Critical Role of Beclin1 in HIV Tat and Morphine-Induced Inflammation and Calcium Release in Glial Cells from Autophagy Deficient Mouse.
ABSTRACT: We previously showed that autophagy is an important component in human immunodeficiency virus (HIV) replication and in the combined morphine-induced neuroinflammation in human astrocytes and microglia. Here we further studied the consequences of autophagy using glial cells of mice partially lacking the essential autophagy gene Atg6 (Beclin1) exposed to HIV Tat and morphine. Tat is known to cause an inflammatory response, increase calcium release, and possibly interact with autophagy pathway proteins. Following Tat exposure, autophagy-deficient (Becn1+/-) glial cells had significantly and consistently reduced levels in the pro-inflammatory cytokine IL-6 and the chemokines RANTES and MCP-1 when compared to Tat-treated cells from control (C57BL/6J) mice, suggesting an association between the inflammatory effects of Tat and Beclin1. Further, differences in RANTES and MCP-1 secretion between C57BL/6J and Becn1+/- glia treated with Tat and morphine also suggest a role of Beclin1 in the morphine-induced enhancement. Analysis of autophagy maturation by immunoblot suggests that Beclin1 may be necessary for Tat, and to a lesser extent morphine-induced arrest of the pathway as demonstrated by accumulation of the adaptor protein p62/SQSTM1 in C57BL/6J glia. Calcium release induced by Tat alone or in combination with morphine in C57BL/6J glia was significantly reduced in Becn1+/- glia while minimal interactive effect of Tat with morphine in the production of reactive oxygen or nitrogen species was detected in glia derived from Becn1+/- or C57BL/6J. Overall, the data establish a role of Beclin1 in Tat and morphine-mediated inflammatory responses and calcium release in glial cells and support the notion that autophagy mediates Tat alone and combined morphine-induced neuropathology.
Project description:Autophagy can protect cancer cells from acute starvation and enhance resistance to chemotherapy. Previously, we reported that autophagy plays a critical role in the survival of dormant, drug resistant ovarian cancer cells using human xenograft models and correlated the up-regulation of autophagy and DIRAS3 expression in clinical samples obtained during "second look" operations. DIRAS3 is an imprinted tumor suppressor gene that encodes a 26 kD GTPase with homology to RAS that inhibits cancer cell proliferation and motility. Re-expression of DIRAS3 in ovarian cancer xenografts also induces dormancy and autophagy. DIRAS3 can bind to Beclin1 forming the Autophagy Initiation Complex that triggers autophagosome formation. Both the N-terminus of DIRAS3 (residues 15-33) and the switch II region of DIRAS3 (residues 93-107) interact directly with BECN1. We have identified an autophagy-inhibiting peptide based on the switch II region of DIRAS3 linked to Tat peptide that is taken up by ovarian cancer cells, binds Beclin1 and inhibits starvation-induced DIRAS3-mediated autophagy.
Project description:The decision of stem cells to proliferate and differentiate is finely controlled. The Caenorhabditis elegans germline provides a tractable system for studying the mechanisms that control stem cell proliferation and homeostasis [1-4]. Autophagy is a conserved cellular recycling process crucial for cellular homeostasis in many different contexts , but its function in germline stem cell proliferation remains poorly understood. Here, we describe a function for autophagy in germline stem cell proliferation. We found that autophagy genes such as bec-1/BECN1/Beclin1, atg-16.2/ATG16L, atg-18/WIPI1/2, and atg-7/ATG7 are required for the late larval expansion of germline stem cell progenitors in the C. elegans gonad. We further show that BEC-1/BECN1/Beclin1 acts independently of the GLP-1/Notch or DAF-7/TGF-? pathways but together with the DAF-2/insulin IGF-1 receptor (IIR) signaling pathway to promote germline stem cell proliferation. Similar to DAF-2/IIR, BEC-1/BECN1/Beclin1, ATG-18/WIPI1/2, and ATG-16.2/ATG16L all promote cell-cycle progression and are negatively regulated by the phosphatase and tensin homolog DAF-18/PTEN. However, whereas BEC-1/BECN1/Beclin1 acts through the transcriptional regulator SKN-1/Nrf1, ATG-18/WIPI1/2 and ATG-16.2/ATG16L exert their function through the DAF-16/FOXO transcription factor. In contrast, ATG-7 functions in concert with the DAF-7/TGF-? pathway to promote germline proliferation and is not required for cell-cycle progression. Finally, we report that BEC-1/BECN1/Beclin1 functions non-cell-autonomously to facilitate cell-cycle progression and stem cell proliferation. Our findings demonstrate a novel non-autonomous role for BEC-1/BECN1/Beclin1 in the control of stem cell proliferation and cell-cycle progression, which may have implications for the understanding and development of therapies against malignant cell growth in the future.
Project description:Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.
Project description:BACKGROUND:Rabies virus (RABV) is reported to encode five phosphoproteins (P), which are involved in viral genomic replication, axonal transport, oxidative stress, interferon antagonism, and autophagy induction. However, the functions of the different P proteins are poorly understood. METHODS:Immunofluorescence staining and western blot were performed to detect the autophagy activity, the form of ring-like structure, and the colocalization of BECN1 and P. Co-immunoprecipitation was performed to detect the interaction between P and BECN1. QRT-PCR and TCID50 assay were performed to detect the replication level of RABV. Small interfering RNA was used to detect the autophagy signaling pathway. RESULTS:We found that P5 attaches to N-terminal residues 1-139 of BECN1 (beclin1) on the BECN1 ring-like structure through amino acid residues 173-222 of P5. Subsequently, we found that P5-induced autophagosomes did not fuse with lysosomes. Becn1 silencing did not recover P5 overexpression-induced promotion of RABV replication. Mechanistically, RABV protein P?N82 (P5) induced incomplete autophagy via the BECN1-mediated signaling pathway. CONCLUSIONS:Our data indicate that P5 binding to the BECN1 ring benefits RABV replication by inducing BECN1 signaling pathway-dependent incomplete autophagy, which provides a potential target for antiviral drugs against RABV. Video abstract.
Project description:Autophagy is an important cellular process that serves as a companion pathway to the ubiquitin-proteasome system to degrade long-lived proteins and organelles to maintain cell homeostasis. Although initially characterized in yeast, autophagy is being realized as an important regulator of development and disease in mammals. Beclin1 (Becn1) is a putative tumor suppressor gene that has been shown to undergo a loss of heterozygosity in 40-75% of human breast, ovarian, and prostate cancers. Because Becn1 is a key regulator of autophagy, we sought to investigate its role in female reproduction by using a conditional knockout approach in mice. We find that pregnant females lacking Becn1 in the ovarian granulosa cell population have a defect in progesterone production and a subsequent preterm labor phenotype. Luteal cells in this model exhibit defective autophagy and a failure to accumulate lipid droplets needed for steroidogenesis. Collectively, we show that Becn1 provides essential functions in the ovary that are essential for mammalian reproduction.
Project description:BECN1/Beclin1 is one of the key proteins in autophagy regulation. However, the biological functions of BECN1 in non-small cell lung cancer (NSCLC) were obscure. Here, we found that neither BECN1 knockdown nor overexpression affected the proliferation of NSCLC cells. Surprisingly, BECN1 overexpression increased cell migration and knocking down BECN1 significantly reduced the migratory ability of NSCLC cells. We further demonstrated that BECN1 could interact with Vimentin and affected its K48-linked ubiquitination. What's more, BECN1 could also interact with ubiquitin-specific peptidase 14 (USP14), the key de-ubiquitinase of Vimentin, and regulated USP14 mediated de-ubiquitination of Vimentin. Thus, our studies revealed an oncosupportive role of BECN1 in the migration of NSCLC cells through regulating the ubiquitination of Vimentin.
Project description:Macroautophagy/autophagy is involved in myeloid cellular repair, destruction, and osteoclast differentiation; conversely, KLF2 (kruppel-like factor 2 [lung]) regulates myeloid cell activation and differentiation. To investigate the specific role of KLF2 in autophagy, osteoclastic differentiation was induced in monocytes in presence or absence of the autophagy inhibitor 3-methyladenine (3-MA), KLF2 inducer geranylgeranyl transferase inhibitor (GGTI298), and adenoviral overexpression of KLF2. We found that the number of autophagic cells and multinucleated osteoclasts were significantly decreased in presence of 3-MA, GGTI298, and KLF2 overexpressed cells indicating involvement of KLF2 in these processes. In addition, autophagy-related protein molecules were significantly decreased after induction of KLF2 during the course of osteoclastic differentiation. Furthermore, induction of arthritis in mice reduced the level of Klf2 in monocytes, and enhanced autophagy during osteoclastic differentiation. Mechanistically, knocking down of KLF2 increased the level of Beclin1 (BECN1) expression, and conversely, KLF2 over-expression reduced the level of BECN1 in monocytes. Moreover, 3-MA and GGTI298 both reduced myeloid cell proliferation concomitantly upregulating senescence-related molecules (CDKN1A/p21 and CDKN1B/p27kip1). We further confirmed epigenetic regulation of Becn1 by modulating Klf2; knocking down of Klf2 increased the levels of histone activation marks H3K9 and H4K8 acetylation in the promoter region of Becn1; and overexpression of Klf2 decreased the levels of H4K8 and H3K9 acetylation. In addition, osteoclastic differentiation also increased levels of H3K9 and H4K8 acetylation in the promoter region of Becn1. Together these findings for the first time revealed that Klf2 critically regulates Becn1-mediated autophagy process during osteoclastogenesis.Abbreviations: ACP5/TRAP: acid phosphatase 5, tartrate resistant; Ad-KLF2: adenoviral construct of KLF2; ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1, autophagy related; C57BL/6: inbred mouse strain C57 black 6; ChIP: chromatin immunoprecipitation; CSF1/MCSF: colony stimulating factor 1 (macrophage); CTSK: cathepsin K; EV: empty vector; GGTI298: geranylgeranyl transferase inhibitor; H3K9Ac: histone H3 lysine 9 acetylation; H4K8Ac: histone H4 lysine 8 acetylation; K/BxN mice: T cell receptor (TCR) transgene KRN and the MHC class II molecule A(g7) generates K/BxN mice; KLF2: kruppel-like factor 2 (lung); 3MA: 3-methyladenine; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MDC: monodansylcadaverine; NFATc1: nuclear factor of activated T cells 1; NFKB: nuclear factor of kappa light polypeptide gene enhancer in B cells; p21/CDKN1A: cyclin dependent kinase inhibitor 1A; p27kip1/CDKN1B: cyclin-dependent kinase inhibitor 1B; PCR: polymerase chain reaction; PtdIns3K: phosphoinositide 3-kinase; RA: rheumatoid arthritis; siKlf2: small interfering KLF2 ribonucleic acid; NS: non-specific; RAW 264.7: abelson murine leukemia virus transformed macrophage cell line; TNFSF11/RANKL: tumor necrosis factor (ligand) superfamily, member 11; TSS: transcriptional start site; UCSC: University of California, Santa Cruz.
Project description:Macroautophagy/autophagy is a multistep cellular process that sequesters cytoplasmic components for lysosomal degradation. BECN1/Beclin1 is a central protein that assembles cofactors for the formation of a BECN1-PIK3C3-PIK3R4 complex to trigger the autophagy protein cascade. Discovering the regulators of BECN1 is important for understanding the mechanism of autophagy induction. Here, we demonstrate that TRIM59, a tripartite motif protein, plays an important role in autophagy regulation in non-small cell lung cancer (NSCLC). On the one hand, TRIM59 regulates the transcription of BECN1 through negatively modulating the NFKB pathway. On the other hand, TRIM59 regulates TRAF6 induced K63-linked ubiquitination of BECN1, thus affecting the formation of the BECN1-PIK3C3 complex. We further demonstrate that TRIM59 can mediate K48-linked ubiquitination of TRAF6 and promote the proteasomal degradation of TRAF6. Taken together, our findings reveal novel dual roles for TRIM59 in autophagy regulation by affecting both the transcription and the ubiquitination of BECN1. Abbreviations: ACTB: actin beta; BECN1: beclin 1; CHX: cycloheximide; CQ: chloroquine; GFP: green fluorescent protein; HA: haemagglutinin tag; His: polyhistidine tag; LC3B: microtubule associated protein 1 light chain 3 beta; NFKB: nuclear factor kappa B; NFKBIA: NFKB inhibitor alpha; NSCLC: non-small cell lung cancer; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RELA: RELA proto-oncogene, NF-kB subunit; SQSTM1: sequestosome 1; tGFP: Turbo green fluorescent protein; TRAF6: TNF receptor associated factor 6; TRIM59: tripartite motif containing 59; B: ubiquitin.
Project description:Alzheimer's disease is not only characterized by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia-mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/- mice exhibited increased expression of IL-1beta and IL-18 compared to wild type microglia. Becn1+/-APPPS1 mice also contained enhanced IL-1beta levels. The investigation of the IL-1beta/IL-18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 and in Becn1+/- microglia. Super resolution microscopy revealed a very close association of NLRP3 aggregates and LC3-positive vesicles. Interestingly, CALCOCO2 colocalised with NLRP3 and its downregulation increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.
Project description:Evidence suggests that the catabolic process of macroautophagy (autophagy hereafter) can either suppress or promote cancer. The essential autophagy gene ATG6/BECN1 encoding the Beclin1 protein has been implicated as a haploinsufficient tumor suppressor in breast, ovarian, and prostate cancers. The proximity of BECN1 to the known breast and ovarian tumor suppressor breast cancer 1, early onset, BRCA1, on chromosome 17q21, has made this determination equivocal. Here, the mutational status of BECN1 was assessed in human tumor sequencing data from The Cancer Genome Atlas (TCGA) and other databases. Large deletions encompassing both BRCA1 and BECN1, and deletions of only BRCA1 but not BECN1, were found in breast and ovarian cancers, consistent with BRCA1 loss being a primary driver mutation in these cancers. Furthermore, there was no evidence for BECN1 mutation or loss in any other cancer, casting doubt on whether BECN1 is a tumor suppressor in most human cancers.Contrary to previous reports, BECN1 is not significantly mutated in human cancer and not a tumor-suppressor gene, as originally thought. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/early/2014/04/01/1541-7786.MCR-13-0614/F1.large.jpg.