NFAT2 regulates COX-2 expression and modulates the integrin repertoire in endothelial cells at the crossroads of angiogenesis and inflammation.

The mechanisms controlling the switch between the pro-angiogenic and pro-inflammatory states of endothelial cells are still poorly understood. In this paper, we show that: (a) COX-2 expression induced by VEGF-A is NFAT2-dependent; and (b) the integrin profile in endothelial cells induced by the pro-angiogenic VEGF-A is distinct from that brought on by the inflammatory cytokine TNF-α. Two groups of integrin subunits specifically upregulated over time by both cytokines were identified using RT-PCR and Western Immunoblotting. The first group included α4, α5, α6, and ß5 subunits that were upregulated by VEGF-A; the second group consisted of αV and ß3 induced by TNF-α. Both cytokines significantly enhanced the expression of ß1 and modulated α2 mRNA. In contrast to TNF-α, VEGF-A induction of integrin subunits depended on the activation of the calcineurin/NFAT pathway. Both calcineurin inhibitors (cyclosporineA and 11R-VIVIT) and downregulation of NFAT2 with specific siRNA decreased induction of integrin subunits. This process of induction could be increased by upregulation of NFAT2 by pBJ5-NFAT2 transfection. This suggests that NFAT2 mediates VEGF-induced upregulation of integrin subunit synthesis by providing a constant supply of newly synthesized "refreshed" mature integrin receptors, particularly α2ß1, α5ß1, α4ß1, α6ß1 and αVß5, which are involved at different stages of angiogenesis.

Bacterial expression, purification and angiogenesis-promoting activity of human thymosin ß4.

Thymosin ß4 (Tß4) is an actin-binding peptide involved in tissue regeneration and angiogenesis. This 43-amino acid peptide is chemically synthesized for research or clinical trials. To overcome the high costs of solid phase synthesis, we developed a genetic engineering procedure of Tß4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6 × HN). The recombinant, non-glycosylated peptide was overexpressed in soluble form and purified by two-step immobilized metal ion affinity chromatography. Use of the pET vector expression system allowed for easy removal of the polyhistidine tag by thrombin. Functional studies revealed that recombinant Tß4 stimulated angiogenesis via activation of the endothelial proteolytic systems, inhibition of endothelial cell adhesion, promotion of migration and capillary tube formation in Matrigel, and that its activity was similar to that observed for the synthetic peptide. The presented study comprises the first evidence that recombinant Tß4 promotes angiogenesis in an in vitro endothelial cell model.

Gene expression pattern in PC12 cells with reduced PMCA2 or PMCA3 isoform: selective up-regulation of calmodulin and neuromodulin.

Cellular calcium homeostasis is controlled predominantly by the plasma membrane calcium pump (PMCA). From four PMCA isoforms, PMCA1 and PMCA4 are ubiquitous, while PMCA2 and PMCA3 are found in excitable cells. We have previously shown that suppression of neuron-specific PMCAs in non-differentiated PC12 cells changed the cell morphology and triggered neuritogenesis. Using the microarrays, real-time PCR and immunodetection, we analyzed the effect of PMCA2 or PMCA3 reduction in PC12 cells on gene expression, with emphasis on calmodulin (CaM), neuromodulin (GAP43) and MAP kinases. In PMCA-suppressed lines total CaM increased, and the calm I and calm II genes appeared to be responsible for this effect. mRNA and protein levels of GAP43 were increased, however, the amount of phosphorylated form was lower than in control cells. Localization of CaM/GAP43 and CaM/pGAP43 differed between control and PMCA-reduced cells. In both PMCA-modified lines, amounts of ERK1/2 increased. While pERK1 decreased, the pERK2 level was similar in all examined lines. PMCA suppression did not change the p38 amount, but the p-p38 diminished. JNK2 protein decreased in both PMCA-reduced cells without changes in pJNK level. Microarray analysis revealed distinct expression patterns of certain genes involved in the regulation of cell cycle, proliferation, migration, differentiation, apoptosis and cell signaling. Suppression of neuron-specific PMCA isoforms affected the phenotype of PC12 cells enabling adaptation to the sustained increase in cytosolic Ca(2+) concentration. This is the first report showing function of PMCA2 and PMCA3 isoforms in the regulation of signaling pathways in PC12 cells.

Functional characteristic of PC12 cells with reduced microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.

Adaptation of microsomal glutathione transferase 1 in PC12 cells with modified PMCA isoforms composition.

Microsomal glutathione transferase 1 (MGST1) is an integral homo-trimeric membrane protein with transferase and peroxidase activities. With glutathione as a co-substrate, it scavenges toxic compounds and may exert anti-apoptotic effect. We examined the effect of suppression of plasma membrane Ca(2+)-ATPase isoforms--PMCA2 or PMCA3 on MGST1 in PC12 cells. GSH level was significantly higher in PMCA2-reduced line, but similar GSSG/GSH ratios in all cell lines suggested an efficient protection or absence of oxidative stress. The ATP concentration decreased in both modified lines, although in PMCA2-suppressed cells the decrease was higher. Total GSTs activity in postmitochondrial fraction increased by 30% in the cells with reduced PMCA3. After treatment with MGST1 activator N-ethylmaleimide (NEM), the activity increased in both transfected lines by 30-40%. Real-time PCR also showed a higher mRNA expression of MGST1 in these lines. Staining with antibody recognizing all cytosolic and membrane-bound GSTs revealed the difference in oligomeric forms of GSTs, and specific anti-MGST1 antibody showed the presence of MGST1 hexamers in the transfected cells. Formation of similar hexamers was detected in the control line after treatment with peroxynitrite. Modification of MGST1 under reduced PMCAs amount may represent an adaptive mechanism that offers protection against the cytotoxicity mediated by increased Ca2+.

Changes in erythrocyte glutathione and plasma membrane calcium pump in preterm newborns treated antenatally with MgSO4.

BACKGROUND: Magnesium is required for the proper activity of many metabolic pathways in every cell type. Mg deficiency gives rise to preterm birth and low body weight that are associated with pathological circumstances, including disturbed ions homeostasis and insufficient antioxidant protection. Antenatal MgSO(4) treatment has been reported to exhibit a protective effect on the developing fetus; however, the molecular mechanism of this protection remains not fully understood. OBJECTIVES: Since Mg ions very rapid cross the placenta, our study tested the hypothesis whether prenatal exposure to MgSO(4) may modify the expression and activity of erythrocyte plasma membrane calcium pump (PMCA), and total erythrocyte glutathione (GSH) concentration in preterm newborns. METHODS: Immunocharacteristics of erythrocyte PMCA in control and MgSO(4)-treated newborns were done using general and isoform-specific antibodies. The hydrolytic activity of PMCA was determined with and without calmodulin. Total GSH erythrocyte levels were analyzed by a quantitative assay using Ellman reagent. All assays were done after delivery and 24 h later. RESULTS: Significant differences were present in PMCA amount, isoform composition, basal activity and sensitivity for stimulation by calmodulin in both groups of neonates. The total GSH content altered during examined time, but only in the control group. CONCLUSIONS: Prenatal treatment with MgSO(4) may exert a significant influence on calcium homeostasis and nonenzymatic antioxidant reserve in the erythrocytes of preterm newborns.

Fast action of neuroactive steroids on plasma membrane calcium pump in PC12 cells.

Calcium ions are essential to proper neurotransmission. Impairment in cytosolic Ca(2+) concentration and Ca(2+) signaling disturbs neuronal activity, leading to pathological consequences. In cells, a high-affinity plasma membrane calcium pump (PMCA) keeps free Ca(2+) in the nanomolar range. Among four genes encoding the enzyme, PMCA2 and 3 are primary in excitable cells. To elucidate the relationship between PMCAs' composition and susceptibility for neurosteroid regulation, we obtained PC12 cells with suppressed neuron-specific isoforms and analyzed the effect of selected steroids on Ca(2+) uptake. Our results indicate that hormones affected Ca(2+) transport activity and that this effect depended on both PMCA isoform composition and steroid structure.

Functional importance of PMCA isoforms in growth and development of PC12 cells.

Intracellular Ca2+ in neuronal cells is an essential regulatory ion responsible for excitability, synaptic plasticity, and neurite outgrowth. Plasma membrane calcium ATPase (PMCA) is the most sensitive enzyme in decreasing of the Ca2+ concentration. The diverse PMCA isoforms composition in the membranes suggests their specific function in the cell, and whereas PMCA1 and 4 appear to be ubiquitous, PMCA2 and 3 are characteristic isoforms for excitable cells. The aim of our study was to elucidate if and how the elimination of neuron-specific isoforms affects the pattern of cell growth and development. We have obtained stable-transfected PC12 cell lines with a suppressed expression of PMCA2, PMCA3, or both neuron-specific isoforms. The modified profile of PMCA generated considerable changes in morphology of examined PC12 lines, suggesting the activation of a differentiation process to pseudoneuronal phenotype. Experiments with Fura-2/AM-loaded cells revealed an increased cytosolic Ca2+ concentration in the cell lines with blocked PMCA2 isoform. The suppression of PMCA2 concomitantly altered expression of sarco/endoplasmic Ca2+-ATPase 2 isoform (SERCA2) at the protein level. Comparative flow cytometry analysis, using Annexin V/PI conjugate, showed the difference in the mean percentage of apoptotic cells in modified PC12 lines. Our data suggest that specific PMCA isoforms presence can regulate the intact cell development; however, it may involve multiple unidentified yet signaling pathways.