Levels Of CypA Research Articles

Extracellular Cyclophilin A, Especially Acetylated, Causes Pulmonary Hypertension by Stimulating Endothelial Apoptosis, Redox Stress, and Inflammation.

Oxidative stress and inflammation play key roles in the development of pulmonary arterial hypertension (PAH). Cyclophilin A (CypA) is secreted in response to oxidative stress and promotes inflammation and cardiovascular disease. Endothelial cell (EC) dysfunction is an early event in the pathogenesis of PAH. We evaluated the role of extracellular CypA in PAH and compared the effects of acetylated CypA (AcK-CypA, increased by oxidative stress) and CypA on EC dysfunction. In transgenic mice that express high levels of CypA in EC specifically, a PAH phenotype was observed at 3 months including increased right ventricular systolic pressure, α-smooth muscle actin expression in small arterioles, and CD45-positive cells in the lungs. Mechanistic analysis using cultured mouse pulmonary microvascular EC and human pulmonary microvascular EC showed that extracellular CypA and AcK-CypA stimulated EC inflammatory signals: increased VCAM1 (vascular cell adhesion molecule 1) and ICAM1 (intercellular adhesion molecule 1), phosphorylation of p65, and degradation of IkB. Extracellular CypA and AcK-CypA increased EC apoptosis measured by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining, Apo-ONE assay, and caspase 3 cleavage. Oxidative stress stimulated CypA and AcK-CypA secretion, which further promoted EC oxidative stress. AcK-CypA, compared with CypA, stimulated greater increases in apoptosis, inflammation, and oxidative stress. MM284, a specific inhibitor of extracellular CypA, attenuated EC apoptosis induced by CypA and AcK-CypA. EC-derived CypA (especially AcK-CypA) causes PAH by a presumptive mechanism involving increased EC apoptosis, inflammation, and oxidative stress. Our results suggest that inhibiting secreted extracellular CypA is a novel therapeutic approach for PAH.

Interaction of cyclophilin A with a novel binding protein, SR-25, and characterization of their expression pattern in Chinese hepatocellular carcinoma patients.

Cyclophilin (Cyp) A has been reported to be overexpressed in the majority of cancer cells, including hepatocellular carcinoma (HCC). However, the biological functions of CypA in HCC are far from being understood. To determine the biological functions of CypA in HCC, the present study screened human fetal liver complementary DNA for proteins interacting with CypA using the yeast two-hybrid system. A nuclear protein, serine/arginine-rich (SR)-25, was isolated as a novel CypA-binding protein that is distinct from those previously described in the literature. Binding assays and co-immunoprecipitation confirmed the physical association between CypA and SR-25. The present study demonstrated that CypA may interact with SR-25 through its peptidyl-prolyl isomerase domain. In addition, CypA may induce the expression of SR-25 in Hep3B cells. The messenger RNA levels of CypA and SR-25 in HCC indicated that there was a significant correlation between the expression of CypA and the expression of SR-25 in HCC. It can be speculated that the interaction between CypA and SR-25 proteins may be involved in potential carcinogenic functions of CypA in HCC. Further studies will focus on elucidating in detail the molecular mechanisms of the interaction between CypA and SR-25.

Evaluation of gingival crevicular fluid cyclophilin a and extracellular matrix metalloproteinase inducer levels in different periodontal diseases

ObjectiveCyclophilin A (CypA) is able to regulate inflammatory responses and matrix metalloproteinase production via its interaction with extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN is the cell surface receptor of CypA. The aim of the present study was to evaluate the gingival crevicular fluid (GCF) CypA and EMMPRIN levels in patients with chronic periodontitis (CP), generalized aggressive periodontitis (G-AgP) and periodontally healthy controls. MethodsTwenty CP patients, 19 G-AgP patients and 20 healthy control subjects were included in the present study. All study participants were non-smokers. Full mouth clinical periodontal parameters including probing depth, clinical attachment level, plaque index, and papilla bleeding index were recorded. GCF CypA and EMMPRIN levels were analyzed by enzyme-linked immunosorbent assay. Data were analyzed statistically with parametric and non-parametric tests. ResultsGCF CypA total amount was higher in the G-AgP group compared to healthy controls (p<0.05), whereas CypA total amounts were similar in CP and healthy controls (p>0.05). No significant difference in GCF CypA total amount between CP and G-AgP was observed (p>0.05). Also, there was no significant difference in GCF EMMPRIN total amounts among the study groups (p>0.05). ConclusionHigher levels of GCF CypA in patients with G-AgP might demonstrate that CypA is associated with the inflammatory infiltrate and alveolar bone destruction of G-AgP. However, GCF CypA level does not seem to be affected by CP. Similar GCF EMMPRIN levels in diseased and healthy groups might suggest that EMMPRIN has role in the turn over of connective tissues in physiological conditions as well as pathological state.

Expression of cyclophilin A/CD147 in carotid atherosclerotic plaque and the intervention of atorvastatin

To explore the expression of CyPA and CD147 in rabbit models of vulnerable carotid atherosclerotic plaque and the therapeutic effect of atorvastatin. Twenty-four male New Zealand rabbits were randomly divided into 3 groups. Eight rabbits were served as a normal diet group (Group A), and the remaining 16 rabbits underwent balloon-induced endothelial injury in the right carotid artery and thereafter were fed on high-cholesterol diet (1% cholesterol) for 12 weeks, then they were divided into 2 groups: a AS group (Group B), an atorvastatin group [Group C, 2.5 mg/(kg.d)]. 4 weeks later, plaque disrupture was triggered by China Russell's viper venom and histamine. Serum levels of TC, TG, LDL-C and HDL-C were measured at different timepoint. The damaged carotid arteries were collected to undergo pathological examination. The macrophage, expression of CyPA and CD147 were detected by immuno-histochemical analysis, and the mRNA levels of CyPA and CD147 were examined by reverse transcription polymerase chain reaction (RT-PCR). Compared with the Group A, the serum levels of TC and LDL-c in the Group B and Group C were significantly increased (all P<0.01). Compared with the Group B, the serum levels of TC and LDL-c in the Group C were reduced significantly after atorvastatin intervention for 4 weeks (all P<0.01). The plaques disruption and thrombosis occurred in 4 out of the 6 rabbits in the Group B, while only 1 rabbit demonstrated plaques disruption and thrombosis in the Group C. Compared with the Group B, the levels of CyPA, CD147 and macrophage in carotid atherosclerotic plaque in the Group C were decreased significantly (all P<0.01). The up-regulation of CyPA and CD147 may be involved in pathogenesis of vulnerable carotid atherosclerotic plaque. Atorvastatin could stabilize the plaque through inhibiting the CyPA and CD147 expression.

Abstract 507: Plasma Cyclophilin A as a Useful Biomarker for Effective Percutaneous Transluminal Pulmonary Angioplasty in Chronic Thromboembolic Pulmonary Hypertension

Background: Cyclophilin A (CyPA, encoded by Ppia) is secreted from pulmonary vascular smooth muscle cells in response to several stimuli including mechanical stretch and hypoxia. Recently, we demonstrated that extracellular CyPA and vascular Basigin (Bsg, encoded by Bsg) are crucial for hypoxia-induced PH by inducing growth factor secretion, inflammatory cell recruitment and VSMC proliferation in mice. Moreover, we demonstrated that high plasma CyPA levels are significantly associated with poor outcome (death or lung transplantation) in patients with pulmonary arterial hypertension (PAH). In this study, we examined plasma levels of CyPA in patients with chronic thromboembolic pulmonary hypertension (CTEPH) before and after percutaneous transluminal pulmonary angioplasty (PTPA), which is recently developed as a catheter-based therapy. Methods and Results: In consecutive 86 PTPA sessions in 24 patients, we examined the relationship between plasma CyPA levels, the severity of CTEPH and hemodynamic improvements after PTPA. We measured plasma CyPA levels by an immunoassay based on the sandwich technique. Plasma CyPA levels (ng/mL) were significantly elevated in patients with CTEPH (13.8±6.0, n=24) compared with those without PH (4.9±4.0, n=7) or healthy controls (4.6±2.8, n=18) (both P&lt;0.001). Moreover, the severity of CTEPH assessed by pulmonary vascular resistance (PVR) correlated with plasma levels of CyPA, IFN-α2, stem cell factor and adipsin (all P&lt;0.05). PTPA significantly reduced mean pulmonary artery pressure (P&lt;0.05), PVR (P&lt;0.01) and improved long-term prognosis compared with historical controls. Importantly, PTPA significantly reduced plasma CyPA levels in patients with CTEPH (5.4±4.1, P&lt;0.001), suggesting that plasma CyPA is useful for evaluation of therapeutic effects of PTPA. Finally, we observed significant increase in anti-inflammatory cytokines (interleukin-4, 7, 13) and reduction in stem cell growth factor-β and adipsin (all P&lt;0.05) after PTPA, suggesting improved metabolic and inflammatory state in CTEPH patients. Conclusions: These results indicate that plasma levels of CyPA are significantly increased in CTEPH patients and could be considered as an effective biomarker for assessment of the effects of PTPA.

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia.

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.

Cyclophilin A secreted from fibroblast-like synoviocytes is involved in the induction of CD147 expression in macrophages of mice with collagen-induced arthritis

BackgroundCyclophilin A (CypA), a member of the immunophilin family, is a ubiquitously distributed intracellular protein. Recent studies have shown that CypA is secreted by cells in response to inflammatory stimuli. Elevated levels of extracellular CypA and its receptor, CD147 have been detected in the synovium of patients with RA. However, the precise process of interaction between CypA and CD147 in the development of RA remains unclear. This study aimed to investigate CypA secretion from fibroblast-like synoviocytes (FLS) isolated from mice with collagen-induced arthritis (CIA) and CypA-induced CD147 expression in mouse macrophages.FindingsCIA was induced by immunization with type II collagen in mice. The expression and localization of CypA and CD147 was investigated by immunoblotting and immunostaining. Both CypA and CD147 were highly expressed in the joints of CIA mice. CD147 was expressed in the infiltrated macrophages in the synovium of CIA mice. In vitro, spontaneous CypA secretion from FLS was detected and this secretion was increased by stimulation with lipopolysaccharide. CypA markedly increased CD147 levels in macrophages.ConclusionsThese findings suggest that an interaction in the synovial joints between extracellular CypA and CD147 expressed by macrophages may be involved in the mechanisms underlying the development of arthritis.

Lentiviral Transduction Efficiency for Human CD34+ Cells Is Affected by TRIM5α Expression and Cell Proliferation

AbstractAbstract 4220HIV-1 based lentiviral vectors have been shown to transduce human hematopoietic stem cells, but transduction efficiency varies among individuals. Innate immune factors, such as TRIM5α and cyclophilin A (CypA), play an important role for restriction of retroviral infection. We previously demonstrated that genetic variations in rhesus TRIM5α affect transduction efficiency with lentiviral vectors for rhesus hematopoietic repopulating cells (ASH 2011), and an inhibitor of CypA, cyclosporine, decreases variation of transduction efficiency for human CD34+ cells (ASGCT 2011). In this study, we sought to evaluate whether the human innate immune factors TRIM5α and CypA could account for the variation in transduction efficiency for human CD34+ cells with HIV-1 based lentiviral vectors.Five human T-cell derived cell lines (CEM, CEMx174, A-3, D1.1, and E6-1) were transduced with a GFP-expressing HIV-1 based lentiviral vector at MOI=0.2, 0.5, and 1.0. RNA levels of TRIM5α and CypA at the time of transduction were evaluated by real time PCR, and transduction efficiency (%GFP) was evaluated by flow cytometry 3 days after transduction. Additionally, cell proliferation was evaluated by fold expansion of cell counts during 3 day culture.All T-cell cell lines revealed an increase of %GFP by MOI escalation, while large variations of %GFP were observed among cell lines (12.5–50.4% at MOI=0.2, 21.3–69.4% at MOI=0.5, and 34.9–84.4% at MOI=1.0). TRIM5α expression levels revealed large differences among all cell lines, while CypA expression levels were similar (815 fold magnitude for TRIM5α, 1.65 fold magnitude for CypA). In all cell lines, %GFP was positively correlated with fold expansion of cell counts (R2=0.87, p<0.05 at MOI=0.2, R2=0.93, p<0.01 at MOI=0.5, and R2=0.95, p<0.01 at MOI=1.0), while %GFP was negatively correlated with relative TRIM5α mRNA levels (R2=0.59, p=0.13 at MOI=0.2, R2=0.72, p=0.07 at MOI=0.5, and R2=0.83, p<0.05 at MOI=1.0). No correlation was observed between %GFP and CypA mRNA levels (R2=0.01, p=0.47 at MOI=0.2, R2=0.10, p=0.52 at MOI=0.5, and R2=0.13, p=0.57 at MOI=1.0).Next, human CD34+ cells from 14 individual donors were transduced with the HIV-1 vector at MOI=50 following a one day prestimulation. As before, RNA levels of TRIM5α and CypA were evaluated at the time of transduction, and %GFP and cell counts were evaluated 3 days after transduction. A large variation of %GFP was observed among individual donors (8.5–35.8%). TRIM5α expression levels also showed a large variation among individual donors, while CypA had similar expression levels in human CD34+ cells (27.7 fold magnitude for TRIM5α, 6.27 fold magnitude for CypA). In donor cells, %GFP was positively correlated with fold expansion of cell counts (R2=0.33, p<0.05) while a negative correlation was seen between %GFP and TRIM5α mRNA levels (R2=0.33, p<0.05) (figure). Again, no correlation was observed between %GFP and CypA mRNA levels (R2=0.02, p=0.65) (figure). Additionally, we sequenced known CypA polymorphisms in coding and promoter regions. Although two polymorphisms in the promoter region were detected (C1868G and A1914G), there was no correlation among %GFP, CypA mRNA levels, and polymorphisms.In conclusion, human TRIM5α mRNA levels were shown to negatively correlate with transduction efficiency of an HIV-1 based lentiviral vector in both human T-cell cell lines and CD34+ cells, while human CypA mRNA levels and CypA polymorphisms do not correlate with transduction efficiency. Additionally, cell proliferation was positively correlated with transduction efficiency. Our findings are important for both understanding and mitigating the variability of transduction efficiency for human CD34+ cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

P-0030 CypA Plays Roles in the Proliferation of Gastric Carcinoma Cells

ABSTRACT Introduction Cyclophilin A (CypA) is a member of the peptidyl-prolyl isomerases, the primary intracellular receptor of the immunosuppressant cyclosporin A (CsA). CypA plays an important role in life activities and participate in a variety of disease processes. Recent evidence points to possible roles for CypA overexpression in tumorigenesis. However gastric carcinoma was rarely involved in CypA research so far. Methods In this research, using immunohistochemistry assay we confirmed the overexpression of CypA in gastric carcinoma tissues at protein level. The mRNA level of CypA in gastric carcinoma cell lines are also detected by Real-Time PCR. Results Compared with normal gastric epithelial cell line GES-1, the mRNA levels of CypA in gastric carcinoma cell lines SGC-7901, MKN-45, BGC-823 and MGC-803 were 1.29, 1.46, 1.54 and 1.55 fold high respectively (Fig.1). To investigate the roles CypA plays in the occurrence and development of gastric carcinoma, we constructed 4 RNA interference vectors: pRNA-CypA shRNA 1-4 and confirmed the positive clone by PCR with pRNA primer, then sequenced the plasmid DNA for additional verification. BGC-823 cells were grown to 70% confluency in 24-well plates and transfected with 0.5ug pRNA-CypA shRNAs using Lipofectamine LTX (Invitrogen). At 48 and 96 hours following transfection the mRNA level of CypA in these cells were detected by Real-Time PCR. The results showed us that after transfection with pRNA-CypA shRNA3, BGC-823 cells yielded ∼80% and ∼30% CypA knockdown at 48h and 96h respectively compared with untreated BGC-823 cells; with pRNA-CypA shRNA2 yielded ∼27% and ∼35% CypA knockdown at 48h and 96h respectively; pRNA-CypA shRNA1 and pRNA-CypA shRNA4 had little RNA interference effects. A transient increase of CypA mRNA level occurred in transfection, and reduced to the normal level at 96h after transfection. We collected the BGC-823 cells at 48h with the best RNAi efficiency by pRNA-CypA shRNA3 plasmid to perform flow cytometric analysis of cell cycle. From the cell cycle distribution, cell populations of S & G2/M phase (M2 & M3) in untreated BGC-823cells, BGC-823cell transfected with pRNA plasmid and CypA knockdown (transfected with pRNA-CypA-shRNA3 plasmid) BGC-823cell were 45.3%, 49.7% and 33.0% respectively. CypA knockdown resulted in an augmentation of cell populations in G0/G1 phase (M1) accompanied with a decrease in S & G2/M phase (M2 & M3). Cell proliferation decreased obviously in CypA knockdown BGC-823 cells, and cell cycle arrested in G0/G1 phase (M1). These results establish the relevance of CypA to gastric carcinoma proliferation in vitro. Conclusion Our results establish the relevance of CypA to gastric carcinoma proliferation in vitro. In further study we will do more experiments to reveal mechanisms how CypA plays roles in occurrence and development of gastric carcinoma and the pathways though which the signals of CypA transfer.

Hepatocytes That Express Variants of Cyclophilin A Are Resistant to HCV Infection and Replication

Hepatitis C virus (HCV) uses several host factors to infect and replicate in human hepatocytes. Cyclophilin A (CypA) is required for viral replication, and CypA inhibitors are in development. We investigated the effects of nonsynonymous single nucleotide polymorphisms (SNPs) in the region of peptidyl-prolyl isomerase A (PPIA) that encodes CypA on HCV infection and replication of human hepatocytes. We used a combination of virologic, biochemical, and genetic approaches to investigate the effects of PPIA variants on HCV replication in cultured Huh-7.5 cells. We reduced levels of CypA in these cells using small hairpin RNAs (shRNAs). Using shRNAs, we showed that CypA was required for replication of HCV in Huh-7.5 cells and identified 3 SNPs in PPIA that protected cells from HCV entry or replication. Levels of HCV RNA were reduced 3-4 log in cells homozygous for the variant alleles; release of new particles was also reduced, but viral entry was not affected. The effects of the variant alleles were recessive and stronger for preventing replication of full-length HCV genomes than subgenomes. CypA inhibitors prevented replication of residual HCV in hepatocytes. The variants appeared to destabilize the CypA protein; the single amino acid changes led to rapid degradation of the protein. We identified variants in PPIA that destabilize its product, CypA, and prevent HCV infection and replication. These findings indicate mechanisms by which some cells might be resistant to HCV infection and that CypA is a good therapeutic target.

Differential Expression of Cyclophilin A and EMMPRIN in Developing Molars of Rats

A complex and intricate cascade of gene expression is essential for late stage tooth development. This study was performed to detect molecules involved in dental hard tissue formation and tooth eruption by comparing gene expression in cap stage molar germs (before eruptive movement and dental hard tissue formation) with that in root formation stage molar germs (after eruptive movement and dental hard tissue formation). DD-PCR revealed that cyclophilin A (Cyp-A), a potent chemoattractant for monocytes as well as a ligand for extracellular matrix metalloproteinase inducer (EMMPRIN) was expressed differentially in the two stages molar germs. The levels of Cyp-A and EMMPRIN mRNA were significantly higher at the root formation stage than at the cap and crown stages of the molar germs. Immunofluorescence showed that Cyp-A and EMMPRIN were expressed strongly in the follicular cells overlaying the occlusal region of the molar germs at the root formation stage. In contrast, their immunoreactivity was weak in the follicular tissues and was not region-specific in molar germs at the cap stage. In addition, the MCP-1 and CSF-1 mRNA levels increased in parallel to that of Cyp-A mRNA and the increased number of osteoclasts at the occlusal region. Immunoreactivity against Cyp-A and EMMPRIN was also observed in the fully differentiated ameloblasts and odontoblasts. This study suggests that Cyp-A and EMMPRIN play roles in the maturation of dental hard tissue and the formation of an eruption pathway.

Cyclophilin A is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147

Although overexpression of cyclophilin A (CypA) is associated with several types of cancer, its role in pancreatic cancer has not been studied. In this study the expression of CypA and its receptor CD147 on pancreatic cancer was determined as well as the effect of exogenous CypA on pancreatic cancer cell proliferation. The expression of CypA and CD147 in human pancreatic cancer cell lines and tissues was determined with real-time reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and immunostaining. Cell proliferation in response to CypA was performed by [3H]thymidine incorporation assay. Phosphorylation of MAPK and cytokine secretion profiles in pancreatic cancer cells were determined by using the Bio-Plex phosphoprotein assay and cytokine assay. Pancreatic cancer cell lines expressed significantly higher levels of CypA and CD147 than normal human pancreatic ductal epithelium (HPDE) cells. Expression of CypA and CD147 was also substantially higher in human pancreatic adenocarcinoma tissues than those in normal pancreatic tissues. Addition of exogenous CypA significantly stimulated pancreatic cancer cell proliferation in a dose-dependent manner and this effect was effectively blocked by pretreatment with anti-CD147 antibody. In addition, CypA activated ERK1/2 and p38 MAPK signaling pathways and increased the secretion of 2 key cytokines IL-5 and IL-17 in Panc-1 cells. The expression of CypA and CD147 was significantly increased in both pancreatic cancer cell lines and tissues. Exogenous CypA promotes pancreatic cancer cell growth, which may be mediated through the interaction with CD147 and the activation of ERK1/2 and p38 MAPKs.

Effect of cyclophilin A on gene expression in human pancreatic cancer cells

Background We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Methods Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Results Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. Conclusions These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

Virus particle core defects caused by mutations in the human immunodeficiency virus capsid N-terminal domain.

The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.

Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Cyclophilin A regulates HIV-1 infectivity, as demonstrated by gene targeting in human T cells.

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds most members of the cyclophilin family of peptidyl-prolyl isomerases. Of 15 known human cyclophilins, cyclophilin A (CypA) has been the focus of investigation because it was detected in HIV-1 virions. To determine whether CypA promotes HIV-1 replication, we deleted the gene encoding CypA (PPIA) in human CD4(+) T cells by homologous recombination. HIV-1 replication in PPIA(-/-) cells was decreased and not inhibited further by cyclosporin or gag mutations that disrupt Gag's interaction with cyclophilins, indicating that no other cyclophilin family members promote HIV-1 replication. The defective replication phenotype was specific for wild-type HIV-1 since HIV-2/SIV isolates, as well as HIV-1 bearing a gag mutation that confers cyclosporin resistance, replicated the same in PPIA(+/+) and PPIA(-/-) cells. Stable re-expression of CypA in PPIA(-/-) cells restored HIV-1 replication to an extent that correlated with steady-state levels of CypA. Finally, virions from PPIA(-/-) cells possessed no obvious biochemical abnormalities but were less infectious than virions from wild-type cells. These data formally demonstrate that CypA regulates the infectivity of HIV-1 virions.

Immunofluorescent localization and immunochemical determination of cyclophilin-A with specific rabbit antisera.

We raised rabbit antisera against homogeneous bovine cyclophilin A (CypA) and we report their use for its immunofluorescent and immunochemical detection without resorting to cyclosporine binding. Indirect immunofluorescence demonstrated that in tissue culture cells CypA is present in the cytoplasm diffusely and also associated with vesicles and the Golgi apparatus. In mitotic cells CypA is increased in amount and redistributed away from cytoplasmic organelles. High levels of CypA were demonstrated in murine splenic erythroblasts and myeloblasts, but they became undetectable during differentiation to mature erythrocytes and granulocytes. Large, often granular, lymphocytes stained very intensely, but small lymphocytes demonstrated variable staining. Dot blot immunoassays demonstrated that murine tissues contain similar amounts of CypA. During CsA treatment murine liver can increase its CypA content much more than spleen. In summary, we demonstrated that cells known to be resistant to the effects of CsA have high levels of CypA. Also tissues that are resistant to CsA can increase their levels more than sensitive tissues upon CsA exposure. Taken together these results suggest that CypA plays a role in cell cycle progression and that sensitivity to CsA may not be simply a reflection of the baseline CypA levels, but may also be affected by the regulation of these levels. Further work is needed in order to delineate the role of CypA in the cell cycle and its relation to the action of CsA.