Cyclophilin A plays an important role in embryo implantation through activating Stat3

Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.

Citron Rho-Interacting Serine/Threonine Kinase Promotes HIF1a-CypA Signaling and Growth of Human Pancreatic Adenocarcinoma

In human pancreatic ductal adenocarcinoma (PDAC), the cyclophilin A (CypA) is overexpressed and promotes the development of PDAC. However, the mechanism underlying cyclophilin A expression remains elusive. Here, we reported that the citron Rho-interacting serine/threonine kinase (CIT) promotes the HIF1a-CypA signaling and growth of PDAC cells. CIT expression was higher in PDAC cells compared with the normal epithelial cells, and clinical data showed that CIT was overexpressed in PDAC tissues and high expression of CIT predicted poor overall and disease-free survival. In PDAC cells, knockdown of CIT expression repressed the rate of proliferation and capacity of colony formation, which were accomplished with an increased percentage of apoptotic cells and cell cycle arrest. The knockdown of CIT in PDAC cells reduced the expression of CypA while overexpression of CIT promoted the expression of CypA. We observed that the effects of CIT on the expression of CypA relied on the transcriptional factor HIF1a, which was previously reported to transcriptionally activate the expression of CypA in PDAC cells. Furthermore, the effects of CIT on apoptosis, cell cycle, proliferation, and colony formation of PDAC cells relied on its role in the regulation of CypA expression. Collectively, our data showed that CIT promoted the activation of HIF1-CypA signaling and enhanced the growth of PDAC cells.

Cyclophilin a signaling induces pericyte-associated blood-brain barrier disruption after subarachnoid hemorrhage

Objective The potential roles and mechanisms of pericytes in maintaining blood–brain barrier (BBB) integrity, which would be helpful for the development of therapeutic strategies for subarachnoid hemorrhage (SAH), remain unclear. We sought to provide evidence on the potential role of pericytes in BBB disruption and possible involvement and mechanism of CypA signaling in both cultured pericytes and SAH models. Methods Three hundred fifty-three adult male C57B6J mice weighing 22 to 30 g, 29 CypA−/− mice, 30 CypA+/+ (flox/flox) mice, and 30 male neonatal C57B6J mice were used to investigate the time course of CypA expression in pericytes after SAH, the intrinsic function and mechanism of CypA in pericytes, and whether the known receptor CD147 mediates these effects. Results Our data demonstrated both intracellular CypA and CypA secretion increased after SAH and could activate CD147 receptor and downstream NF-κB pathway to induce MMP9 expression and proteolytic functions for degradation of endothelium tight junction proteins and basal membranes. CypA served as autocrine or paracrine ligand for its receptor, CD147. Although CypA could be endocytosed by pericytes, specific endocytosis inhibitor chlorpromazine did not have any effect on MMP9 activation. However, specific knockdown of CD147 could reverse the harmful effects of CypA expression in pericytes on the BBB integrity after SAH. Conclusions This study demonstrated for the first time that CypA mediated the harmful effects of pericytes on BBB disruption after SAH, which potentially mediated by CD147/NF-κB/MMP9 signal, and junction protein degradation in the brain. By targeting CypA and pericytes, this study may provide new insights on the management of SAH patients.

Cyclophilin A in Arrhythmogenic Cardiomyopathy Cardiac Remodeling

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder characterized by the progressive substitution of functional myocardium with noncontractile fibro-fatty tissue contributing to ventricular arrhythmias and sudden cardiac death. Cyclophilin A (CyPA) is a ubiquitous protein involved in several pathological mechanisms, which also characterize ACM (i.e., fibrosis, inflammation, and adipogenesis). Nevertheless, the involvement of CyPA in ACM cardiac remodeling has not been investigated yet. Thus, we first evaluated CyPA expression levels in the right ventricle (RV) tissue specimens obtained from ACM patients and healthy controls (HC) by immunohistochemistry. Then, we took advantage of ACM- and HC-derived cardiac mesenchymal stromal cells (C-MSC) to assess CyPA modulation during adipogenic differentiation. Interestingly, CyPA was more expressed in the RV sections obtained from ACM vs. HC subjects and positively correlated with the adipose replacement extent. Moreover, CyPA was upregulated at early stages of C-MSC adipogenic differentiation and was secreted at higher level over time in ACM- derived C-MSC. Our study provides novel ex vivo and in vitro information on CyPA expression in ACM remodeling paving the way for future C-MSC-based mechanistic and therapeutic investigations.

Impact of cyclophilin A on survival in gastric cancer patients who received platinum-based chemotherapy.

73 Background: Cyclophilin A (CypA), which is a protein that mediates protein folding and trafficking, has known to be associated with cancer progression and drug resistance such as cisplatin. However, the actual effect in cancer patients has not been elucidated. Methods: We collected clinical data from the cohort of 289 gastric cancer patients. Subjects were selected as following criteria: 1) adjuvant or first-line palliative chemotherapy containing platinum, 2) available FFPE tissue for immunohistochemical stain for CypA. We divided the population into two groups; Group A was patients who received adjuvant chemotherapy and Group P was those of palliative chemotherapy-treated group. The score of CypA immunohistochemistry was calculated depending on the intensity of stain and the percentage of stained cells. We performed survival analysis based on CypA expression. Results: 1) Group A (N = 147) Patients with lower expression of CypA ( < vs. ≥ median IHC score) showed significantly worse progression-free survival (PFS, 2.4 years vs NR, p = 0.043), and overall survival (OS, 3.4 months vs. NR, p = 0.002). In subgroup analysis, level of CypA expression discriminated PFS significantly in patients with age less than 65, poorly differentiated histology, signet ring cell type, and OS for those with use of oxaliplatin, age less than 65, stage III, poorly differentiated histology, diffuse type of Lauren classification, and signet ring cell type favoring higher expression of CypA. 2) Group P (N = 142) There was no relationship between expression level of CypA and PFS/OS in patients with palliative settings. However, in patients who were treated with oxaliplatin-containing chemotherapy, lower expression of CypA showed worse OS (8.3 vs 11.1 months, p = 0.003) Conclusions: Lower level of CypA expression was associated with worse PFS and OS in patients with adjuvant setting, especially in case of advanced stage, and high grade histology. Although CypA did not have significant effect on survivals in palliative chemotherapy-received patients, those who received oxaliplatin showed worse OS with lower CypA.

Abstract 523: Acetylation of Cyclophilin A in Response to Oxidative Stress Enhances Its Expression and Secretion

Objective: Cyclophilin A (CyPA) is a Secreted OXidative stress-induced Factor (SOXF) secreted by cardiovascular cells in response to Angiotensin II (Ang II) and reactive oxygen species (ROS). Extracellular CyPA is a proinflammatory mediator that regulates vascular remodeling, abdominal aortic aneurysm, atherosclerosis and cardiac hypertrophy. Post-translational modification of CyPA by acetylation in response to ROS has been described. Moreover, acetylation of CyPA is important in HIV pathogenesis. The mechanism and regulation of CyPA acetylation as well as its role in cardiovascular diseases are currently unknown. We hypothesized that Ang II regulates oxidative stress-induced CyPA acetylation that alters its expression and/or secretion in vascular smooth muscle cells. Methods and results: Ang II (1μM) increased acetylation of CyPA (Acyl-CyPA) in a time dependent manner, with a peak at 8hr (3.5±0.6 fold increase) in rat aortic smooth muscle cells (RASMC) as shown by Western blot. Mouse aortic smooth muscle cells from mice lacking CyPA (CyPA-/-) and wild type controls (WT) confirmed that Ang II induced acetylation reactivity coincided exactly with CyPA reactivity. In AT1R and CyPA cotransfected HeLa cells, Ang II increased Acyl-CyPA in a time dependent manner consistent with that in RASMC. The ROS scavengers Tiron or N-acetylcysteine significantly inhibited Ang II induced Acyl-CyPA in a dose dependent manner in RASMC. Ang II-induced CyPA acetylation was enhanced by 2 hr pretreatment with histone deacetylase inhibitor trichostatin (TSA) or sirtinol in a dose dependent manner. Similarly, Ang II-induced CyPA secretion was enhanced by pretreatment with TSA (1μM) in a time dependent manner. Moreover, acetyltransferase p300 and PCAF (p300/CBP-asociated factor) inhibitor anacardic acid (6-nonadecyl salicylic acid) dramatically inhibited CyPA expression, and Ang II induced Acyl-CyPA in a dose dependent manner. Conclusion: These results suggest that Ang II-induced CyPA acetylation is oxidative stress dependent, and that acetylation enhanced CyPA expression and secretion. Detailed mechanistic studies of the regulation of CyPA acetylation will help to identify a future therapeutic target for CyPA regulated cardiovascular diseases.

Cyclophilin A is required for efficient human cytomegalovirus DNA replication and reactivation

Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.

Cyclophilin A Is an Essential Cofactor for Hepatitis C Virus Infection and the Principal Mediator of Cyclosporine Resistance In Vitro

ABSTRACT Cyclosporine (CsA) and its derivatives potently suppress hepatitis C virus (HCV) replication. Recently, CsA-resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA-resistant replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing of CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of an siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.