Cyclophilin A differentially activates monocytes and endothelial cells: Role of purity, activity, and endotoxin contamination in commercial preparations
Introduction
Cyclophilin A (CyPA) belongs to the immunophilin family and is an abundantly expressed cytosolic protein found in most prokaryotes and eukaryotes [1], [2]. Intracellular CyPA possesses multiple functions, among which peptidyl prolyl cis-trans isomerase (PPIase-) activity is considered to be important for protein folding [3], [4]. Furthermore, CyPA is the cytoplasmatic host cell receptor for the immunosuppressive drug cyclosporine A (CsA), which is commonly employed to prevent organ rejection after transplantation [5], [6]. CsA inhibits PPIase activity of CyPA; this effect, however, is thought to occur independent of its immunosuppressive action.
Although CyPA was initially assumed to exist only intracellularly, numerous observations demonstrate that it can also be released into the extracellular space [7]. Indeed, CyPA is secreted from monocytes [7], endothelial cells [8], and vascular smooth muscle cells [9] in response to a variety of inflammatory stimuli; furthermore, extracellular CyPA is detected in the inflammatory microenvironment of the atherosclerotic vessel wall [10]. Extracellular CyPA has been reported to trigger endothelial cell activation and to promote endothelial cell migration in response to inflammatory stimuli [8], [10], hinting at a possible role for CyPA as an inflammatory mediator. In particular, CyPA released in response to increased levels of reactive oxygen species was described to activate the endothelium by inducing the expression of VCAM-1 and E-selectin, thereby favoring the development of atherosclerotic lesions [10].
In this study, we attempted to elucidate the mechanisms by which extracellular CyPA triggers endothelial activation.
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Cell culture
Human aortic endothelial cells (HAEC, Clonetics #CC-2535) were cultured as described [11]. Cells were grown in EGM-2 (Clonetics #CC-4176) medium supplemented with 10% fetal calf serum (FCS, Biowhittaker #14-471F). Subconfluent cells were rendered quiescent for 24 h in EBM-2 (Clonetics #CC-3156) with 0.5% FCS before stimulation with CyPA (BIOMOL #SE-105, Sigma #C3805, Calbiochem #239777) or TNF-α (R&D #210-TA). The monocytic cell line THP-1 was grown in RPMI 1640 medium (GIBCO #21875-034)
Purity and activity of recombinant CyPA
The purity of an in-house preparation of recombinant CyPA, as assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Coomassie blue staining, was > 95%. The E280/E260 absorption ratio of 1.35 of this preparation indeed indicates only minor amounts of non-proteinaceous impurities (Fig. 1A). This preparation displayed high PPIase activity reaching a specificity constant (kcat/Km) of 9.6 × 106 M−1 s−1 (Fig. 1B); moreover, the preparation contained no LPS (Fig. 2B).
Compared to the
Discussion
This study demonstrates that pure and active CyPA is a potent chemoattractant for monocytes; in contrast, CyPA fails to activate endothelial cells as assessed by induction of TF and VCAM-1 expression as well as by endothelial cell migration.
Pure and active CyPA did not induce endothelial TF and VCAM-1 expression; an effect on these proteins was only observed when the samples were contaminated by significant concentrations of LPS. These data stand in contrast to earlier studies demonstrating
Acknowledgments
This study was supported by the Swiss National Science Foundation (grant no. 3200B0-113328/1 to FCT and grant no. 3100-068118.02/1 to TFL), the Bonizzi-Theler Foundation, the Velux Foundation, the Wolfermann Nägeli Foundation, the Center for Integrative Human Physiology of the University of Zurich, and the Swiss Heart Foundation.
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