Active site residues of cyclophilin A are crucial for its signaling activity via CD147

Cyclophilin A (CyPA), a ubiquitously distributed intracellular protein, is a peptidylprolyl cis-trans-isomerase and the major target of the potent immunosuppressive drug cyclosporin A. Although expressed predominantly as an intracellular molecule, CyPA is secreted by cells in response to inflammatory stimuli and is a potent neutrophil and eosinophil chemoattractant in vitro and in vivo. The mechanisms underlying CyPA-mediated signaling and chemotaxis are unknown. Here, we identified CD147 as a cell surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation. Both signaling and chemotactic activities of CyPA depended also on the presence of heparans, which served as primary binding sites for CyPA on target cells. The proline 180 and glycine 181 residues in the extracellular domain of CD147 were critical for signaling and chemotactic activities mediated by CD147. Also crucial were active site residues of CyPA, because rotamase-defective CyPA mutants failed to initiate signaling events. These results establish cyclophilins as natural ligands for CD147 and suggest an unusual, rotamase-dependent mechanism of signaling.     

CD147 is a signaling receptor for cyclophilin B.

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins.     

Structural and functional characterization of the interaction between cyclophilin B and a heparin-derived oligosaccharide

The chemotaxis and integrin-mediated adhesion of T lymphocytes triggered by secreted cyclophilin B (CypB) depend on interactions with both cell surface heparan sulfate proteoglycans (HSPG) and the extracellular domain of the CD147 membrane receptor. Here, we use NMR spectroscopy to characterize the interaction of CypB with heparin-derived oligosaccharides. Chemical shift perturbation experiments allowed the precise definition of the heparan sulfate (HS) binding site of CypB. The N-terminal extremity of CypB, which contains a consensus sequence for heparin-binding proteins was modeled on the basis of our experimental NMR data. Because the HS binding site extends toward the CypB catalytic pocket, we measured its peptidyl-prolyl cis-trans isomerase (PPIase) activity in the absence or presence of a HS oligosaccharide toward a CD147-derived peptide. We report the first direct evidence that CypB is enzymatically active on CD147, as it is able to accelerate the cis/trans isomerization of the Asp(179)-Pro(180) bond in a CD147-derived peptide. However, HS binding has no significant influence on this PPIase activity. We thus conclude that the glycanic moiety of HSPG serves as anchor for CypB at the cell surface, and that the signal could be transduced by CypB via its PPIase activity toward CD147.     

A conserved tandem cyclophilin-binding site in hepatitis C virus nonstructural protein 5A regulates Alisporivir susceptibility

Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilin-binding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir.     

Extracellular cyclophilins contribute to the regulation of inflammatory responses

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.     

Expression of CD147 (EMMPRIN) on neutrophils in rheumatoid arthritis enhances chemotaxis,matrix metalloproteinase production and invasiveness of synoviocytes

The occurrence of neutrophils at the pannus‐cartilage border is an important phenomenon for understanding the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are predominant enzymes responsible for the cartilage degradation. The present article studied the expression of CD147 on neutrophils and its potential role in neutrophil chemotaxis, MMPs production and the invasiveness of fibroblast‐like synoviocytes (FLS). The results of flow cytometry revealed that the mean fluorescence intensity of CD147 expression on neutrophils of peripheral blood from RA patients was higher than that in healthy individual. The potential role of CD147 in cyclophilin A (CyPA)‐mediated cell migration was studied using chemotaxis assay and it was found that the addition of anti‐CD147 antibody significantly decreased the chemotactic index of the neutrophils. Significantly elevated release and activation of MMPs were seen in the co‐culture of neutrophil and FLS compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays were also observed in the co‐cultured cells. The addition of anti‐CD147 antibody had some inhibitory effect, not only on MMP production but also on cell invasion in the co‐culture model. Our study demonstrates that the increased expression of CD147 on neutrophils in RA may be responsible for CyPA‐mediated neutrophil migration into the joints, elevated MMPs secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. Better knowledge of these findings will hopefully provide a new insight into the pathogenesis of RA.     

Functional analysis of Leishmania major cyclophilin

A potent immunosuppressive drug cyclosporin A (CsA) is known to inhibit human cell infection by the pathogenic protozoan parasite Leishmania major both in vitro and in vivo. The proposed mechanism of action involves CsA binding to Leishmania major-expressed cyclophilin and subsequent down-regulation of signaling events necessary for establishing productive infection. Recently, we identified a ubiquitously expressed membrane protein, CD147, as a signaling receptor for extracellular cyclophilins in mammalian cells. Here we demonstrate that, while being enzymatically active, the Leishmania cyclophilin, unlike its human homologue, does not interact with CD147 on the cell surface of target cells. CD147 facilitates neither Leishmania binding nor infection. Primary structure and biochemical analyses revealed that the parasite's cyclophilin is defective in heparan binding, an event required for signaling interaction between CD147 and human cyclophilin. When the heparan-binding motif was reconstituted in Leishmania cyclophilin, it regained the CD147-dependent signaling activity. These results underscore a critical role of cyclophilin-heparan interactions in CD147-mediated signaling events and argue against the role of Leishmania cyclophilin in parasite binding to target cells.     

Identification of simian cyclophilin A as a calreticulin-binding protein in yeast two-hybrid screen and demonstration of cyclophilin A interaction with calreticulin

Cyclophilin A (CyPA) was identified as one of the calreticulin (CR)-binding proteins in a yeast two-hybrid screen utilizing simian cDNA expression-library. The simian CyPA protein had 96% identity with that of human, differing only at eight amino acid residues. We further established CyPA–CR interaction by incubation of glutathione transferase-fused CyPA (GST-CyPA) and CR proteins with CV-1 cyto-lysates, followed by CR and CyPA-specific immuno-blot analysis. The immunosuppressive drug cyclosporin A, a CyPA ligand, did not inhibit CyPA–CR interaction. Our results established a new property of CyPA binding activity to CR. Since CR is a Ca²⁺-binding protein, CR–CyPA interactions may be important in signaling pathways for induction of Ca²⁺-dependent cellular processes.     

Identification of two forms of cyclophilin from the hard tick Haemaphysalis longicornis

We have shown here the identification and characterization of two cyclophilin, cyclophilin A (CyPA) and B (CyPB), from the ixodid tick, Haemaphysalis longicornis. Both CyPA and CyPB contain the conserved peptidyl-prolyl isomerase (PPIase) domain, with CyPA consisting of 188 amino acids and CyPB of 216 amino acids. CyPA and CyPB share 50–67% amino acid sequence identity with the cyclophilins of other organisms, and are found in multiple organs throughout the developmental stages of ixodid ticks. In addition, recombinant CyPA and CyPB exhibited PPIase activity that could be inhibited by the cyclic peptides cyclosporin A (CsA). Silencing of CyPA through RNA interference has led to a significant reduction in the body weight of engorged ticks and their failure to lay eggs, in contrast to CyPB whose silencing did not result in any detectable phenotypic changes. Our results indicate that CyPA represents the major cyclophilin protein in H. longicornis involved in blood ingestion, viability, and oocyte development. This is the first report of cyclophilin proteins from ixodid and argasid ticks.     

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.     

Hypoxia followed by reoxygenation induces secretion of cyclophilin A from cultured rat cardiac myocytes

We previously reported that hypoxia followed by reoxygenation (hypoxia/reoxygenation) rapidly activated intracellular signaling such as mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated protein kinase (ERK) 1/2, p38MAPK, and stress-activated protein kinases (SAPKs). To investigate the humoral factors which mediate cardiac response to hypoxia/reoxygenation, we analyzed the conditioned media from cardiac myocytes subjected to hypoxia/reoxygenation by two-dimensional electrophoresis and mass spectrometry. We identified cyclophilin A (CyPA) as one of the proteins secreted from cardiac myocytes in response to hypoxia/reoxygenation. Hypoxia/reoxygenation induced the expression of CyPA and its cell surface receptor CD147 on cardiac myocytes in vitro. This was also confirmed by ischemia/reperfusion in vivo. Recombinant human (rh) CyPA activated ERK1/2, p38MAPK, SAPKs, and Akt in cultured cardiac myocytes. Furthermore, CyPA significantly increased Bcl-2 in cardiac myocytes. These data strongly suggested that CyPA is released from cardiac myocytes in response to hypoxia/reoxygenation and may protect cardiac myocytes from oxidative stress-induced apoptosis.     

Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production

Monocytes/macrophages play an important role in rheumatoid arthritis (RA) pathogenesis. They can activate fibroblasts through many molecules, including IL-1 and tumor necrosis factor-alpha, but there have been very few reports on the role of CD147 in RA. In our study, the results of flow cytometry reveal that the mean fluorescence intensity (MFI) of CD147 expression on CD14+ monocytes of peripheral blood from RA patients was higher than that in normal control and ankylosing spondylitis (AS) patients. The MFI of CD147 expression on the CD14+ monocytes in RA synovial fluid was higher than that in RA peripheral blood. Immunohistochemical staining shows that CD147 expression in RA synovium correlated with matrix metalloproteinase (MMP)-1 expression. A double immunofluorescent assay shows that CD147 was expressed on CD68+ cells in RA synovium. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using a chemotaxis assay in vitro and it was found that the addition of anti-CD147 antibody or a CD147 antagonistic peptide significantly decreased the chemotactic index of the mononuclear cells. The role of CD147 in MMP production and cell invasion in vitro were studied through the co-culture of human CD14+ monocytes or monocytic line THP-1 cells and human fibroblasts, as well as by gel zymography and an invasion assay. Significantly elevated release and activation of MMP-9 and/or MMP-2 were seen in the co-culture of human monocytes/THP-1 cells and fibroblasts compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays was also observed in the co-cultured cells. The addition of CD147 antagonistic peptide had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture. Our study demonstrates that the increased expression of CD147 on monocytes/macrophages in RA may be responsible for elevated MMP secretion, cell invasion and CyPA-mediated cell migration into the joints, all of which may contribute to the cartilage and bone destruction of RA. These findings, together with a better understanding of CD147, CyPA and RA, will help in the development of innovative therapeutic interventions for RA.     

Novel approach to inhibit asthma-mediated lung inflammation using anti-CD147 intervention

Extracellular cyclophilins have been well described as chemotactic factors for various leukocyte subsets. This chemotactic capacity is dependent upon interaction of cyclophilins with the cell surface signaling receptor CD147. Elevated levels of extracellular cyclophilins have been documented in several inflammatory diseases. We propose that extracellular cyclophilins, via interaction with CD147, may contribute to the recruitment of leukocytes from the periphery into tissues during inflammatory responses. In this study, we examined whether extracellular cyclophilin-CD147 interactions might influence leukocyte recruitment in the inflammatory disease allergic asthma. Using a mouse model of asthmatic inflammation, we show that 1) extracellular cyclophilins are elevated in the airways of asthmatic mice; 2) mouse eosinophils and CD4+ T cells express CD147, which is up-regulated on CD4+ T cells upon activation; 3) cyclophilins induce CD147-dependent chemotaxis of activated CD4+ T cells in vitro; 4) in vivo treatment with anti-CD147 mAb significantly reduces (by up to 50%) the accumulation of eosinophils and effector/memory CD4+ T lymphocytes, as well as Ag-specific Th2 cytokine secretion, in lung tissues; and 5) anti-CD147 treatment significantly reduces airway epithelial mucin production and bronchial hyperreactivity to methacholine challenge. These findings provide a novel mechanism whereby asthmatic lung inflammation may be reduced by targeting cyclophilin-CD147 interactions.     

Cyclophilin a increases CD68<Superscript>+</Superscript> cell infiltration in rat experimental periodontitis

Cyclophilin A (CyPA) is a potent chemokine, which can directly induce leukocyte chemotaxis and contribute to the pathogenesis of inflammation-mediated diseases. This study is to observe the expression and distribution of CyPA and CD68⁺ cells in the histopathogenesis of rat ligation-induced experimental periodontitis, and assess the role of CyPA in CD68⁺ cell infiltration in rat experimental periodontitis. Experimental periodontitis was induced by ligation according to our previous method. CyPA expression in gingival tissues was detected by western blotting. Immunohistochemistry was applied for CyPA and CD68 distribution. For further certifying the role of CyPA in CD68⁺ cell infiltration, the right mandibular first molar received 0.1 μM CyPA locally by gingival injection every 2 days (L?+?C group), while the left mandibular first molar received saline as a control group (L group). The number of CD68⁺ cells in the experimental periodontitis was observed by immunohistochemistry. Alveolar bone destruction was assessed by micro-computerized tomography (micro-CT). Osteoclast was observed through TRAP staining. Nuclear factor (NF)-κB phospho-p65 (p p65) and phosphor-IκBα (p IκBα) expressions were detected to investigate NF-κB activation. CyPA showed an increasing trend at 1–6 weeks after ligation. CyPA and CD68⁺ cells were present in the gingival inflammatory infiltration, and participated in alveolar bone destruction. In the L?+?C group, the number of CD68⁺ cells was increased compared with the L group, and greater alveolar bone destruction was observed. NF-κB p p65 and p IκBα expressions were upregulated in the L?+?C group compared with the L group indicating NF-κB activation. CyPA increases CD68⁺ cell infiltration in rat experimental periodontitis, suggesting CyPA might be an anti-inflammatory therapeutic target.     

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding

Porcine kidney 18 kD peptidyl-prolyl cis-trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 microM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration-dependent manner. PPIase exhibited chaperone-like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.     

Cyclophilin A: a key player for human disease

Cyclophilin A (CyPA) is a ubiquitously distributed protein belonging to the immunophilin family. CyPA has peptidyl prolyl cis-trans isomerase (PPIase) activity, which regulates protein folding and trafficking. Although CyPA was initially believed to function primarily as an intracellular protein, recent studies have revealed that it can be secreted by cells in response to inflammatory stimuli. Current research in animal models and humans has provided compelling evidences supporting the critical function of CyPA in several human diseases. This review discusses recently available data about CyPA in cardiovascular diseases, viral infections, neurodegeneration, cancer, rheumatoid arthritis, sepsis, asthma, periodontitis and aging. It is believed that further elucidations of the role of CyPA will provide a better understanding of the molecular mechanisms underlying these diseases and will help develop novel pharmacological therapies.     

Evidences of monomer, dimer and trimer of recombinant human cyclophilin A

Cyclophilin A (CyPA) is a cytosolic receptor of immunosuppressive drug cyclosporin A (CsA) which possesses peptidyl-prodyl cis/trans isomerase (PPIase) activity. The recombinant human CyPA (rhCyPA) gene has been expressed in E. coli M15. Purification was performed using salting-out, as well as Sephacryl S-100 and DEAE-Sepharose CL-6B column chromatography. The molecular weight is about 18 kDa, confirmed by SDS-PAGE and mass spectrum. The results of Native-PAGE and immunoblotting showed the existence of three bands, which agreed well with the gel filtration results. The molecular mass of the three bands determined via CTAB gel electrophoresis and SDS-PAGE (rhCyPA cross-linked with glutaraldehyde) was 18 kDa, 36 kDa and 54 kDa respectively. Further more, the native rhCyPA and the cross-linked rhCyPA had the similar chromatographic behavior in gel filtration. All of the evidences above suggest that rhCyPA exists in forms of monomer, dimer and trimer. Moreover, we observed that even at low protein concentrations CyPA largely occurs as a dimer in solution, and enzyme kinetic parameters showed that activity of dimer was much higher than monomer or trimer, which probably have some biological significances.     

Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis-trans isomerase activity

Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis-trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsA-induced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal's resistance toward CsA-induced nephrotoxicity.