A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1

Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy.Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1), antiaging (2–4), development (1, 5), protection of the genome (6), and regulation of cell size (7). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8–10), cardiomyopathy (11), and neurodegenerative disorders (12).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg)² are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13, 14).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5, 16–20). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17, 21), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24, 25). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26, 27). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28, 29). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.     

Trafficking of Crumbs3 during Cytokinesis Is Crucial for Lumen Formation

Although lumen generation has been extensively studied through so-called cyst-formation assays in Madin-Darby canine kidney (MDCK) cells, an underlying mechanism that leads to the initial appearance of a solitary lumen remains elusive. Lumen formation is thought to take place at early stages in aggregates containing only a few cells. Evolutionarily conserved polarity protein complexes, namely the Crumbs, Par, and Scribble complexes, establish apicobasal polarity in epithelial cells, and interference with their function impairs the regulated formation of solitary epithelial lumina. Here, we demonstrate that MDCK cells form solitary lumina during their first cell division. Before mitosis, Crumbs3a becomes internalized and concentrated in Rab11-positive recycling endosomes. These compartments become partitioned in both daughter cells and are delivered to the site of cytokinesis, thus forming the first apical membrane, which will eventually form a lumen. Endosome trafficking in this context appears to depend on the mitotic spindle apparatus and midzone microtubules. Furthermore, we show that this early lumen formation is regulated by the apical polarity complexes because Crumbs3 assists in the recruitment of aPKC to the forming apical membrane and interference with their function can lead to the formation of a no-lumen or multiple-lumen phenotype at the two-cell stage.     

Hydrogen sulfide and the vasculature: a novel vasculoprotective entity and regulator of nitric oxide bioavailability?

Hydrogen sulfide (H₂S) is a well known and pungent toxic gas that has recently been shown to be synthesised in man from the amino acids cystathionine, homocysteine and cysteine by at least two distinct enzymes; cystathionine-γ-lyase and cystathionine-β-synthase. In the past few years, H₂S has emerged as a novel and increasingly important mediator in the cardiovascular system but delineating the precise physiology and pathophysiology of H₂S is proving to be complex and difficult to unravel with disparate findings reported with cell types, tissue types and animal species reported. Therefore, in this review we summarize the mechanisms by which H₂S has been proposed to regulate blood pressure and cardiac function, discuss the mechanistic discrepancies reported in the literature as well as the therapeutic potential of H₂S. We also examine the methods of H₂S detection in biological fluids, processes for H₂S removal and discuss the reported blood levels of H₂S in man and animal models of cardiovascular pathology. We also highlight the complex interaction of H₂S with nitric oxide in regulating cardiovascular function in health and disease.     

Mammalian lin-7 stabilizes polarity protein complexes

Mammalian Lin-7 forms a complex with several proteins, including PALS1, that have a role in polarity determination in epithelial cells. In this study we have found that loss of Lin-7 protein from the polarized epithelial cell line Madin-Darby canine kidney II by small hairpin RNA results in defects in tight junction formation as indicated by lowered transepithelial electrical resistance and mislocalization of the tight junction protein ZO-1 after calcium switch. The knock down of Lin-7 also resulted in the loss of expression of several Lin-7 binding partners, including PALS1 and the polarity protein PATJ. The effects of Lin-7 knock down were rescued by the exogenous expression of murine Lin-7 constructs that contained the L27 domain, but not the PDZ domain alone. Furthermore, exogenously expressed PALS1, but not other Lin-7 binding partners, also rescued the effects of Lin-7 knock down, including the restoration of PATJ protein in rescued cell lines. Finally, the effects of Lin-7 knock down appeared to be due to instability of PALS1 protein in the absence of Lin-7, as indicated by an increased rate of PALS1 protein degradation. Taken together, these results indicate that Lin-7 functions in tight junction formation by stabilizing its membrane-associated guanylate kinase binding partner PALS1.     

Gastroesophageal Reflux in Relation to Adenocarcinomas of the Esophagus: A Pooled Analysis from the Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON)

Background

Previous studies have evidenced an association between gastroesophageal reflux and esophageal adenocarcinoma (EA). It is unknown to what extent these associations vary by population, age, sex, body mass index, and cigarette smoking, or whether duration and frequency of symptoms interact in predicting risk. The Barrett’s and Esophageal Adenocarcinoma Consortium (BEACON) allowed an in-depth assessment of these issues.

Methods

Detailed information on heartburn and regurgitation symptoms and covariates were available from five BEACON case-control studies of EA and esophagogastric junction adenocarcinoma (EGJA). We conducted single-study multivariable logistic regressions followed by random-effects meta-analysis. Stratified analyses, meta-regressions, and sensitivity analyses were also conducted.

Results

Five studies provided 1,128 EA cases, 1,229 EGJA cases, and 4,057 controls for analysis. All summary estimates indicated positive, significant associations between heartburn/regurgitation symptoms and EA. Increasing heartburn duration was associated with increasing EA risk; odds ratios were 2.80, 3.85, and 6.24 for symptom durations of <10 years, 10 to <20 years, and ≥20 years. Associations with EGJA were slighter weaker, but still statistically significant for those with the highest exposure. Both frequency and duration of heartburn/regurgitation symptoms were independently associated with higher risk. We observed similar strengths of associations when stratified by age, sex, cigarette smoking, and body mass index.

Conclusions

This analysis indicates that the association between heartburn/regurgitation symptoms and EA is strong, increases with increased duration and/or frequency, and is consistent across major risk factors. Weaker associations for EGJA suggest that this cancer site has a dissimilar pathogenesis or represents a mixed population of patients.     

Lorvotuzumab mertansine,a CD56-targeting antibody-drug conjugate with potent antitumor activity against small cell lung cancer in human xenograft models

Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5–10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.     

Differentiation-dependent suppression of platelet-derived growth factor signaling in cultured adipocytes.

A critical component of vertebrate cellular differentiation is the acquisition of sensitivity to a restricted subset of peptide hormones and growth factors. This accounts for the unique capability of insulin (and possibly insulin-like growth factor-1), but not other growth factors, to stimulate glucose uptake and anabolic metabolism in heart, skeletal muscle, and adipose tissue. This selectivity is faithfully recapitulated in the cultured adipocyte line, 3T3-L1, which responds to insulin, but not platelet-derived growth factor (PDGF), with increased hexose uptake. The serine/threonine protein kinases Akt1 and Akt2, which have been implicated as mediators of insulin-stimulated glucose uptake, as well as glycogen, lipid, and protein synthesis, were shown to mirror this selectivity in this tissue culture system. This was particularly apparent in 3T3-L1 adipocytes overexpressing an epitope-tagged form of Akt2 in which insulin activated Akt2 10-fold better than PDGF. Similarly, in 3T3-L1 adipocytes, only insulin stimulated phosphorylation of Akt's endogenous substrate, GSK-3beta. Other signaling molecules, including phosphatidylinositol 3-kinase, pp70 S6-kinase, mitogen-activated protein kinase, and PHAS-1/4EBP-1, did not demonstrate this selective responsiveness to insulin but were instead activated comparably by both insulin and PDGF. Moreover, concurrent treatment with PDGF and insulin did not diminish activation of phosphatidylinositol 3-kinase, Akt, or glucose transport, indicating that PDGF did not simultaneously activate an inhibitory mechanism. Interestingly, PDGF and insulin comparably stimulated both Akt isoforms, as well as numerous other signaling molecules, in undifferentiated 3T3-L1 preadipocytes. Collectively, these data suggest that differential activation of Akt in adipocytes may contribute to insulin's exclusive mediation of the metabolic events involved in glucose metabolism. Moreover, they suggest a novel mechanism by which differentiation-dependent hormone selectivity is conferred through the suppression of specific signaling pathways operational in undifferentiated cell types.     

Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.     

Sex differences in the proportion of esophageal squamous cell carcinoma cases attributable to tobacco smoking and alcohol consumption

Objective: Alcohol and tobacco are the two major established environmental factors associated with squamous cell carcinoma of the esophagus (ESCC). However, the prevalence of these exposures differs substantially between men and women. Moreover, the prevalence of smoking has declined in recent years, whereas per capita consumption of alcohol has remained steady in both sexes. Quantifying the burden of ESCC attributable to these causal factors is necessary to inform potential preventive strategies. Methods: We estimated the population attributable fraction (PAF) of ESCC due to smoking and alcohol, using data from an Australian population based case–control study (305 ESCC cases, 1554 controls). Results: Estimated PAF for ESCC were 49% (95% CI: 38–60) and 32% (95% CI: 25–40) due to smoking and heavy alcohol consumption respectively. More than 75% of the ESCC burden in men could be attributed to smokers with heavy alcohol consumption. The highest burden was among ≥30 pack years smokers who also consumed alcohol heavily (>17 drinks/week); this differed significantly between men (PAF 36%, 95% CI 29–44) and women (PAF 5%, 95% CI 2–10). Among women only, low intakes of fruit and vegetables accounted for about 9% of the ESCC burden. Conclusion: The burden of ESCC attributable to smoking combined with heavy alcohol consumption is remarkably high in men. In women, the burden of ESCC due to these factors is lower, and poor nutrition may also play a role.     

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.