Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.     

A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.     

Upregulation of Glypicans in Hippo mutants alters the coordinated activity of morphogens

The function of the conserved Drosophila Hippo signaling pathway has been shown to be required to limit cell proliferation. Several studies have identified different target genes of this pathway that could modulate this function. However, the ectopic expression of these genes cannot account for all of the hyperplasic and pattern defects displayed by Hippo signaling mutants. We have recently identified two new targets of the Hippo pathway, the heparan sulfate proteoglycans (HSPGs) encoded by division abnormally delayed (dally) and dally-like protein (dlp). The function of these glypicans is required to modulate the activity of different signaling pathways triggered by diffusable ligands. Thus, our results link the function of the Hippo pathway with the control of the activity of several signaling pathways required for the definition of the size and pattern of an organ.     

Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal

Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders.     

Oxidative stress is involved in age-dependent spermatogenic damage of Immp2l mutant mice

Mitochondrial reactive oxygen species (ROS) have been implicated in spermatogenic damage, although direct in vivo evidence is lacking. We recently generated a mouse in which the inner mitochondrial membrane peptidase 2-like (Immp2l) gene is mutated. This Immp2l mutation impairs the processing of signal peptide sequences from mitochondrial cytochrome c₁ and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion, which causes age-dependent spermatogenic damage. Here we confirm age-dependent spermatogenic damage in a new cohort of mutants, which started at the age of 10.5 months. Compared with age-matched controls, protein carbonyl content was normal in testes of 2- to 5-month-old mutants, but significantly elevated in testes of 13-month-old mutants, indicating elevated oxidative stress in the testes at the time of impaired spermatogenesis. Testicular expression of superoxide dismutases was not different between control and mutant mice, whereas that of catalase was increased in young and old mutants. The expression of cytosolic glutathione peroxidase 4 (phospholipid hydroperoxidase) in testes was significantly reduced in 13-month-old mutants, concomitant with impaired spermatogenesis. Apoptosis of all testicular populations was increased in mutant mice with spermatogenic damage. The mitochondrial DNA (mtDNA) mutation rate in germ cells of mutant mice with impaired spermatogenesis was unchanged, excluding a major role of mtDNA mutation in ROS-mediated spermatogenic damage. Our data show that increased mitochondrial ROS are one of the driving forces for spermatogenic impairment.     

Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. In order to evaluate the role of PINCH in integrin-mediated cellular events, we have tested directly the in vivo function of PINCH in Drosophila melanogaster. We demonstrate that the steamer duck (stck) alleles that were first identified in a screen for potential integrin effectors represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with betaPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, we show that integrins are required for proper localization of PINCH at the myotendinous junction. The integrin-linked kinase, ILK, is also essential for integrin function. We demonstrate that Drosophila PINCH and ILK are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, ILK localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of ILK. Our results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion.     

Mitochondrial dysfunction in insulin resistance: differential contributions of chronic insulin and saturated fatty acid exposure in muscle cells

Mitochondrial dysfunction has been associated with insulin resistance, obesity and diabetes. Hyperinsulinaemia and hyperlipidaemia are hallmarks of the insulin-resistant state. We sought to determine the contributions of high insulin and saturated fatty acid exposure to mitochondrial function and biogenesis in cultured myocytes. Differentiated C2C12 myotubes were left untreated or exposed to chronic high insulin or high palmitate. Mitochondrial function was determined assessing: oxygen consumption, mitochondrial membrane potential, ATP content and ROS (reactive oxygen species) production. We also determined the expression of several mitochondrial genes. Chronic insulin treatment of myotubes caused insulin resistance with reduced PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) signalling. Insulin treatment increased oxygen consumption but reduced mitochondrial membrane potential and ROS production. ATP cellular levels were maintained through an increased glycolytic rate. The expression of mitochondrial OXPHOS (oxidative phosphorylation) subunits or Mfn-2 (mitofusin 2) were not significantly altered in comparison with untreated cells, whereas expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) and UCPs (uncoupling proteins) were reduced. In contrast, saturated fatty acid exposure caused insulin resistance, reducing PI3K (phosphoinositide 3-kinase) and ERK (extracellular-signal-regulated kinase) activation while increasing activation of stress kinases JNK (c-Jun N-terminal kinase) and p38. Fatty acids reduced oxygen consumption and mitochondrial membrane potential while up-regulating the expression of mitochondrial ETC (electron chain complex) protein subunits and UCP proteins. Mfn-2 expression was not modified by palmitate. Palmitate-treated cells also showed a reduced glycolytic rate. Taken together, our findings indicate that chronic insulin and fatty acid-induced insulin resistance differentially affect mitochondrial function. In both conditions, cells were able to maintain ATP levels despite the loss of membrane potential; however, different protein expression suggests different adaptation mechanisms.     

Genistein对卵巢癌铂类耐药细胞株CP70增殖凋亡的影响及与细胞内ROS的相关性

目的:探讨Genistein对卵巢癌铂类耐药细胞CP70增殖、凋亡的影响及与细胞内活性氧水平的关系。方法:采用MTT法检测Genistein对CP70细胞增殖的影响;流式细胞仪分析不同药物处理后对细胞凋亡的影响,线粒体膜电位及细胞内ROS水平的变化情况。结果:Genistein对CP70细胞增殖表现出剂量和时间依赖性的抑制作用,并能诱导其凋亡;Genistein作用于CP70细胞后,可使其线粒体膜电位降低,并引发了细胞内ROS水平的显著升高;ROS抑制剂NAC预处理CP70细胞后,有效抑制了ROS的产生,并降低了细胞凋亡率,与未加NAC组相比差异有显著性(P0.05)。结论:Genistein能抑制铂类耐药卵巢癌细胞CP70的增殖,并促进其凋亡,这与细胞内ROS水平的升高有关,可能是Genistein抗肿瘤诱导细胞凋亡的机制之一。     

Docosahexaenoic acid-induced unfolded protein response, cell cycle arrest, and apoptosis in vascular smooth muscle cells are triggered by Ca²⁺-dependent induction of oxidative stress

Proliferation of vascular smooth muscle cells is a characteristic of pathological vascular remodeling and represents a significant therapeutic challenge in several cardiovascular diseases. Docosahexaenoic acid (DHA), a member of the n-3 polyunsaturated fatty acids, was shown to inhibit proliferation of numerous cell types, implicating several different mechanisms. In this study we examined the molecular events underlying the inhibitory effects of DHA on proliferation of primary human smooth muscle cells isolated from small pulmonary artery (hPASMCs). DHA concentration-dependently inhibited hPASMC proliferation, induced G1 cell cycle arrest, and decreased cyclin D1 protein expression. DHA activated the unfolded protein response (UPR), evidenced by increased mRNA expression of HSPA5, increased phosphorylation of eukaryotic initiation factor 2α, and splicing of X-box binding protein 1. DHA altered cellular lipid composition and led to increased reactive oxygen species (ROS) production. DHA-induced ROS were dependent on both intracellular Ca(2+) release and entry of extracellular Ca(2+). Overall cellular ROS and mitochondrial ROS were decreased by RU360, a specific inhibitor of mitochondrial Ca(2+) uptake. DHA-induced mitochondrial dysfunction was evidenced by decreased mitochondrial membrane potential and decreased cellular ATP content. DHA triggered apoptosis as found by increased numbers of cleaved caspase-3- and TUNEL-positive cells. The free radical scavenger Tempol counteracted DHA-induced ROS, cell cycle arrest, induction of UPR, and apoptosis. We conclude that Ca(2+)-dependent oxidative stress is the central and initial event responsible for induction of UPR, cell cycle arrest, and apoptosis in DHA-treated hPASMCs.     

Use of a Drosophila model to identify genes regulating Plasmodium growth in the mosquito

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.     

Identification of Drosophila mutants altering defense of and endurance to Listeria monocytogenes infection

We extended the use of Drosophila beyond being a model for signaling pathways required for pattern recognition immune signaling and show that the fly can be used to identify genes required for pathogenesis and host-pathogen interactions. We performed a forward genetic screen to identify Drosophila mutations altering sensitivity to the intracellular pathogen Listeria monocytogenes. We recovered 18 mutants with increased susceptibility to infection, none of which were previously shown to function in a Drosophila immune response. Using secondary screens, we divided these mutants into two groups: In the first group, mutants have reduced endurance to infections but show no change in bacterial growth. This is a new fly immunity phenotype that is not commonly studied. In the second group, mutants have a typical defense defect in which bacterial growth is increased and survival is decreased. By further challenging mutant flies with L. monocytogenes mutants, we identified subgroups of fly mutants that affect specific stages of the L. monocytogenes life cycle, exit from the vacuole, or actin-based movement. There is no overlap between our genes and the hundreds of genes identified in Drosophila S2 cells fighting L. monocytogenes infection, using genomewide RNAi screens in vitro. By using a whole-animal model and screening for host survival, we revealed genes involved in physiologies different from those that were found in previous screens, which all had defects in defensive immune signaling.