SUMOylation regulates nucleo-cytoplasmic shuttling of Elk-1

The transcription factor Elk-1 is a nuclear target of mitogen-activated protein kinases and regulates immediate early gene activation by extracellular signals. We show that Elk-1 is also conjugated to SUMO on either lysines 230, 249, or 254. Mutation of all three sites is necessary to fully block SUMOylation in vitro and in vivo. This Elk-1 mutant, Elk-1(3R), shuttles more rapidly to nuclei of Balb/C cells fused to transfected HeLa cells. Coexpression of SUMO-1 or -2 strongly reduces shuttling by Elk-1 without affecting that of Elk-1(3R), indicating that SUMOylation regulates nuclear retention of Elk-1. Accordingly, overexpression of Elk-1(3R) in PC12 cells, where cytoplasmic relocalization of Elk-1 has been linked to differentiation, enhances neurite extension relative to Elk-1. The effect of Elk-1, but not of the 3R mutant, was blocked upon cotransfection with SUMO-1 or -2 and enhanced by coexpression with mutant Ubc-9. Thus, SUMO conjugation is a novel regulator of Elk-1 function through the control of its nuclear-cytoplasmic shuttling

Calpain-5 gene variants are associated with diastolic blood pressure and cholesterol levels

BACKGROUND: Genes implicated in common complex disorders such as obesity, type 2 diabetes mellitus (T2DM) or cardiovascular diseases are not disease specific, since clinically related disorders also share genetic components. Cysteine protease Calpain 10 (CAPN10) has been associated with T2DM, hypertension, hypercholesterolemia, increased body mass index (BMI) and polycystic ovary syndrome (PCOS), a reproductive disorder of women in which isunlin resistance seems to play a pathogenic role. The calpain 5 gene (CAPN5) encodes a protein homologue of CAPN10. CAPN5 has been previously associated with PCOS by our group. In this new study, we have analysed the association of four CAPN5 gene variants(rs948976A>G, rs4945140G>A, rs2233546C>T and rs2233549G>A) with several cardiovascular risk factors related to metabolic syndrome in general population. METHODS: Anthropometric measurements, blood pressure, insulin, glucose and lipid profiles were determined in 606 individuals randomly chosen from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille), recruited to investigate the prevalence of anthropometric and physiological parameters related to obesity and other components of the metabolic syndrome. Genotypes at the four polymorphic loci in CAPN5 gene were detected by polymerase chain reaction (PCR). RESULTS: Genotype association analysis was significant for BMI (p ≤ 0.041), diastolic blood pressure (p = 0.015) and HDL-cholesterol levels (p = 0.025). Different CAPN5 haplotypes were also associated with diastolic blood pressure (DBP) (0.0005 ≤ p ≤ 0.006) and total cholesterol levels (0.001 ≤ p ≤ 0.029). In addition, the AACA haplotype, over-represented in obese individuals, is also more frequent in individuals with metabolic syndrome defined by ATPIII criteria (p = 0.029). CONCLUSION: As its homologue CAPN10, CAPN5 seems to influence traits related to increased risk for cardiovascular diseases. Our results also may suggest CAPN5 as a candidate gene for metabolic syndrome

A Novel Mouse c-fos Intronic Promoter That Responds to CREB and AP-1 Is Developmentally Regulated In Vivo

BACKGROUND: The c-fos proto-oncogene is an archetype for rapid and integrative transcriptional activation. Innumerable studies have focused on the canonical promoter, located upstream from the transcriptional start site. However, several regulatory sequences have been found in the first intron. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely conserved region in c-fos first intron that contains a putative TATA box, and functional TRE and CRE sites. This fragment drives reporter gene activation in fibroblasts, which is enhanced by increasing intracellular calcium and cAMP and by cotransfection of CREB or c-Fos/c-Jun expression vectors. We produced transgenic mice expressing a lacZ reporter controlled by the intronic promoter. Lac Z expression of this promoter is restricted to the developing central nervous system (CNS) and the mesenchyme of developing mammary buds in embryos 12.5 days post-conception, and to brain tissue in adults. RT-QPCR analysis of tissue mRNA, including the anlage of the mammary gland and the CNS, confirms the existence of a novel, nested mRNA initiated in the first intron. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence for a novel, developmentally regulated promoter in the first intron of the c-fos gene

A Crucial Role for Infected-Cell/Antibody Immune Complexes in the Enhancement of Endogenous Antiviral Immunity by Short Passive Immunotherapy

Antiviral monoclonal antibodies (mAbs) represent promising therapeutics. However, most mAbs-based immunotherapies conducted so far have only considered the blunting of viral propagation and not other possible therapeutic effects independent of virus neutralization, namely the modulation of the endogenous immune response. As induction of long-term antiviral immunity still remains a paramount challenge for treating chronic infections, we have asked here whether neutralizing mAbs can, in addition to blunting viral propagation, exert immunomodulatory effects with protective outcomes. Supporting this idea, we report here that mice infected with the FrCasE murine retrovirus on day 8 after birth die of leukemia within 4–5 months and mount a non-protective immune response, whereas those rapidly subjected to short immunotherapy with a neutralizing mAb survive healthy and mount a long-lasting protective antiviral immunity with strong humoral and cellular immune responses. Interestingly, the administered mAb mediates lysis of infected cells through an antibody-dependent cell cytotoxicity (ADCC) mechanism. In addition, it forms immune complexes (ICs) with infected cells that enhance antiviral CTL responses through FcγR-mediated binding to dendritic cells (DCs). Importantly, the endogenous antiviral antibodies generated in mAb-treated mice also display the same properties, allowing containment of viral propagation and enhancement of memory cellular responses after disappearance of the administered mAb. Thus, our data demonstrate that neutralizing antiviral mAbs can act as immunomodulatory agents capable of stimulating a protective immunity lasting long after the end of the treatment. They also show an important role of infected-cells/antibody complexes in the induction and the maintenance of protective immunity through enhancement of both primary and memory antiviral T-cell responses. They also indicate that targeting infected cells, and not just viruses, by antibodies can be crucial for elicitation of efficient, long-lasting antiviral T-cell responses. This must be considered when designing antiviral mAb-based immunotherapies

Single amino acid change in gp41 region of HIV-1 alters bystander apoptosis and CD4 decline in humanized mice

<p>Abstract</p> <p>Background</p> <p>The mechanism by which HIV infection leads to a selective depletion of CD4 cells leading to immunodeficiency remains highly debated. Whether the loss of CD4 cells is a direct consequence of virus infection or bystander apoptosis of uninfected cells is also uncertain.</p> <p>Results</p> <p>We have addressed this issue in the humanized mouse model of HIV infection using a HIV variant with a point mutation in the gp41 region of the Env glycoprotein that alters its fusogenic activity. We demonstrate here that a single amino acid change (V38E) altering the cell-to-cell fusion activity of the Env minimizes CD4 loss in humanized mice without altering viral replication. This differential pathogenesis was associated with a lack of bystander apoptosis induction by V38E virus even in the presence of similar levels of infected cells. Interestingly, immune activation was observed with both WT and V38E infection suggesting that the two phenomena are likely not interdependent in the mouse model.</p> <p>Conclusions</p> <p>We conclude that Env fusion activity is one of the determinants of HIV pathogenesis and it may be possible to attenuate HIV by targeting gp41.</p

The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets

Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors

<p>Abstract</p> <p>Background</p> <p>Chondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established - namely, the Swarm Rat Chondrosarcoma (SRC) - and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy.</p> <p>Methods</p> <p>To examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC.</p> <p>Results</p> <p>The site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression.</p> <p>Conclusion</p> <p>This report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-β4 may have a role in chondrosarcoma metastasis.</p