aberrant functioning of these pathways appears crucial for most cancer cells to evade apoptosis, invade surrounding tissues, and metastasize. Internalization of receptor-ligand complexes by CDE begins a sequence of events that can terminate, initiate, or modulate downstream signaling; the consequences of signaling through these downstream pathways may be inherently different in cancer and normal cells, a view supported by numerous basic and clinical observations. In this review, we will discuss the GS-E transport activity of RLIP, which determines the rate of ligand endocytosis, and how the inhibition and/or depletion of RLIP globally disrupts in ligand-receptor signaling.     

RLIP: An existential requirement for breast carcinogenesis

Breast cancer (BC) is the most common cancer among women worldwide. Due to its complexity in nature, effective BC treatment can encounter many challenges. The human RALBP1 gene encodes a 76-kDa splice variant protein, RLIP (ral-binding protein1, RalBP1), a stress-protective mercapturic acid pathway (MAP) transporter protein, that also plays a key role in regulating clathrin-dependent endocytosis (CDE) as a Ral effector. Growing evidence shows that targeting RLIP may be an effective strategy in cancer therapy, as RLIP is over-expressed in multiple cancers and is known to induce resistance to apoptosis and chemotherapeutic drugs. Recent studies demonstrated that RLIP is expressed in human BC tissues, as well as BC cell lines. Knockdown of RLIP resulted in apoptotic death of BC cells in vitro, and targeted inhibition and depletion of RLIP resulted in regression of BC in xenograft studies of nude mice. Signaling studies showed that RLIP depletion inhibited endocytosis and differentially regulated signaling to Akt, Myc, and ERK1/2. However, the proliferation and multi-specific transport mechanisms that promote RLIP-mediated cell death in BC are not well understood. In this review, we will discuss a missing but an essentially determining and connecting piece of the puzzle on the understanding of proliferation and transport mechanisms by focused analyses of the apoptotic, drug- and radiation-sensitivity regulated by RLIP, a stress-responsive non-ATP-binding cassette (ABC), high capacity MAP transporter, in breast cancer.     

RLIP76 Protein Knockdown Attenuates Obesity Due to a High-fat Diet

Feeding a Western high-fat diet (HFD) to C57BL/6 mice induces obesity, associated with a chronic inflammatory state, lipid transport, and metabolic derangements, and organ system effects that particularly prominent in the kidneys. Here, we report that RLIP76 homozygous knock-out (RLIP76^−/−) mice are highly resistant to obesity as well as these other features of metabolic syndrome caused by HFD. The normal increase in pro-inflammatory and fibrotic markers associated with HFD induced obesity in wild-type C57B mice was broadly and nearly completely abrogated in RLIP76^−/− mice. This is a particularly striking finding because chemical markers of oxidative stress including lipid hydroperoxides and alkenals were significantly higher in RLIP76^−/− mice. Whereas HFD caused marked suppression of AMPK in wild-type C57B mice, RLIP76^−/− mice had baseline activation of AMP-activated protein kinase, which was not further affected by HFD. The baseline renal function was reduced in RLIP76^−/− mice as compared with wild-type, but was unaffected by HFD, in marked contrast to severe renal impairment and glomerulopathy in the wild-type mice given HFD. Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines, and provide a novel mechanism for targeted therapy of obesity and metabolic syndrome.     

Platelet derived growth factor B and epithelial mesenchymal transition of peritoneal mesothelial cells

Platelet derived growth factor (PDGF) is involved in wound healing in various organ systems. Its potential role in the context of peritoneal injury following long-term peritoneal dialysis is unclear. We used an adenovirus expressing the B chain of PDGF (AdPDGF-B) to assess its effect on pro-fibrotic pathways in the peritoneal membrane. To assess the transforming growth factor (TGF) β independent effects of PDGF, we over-expressed PDGF-B in the peritoneum of either wild-type mice (Smad3^+/+) or those with a deletion of the TGFβ signaling protein Smad3 (Smad3^?/?). PDGF-B induced sustained angiogenesis in both Smad3^+/+ and Smad3^?/? mice. Despite increased collagen gene expression, collagen accumulation was transient and fibrogenesis was associated with induction of collagenase activity. We observed epithelial to mesenchymal transition (EMT) involving the peritoneal mesothelial cells, as shown by increased SNAIL and decreased E-Cadherin expression with evidence of mesothelial cells expressing both epithelial and mesenchymal markers. Unlike TGFβ-induced EMT, PDGF-B exposure did not lead to mobilization of the mesothelial cells; they remained as a single monolayer throughout the observation period. This “non-invasive” EMT phenomenon is a novel finding and may have implications concerning the role of EMT in peritoneal fibrosis and injury to other organ systems. The observed effects were similar in Smad3^?/? and Smad3^+/+ animals, suggesting that the PDGF-B effects were independent of TGFβ or Smad signaling.     

Cardiac myocyte KLF5 regulates body weight via alteration of cardiac FGF21

Cardiac metabolism affects systemic energetic balance. Previously, we showed that Krüppel-like factor (KLF)-5 regulates cardiomyocyte PPARα and fatty acid oxidation-related gene expression in diabetes. We surprisingly found that cardiomyocyte-specific KLF5 knockout mice (αMHC-KLF5^?/?) have accelerated diet-induced obesity, associated with increased white adipose tissue (WAT). Alterations in cardiac expression of the mediator complex subunit 13 (Med13) modulates obesity. αMHC-KLF5^?/? mice had reduced cardiac Med13 expression likely because KLF5 upregulates Med13 expression in cardiomyocytes. We then investigated potential mechanisms that mediate cross-talk between cardiomyocytes and WAT. High fat diet-fed αMHC-KLF5^?/? mice had increased levels of cardiac and plasma FGF21, while food intake, activity, plasma leptin, and natriuretic peptides expression were unchanged. Consistent with studies reporting that FGF21 signaling in WAT decreases sumoylation-driven PPARγ inactivation, αMHC-KLF5^?/? mice had less SUMO-PPARγ in WAT. Increased diet-induced obesity found in αMHC-KLF5^?/? mice was absent in αMHC-[KLF5^?/?;FGF21^?/?] double knockout mice, as well as in αMHC-FGF21^?/? mice that we generated. Thus, cardiomyocyte-derived FGF21 is a component of pro-adipogenic crosstalk between heart and WAT.     

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh^?/?/ogg1^?/? double knockout mouse.In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1 month) and adult (6 months) wt and myh^?/?/ogg1^?/? mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh^?/?/ogg1^?/? mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh^?/?/ogg1^?/? mouse. The reduced respiration capacity correlated with a specific reduction (?25%) in complex I biochemical activity in brain mitochondria.Our results demonstrate that mtDNA mutations are not associated with cancer development in myh^?/?/ogg1^?/? mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.     

SR-A deficiency reduces myocardial ischemia/reperfusion injury; involvement of increased microRNA-125b expression in macrophages

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A^?/? and WT mice were subjected to ischemia (45 min) followed by reperfusion for up to 7 days. SR-A^?/? mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A^?/? heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A^?/?) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A^?/? macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A^?/? macrophages. The levels of miR-125b in SR-A^?/? macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A^?/? macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.     

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome

Purpose

Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76^−/− mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76^−/− mice.

Research Design and Methods

Blood glucose (BG) and lipid measurements were performed in RLIP76^+/+ and RLIP76^−/− mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining.

Results

The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76^−/− mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK.

Conclusions/Significance

All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76^−/− mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.     

Sphk2−/− mice are protected from obesity and insulin resistance

Sphingosine kinases phosphorylate sphingosine to sphingosine 1?phosphate (S1P), which functions as a signaling molecule. We have previously shown that sphingosine kinase 2 (Sphk2) is important for insulin secretion. To obtain a better understanding of the role of Sphk2 in glucose and lipid metabolism, we have characterized 20- and 52-week old Sphk2^?/? mice using glucose and insulin tolerance tests and by analyzing metabolic gene expression in adipose tissue. A detailed metabolic characterization of these mice revealed that aging Sphk2^?/? mice are protected from metabolic decline and obesity compared to WT mice. Specifically, we found that 52-week old male Sphk2^?/? mice had decreased weight and fat mass, and increased glucose tolerance and insulin sensitivity compared to control mice. Indirect calorimetry studies demonstrated an increased energy expenditure and food intake in 52-week old male Sphk2^?/? versus control mice. Furthermore, expression of adiponectin gene in adipose tissue was increased and the plasma levels of adiponectin elevated in aged Sphk2^?/? mice compared to WT. Analysis of lipid metabolic gene expression in adipose tissue showed increased expression of the Atgl gene, which was associated with increased Atgl protein levels. Atgl encodes for the adipocyte triglyceride lipase, which catalyzes the rate-limiting step of lipolysis. In summary, these data suggest that mice lacking the Sphk2 gene are protected from obesity and insulin resistance during aging. The beneficial metabolic effects observed in aged Sphk2^?/? mice may be in part due to enhanced lipolysis by Atgl and increased levels of adiponectin, which has lipid- and glucose-lowering effects.     

RLIP76 regulates PI3K/Akt signaling and chemo-radiotherapy resistance in pancreatic cancer

Purpose

Pancreatic cancer is an aggressive malignancy with characteristic metastatic course of disease and resistance to conventional chemo-radiotherapy. RLIP76 is a multi-functional cell membrane protein that functions as a major mercapturic acid pathway transporter as well as key regulator of receptor-ligand complexes. In this regard, we investigated the significance of targeting RLIP76 on PI3K/Akt pathway and mechanisms regulating response to chemo-radiotherapy.

Research Design and Methods

Cell survival was assessed by MTT and colony forming assays. Cellular levels of proteins and phosphorylation was determined by Western blot analyses. The impact on apoptosis was determined by TUNEL assay. The anti-cancer effects of RLIP76 targeted interventions in vivo were determined using mice xenograft model of the pancreatic cancer. The regulation of doxorubicin transport and radiation sensitivity were determined by transport studies and colony forming assays, respectively.

Results

Our current studies reveal an encompassing model for the role of RLIP76 in regulating the levels of fundamental proteins like PI3K, Akt, E-cadherin, CDK4, Bcl2 and PCNA which are of specific importance in the signal transduction from critical upstream signaling cascades that determine the proliferation, apoptosis and differentiation of pancreatic cancer cells. RLIP76 depletion also caused marked and sustained regression of established human BxPC-3 pancreatic cancer tumors in nude mouse xenograft model. RLIP76 turned out to be a major regulator of drug transport along with contributing to the radiation resistance in pancreatic cancer.

Conclusions/Significance

RLIP76 represents a mechanistically significant target for developing effective interventions in aggressive and refractory pancreatic cancers.     

Loss of mitochondrial protease ClpP protects mice from diet‐induced obesity and insulin resistance

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPR^mt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPR^mt on metabolism, ClpP knockout (ClpP^?/?) mice were analyzed. ClpP^?/? mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole‐body energy expenditure and markers of mitochondrial biogenesis are selectively up‐regulated in the white adipose tissue (WAT) of ClpP^?/? mice. When challenged with a metabolic stress such as high‐fat diet, despite similar caloric intake, ClpP^?/? mice are protected from diet‐induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPR^mt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial.     

Perlecan deficiency causes muscle hypertrophy,a decrease in myostatin expression,and changes in muscle fiber composition

Perlecan is a component of the basement membrane that surrounds skeletal muscle. The aim of the present study is to identify the role of perlecan in skeletal muscle hypertrophy and myostatin signaling, with and without mechanical stress, using a mouse model (Hspg2^?/?-Tg) deficient in skeletal muscle perlecan. We found that myosin heavy chain (MHC) type IIb fibers in the tibialis anterior (TA) muscle of Hspg2^?/?-Tg mice had a significantly increased fiber cross-sectional area (CSA) compared to control (WT-Tg) mice. Hspg2^?/?-Tg mice also had an increased number of type IIx fibers in the TA muscle. Myostatin and its type I receptor (ALK4) expression was substantially decreased in the Hspg2^?/?-Tg TA muscle. Myostatin-induced Smad activation was also reduced in a culture of myotubes from the Hspg2^?/?-Tg muscle, suggesting that myostatin expression and its signaling were decreased in the Hspg2^?/?-Tg muscle. To examine the effects of mechanical overload or unload on fast and slow muscles in Hspg2^?/?-Tg mice, we performed tenotomy of the plantaris (fast) muscle and the soleus (slow) muscle. Mechanical overload on the plantaris muscle of Hspg2^?/?-Tg mice significantly increased wet weights compared to those of control mice, and unloaded plantaris muscles of Hspg2^?/?-Tg mice caused less decrease in wet weights compared to those of control mice. The decrease in myostatin expression was significantly profound in the overloaded plantaris muscle of Hspg2^?/?-Tg mice, compared with that of control mice. In contrast, overloading the soleus muscle caused no changes in either type of muscle. These results suggest that perlecan is critical for maintaining fast muscle mass and fiber composition, and for regulating myostatin signaling.     

Deficiency of CCAAT/enhancer‐binding protein homologous protein (CHOP) prevents diet‐induced aortic valve calcification in vivo

Aortic valve (AoV) calcification is common in aged populations. Its subsequent aortic stenosis has been linked with increased morbidity, but still has no effective pharmacological intervention. Our previous data show endoplasmic reticulum (ER) stress is involved in AoV calcification. Here, we investigated whether deficiency of ER stress downstream effector CCAAT/enhancer‐binding protein homology protein (CHOP) may prevent development of AoV calcification. AoV calcification was evaluated in Apoe^?/? mice (n = 10) or in mice with dual deficiencies of ApoE and CHOP (Apoe^?/?CHOP^?/?, n = 10) fed with Western diet for 24 weeks. Histological and echocardiographic analysis showed that genetic ablation of CHOP attenuated AoV calcification, pro‐calcification signaling activation, and apoptosis in the leaflets of Apoe^?/? mice. In cultured human aortic valvular interstitial cells (VIC), we found oxidized low‐density lipoprotein (oxLDL) promoted apoptosis and osteoblastic differentiation of VIC via CHOP activation. Using conditioned media (CM) from oxLDL‐treated VIC, we further identified that oxLDL triggered osteoblastic differentiation of VIC via paracrine pathway, while depletion of apoptotic bodies (ABs) in CM suppressed the effect. CM from oxLDL‐exposed CHOP‐silenced cells prevented osteoblastic differentiation of VIC, while depletion of ABs did not further enhance this protective effect. Overall, our study indicates that CHOP deficiency protects against Western diet‐induced AoV calcification in Apoe^?/? mice. CHOP deficiency prevents oxLDL‐induced VIC osteoblastic differentiation via preventing VIC‐derived ABs releasing.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Preferential killing of p53-deficient cancer cells by reversine

Reversine is a small synthetic molecule that inhibits multiple mitotic kinases, including MPS1 as well as Aurora kinase A and B (AURKA and AURKB). Here, we investigated the effects of reversine on p53-deficient vs p53-proficient cancer cells. We found that low doses (~0.5 µM) of reversine, which selectively inhibit MPS1 and hence impair the spindle assembly checkpoint, kill human TP53^?/? colon carcinoma cells less efficiently than their wild-type counterparts. In sharp contrast, high doses (~5 µM) of reversine induced hyperploidization and apoptosis to a much larger extent in TP53^?/? than in TP53^+/+ cells. Such a selective cytotoxicity could not be reproduced by the knockdown of MPS1, AURKA and AURKB, neither alone nor in combination, suggesting that it involves multiple (rather than a few) molecular targets of reversine. Videomicroscopy-based cell fate profiling revealed that, in response to high-dose reversine, TP53^?/? (but not TP53^+/+) cells undergo several consecutive rounds of abortive mitosis, resulting in the generation of hyperpolyploid cells that are prone to succumb to apoptosis upon the activation of mitotic catastrophe. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53^?/? cells to the toxic effects of high-dose reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53^?/? cells in response to high-dose reversine. Altogether, these results suggest that p53-deficient cells are particularly sensitive to the simultaneous inhibition of multiple kinases, including MPS1, as it occurs in response to high-dose reversine.     

TLR9 limits enteric antimicrobial responses and promotes microbiota‐based colonisation resistance during Citrobacter rodentium infection

Mammalian cells express an array of toll‐like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human‐specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C. rodentium. Infected Tlr9^?/? mice suffered exaggerated intestinal damage and carried significantly higher (10–100 fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C. rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF‐κB signalling in the intestines of Tlr9^?/? mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9^?/? mice as compared with WT mice. Notably, antibiotic‐based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9^?/? mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota‐mediated colonisation resistance against C. rodentium infection.     

NLRC3 deficiency promotes cutaneous wound healing due to the inhibition of p53 signaling

Cutaneous wound healing is a complicated process that is characterized by an initial inflammatory phase followed by a proliferative phase. NLRC3 plays important roles in innate immunity, inflammatory regulation and tumor cell growth. However, the function of NLRC3 in wound healing remains unclear. Here, we investigated the function of NLRC3 in acute cutaneous wound healing using Nlrc3 gene knockout (Nlrc3^?/?) mice. Our results demonstrated that skin wound repair in Nlrc3^?/? mice was significantly accelerated compared with that in wild-type (WT) mice. NLRC3 deficiency promoted the inflammatory and proliferative phases in wounds enhanced the inflammatory response and increased re-epithelialization and granulation tissue formation, and these phenotypes were primarily ascribed to regulatory effects on p53 signaling. Mechanistically, we uncovered novel crosstalk between NLRC3 and p53 signaling and revealed that NLRC3 could mediate the ubiquitination and degradation of p53 in an Hsp90-dependent manner. In conclusion, our study suggests that NLRC3 is a critical negative regulator of the inflammatory response and cell proliferation during wound healing and that blocking NLRC3 may represent a potential approach for accelerating wound healing.     

The essential function of CARD9 in diet‐induced inflammation and metabolic disorders in mice

Inflammation and metabolic disorder are common pathophysiological conditions, which play a vital role in the development of obesity and type 2 diabetes. The purpose of this study was to explore the effects of caspase recruitment domain (CARD) 9 in the high fat diet (HFD)‐treated mice and attempt to find a molecular therapeutic target for obesity development and treatment. Sixteen male CARD9^?/? and corresponding male WT mice were fed with normal diet or high fat diet, respectively, for 12 weeks. Glucose tolerance, insulin resistance, oxygen consumption and heat production of the mice were detected. The CARD9/MAPK pathway‐related gene and protein were determined in insulin‐responsive organs using Western blotting and quantitative PCR. The results showed that HFD‐induced insulin resistance and impairment of glucose tolerance were more severe in WT mice than that in the CARD9^?/? mice. CARD9 absence significantly modified O₂ consumption, CO₂ production and heat production. CARD9^?/? mice displayed the lower expression of p38 MAPK, JNK and ERK when compared to the WT mice in both HFD‐ and ND‐treated groups. HFD induced the increase of p38 MAPK, JNK and ERK in WT mice but not in the CARD9^?/? mice. The results indicated that CARD9 absence could be a vital protective factor in diet‐induced obesity via the CARD9/MAPK pathway, which may provide new insights into the development of gene knockout to improving diet‐induced obesity and metabolism disorder.