Glomerular visceral epithelial cells (podocytes) play a critical role in the pathogenesis of human immunodeficiency virus (HIV)-associated nephropathy. A key question concerns the mechanism(s) by ...which the HIV-1 genome alters the phenotype of the highly specialized, terminally differentiated podocytes. Here, using an in vitro system of conditionally immortalized differentiated human podocytes (CIDHPs), we document a pivotal role for the p66ShcA protein in HIV-1-induced reactive oxygen species generation and CIDHP apoptosis. CIDHP transfected with truncated HIV-1 construct (NL4-3) exhibit increased reactive oxygen species metabolism, DNA strand breaks, and a 5-fold increase in apoptosis, whereas the opposite was true for NL4-3/CIDHP co-transfected with mu-36p66ShcA (mu-36) dominant negative expression vector or isoform-specific p66-small interfering RNA. Phosphorylation at Ser-36 of the wild type p66ShcA protein, required for p66ShcA redox function and inhibition of the potent stress response regulator Foxo3a, was unchanged in mu-36/NL4-3/CIDHP but increased in NL4-3/CIDHP. Acute knockdown of Foxo3a by small interfering RNA induced a 50% increase in mu-36/NL4-3/CIDHP apoptosis, indicating that Foxo3a-dependent responses promote the survival phenotype in mu-36 cells. We conclude that inhibition of p66ShcA redox activity prevents generation of HIV-1 stress signals and activation of the CIDHP apoptosis program.
Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has ...transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigen-positive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorage-independent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus.
Glioblastoma multiforme is characterized by rapid proliferation, aggressive metastatic potential, and resistance to radio- and chemotherapy. The matricellular protein CYR61 regulates cellular ...proliferation and migration and is highly expressed in Glioblastomas. MicroRNAs are 22-nucleotides long RNAs that regulate gene expression post-transcriptionally. Here, we utilized the LN229 glioblastoma cell line and found that CYR61 is a target of miR-136, miR-155, and miR-634. Over-expression of miR-136 and miR-634 miRNAs negatively affected proliferation, but not migration, while expression of miR-155 reduced migration but did not affect the proliferation of LN229 cells. Investigation of the molecular mechanisms affected by expression of miR-634 revealed an increased phosphorylation of p70S6 kinase, suggesting an induction of the mammalian target of rapamycin (mTOR) complex 1 pathway. Additionally, in miR-634 overexpressing cells, TSC2, a negative regulator of mTOR signaling, was found to be decreased. Altogether, our study provides insights on the differential roles of miRs-136, -155, and -634 in regulating glioblastoma cell growth and migration, and how microRNAs could be manipulated to decrease the aggressiveness and metastatic potential of tumor cells.
The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which ...target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.
Alcohol use disorders (AUD) exacerbate neurocognitive dysfunction in Human Immunodeficiency Virus (HIV+) patients. We have shown that chronic binge alcohol (CBA) administration (13-14 g EtOH/kg/wk) ...prior to and during simian immunodeficiency virus (SIV) infection in rhesus macaques unmasks learning deficits in operant learning and memory tasks. The underlying mechanisms of neurocognitive alterations due to alcohol and SIV are not known. This exploratory study examined the CBA-induced differential expression of hippocampal genes in SIV-infected (CBA/SIV+;
= 2) macaques in contrast to those of sucrose administered, SIV-infected (SUC/SIV+;
= 2) macaques. Transcriptomes of hippocampal samples dissected from brains obtained at necropsy (16 months post-SIV inoculation) were analyzed to determine differentially expressed genes. MetaCore from Thomson Reuters revealed enrichment of genes involved in inflammation, immune responses, and neurodevelopment. Functional relevance of these alterations was examined in vitro by exposing murine neural progenitor cells (NPCs) to ethanol (EtOH) and HIV trans-activator of transcription (Tat) protein. EtOH impaired NPC differentiation as indicated by decreased βIII tubulin expression. These findings suggest a role for neuroinflammation and neurogenesis in CBA/SIV neuropathogenesis and warrant further investigation of their potential contribution to CBA-mediated neurobehavioral deficits.
Medulloblastomas represent about 25% of all pediatric intracranial neoplasms. These highly malignant tumors arise from the
cerebellum affecting mainly children between ages 5 and 15. Although the ...etiology of medulloblastomas has not yet been elucidated,
several reports suggest that insulin-like growth factor I (IGF-I) may contribute to the development of these tumors. Results
of this study show that the majority of cases examined were characterized by the abundant presence of the receptor for IGF-I
(IGF-IR) protein (16 of 20 cases), and its major signaling molecule, insulin receptor substrate 1 (IRS-1; 15 of 20). Protein
levels for IGF-IR and IRS-1, determined by Western blot and immunohistochemistry, were significantly higher in medulloblastoma
biopsies than in control cerebellar tissue. By immunohistochemistry, 10 of 17 biopsies examined were also positive for the
anti-pY1316 antibody staining that specifically recognizes the phosphorylated (active) form of the IGF-IR. These findings
correlate with the fact that phosphorylated forms of the downstream-signaling molecules Erk-1, Erk-2, and Akt/protein kinase
B were found in medulloblastoma biopsies but not in control cerebellar tissue. Importantly, there is a strong inverse correlation
between biopsies that are positive for anti-pY1316 and for anti-Trk-C immunoreactivity. These observations direct our attention
to the IGF-IR system as a potential therapeutic target in medulloblastomas and suggest a possibility of using the anti-pY1316
antibody as a potential prognostic marker for medulloblastomas.
Cell cultures constitute an important tool for research as a way to reproduce pathological processes in a controlled system. However, the culture of brain-derived cells in monolayer presents ...significant challenges that obscure the fidelity of in vitro results. After a few number of passages, glial and neuronal cells begin to lose their morphological characteristics, and most importantly, their specific cellular markers and phenotype. In recent years, the discovery of neural progenitor cells, and the methodology to culture them in suspension maintaining their potentiality while still retaining the ability to differentiate into astrocytes, oligodendrocytes and neurons has been a significant contribution to the fields of neuroscience and neuropathology.In the brain, progenitor cells are located in the Germinal Matrix, the subventricular zone in what later would become the basal ganglia, and play an essential role in the homeostasis of the brain by providing the source to replace differentiated cells that have been lost or damaged by different pathological processes, such as senescence, injury, genetic conditions, or disease. The discovery of these neural stem cells in an organ traditionally thought to have limited or no regenerative capacity has opened the door to the development of novel treatments, which include cell replacement therapy. Here we describe the culture and differentiation of neural progenitor cells into neurospheres, and the phenotyping of the resulting cells using immunocytochemistry . The immunocytological methods outlined are not restricted to the analysis of neurosphere-derived cultures but are also applicable for cell typing of primary glial or cell line-derived samples.
Development and progression of melanoma can be accelerated by intensification of particular metabolic pathways, such as aerobic glycolysis and avid amino acid catabolism, and is accompanied by ...aberrant immune responses within the tumor microenvironment. Contrary to other cancer types, melanoma reveals some unique tissue‐specific features, such as melanogenesis, which is intertwined with metabolism. Nuclear peroxisome proliferator‐activated receptors (PPARs) take part in regulation of systemic and cellular metabolism, inflammation and melanogenesis. They appear as a focal regulatory point for these three distinct processes by occupying the intersection among AMP‐dependent protein kinase (AMPK), mammalian target of rapamycin (mTOR) and PPAR gamma coactivator 1‐alpha (PGC‐1α) signalling pathways. When deregulated, they may accelerate melanoma malignant growth. Presenting the contribution of PPARα and PPARγ in melanoma biology, we attempt to ask how two contrasting metabolic states: obesity and fasting, can change progression of the disease and possible outcome of the treatment. This short essay is aimed to provoke a discussion about some practical implications for melanoma prevention and treatment, especially: how metabolic manipulation may be exploited to overcome immunosuppression and support immune checkpoint blockade efficacy.
Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by ...silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.
PDF
