Cyclophilin a plays distinct roles in human immunodeficiency virus type 1 entry and postentry events, as revealed by spinoculation.
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Cyclophilin A (CypA) is necessary for effective human immunodeficiency virus type 1 (HIV-1) replication. However, the functions of CypA and the precise steps at which CypA acts in the HIV-1 life cycle remain to be determined. By using a methodology that bypasses the need for attachment factors-spinoculation-we present evidence that CypA participates in both entry and postentry events. (+info)
Active site residues of cyclophilin A are crucial for its signaling activity via CD147.
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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. (+info)
Anti-inflammatory effects of a cyclosporine receptor-binding compound, D-43787.
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By virtue of its binding to cyclophilin, the cellular receptor for cyclosporine (CsA), we could identify a new compound D-43787 [N-[(1-tert-butyloxycarbonyl)-indolin-2-(S)-carbonyl]-indolin-2-(S)-carbonacid-[N -epsilon-benzyloxycarbonyl)-2-(S)-lysin methylester]-amide] exhibiting immunomodulating properties. It inhibited cell proliferation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)/ionomycin and anti-CD3/CD28 with an IC(50) of 0.3 microM. The protein phosphatase calcineurin, which is the target of the CsA-cyclophilin complex, is not inhibited by D-43787. It inhibited T helper cell (Th) 2 cytokines interleukin (IL)-4, -5, and -13 more effectively than the Th1 cytokine interferon (IFN)-gamma in human primary T cells. The IC(50) for IL-5 and IL-13 in TPA/ionomycin-stimulated peripheral blood mononuclear cells (PBMC) is 0.7 +/- 0.1 and 0.5 +/- 0.1 microM, respectively, whereas the IC(50) for IFN-gamma is 2.0 +/- 0.4 microM. When PBMC were stimulated with anti-CD3/CD28, the IC(50) for IL-4, -5, and -13 were 1.5 +/- 0.2, 1.8 +/- 0.2, and 1.9 +/- 0.4 microM, respectively. IFN-gamma was only partially inhibited under these conditions. This effect was even more pronounced in pure CD4(+) T cells. Pretreatment of human monocytes with D-43787 inhibited lipopolysaccharide-induced proinflammatory cytokines IL-6 and TNFalpha with an IC(50) of 1.2 +/- 0.1 and 4.7 +/- 0.9 microM, respectively. In vivo, D-43787 potently inhibited late-phase eosinophilia in actively sensitized and challenged guinea pigs (10 mg/kg, i.p.: 51%) and Brown-Norway rats (1 mg/kg, intrapulmonary: 66% 30 mg/kg, i.p.: 50%). In adjuvant-induced arthritis, D-43787 (10-40 mg/kg, b.i.d., i.p.) dose dependently reduced edema development on both hind paws. The potency of D-43787 was comparable with that of indomethacin and dexamethasone. In conclusion, we characterized a novel Th2 selective immunosuppressive drug with possible anti-asthmatic/anti-inflammatory effects. Its mode of action is distinct from that of CsA. (+info)
Localization of calcineurin/NFAT in human skin and psoriasis and inhibition of calcineurin/NFAT activation in human keratinocytes by cyclosporin A.
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Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis. (+info)
Differential dependence of the infectivity of HIV-1 group O isolates on the cellular protein cyclophilin A.
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The cellular protein Cyclophilin A (Cyp A) is packaged into human immunodeficiency virus type 1 (HIV-1) particles through a specific interaction with the capsid domain of the Gag polyprotein. Inhibition of Cyp A incorporation by mutagenesis or cyclosporin treatment severely affects infectivity of all HIV-1 M subtypes tested. In contrast, the closely related lentiviruses HIV-2 and simian immunodeficiency virus (SIV) do not package Cyp A and are not inhibited by cyclosporin. For the HIV-1 group O isolate MVP5180, it was found that Cyp A incorporation and Cyp A dependence of infectivity did not correlate. This virus incorporates Cyp A but is not sensitive to treatment with cyclosporin A. For a more detailed study concerning the relationship between Cyp A incorporation and Cyp A dependence, we have analyzed five group O isolates for their ability to incorporate Cyp A and their sensitivity to cyclosporin treatment. All group O viruses incorporated Cyp A in comparable amounts as the M-group HIV-1 strain NL4-3. Furthermore, Cyp A incorporation was inhibited by cyclosporin in all cases. However, while isolate MVP 5180 was confirmed to replicate independent of Cyp A, three of the other four isolates were sensitive to cyclosporin treatment. Sequence analysis of the Cyp A binding regions revealed that the proline-rich motif, which is responsible for Cyp A incorporation, was conserved in all four isolates, while some sequence variations were detected in other positions close to this region. These results suggest that Cyp A dependence of replication is influenced by regions outside the Cyp-binding loop and may aid in determination of Cyp A function in HIV-1 replication. (+info)
Tissue distribution of calcineurin and its sensitivity to inhibition by cyclosporine.
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The immunosuppressive activity of cyclosporine is mediated by inhibiting calcineurin phosphatase. However, calcineurin is widely distributed in other tissues. We examined the degree of calcineurin inhibition by cyclosporine in various tissues. In vitro, the cyclosporine concentration inhibiting 50% (IC50) of calcineurin was to approximately 10 ng/mL in human and mouse leukocytes suspensions. In vitro and in vivo IC50s of cyclosporine in homogenates of mouse kidney, heart, liver, testis, and spleen were also comparable (9-48 ng/mL). The maximum calcineurin inhibition by cyclosporine varied, from 83 to 95% of calcineurin activity in spleen, kidney, liver, and testis to 60% in heart and only 10% in brain. Maximum calcineurin inhibition was increased by the addition of cyclophilin A, indicating that cyclophilin concentrations were limiting in some tissues, at least in this assay. Western analysis of mouse tissues showed significantly less cyclophilin in heart than other tissues. cyclosporine concentrations per weight of tissue protein were highest in kidney and liver and lowest in brain and testis after oral dosing, with intermediate levels in spleen, heart, and whole blood. Thus each cyclosporine dose produces rapid and wide-spread inhibition of calcineurin in tissues, with differences in total susceptibility of each tissue. (+info)
Cyclosporin A blocks muscle differentiation by inducing oxidative stress and inhibiting the peptidyl-prolyl-cis-trans isomerase activity of cyclophilin A: cyclophilin A protects myoblasts from cyclosporin A-induced cytotoxicity.
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Allogenic myoblast transplantation (AMT) is under investigation for treatment of severe genetic myopathies. Data regarding the role of cyclosporine (CsA) and FK-506 in AMT have shown that CsA is less effective than FK-506. For this study, we investigated mechanisms of CsA toxicity during AMT and showed that a high level of reactive oxygen species (ROS) generated by CsA, mediated partly by inhibition of the peptidylprolyl-cis-trans-isomerase (PPIase)-like activity of cyclophilin A (CypA), blocked differentiation and induced apoptosis at an early stage of muscle differentiation. Inhibition of the PPIase-like activity of CypA alone also blocked muscle differentiation. However, CsA toxicity did not depend on the inhibition of calcineurin activity during muscle differentiation. Together, these data suggest that CsA-mediated inhibition of the PPIase-like activity of CypA and the high level of ROS generation contributed to the low efficacy of CsA in AMT. In addition, we showed that a reduction of oxidative stress protected cells from CsA-induced apoptosis, and myoblasts that had survived after preexposure to CsA not only proliferated and differentiated reversibly but also gained resistance to subsequent CsA exposure. Thus, administration of antioxidants or overexpression of CypA either exogenously or endogenously during CsA treatment has the potential to improve the success of this treatment in AMT. (+info)
Three isoforms of cyclophilin A associated with human immunodeficiency virus type 1 were found by proteomics by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry.
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Human immunodeficiency virus type 1 (HIV-1) strain LAV-1 (HIV-1(LAV-1)) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified HIV-1(LAV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained. The 2D gel electrophoresis image suggested that 24 proteins can be identified inside the virion. Furthermore, the stained protein spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Peptide mass fingerprinting data suggested that two isoforms of cyclophilin A (CyPA), one with an isoelectric point (pI) of 6.40 and one with a pI of 6.53, are inside the viral membrane; that another isoform, with a pI of 6.88, is outside the viral membrane; and that the CyPA isoform with a pI of 6.53 is N acetylated. The mechanisms that permit the redistribution of CyPA on the viral surface have not yet been clarified, but it is surmised that the CyPA isoform with a pI of 6.88 may play a critical role in the attachment of virions to the surface of target cells and that both CyPA isoforms with pIs of 6.40 and 6.53 may regulate the conformation of the HIV-1 capsid protein. (+info)