Elsevier

Brain Research

Volume 1397, 23 June 2011, Pages 55-65
Brain Research

Research Report
Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages

https://doi.org/10.1016/j.brainres.2011.03.036Get rights and content

Abstract

Experimental cerebral ischemia has been reportedly alleviated by the immunosuppressive agent cyclosporin A (CsA). Cyclophilin C-associated protein (CyCAP) was proposed to be an endogenous equivalent of CsA; CsA- and CyCAP-targeting protein cyclophilin C have attracted extensive attention regarding their ischemia-alleviating mechanisms. In this study we have introduced the specific CyCAP antibody for evaluating its distribution in the rat ischemic brain after middle cerebral artery occlusion. During the recovery of cerebral ischemia in rats, CyCAP was highly expressed in the activated microglia/macrophages in the ischemic lesion. However, it remains unknown what roles CyCAP plays in the activation of macrophage phagocytosis. Thus, we studied CyCAP function using a RAW264.7 macrophage cell line. When we expressed CyCAP-GFP and cyclophilin C-FLAG in RAW264.7 cells, we found that CyCAP and cyclophilin C make a complex, which is competitively inhibited by CsA. Consistently, in immunoprecipitates by anti-calcineurin antibody, cyclophilin C and CyCAP were detected, and CyCAP pulled down NFATc1, suggesting that both CyCAP and cyclophilin C form a complex with calcineurin and NFATc1. When CyCAP was adenovirally overexpressed in RAW cells, NFAT staining increased over the nucleus. Furthermore, calcineurin and IL-2 were increased with time. Thus, CyCAP appears to control macrophage functions by activating NFAT and the resultant IL-2 production. With a protein phosphatase inhibitor PhoSTOP, NFAT was localized more to the cytoplasm, and phagocytosis was decreased strikingly. Thus, we suggest that in a CyCAP-cyclophilin C pathway for macrophage activation, calcineurin phosphatase activity is essential for the phagocytosis activity via dephosphorylation of NFATc1.

Research highlights

► Experimental cerebral ischemia has been reportedly alleviated by cyclosporin A. ► Its endogenous equivalent is cyclophilin C-associated protein (CyCAP). ► CyCAP is expressed in microglia/macrophages in the penumbral zone. ► CyCAP maintains macrophage functions active for the recovery from penumbral ischemia. ► CyCAP works through the cyclophilin C/calcineurin/NFAT pathway.

Introduction

Cerebral ischemia induced the innate immune response where microglia, the resident macrophages in the brain, play a critical role. Microglial activation is the initial step in the central nervous system for pathological events after an ischemic insult, although whether the activated microglia exert neuroprotective or harmful efficacy remains to be elucidated. Notably, there is strong evidence that the immunosuppressive drugs cyclosporin A (CsA) and FK506 exert a neuroprotective effect on cerebral ischemia in animal models (McCarter et al., 2001, Sharkey and Butcher, 1994, Shiga et al., 1992) by binding with immunophilins, which are protein receptors for CsA and FK506. The cellular role of immunophilins remains somewhat unclear, but immunophilins including cyclophilins and FK506-binding proteins, which bind to CsA and FK506, respectively, are thought to be important mediators of neuroprotection (Guo et al., 2001). One of the immunophilins, cyclophilin C, is a CsA-targeting peptidylprolyl isomerase protein which is highly expressed in the kidney (Grinyo and Cruzado, 2004). Cyclophilin C has its own endogenous ligand, cyclophilin C-associated protein (CyCAP), whose cDNA was cloned from the murine bone marrow-derived stromal cell line AC-6, and was characterized as a cyclophilin C-binding protein (Friedman and Weissman, 1991, Friedman et al., 1993, Ke et al., 1993). CyCAP is regarded as a candidate for the natural cellular ligand for cyclophilin C because the interaction is inhibited by CsA (Friedman and Weissman, 1991). Moreover, CyCAP has a protective effect in its response to endotoxins by down-modulating the proinflammatory response in vivo (Trahey and Weissman, 1999). However, little is known about the role CyCAP and cyclophilin C in controlling the phagocytic function of microglia/macrophages.

Previously we have evaluated mRNA expression for CyCAP and cyclophilin C spatially and temporally after middle cerebral artery (MCA) occlusion (Shimizu et al., 2005). There is an increase of CyCAP mRNA and cyclophilin C mRNA in the activated microglia/macrophages in the core as well as neurons of the peri-infarct zone after MCA occlusion in rats. However, from the cellular distribution of CyCAP and cyclophilin C mRNAs, it was hard to elucidate their molecular interaction and the subsequent molecular events in neurons and microglia during ischemic events. Thus, in the present study, we first produced anti-CyCAP antibody to study CyCAP and cycliphilin C expression immunohistochemically in the ischemic brain for mapping their spatial distribution after MCA occlusion. Secondly, we explored the action mechanism of CyCAP and cyclophilin C in relation to Ca2+/calmodulin-dependent phosphatase calcineurin (protein phosphatase 2B) activity (Friedman and Weissman, 1991, Liu et al., 1991). Furthermore, one of the dephosphorylation targets of calcineurin, nuclear factor of activated T cells (NFAT), was investigated for its translocation to the nucleus, where it activates interleulin 2 (IL-2) production (Macian, 2005). We found that the CyCAP and cyclophilin C interaction causes the activation of microglia/macrophage functions via a calcineurin/NFAT pathway for alleviating brain ischemic damage.

Section snippets

Pathological changes after focal cerebral ischemia

We initially confirmed the specificity of anti-CyCAP antibodies raised in three rabbits. One of the three rabbits produced antibody that immunoblotted a 77 kDa-size band on the gel, which was erased by the peptide used for CyCAP antibody preparation (the sequence is shown in the immunostaining of the Experimental procedures section: Supplementary Fig. 1). We used this antibody for immunoblotting and immunostaining in this study.

After the occlusion of the MCA in rats, ischemic damages were

Discussion

CyCAP is postulated to be a candidate ligand for cyclophilin C because association of CyCAP with cyclophilin C is inhibited by CsA (Friedman and Weissman, 1991). We found that the CyCAP binding to cyclophilin C is competitively inhibited by CsA (Fig. 3B). CsA binds not only to cyclophilin C but also to other cyclophilin subtypes such as cyclophilin A, B, and D. In terms of immunosuppressive action by CsA, cyclophilin A is reportedly most effective (Bram et al., 1993, Colgan et al., 2005). Thus,

General surgical preparation

Adult male Sprague–Dawley rats (300 to 400 g) had free access to a laboratory pellet diet and water until the day of surgery. The surgical procedure was carried out with approval of the Guideline Committee for the Care and Use of Laboratory Animals, Gunma University. Anesthesia was induced with 5% halothane and subsequently maintained with 1.2–1.7% halothane in a mixture with a nitrous oxide:oxygen (2:1). The rats were intubated transorally and artificially ventilated via a small animal

Acknowledgments

We thank Ms. M. Kosaki and Ms. M. Hosoi for their secretarial and technical assistance.

This work was supported by grants-in-aid from the Ministry of Education, Culture, Science, Sports, and Technology.

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