Integrin clustering enables anandamide-induced Ca2+ signaling in endothelial cells via GPR55 by protection against CB1-receptor-triggered repression.

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 NFkappaB 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.

T-cadherin mediates low-density lipoprotein-initiated cell proliferation via the Ca(2+)-tyrosine kinase-Erk1/2 pathway.

The GPI-anchored protein T-cadherin was found to be an atypical LDL binding site that is expressed in various types of cells, including endothelial cells, smooth muscle cells, and neurons. Notably, the expression of T-cadherin was reduced in numerous types of cancers, although it was up-regulated in tumor-penetrating blood vessels, atherosclerotic lesions, and during neointima formation. Despite these intriguing findings, our knowledge of the physiological role and the signal transduction pathways associated with this protein is limited. Therefore, T-cadherin was overexpressed in the human umbilical vein-derived endothelial cell line EA.hy926, the human embryonic kidney cell line HEK293, and LDL-initiated signal transduction, and its consequences were elucidated. Our data revealed that T-cadherin serves as a receptor specifically for LDL. Following LDL binding to T-cadherin, mitogenic signal transduction was initiated that involved activation of PLC and IP3 formation, which subsequently yielded intracellular Ca2+ mobilization. Downstream to these early phenomena, activation of tyrosine kinase(s) Erk 1/2 kinase, and the translocation of NF kappa B toward the nucleus were found. Finally, overexpression of T-cadherin in HEK293 cells resulted in accelerated cell proliferation in an LDL-dependent manner, although cell viability was not influenced. Because LDL uptake was not facilitated by T-cadherin, our data suggest that T-cadherin serves as a signaling receptor for LDL that facilitates an LDL-dependent mitogenic signal in the vasculature.

Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts.

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 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 identified Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Anandamide initiates Ca(2+) signaling via CB2 receptor linked to phospholipase C in calf pulmonary endothelial cells.

The endocannabinoid anandamide has been reported to affect neuronal cells, immune cells and smooth muscle cells via either CB1 or CB2 receptors. In endothelial cells, the receptors involved in activating signal transduction are still unclear, despite the fact that anandamide is produced in this cell type. The present study was designed to explore in detail the effect of this endocannabinoid on Ca2+ signaling in single cells of a calf pulmonary endothelial cell line. Anandamide initiated a transient Ca2+ elevation that was prevented by the CB2 receptor antagonist SR144528, but not by the CB1 antagonist SR141716A. These data were confirmed by molecular identification of the bovine CB2 receptor in these endothelial cells by partial sequencing. The phospholipase C inhibitor 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5dione and the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenylborate prevented Ca2+ signaling in response to anandamide. Using an improved cameleon probe targeted to the endoplasmic reticulum (ER), fura-2 and ratiometric-pericam, which is targeted to the mitochondria, anandamide was found to induce Ca2+ depletion of the ER accompanied by the activation of capacitative Ca2+ entry (CCE) and a transient elevation of mitochondrial Ca2+. These data demonstrate that anandamide stimulates the endothelial cells used in this study via CB2 receptor-mediated activation of phospholipase C, formation of inositol 1,4,5-trisphosphate, Ca2+ release from the ER and subsequent activation of CCE. Moreover, the cytosolic Ca2+ elevation was accompanied by a transient Ca2+ increase in the mitochondria. Thus, in addition to its actions on smooth muscle cells, anandamide also acts as a powerful stimulus for endothelial cells.

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering, sore-operated Ca2+ entry, and Ca2+ store refilling.

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.