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/migration-related 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 Ca²⁺ 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.

To analyze the functional consequences of coassembly of transient receptor potential 1 (Trp1) and Trp3 channel proteins, we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated conductance that was detectable only in Ca(2+)-free extracellular solution. Trp3 expression gave rise to an OAG-activated conductance that was suppressed but clearly detectable at physiological Ca(2+) concentrations. Coexpression of both species resulted in a constitutively active, OAG-sensitive conductance, which exhibited distinctive cation selectivity and high sensitivity to inhibition by intracellular Ca(2+). Trp1-expressing cells displayed only modest carbachol-induced Ca(2+) entry and lacked OAG-induced Sr(2+) entry, whereas Trp3-expressing cells responded to both agents with a substantial divalent cation entry. Coexpression of Trp1 plus Trp3 suppressed carbachol-induced Ca(2+) entry compared with Trp3 expression and abolished OAG-induced Sr(2+) entry signals. We concluded that coassembly of Trp1 and Trp3 resulted in the formation of oligomeric Trp channels that are subject to regulation by phospholipase C and Ca(2+). The distinguished Ca(2+) sensitivity of these Trp1/Trp3 hetero-oligomers appeared to limit Trp-mediated Ca(2+) signals and may be of importance for negative feedback control of Trp function in mammalian cells.

Activation of endothelial cells by lipid oxidation products is a key event in the initiation and progression of the atherosclerotic lesion. Minimally modified low-density lipoprotein (MM-LDL) induces the expression of certain inflammatory molecules such as tissue factor (TF) in endothelial cells. This study examined intracellular signaling pathways leading to TF up-regulation by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a biologically active component of MM-LDL. OxPAPC induced TF activity and protein expression in human umbilical vein endothelial cells (HUVECs). However, OxPAPC neither induced phosphorylation or degradation of IκBα nor DNA binding of nuclear factor-κB (NF-κB). Furthermore, OxPAPC-induced TF expression was not inhibited by overexpression of IκBα. These results strongly indicate that OxPAPC-induced TF expression is independent of the classical NF-κB pathway. However, OxPAPC stimulated phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and expression of early growth response factor 1 (EGR-1). Inhibitors of mitogen-activated kinase/ERK (MEK) or protein kinase C (PKC) blocked elevation of both EGR-1 and TF. Furthermore, overexpression of NAB2, a corepressor of EGR-1, inhibited effects of OxPAPC. In addition, OxPAPC induced rapid and reversible elevation of free cytosolic Ca⁺⁺ levels and nuclear factor of activated T cells (NFAT)/DNA binding. Induction of TF expression by OxPAPC was partially inhibited by cyclosporin A, known to block calcineurin, a Ca⁺⁺-dependent phosphatase upstream of NFAT. Treatment of OxPAPC with phospholipase A₂ destroyed its biologic activity and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine was identified as one biologically active component of OxPAPC that induces TF expression. Together, the results demonstrate that OxPAPC induces TF expression in HUVECs through activation of PKC/ERK/EGR-1 and Ca⁺⁺/calcineurin/NFAT pathways rather than by NF-κB–mediated transcription. Thus, oxidized phospholipids may contribute to inflammation by activating pathways alternative to the classical NF-κB pathway.

Changes in intra- and extracellular potassium ion (K⁺) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K⁺ changes is severely limited by the lack of practicable detection methods. We developed K⁺-sensitive genetically encoded, Förster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K⁺ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K⁺ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K⁺ signals. Hitherto unknown Ca²⁺-triggered, organelle-specific K⁺ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K⁺ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K⁺ assays and open new avenues for live-cell K⁺ imaging.