[Abstract unavailable

Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migrationrelated genes. In this 'invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca 2+ levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identifed Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Mitochondrial Ca(2+) uptake is crucial for the regulation of the rate of oxidative phosphorylation, the modulation of spatio-temporal cytosolic Ca(2+) signals and apoptosis. Although the phenomenon of mitochondrial Ca(2+) sequestration, its characteristics and physiological consequences have been convincingly reported, the actual protein(s) involved in this process are unknown. Here, we show that the uncoupling proteins 2 and 3 (UCP2 and UCP3) are essential for mitochondrial Ca(2+) uptake. Using overexpression, knockdown (small interfering RNA) and mutagenesis experiments, we demonstrate that UCP2 and UCP3 are elementary for mitochondrial Ca(2+) sequestration in response to cell stimulation under physiological conditions - observations supported by isolated liver mitochondria of Ucp2(-/-) mice lacking ruthenium red-sensitive Ca(2+) uptake. Our results reveal a novel molecular function for UCP2 and UCP3, and may provide the molecular mechanism for their reported effects. Moreover, the identification of proteins fundemental for mitochondrial Ca(2+) uptake expands our knowledge of the physiological role for mitochondrial Ca(2+) sequestration.

No abstract

Summary Although the endocannabinoid anandamide is frequently described to act predominantly in the cardiovascular system, the molecular mechanisms of its signaling remained unclear. In human endothelial cells, two receptors for anandamide were found, which were characterized as cannabinoid 1 receptor (CB1R; CNR1) and G-protein-coupled receptor 55 (GPR55). Both receptors trigger distinct signaling pathways. It crucially depends on the activation status of integrins which signaling cascade becomes promoted upon anandamide stimulation. Under conditions of inactive integrins, anandamide initiates CB1R-derived signaling, including Gi-protein-mediated activation of spleen tyrosine kinase (Syk), resulting in NFκB translocation. Furthermore, Syk inhibits phosphoinositide 3-kinase (PI3K) that represents a key protein in the transduction of GPR55-originated signaling. However, once integrins are clustered, CB1R splits from integrins and, thus, Syk cannot further inhibit GPR55-triggered signaling resulting in intracellular Ca2+ mobilization from the endoplasmic reticulum (ER) via a PI3K-Bmx-phospholipase C (PLC) pathway and activation of nuclear factor of activated T-cells. Altogether, these data demonstrate that the physiological effects of anandamide on endothelial cells depend on the status of integrin clustering.

IntroductionTissue factor (TF) is a cell surface receptor initiating blood coagulation, 1 thereby promoting thrombotic events in atherosclerosis, sepsis, and cancer. 2,3 Enhanced endothelial TF expression has been demonstrated in atherosclerotic plaques, 4,5 a process that may account for thrombotic events associated with early and advanced atherosclerosis. TF expression in endothelial cells (ECs) can be induced by a variety of agonists, including inflammatory cytokines, angiogenic growth factors, infectious agents, and minimally modified low-density lipoprotein (MM-LDL). 1,6 MM-LDL regulates TF expression at the level of transcription 5 ; however, the signaling pathways and transcription factors involved in this process are not known. Some of the effects of MM-LDL can be mimicked by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC). 7 Three biologically active components of oxidized PAPC (Ox-PAPC) have been structurally identified as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 8 and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC). 9 Which of these components of MM-LDL is responsible for induction of TF is not known.In contrast to interleukin-1 (IL-1) or tumor necrosis factor ␣ (TNF-␣), MM-LDL neither up-regulates E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), nor stimulates neutrophil binding to human ECs. 7 This suggests that classical inflammatory agonists and MM-LDL activate different signaling mechanisms. The major pathway induced by inflammatory cytokines activates transcription factors of the nuclear factor-B (NF-B) family. 10 Whether NF-B is activated by MM-LDL is a subject of controversy. 6,11 In fact, it was shown that MM-LDL and some of its components were capable of down-regulating NF-B-mediated transcription induced by inflammatory cytokines. 12 Thus, the role of the NF-B pathway in inflammatory activation of ECs by oxidized lipids requires further investigation.Apart from NF-B, 13 transcription of the TF gene can be promoted by early growth response factor 1 (EGR-1) and nuclear factor of activated T cells (NFAT). 14,15 Whereas inflammatory cytokines induce NF-B as well as EGR-1, vascular endothelial In the present study, we investigated signaling pathways and transcription factors mediating induction of TF expression in human ECs by biologically active oxidized phospholipids. We show that expression of TF is elevated by OxPAPC, and that this induction was mainly mediated by EGR-1-and NFAT-dependent transcription, but was independent of NF-B activation. Upstream mechanisms activated by OxPAPC were elevation of cytosolic Ca ϩϩ , activation of protein kinase C (PKC), and the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAP kinase cascade. Materials and methods MaterialsCyclosporin A was purchased from Novartis (Vienna, Austria); TNF-␣ from Genzyme (Cambridge...

Causes of autosomal-recessive intellectual disability (ID) have, until very recently, been under researched because of the high degree of genetic heterogeneity. However, now that genome-wide approaches can be applied to single multiplex consanguineous families, the identification of genes harboring disease-causing mutations by autozygosity mapping is expanding rapidly. Here, we have mapped a disease locus in a consanguineous Pakistani family affected by ID and distal myopathy. We genotyped family members on genome-wide SNP microarrays and used the data to determine a single 2.5 Mb homozygosity-by-descent (HBD) locus in region 5p15.32-p15.31; we identified the missense change c.2035G>A (p.Gly679Arg) at a conserved residue within NSUN2. This gene encodes a methyltransferase that catalyzes formation of 5-methylcytosine at C34 of tRNA-leu(CAA) and plays a role in spindle assembly during mitosis as well as chromosome segregation. In mouse brains, we show that NSUN2 localizes to the nucleolus of Purkinje cells in the cerebellum. The effects of the mutation were confirmed by the transfection of wild-type and mutant constructs into cells and subsequent immunohistochemistry. We show that mutation to arginine at this residue causes NSUN2 to fail to localize within the nucleolus. The ID combined with a unique profile of comorbid features presented here makes this an important genetic discovery, and the involvement of NSUN2 highlights the role of RNA methyltransferase in human neurocognitive development.

Adipose triglyceride lipase (ATGL) catalyzes the first step in the hydrolysis of triacylglycerol (TG) generating diacylglycerol and free fatty acids. The enzyme requires the activator protein CGI-58 (or ABHD5) for full enzymatic activity. Defective ATGL function causes a recessively inherited disorder named neutral lipid storage disease that is characterized by systemic TG accumulation and myopathy. In this study, we investigated the functional defects associated with mutations in the ATGL gene that cause neutral lipid storage disease. We show that these mutations lead to the expression of either inactive enzymes localizing to lipid droplets (LDs) or enzymatically active lipases with defective LD binding. Additionally, our studies assign important regulatory functions to the C-terminal part of ATGL. Truncated mutant ATGL variants lacking ϳ220 amino acids of the C-terminal protein region do not localize to LDs. Interestingly, however, these mutants exhibit substantially increased TG hydrolase activity in vitro (up to 20-fold) compared with the wild-type enzyme, indicating that the C-terminal region suppresses enzyme activity. Protein-protein interaction studies revealed an increased binding of truncated ATGL to CGI-58, suggesting that the C-terminal part interferes with CGI-58 interaction and enzyme activation. Compared with the human enzyme, the C-terminal region of mouse ATGL is much less effective in suppressing enzyme activity, implicating species-dependent differences in enzyme regulation. Together, our results demonstrate that the C-terminal region of ATGL is essential for proper localization of the enzyme and suppresses enzyme activity.