Distinct role of the intracellular C-terminus for subcellular expression, shedding and function of the murine transmembrane chemokine CX3CL1.

The transmembrane chemokine CX3CL1 is expressed on the endothelial surface and promotes leukocyte adhesion and transmigration by receptor interaction via its extracellular chemokine domain. Since little is known about its intracellular C-terminus, we examined the consequences of C-terminal truncation on cellular distribution, proteolytic shedding and function of murine CX3CL1. Full length murine CX3CL1 was expressed and shed by the metalloproteinase ADAM10 as described for human CX3CL1. Truncation of murine CX3CL1 led to reduced maturation and impaired trafficking to the surface. Truncation of CX3CL1 also abrogated localization to early endosomal vesicles, but increased shedding from the surface by ADAM10. Once truncated CX3CL1 was expressed on the surface, it mediated cell contact and induced leukocyte transmigration similar as full length CX3CL1. These data suggest that the C-terminus of CX3CL1 carries important determinants for cellular trafficking but not for function of the chemokine during leukocyte recruitment.

The angiotensin-calcineurin-NFAT pathway mediates stretch-induced up-regulation of matrix metalloproteinases-2/-9 in atrial myocytes.

AIM: During atrial fibrillation, arterial hypertension and systolic or diastolic heart failure, atrial myocytes are exposed to increased baseline stretch. Atrial stretch has been shown to induce cellular hypertrophy and extracellular matrix remodeling (ECM) via angiotensin-II dependent pathways and the matrix metalloproteinases system (MMPs). We hypothesized that atrial myocytes exposed to static stretch may increase their ECM remodeling activity via up-regulation of MMP-2/-9. We then tested the hypothesis that the membrane bound angiotensin-II type 1 (AT1) receptor and the intracellular calcineurin (Cn)-NFAT signaling pathway are potential mediators of stretch-induced MMP alterations, since Cn-NFAT is one important contributor to myocyte hypertrophy. METHODS AND RESULTS: Neonatal rat atrial myocytes (NRAM) were cultured under conditions of static stretch by 21%. The differential effects of selective AT1 receptor blockade by losartan, Cn blockade by Cyclosporine-A (CsA) or NFAT inhibition by 11R-VIVIT (VIV), were analyzed. Stretch resulted in a significant up-regulation of active-MMP-2/-9 protein amount (active-MMP-2 ng/microg: control 8.95 +/- 0.64 vs. stretch 13.11 +/- 0.74 / active-MMP-9 ng/microg: control 1.45 +/- 0.18 vs. stretch 1.94 +/- 0.21, all n = 5) and enzyme activity (MMP-2 in %: control 1 +/- 0.0 vs. stretch 1.87 +/- 0.25, n = 7) associated with a significant increase of the membrane-type-1-MMP (MT1-MMP) protein expression (MT1-MMP in %: control 1 +/- 0.0 vs. stretch 2.17 +/- 0.21, n = 8). These observations were accompanied by an activation of the Cn-NFAT pathway (Cn-activity in nmol PO(4) release/20 microg protein/30 min: control 0.37 +/- 0.08 vs. stretch 0.65 +/- 0.09, n = 3 / NFATc1-DNA binding activity in %: control 1 +/- 0.0 vs. stretch 1.53 +/- 0.17, n = 3). Losartan, CsA or VIV abolished stretch-induced alterations in MMP-2/-9 and MT1-MMP expression and enzyme activity by normalizing the Cn-activity and the DNA binding activity of NFATc1. CONCLUSION: Our results present new insights in molecular mechanisms of ECM remodeling activity of atrial myocytes exposed to static stretch. The AT1-Cn-NFAT pathway is a potential mediator of MMP activation.

Regulation of nerve growth factor in the heart: the role of the calcineurin-NFAT pathway.

A heightened sympathetic tone accelerates the development of lethal arrhythmias after myocardial infarction (MI) and the progression of heart failure (HF). Cardiomyocytes control their local neural milieu by expression of nerve growth factor (NGF), which triggers sympathetic neural growth (sympathetic nerve sprouting: SNS). The molecular mechanisms that regulate NGF expression are largely unknown. During HF or MI the myocytes are exposed to increased mechanical load and adrenergic stimulation. Both stimuli induce myocyte hypertrophy. The angiotensin-II-calcineurin-NFAT (nuclear factor of activated t-cells) pathway is a well characterized signaling cascade in the pathogenesis of myocyte hypertrophy. The present study aims to investigate the molecular mechanisms by which mechanical stretch and/or alpha-1-adrenergic stimulation affect NGF expression in neonatal rat ventricular myocytes. Both stimuli resulted in a down-regulation of NGF gene and protein expression. Angiotensin-II type 1 receptor blockade with losartan blunted the stretch-induced NGF down-regulation. Specific calcineurin inhibition with cyclosporine A and FK506 or NFAT inhibition with 11R-VIVIT reversed the stretch or alpha-1-adrenergic induced decrease of NGF. Calcineurin over-expression increased NFAT-DNA binding activity and decreased NGF expression. The magnitude of NGF decrease was sufficient to reduce neurite outgrowth of cultured sympathetic neurons. In conclusion, mechanical stretch and alpha-1-adrenergic stimulation contribute to a decrease of cardiomyocyte NGF expression via the calcineurin-NFAT pathway. To evaluate if the calcineurin-NFAT is critically involved in the pathogenesis of SNS further in-vivo studies in models of HF and MI are required. Nevertheless, the calcineurin-NFAT pathway may provide promising starting points for new pharmacological strategies to prevent SNS in the heart.

Regulated shedding of transmembrane chemokines by the disintegrin and metalloproteinase 10 facilitates detachment of adherent leukocytes.

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.