Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53

Statins inhibit the proximal steps of cholesterol biosynthesis, and are linked to health benefits in various conditions, including cancer and lung disease. We have previously investigated apoptotic pathways triggered by statins in airway mesenchymal cells, and identified reduced prenylation of small GTPases as a primary effector mechanism leading to p53-mediated cell death. Here, we extend our studies of statin-induced cell death by assessing endpoints of both apoptosis and autophagy, and investigating their interplay and coincident regulation. Using primary cultured human airway smooth muscle (HASM) and human airway fibroblasts (HAF), autophagy, and autophagosome formation and flux were assessed by transmission electron microscopy, cytochemistry (lysosome number and co-localization with LC3) and immunoblotting (LC3 lipidation and Atg12-5 complex formation). Chemical inhibition of autophagy increased simvastatin-induced caspase activation and cell death. Similarly, Atg5 silencing with shRNA, thus preventing Atg5-12 complex formation, increased pro-apoptotic effects of simvastatin. Simvastatin concomitantly increased p53-dependent expression of p53 up-regulated modulator of apoptosis (PUMA), NOXA, and damage-regulated autophagy modulator (DRAM). Notably both mevalonate cascade inhibition-induced autophagy and apoptosis were p53 dependent: simvastatin increased nuclear p53 accumulation, and both cyclic pifithrin-α and p53 shRNAi partially inhibited NOXA, PUMA expression and caspase-3/7 cleavage (apoptosis) and DRAM expression, Atg5-12 complex formation, LC3 lipidation, and autophagosome formation (autophagy). Furthermore, the autophagy response is induced rapidly, significantly delaying apoptosis, suggesting the existence of a temporally coordinated p53 regulation network. These findings are relevant for the development of statin-based therapeutic approaches in obstructive airway disease.     

Hemispheric asymmetry in white matter connectivity of the temporoparietal junction with the insula and prefrontal cortex

The temporoparietal junction (TPJ) is a key node in the brain's ventral attention network (VAN) that is involved in spatial awareness and detection of salient sensory stimuli, including pain. The anatomical basis of this network's right-lateralized organization is poorly understood. Here we used diffusion-weighted MRI and probabilistic tractography to compare the strength of white matter connections emanating from the right versus left TPJ to target regions in both hemispheres. Symmetry of structural connectivity was evaluated for connections between TPJ and target regions that are key cortical nodes in the right VAN (insula and inferior frontal gyrus) as well as target regions that are involved in salience and/or pain (putamen, cingulate cortex, thalamus). We found a rightward asymmetry in connectivity strength between the TPJ and insula in healthy human subjects who were scanned with two different sets of diffusion-weighted MRI acquisition parameters. This rightward asymmetry in TPJ-insula connectivity was stronger in females than in males. There was also a leftward asymmetry in connectivity strength between the TPJ and inferior frontal gyrus, consistent with previously described lateralization of language pathways. The rightward lateralization of the pathway between the TPJ and insula supports previous findings on the roles of these regions in stimulus-driven attention, sensory awareness, interoception and pain. The findings also have implications for our understanding of acute and chronic pains and stroke-induced spatial hemineglect.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

NF-kappaB activation represses tumor necrosis factor-alpha-induced autophagy

Activation of NF-kappaB and autophagy are two processes involved in the regulation of cell death, but the possible cross-talk between these two signaling pathways is largely unknown. Here, we show that NF-kappaB activation mediates repression of autophagy in tumor necrosis factor-alpha (TNFalpha)-treated Ewing sarcoma cells. This repression is associated with an NF-kappaB-dependent activation of the autophagy inhibitor mTOR. In contrast, in cells lacking NF-kappaB activation, TNFalpha treatment up-regulates the expression of the autophagy-promoting protein Beclin 1 and subsequently induces the accumulation of autophagic vacuoles. Both of these responses are dependent on reactive oxygen species (ROS) production and can be mimicked in NF-kappaB-competent cells by the addition of H2O2. Small interfering RNA-mediated knockdown of beclin 1 and atg7 expression, two autophagy-related genes, reduced TNFalpha- and reactive oxygen species-induced apoptosis in cells lacking NF-kappaB activation and in NF-kappaB-competent cells, respectively. These findings demonstrate that autophagy may amplify apoptosis when associated with a death signaling pathway. They are also evidence that inhibition of autophagy is a novel mechanism of the antiapoptotic function of NF-kappaB activation. We suggest that stimulation of autophagy may be a potential way bypassing the resistance of cancer cells to anti-cancer agents that activate NF-kappaB.     

Autophagy signaling and the cogwheels of cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haploinsufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Effects of sodium benzoate,a commonly used food preservative,on learning,memory, and oxidative stress in brain of mice

Sodium benzoate (SB) is a widely used preservative and antimicrobial substance in many foods and soft drinks. However, this compound is generally recognized as safe food additives, but evidence has suggested that a high intake of SB may link to attention deficit‐hyperactivity disorder in children. Present study investigate the effects of oral administration of different concentrations of SB (0.56, 1.125, and 2.25 mg/mL) for 4 weeks, on the learning and memory performance tests, and also the levels of malondialdehyde (MDA), reduced glutathione (GSH), and acetylcholinesterase activity (AChE) in the mouse brain. The results showed that SB significantly impaired memory and motor coordination. Moreover, SB decreased reduced GSH and increased the MDA level in the brain significantly (P < 0.001). However, nonsignificant alteration was observed in the AChE activity. These findings suggest that short‐term consumption of SB can impair memory performance and increased brain oxidative stress in mice.     

The mitochondria-targeted antioxidant MitoQ decreases features of the metabolic syndrome in ATM+/-/ApoE-/- mice

A number of recent studies suggest that mitochondrial oxidative damage may be associated with atherosclerosis and the metabolic syndrome. However, much of the evidence linking mitochondrial oxidative damage and excess reactive oxygen species (ROS) with these pathologies is circumstantial. Consequently the importance of mitochondrial ROS in the etiology of these disorders is unclear. Furthermore, the potential of decreasing mitochondrial ROS as a therapy for these indications is not known. We assessed the impact of decreasing mitochondrial oxidative damage and ROS with the mitochondria-targeted antioxidant MitoQ in models of atherosclerosis and the metabolic syndrome (fat-fed ApoE(-/-) mice and ATM(+/-)/ApoE(-/-) mice, which are also haploinsufficient for the protein kinase, ataxia telangiectasia mutated (ATM). MitoQ administered orally for 14weeks prevented the increased adiposity, hypercholesterolemia, and hypertriglyceridemia associated with the metabolic syndrome. MitoQ also corrected hyperglycemia and hepatic steatosis, induced changes in multiple metabolically relevant lipid species, and decreased DNA oxidative damage (8-oxo-G) in multiple organs. Although MitoQ did not affect overall atherosclerotic plaque area in fat-fed ATM(+/+)/ApoE(-/-) and ATM(+/-)/ApoE(-/-) mice, MitoQ reduced the macrophage content and cell proliferation within plaques and 8-oxo-G. MitoQ also significantly reduced mtDNA oxidative damage in the liver. Our data suggest that MitoQ inhibits the development of multiple features of the metabolic syndrome in these mice by affecting redox signaling pathways that depend on mitochondrial ROS such as hydrogen peroxide. These findings strengthen the growing view that elevated mitochondrial ROS contributes to the etiology of the metabolic syndrome and suggest a potential therapeutic role for mitochondria-targeted antioxidants.     

Network of three specific microRNAs influence type 2 diabetes through inducing insulin resistance in muscle cell lines

Insulin resistance has been implicated as one of the best predictors for type 2 diabetes. Growing evidence propose the involvement of microRNAs (miRNAs) as short regulatory molecules in modulating and inducing resistance. In this regard, we have investigated the role of three selected miRNAs in insulin resistance development (miR-135, miR-202, and miR-214), via assessing glucose uptake levels in C2C12 and L6 muscle cell lines. Interestingly, miRNA-transfected cells demonstrated a significantly different glucose uptake compared to the positive control cells. In addition, we evaluated the expression levels of three putative miRNA target genes (Rho-associated coiled-coil containing protein kinase 1, serine/threonine kinase 2, and vesicle-associated membrane protein 2) in transfected cells, recruiting luciferase assay. Our results indicated the targeting and downregulation of Rho-associated coiled-coil containing protein kinase 1 and serine/threonine kinase 2 genes in all miR-transfected cell lines ( P ≤ 0.05), but not for vesicle-associated membrane protein 2. MiRNA upregulation led to the poor stimulation of glucose uptake through insulin and developed insulin-resistant phenotype in both muscle cell lines. Our study showed the role of three miRNAs in the induction of insulin resistance in cell lines and making them prone to type 2 diabetes development.     

Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens

Pathogens or their toxins, including influenza virus, Pseudomonas, and anthrax toxins, require processing by host proprotein convertases (PCs) to enter host cells and to cause disease. Conversely, inhibiting PCs is likely to protect host cells from multiple furin-dependent, but otherwise unrelated, pathogens. To determine if this concept is correct, we designed specific nanomolar inhibitors of PCs modeled from the extended cleavage motif TPQRERRRKKR downward arrowGL of the avian influenza H5N1 hemagglutinin. We then confirmed the efficacy of the inhibitory peptides in vitro against the fluorescent peptide, anthrax protective antigen (PA83), and influenza hemagglutinin substrates and also in mice in vivo against two unrelated toxins, anthrax and Pseudomonas exotoxin. Peptides with Phe/Tyr at P1' were more selective for furin. Peptides with P1' Thr were potent against multiple PCs. Our strategy of basing the peptide sequence on a furin cleavage motif known for an avian flu virus shows the power of starting inhibitor design with a known substrate. Our results confirm that inhibiting furin-like PCs protects the host from the distinct furin-dependent infections and lay a foundation for novel, host cell-focused therapies against acute diseases.