Cell Cycle Analysis of ER Stress and Autophagy

Cell cycle-arresting drugs, thapsigargin (Tg) and chloroquine (CQ), are employed to study endoplasmic reticulum (ER) stress and the autophagic process using cell lines without measuring the cell cycle of such cells. The potential cell cycle-dependent aspect of such processes in cell lines may impact upon the degree of ER stress and autophagy measured. ER stress is known to be caused by a build-up of misfolded proteins within the ER, which may then undergo ER phagy or reticulophagy. The cell cycle-dependent nature of all these processes is not well studied, so we investigated ER stress and autophagy by use of a combination of flow cytometric assays. These included cell cycle-dependent measurement of reticulophagy, misfolded protein levels and autophagic marker LC3-II in K562 and Jurkat cells. ER stress-inducing drug Tg caused significant reticulophagy in both cell types. This was cell cycle dependent in K562 cells only, with proliferating cells undergoing more reticulophagy. In contrast, autophagy-initiating drug CQ caused reticulophagy at higher doses in Jurkat cells, whereas K562 cells showed a cell cycle-dependent elongation of the ER, which was less pronounced in proliferating cells. The level of cellular misfolded protein in response to both drugs was high in K562 cells when either undergoing reticulophagy or elongation in a non-cell cycle-dependent manner, whereas the misfolded protein levels in Jurkat cells in response to both drugs were lower than those observed in K562 cells. Both cell lines employed in this study showed no increase of LC3-II above controls in response to Tg treatment. However, CQ induced a cell cycle-dependent increase of LC3-II in both cell types. Thus, the type of cell employed and the cell cycle dependent modulation of thebiological processes involved in ER stress and autophagy should be considered when designing studies in ER stress and autophagy

Flow Cytometry Reveals the Nature of Oncotic Cells

The term necrosis is commonly applied to cells that have died via a non-specific pathway or mechanism but strictly is the description of the degradation processes involved once the plasma membrane of the cell has lost integrity. The signalling pathways potentially involved in accidental cell death (ACD) or oncosis are under-studied. In this study, the flow cytometric analysis of the intracellular antigens involved in regulated cell death (RCD) revealed the phenotypic nature of cells undergoing oncosis or necrosis. Sodium azide induced oncosis but also classic apoptosis, which was blocked by zVAD (z-Vla-Ala-Asp(OMe)-fluoromethylketone). Oncotic cells were found to be viability+ve/caspase-3–ve/RIP3+ve/–ve (Receptor-interacting serine/threonine protein kinase 3). These two cell populations also displayed a DNA damage response (DDR) phenotype pH2AX+ve/PARP–ve, cleaved PARP induced caspase independent apoptosis H2AX–ve/PARP+ve and hyper-activation or parthanatos H2AX+ve/PARP+ve. Oncotic cells with phenotype cell viability+ve/RIP3–ve/caspase-3–ve showed increased DDR and parthanatos. Necrostatin-1 down-regulated DDR in oncotic cells and increased sodium azide induced apoptosis. This flow cytometric approach to cell death research highlights the link between ACD and the RCD processes of programmed apoptosis and necrosis

Compromised Function of Regulatory T Cells in Rheumatoid Arthritis and Reversal by Anti-TNFα Therapy

Regulatory T cells have been clearly implicated in the control of disease in murine models of autoimmunity. The paucity of data regarding the role of these lymphocytes in human autoimmune disease has prompted us to examine their function in patients with rheumatoid arthritis (RA). Regulatory (CD4+CD25+) T cells isolated from patients with active RA displayed an anergic phenotype upon stimulation with anti-CD3 and anti-CD28 antibodies, and suppressed the proliferation of effector T cells in vitro. However, they were unable to suppress proinflammatory cytokine secretion from activated T cells and monocytes, or to convey a suppressive phenotype to effector CD4+CD25− T cells. Treatment with antitumor necrosis factor α (TNFα; Infliximab) restored the capacity of regulatory T cells to inhibit cytokine production and to convey a suppressive phenotype to “conventional” T cells. Furthermore, anti-TNFα treatment led to a significant rise in the number of peripheral blood regulatory T cells in RA patients responding to this treatment, which correlated with a reduction in C reactive protein. These data are the first to demonstrate that regulatory T cells are functionally compromised in RA, and indicate that modulation of regulatory T cells by anti-TNFα therapy may be a further mechanism by which this disease is ameliorated

Binding of Ikaros to the λ5 promoter silences transcription through a mechanism that does not require heterochromatin formation

The Ikaros family of proteins are DNA binding factors required for correct development of B and T lymphocytes. Cytogenetic studies have shown that these proteins form complexes with pericentromeric heterochromatin in B cells, and the colocalization of transcriptionally silent genes with these complexes suggests that Ikaros could silence transcription by recruiting genes to heterochromatin. Here we show that a site in the λ5 promoter that binds Ikaros and Aiolos is required for silencing of λ5 expression in activated mature B cells. Analysis of methylation and nuclease accessibility indicates that the silenced λ5 gene is not heterochromatinized in B cells, despite being associated with pericentromeric heterochromatin clusters. We also found that a promoter mutation, which affects Ikaros-mediated silencing of λ5 expression, is not rescued in a transgenic line that has the gene integrated into pericentromeric heterochromatin. Our results indicate that the Ikaros proteins initiate silencing of λ5 expression through a direct effect on the promoter with localization to pericentromeric heterochromatin likely to affect the action of Ikaros on regulatory sequences rather than causing heterochromatinization of the gene

iASPP is a novel autophagy inhibitor in keratinocytes

The protein iASPP (encoded by PPP1R13L) is an evolutionarily conserved p53 inhibitor, the expression of which is often upregulated in human cancers. We have recently shown that iASPP is a crucial regulator of epidermal homeostasis. Here, we report that iASPP also acts as autophagy inhibitor in keratinocytes. Our data show that depletion of iASPP protects keratinocytes from apoptosis by modulating the expression of Noxa (also known as PMAIP1). In our model, iASPP expression can affect the fission-fusion cycle, mass and shape of mitochondria. iASPP-silenced keratinocytes display disorganization of cytosolic compartments and increased metabolic stress caused by deregulation of mTORC1 signaling. Moreover, increased levels of lipidated LC3 protein confirmed the activation of autophagy in iASPP-depleted cells. We have identified a novel mechanism modulating autophagy in keratinocytes that relies upon iASPP expression specifically reducing the interaction of Atg5-Atg12 with Atg16L1, an interaction that is essential for autophagosome formation or maturation. Using organotypic culture, we further explored the link between autophagy and differentiation, and we showed that impairing autophagy affects epidermal terminal differentiation. Our data provide an alternative mechanism to explain how epithelial integrity is maintained against environmental stressors and might also improve the understanding of the etiology of skin diseases that are characterized by defects in differentiation and DNA damage responses.15 page(s