BACKGROUND: CD147, as a cellular receptor for cyclophilin A (CypA), is a multifunctional protein involved in tumor invasion, inflammation, tissue remodeling, neural function, and reproduction. Recent observations showing the expression of CD147 in leukocytes indicate that this molecule may have roles in inflammation. METHODS: In order to investigate the role of CD147 and its ligand in the pathogenesis of atherosclerosis, human atherosclerotic plaques were analyzed for the expression pattern of CD147 and CypA. The cellular responses and signaling molecules activated by the stimulation of CD147 were then investigated in the human macrophage cell line, THP-1, which expresses high basal level of CD147 on the cell surface. RESULTS: Staining of both CD147 and CypA was detected in endothelial cell layers facing the lumen and macrophage-rich areas. Stimulation of CD147 with its specific monoclonal antibody induced the expression of matrix metalloproteinase (MMP)-9 in THP-1 cells and it was suppressed by inhibitors of both ERK and NF-kappaB. Accordingly, the stimulation of CD147 was observed to induce phosphorylation of ERK, phosphorylation-associated degradation of IkappaB, and nuclear translocation of NF-kappaB p65 and p50 subunits. CONCLUSION: These results suggest that CD147 mediates the inflammatory activation of macrophages that leads to the induction of MMP-9 expression, which could play a role in the pathogenesis of inflammatory diseases such as atherosclerosis.
Atherosclerosis ; Cell Line ; Cyclophilin A ; Endothelial Cells ; Humans ; Inflammation ; Leukocytes ; Macrophages ; NF-kappa B ; Phosphorylation ; Plaque, Atherosclerotic ; Reproduction

Cyclophilin A (CyPA) is a 17 kDa, ubiquitously expressed multifunctional protein that possesses peptidylprolyl cis-trans isomerase activity and scaffold function. Its expression is increased in inflammatory conditions including rheumatoid arthritis, autoimmune disease and cancer. Intracellular CyPA regulates protein trafficking, signal transduction, transcription regulation and the activity of certain other proteins. Secreted CyPA activates cardiovascular cells resulting in a variety of cardiovascular diseases; including vascular remodeling, abdominal aortic aneurysms formation, atherosclerosis, cardiac hypertrophy and myocardial ischemic reperfusion injury.
Aortic Aneurysm, Abdominal ; Arthritis, Rheumatoid ; Atherosclerosis ; Autoimmune Diseases ; Cardiomegaly ; Cardiovascular Diseases ; Cyclophilin A ; Cyclophilins ; Myocardial Reperfusion Injury ; Oxidative Stress ; Protein Transport ; Proteins ; Quaternary Ammonium Compounds ; Signal Transduction

OBJECTIVETo construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer.

METHODSFirst, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM.

RESULTSIt was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05).

CONCLUSIONLentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.

Animals ; Carcinoma, Non-Small-Cell Lung ; genetics ; Cell Line, Tumor ; Cyclophilin A ; genetics ; Gene Silencing ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Lung Neoplasms ; genetics ; Mice ; RNA, Small Interfering

BACKGROUNDbeta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease (AD). Possible mechanisms underlying Abeta-induced neuronal cytotoxicity include excessive production of reactive oxidative species (ROS) and apoptosis. Cyclophilin A (CypA), exhibits antioxidant properties and protects neurons against oxidative stress induced injury. This study was conducted to demonstrate whether CyPA added to cultured PC12 cells could alleviate Abeta-induced oxidative stress and protect them from apoptosis.

METHODSPC12 cells were pre-incubated for 30 minutes with recombinant human cyclophilin A (rhCyPA) in 0.1 nmol/L, 1.0 nmol/L, 10 nmol/L and 100 nmol/L and then incubated with 10 micromol/L Abeta(25-35). In every group, cell viability, apoptotic morphology, apoptotic rate, intracellular ROS accumulation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of PC12 cells and mitochondrial transmembrane potential were detected. Subsequently, the expression of the active form of caspase-3 was determined by Western blotting.

RESULTSIt was shown that cultures treated with 1.0 nmol/L, 10 nmol/L or 100 nmol/L rhCyPA + Abeta(25-35) had significantly higher cell viability and a lower rate of apoptosis compared with the cultures exposed only to Abeta(25-35). In addition, rhCyPA attenuated Abeta(25-35)-induced overproduction of intracellular ROS and Abeta(25-35)-induced a decrease in activity of the key antioxidant enzymes SOD and GSH-Px. Furthermore, rhCyPA also attenuated Abeta(25-35)-induced mitochondrial dysfunction and the activation of caspase-3.

CONCLUSIONCyPA may act as an ROS scavenger, and prevent Abeta(25-35)-induced neurotoxicity through attenuating oxidative stress induced by Abeta(25-35).

Amyloid beta-Peptides ; pharmacology ; Animals ; Caspase 3 ; metabolism ; Cyclophilin A ; pharmacology ; Glutathione Peroxidase ; metabolism ; Humans ; Oxidative Stress ; drug effects ; PC12 Cells ; Peptide Fragments ; pharmacology ; Rats ; Superoxide Dismutase ; metabolism

Hepatocellular carcinoma (HCC) related to hepatitis B virus (HBV) and hepatitis C virus (HCV) infections is thought to account for more than 80% of primary liver cancers. Both HBV and HCV can establish chronic liver inflammatory infections, altering hepatocyte and liver physiology with potential liver disease progression and HCC development. Cyclophilin A (CypA) has been identified as an essential host factor for the HCV replication by physically interacting with the HCV non structural protein NS5A that in turn interacts with RNA-dependent RNA polymerase NS5B. CypA, a cytosolic binding protein of the immunosuppressive drug cyclosporine A, is overexpressed in many cancer types and often associated with malignant transformation. Therefore, CypA can be a good target for molecular cancer therapy. Because of antiviral activity, the CypA inhibitors have been tested for the treatment of chronic hepatitis C. Nonimmunosuppressive Cyp inhibitors such as NIM811, SCY-635, and Alisporivir have attracted more interests for appropriating CypA for antiviral chemotherapeutic target on HCV infection. This review describes CypA inhibitors as a potential HCC treatment tool that is contrived by their obstructing chronic HCV infection and summarizes roles of CypA in cancer development.
Carcinoma, Hepatocellular* ; Carrier Proteins ; Cyclophilin A* ; Cyclophilins ; Cyclosporine ; Cyclosporins ; Cytosol ; Hepacivirus* ; Hepatitis B virus ; Hepatitis C* ; Hepatitis C, Chronic ; Hepatitis ; Hepatocytes ; Liver ; Liver Diseases ; Liver Neoplasms ; RNA Replicase

OBJECTIVE: To study differential expression of MRP8, CypA protein in the patients of laryngeal squamous cell carcinoma (LSCC) and the relationship in the development of LSCC. METHOD: Immunohistochemistry was used to detect the expression of MRP8,CypA protein in LSCC tissues of 41 cases and matched paraneoplastic normal tissues of 41 cases,with results compared to the clinical data to determine significance. RESULT: The expression of MRP8, CypA protein in carcinoma and normal tissues and composition of different positive grades were in statistical significance (P < 0.01). The expression levels of MRP8 were no significant correlations were identified against any parameter (age,sex and cervical lymphatic metastasis) examined (P > 0.05), but related to pathological stage (P < 0.05). CONCLUSION MRP8 protein is on intimate terms with different pathological differentiation stage of LSCC. MRP8, CypA protein may play an important role in the development and progression of LSCC.
Adult ; Aged ; Calgranulin A ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Cyclophilin A ; metabolism ; Female ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Prognosis

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L^-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Atractyloside/pharmacology* ; Cyclophilin D ; Male ; Matrix Metalloproteinases ; Mice ; Mitochondrial Membrane Transport Proteins/metabolism* ; Mitochondrial Permeability Transition Pore ; RNA, Messenger ; Rats

The effects of the an immunosuppressive drug cyclosporine A (CsA), on the salivary gland are largely unknown, even though clinical trials for the stimulation of salivation using CsA have been attempted. Cyclophilin A (CypA) is known to be a binding protein for CsA. CypA has cell proliferation and tissue matrix change activities. In our present study, the presence of CypA in the gland and effects of CsA on CypA expression were investigated by immunohistochemistry, immunoblotting and RT-PCR analyses. CypA was immunohistochemically detected in various kinds of ducts in the submandibular glands of Sprague Dawley rats. The CypA mRNA level was highest at postnatal day 1 and gradually decreased in a time-dependent manner up to adulthood. The expression of CypA increased after a 10 day subcutaneous administration of CsA in postnatal day 1 rats. Surgical sections of the chorda-lingual nerve with impaired salivation showed no changes in CypA expression. A cell proliferation assay using PCNA anti-serum showed increased cell division following CsA treatment. These results suggest that CsA and CypA may act on ductal cells to regulate saliva composition rather than salivation levels.
Animals ; Benzeneacetamides ; Carrier Proteins ; Cell Division ; Cell Proliferation ; Cyclophilin A ; Cyclosporine ; Immunoblotting ; Immunohistochemistry ; Piperidones ; Proliferating Cell Nuclear Antigen ; Rats ; Rats, Sprague-Dawley ; RNA, Messenger ; Saliva ; Salivary Glands ; Salivation ; Submandibular Gland

OBJECTIVETo investigate the role of CD147 in the proliferation, activation and chemotaxis of Jurkat cell induced by cyclophilin A (CyPA).

METHODSCD147 mRNA and protein level siRNA transfected Jurkat cells were identified by RT-PCR and Western blot respectively. Jurkat cell, Jurkat-vector cell and Jurkat-CD147 siRNA cells were treated with different concentrations of CyPA (0.01 nmol/L, 0.1 nmol/L, 1 nmol/L, 10 nmol/L) or PBS for 24 h and 48 h. Proliferation level was detected by MTT assay. CD25 was measured by flow cytometry. Transwell chamber was used to detect the chemotaxis. The effect of CyPA on the adhesive potential of Jurkat cell was studied by cell-matrix adhesion assay.

RESULTSCD147 mRNA and protein level siRNA transfected cells were decreased significantly than that of control cells. CyPA stimulated the proliferation of Jurkat cell in a dose-dependent manner, its effect peaked at 10 nmol/L CyPA. Blockage of CD147 expression decreased the proliferation level of Jurkat cell induced by CyPA. CyPA increased the activation rate of Jurkat cell, and blockage CD147 expression decreased the activation rate of the cell induced by CypA. CyPA showed a chemotactic activity on Jurkat cell, the chemotaxis index being 2.32, and the chemotactic ability was decreased after inhibition of CD147 expression. CyPA had no effect on adhesion of Jurkat cell to extracellular matrix.

CONCLUSIONCD147 plays a role in the proliferation, activation and chemotaxis of Jurkat cell induced by CyPA.

Basigin ; genetics ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Chemotaxis ; drug effects ; Cyclophilin A ; pharmacology ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Jurkat Cells ; RNA, Small Interfering ; genetics ; Transfection

The changes of serum cyclophilin A (CyPA), its receptor CD147 and the downstream signaling pathway during the process of cardiac hypertrophy remain unknown. The present study aims to investigate the relationships between CyPA-CD147-ERK1/2-cyclin D2 signaling pathway and the development of cardiac hypertrophy. Left ventricular hypertrophy was prepared by 2-kidney, 2-clip in Sprague-Dawley rats and observed for 1 week, 4 and 8 weeks. Left ventricular hypertrophy was evaluated by ratio of left ventricular heart weight to body weight (LVW/BW) and cardiomyocyte cross sectional area (CSA). CyPA levels in serum were determined with a rat CyPA ELISA kit. Expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular myocytes were determined by Western blot and immunostaining. Compared with sham groups, systolic blood pressure reached hypertensive levels at 4 weeks in 2K2C groups. LVW/BW and CSA in 2K2C groups were significantly increased at 4 and 8 weeks after clipping. ELISA results indicated a prominent increase in serum CyPA level associated with the degree of left ventricular hypertrophy. Western blot revealed that the expressions of CyPA, CD147, phospho-ERK1/2 and cyclin D2 in left ventricular tissues were also remarkably increased as the cardiac hypertrophy developed. The results of the present study demonstrates that serum CyPA and CyPA-CD147-ERK1/2-cyclin D2 signaling pathway in ventricular tissues are time-dependently upregulated and activated with the process of left ventricular hypertrophy. These data suggest that CyPA-CD147 signaling cascade might play a role in the pathogenesis of left ventricular hypertrophy, and CyPA might be a prognosticator of the degree of left ventricular hypertrophy.
Animals ; Basigin ; metabolism ; Blood Pressure ; Cyclin D2 ; Cyclophilin A ; metabolism ; Hypertension ; Hypertrophy, Left Ventricular ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Myocytes, Cardiac ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Up-Regulation