Selective inhibition of plasma membrane calcium ATPase 4 improves angiogenesis and vascular reperfusion

Aims Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. Methods and results We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. Conclusions Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors

Structural and functional characterization of the human CD36 gene promoter: identification of a proximal PEBP2/CBF site.

CD36 is a cell surface glycoprotein composed of a single polypeptide chain, which interacts with thrombospondin, collagens type I and IV, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and erythrocytes parasitized with Plasmodium falciparum. Its expression is restricted to a few cell types, including monocyte/macrophages. In these cells, CD36 is involved in phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low density lipoprotein. To study the molecular mechanisms that control the transcription of the CD36 gene in monocytic cells we have isolated and analyzed the CD36 promoter. Transient expression experiments of 5'-deletion fragments of the CD36 promoter coupled to luciferase demonstrated that as few as 158 base pairs upstream from the transcription initiation site were sufficient to direct the monocyte-specific transcription of the reporter gene. Within the above region, the fragment spanning nucleotides -158 to -90 was required for optimal transcription in monocytic cells. Biochemical analysis of the region -158/-90 revealed a binding site for transcription factors of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family at position -103. Disruption of the PEBP2/CBF site markedly diminished the role of the PEBP2/CBF factors in the constitutive transcription of the CD36 gene. The involvement of members of the PEBP2/CBF family in chromosome translocations associated with acute myeloid leukemia, and in the transcriptional regulation of the myeloid-specific genes encoding for myeloperoxidase, elastase, and the colony-stimulating factor receptor, highlights the relevance of the regulation of the CD36 gene promoter in monocytic cells by members of the PEBP2/CBF family

Organización estructural del gen que codifica la glicoproteina de membrana cd36. Caracterización estructural y funcional de su región promotora

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 13-12-199

Organizacion estructural del gen que codifica la glicoproteina de membrana cd36 Caracterizacion estructural y funcional de su region promotora

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

JNK (c-Jun NH2-terminal kinase) is a target for antioxidants in T lymphocytes.

AP-1 has been shown to behave as a redox-sensitive transcription factor that can be activated by both oxidant and antioxidant stimuli. However, the mechanisms involved in the activation of AP-1 by antioxidants are largely unknown. In this study we show that the structurally unrelated antioxidant agents pyrrolidine dithiocarbamate (PDTC), butylated hydroxyanisole, and Nacetylcysteine activated JNK (c-Jun NH2-terminal kinase) in Jurkat T cells. This activation differed substantially from that mediated by phorbol 12-myristate 13-acetate (PMA) and Ca2+ ionophore or produced by costimulation with antibodies against the T cell receptor-CD3 complex and to CD28. The activation of JNK by classical T cell stimuli was transient, whereas that mediated by PDTC and butylated hydroxyanisole (but not N-acetylcysteine) was sustained. The kinetics of JNK activation correlated with the expression of c-jun which was transient after stimulation with PMA plus ionophore and prolonged in response to PDTC, which also transiently induced c-fos. In addition, JNK activation by PMA plus ionophore was sensitive to inhibitors of signaling pathways involving Ca2+, protein kinase C, and tyrosine phosphorylation, which failed to inhibit the activation mediated by PDTC. Transfection of trans-dominant negative expression vectors of ras and raf, together with AP-1-dependent reporter constructs, as well as Western blot analysis using anti-ERK (extracellular signal-regulated kinase) antibodies, indicated that the Ras/Raf/ERK pathway did not appear to mediate the effect of the antioxidant. However, the combined treatment with PDTC and PMA, two agents that synergize on AP-1 activation, resulted in the persistent phosphorylation of ERK-2. In conclusion, our results identify JNK as a target of antioxidant agents which can be regulated differentially under oxidant and antioxidant conditions

Structural organization of the gene for human CD36 glycoprotein.

The cell-surface glycoprotein CD36 interacts with a large variety of ligands, including collagen types I and IV, thrombospondin, erythrocytes parasitized with Plasmodium falciparum, platelet-agglutinating protein p37, oxidized low density lipoprotein, and long-chain fatty acids. Its expression is restricted to platelets, monocytes, adipocytes, and some endothelial and epithelial cells and is regulated during cell activation, differentiation, and development. CD36 belongs to a novel gene family of structurally related glycoproteins that includes CLA-1 and the lysosomal membrane glycoprotein LIMPII. To advance our knowledge on the genomic organization and the regulation of the cellular expression of the genes of this family, we have investigated the structural organization of the human CD36 gene and of its 5'-proximal flanking region. The CD36 gene is encoded by 15 exons that extend more than 32 kilobases on the human genome. Interestingly, the CD36 mRNA 5'-untranslated region is encoded by three exons. The 3'-untranslated region is contained in two exons, whose expression pattern can originate two mRNA forms. The cytoplasmic and transmembrane regions predicted at both terminal ends of the polypeptide chain are encoded by single exons, while the extracellular domain is encoded by 11 exons. The transcription initiation site of the CD36 gene is located 289 nucleotides upstream from the translational start codon. Sequence analysis of the proximal 5'-flanking region of the gene reveals the existence of a TATA box appropriately located with respect to the transcription initiation site and several potential cis-regulatory elements that might contribute to the transcriptional regulation of the CD36 gene. Delineation of the structural organization of the CD36 gene may help in defining the boundaries of relevant structural and/or functional domains in CD36 and, by extension, in the other members of the family

P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation.

Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naive cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic

Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.

Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified kappaB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-kappaB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-kappaB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression

CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose.

Metadata onlyThe cel1 gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases. The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. The fusion protein was used to raise a CEL1-specific antibody. CEL1 was detected as an extracellular 49.8 kDa protein in A. bisporus cellulose-grown cultures, where it bound strongly to cellulose. CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a beta-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase. CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose

Cardiomyocyte calcineurin is required for the onset and progression of cardiac hypertrophy and fibrosis in adult mice

Previous studies have demonstrated that activation of calcineurin induces pathological cardiac hypertrophy (CH). In these studies, loss-of-function was mostly achieved by systemic administration of the calcineurin inhibitor cyclosporin A. The lack of conditional knockout models for calcineurin function has impeded progress toward defining the role of this protein during the onset and the development of CH in adults. Here, we exploited a mouse model of CH based on the infusion of a hypertensive dose of angiotensin II (AngII) to model the role of calcineurin in CH in adulthood. AngII-induced CH in adult mice was reduced by treatment with cyclosporin A, without affecting the associated increase in blood pressure, and also by induction of calcineurin deletion in adult mouse cardiomyocytes, indicating that cardiomyocyte calcineurin is required for AngII-induced CH. Surprisingly, cardiac-specific deletion of calcineurin, but not treatment of mice with cyclosporin A, significantly reduced AngII-induced cardiac fibrosis and apoptosis. Analysis of profibrotic genes revealed that AngII-induced expression of Tgfβ family members and Lox was not inhibited by cyclosporin A but was markedly reduced by cardiac-specific calcineurin deletion. These results show that AngII induces a direct, calcineurin-dependent prohypertrophic effect in cardiomyocytes, as well as a systemic hypertensive effect that is independent of calcineurin activity.Spanish Ministerio de Economia, Industria y Competitividad (MEIC); Pro-CNIC Foundation; Severo Ochoa Center of Excellence (MEIC award) [SEV-2015-0505]; MEIC [SAF2012-34296, SAF2015-636333R]; Fundacio La Marato TV3 [CNIC is supported by the Spanish Ministerio de Economia, Industria y Competitividad (MEIC) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505). Support was also provided by grants from MEIC (SAF2012-34296 and SAF2015-636333R to JMR), Fundacio La Marato TV3 (20151330 to JMR), Ministerio de Sanidad CIBERCV (CB16/11/00264 to JMR), Comunidad de Madrid (AORTASANA-CM; B2017/BMD-3676 to JMR), and Red de Investigacion Cardiovascular (RIC) (RD12/0042/0022 to JMR and RD12/0042/0056 to LJJ-B), and an FPU fellowship (AP 2009-1713 to NL-V). We thank Dr S. Bartlett for English language editing, Dr Fatima Sanchez Cabo for statistical advice, Dr M. R. Campanero, Dr J. F. Nistal, and Dr B. Ibanez for critical reading of the manuscript. We also thank Dr G. R. Crabtree for providing Cnb1Delta/flox mice, A. Peral for technical assistance, and the ultrasonographers A.V. Alonso and L. Flores for technical support.]; Ministerio de Sanidad CIBERCV [CB16/11/00264]; Comunidad de Madrid (AORTASANA-CM) [B2017/BMD-3676]; Red de Investigacion Cardiovascular (RIC) [RD12/0042/0022, RD12/0042/0056]; FPU fellowship [AP 2009-1713]S