Cyclophilin A (CypA) Interacts with NF-κB Subunit, p65/RelA, and Contributes to NF-κB Activation Signaling

and Contributes to NF-kB Activation Signaling. PLoS ONE 9(8): e96211. doi:10.1371/journal.pone.0096211 Cyclophilin A (CypA) Interacts with NF-kB Subunit, p65/ RelA, and Contributes to NF-kB Activation Signaling Shan Sun 0 1 Mian Guo 0 1 James Beiji Zhang 0 1 Albert Ha 0 1 Kazunari K. Yokoyama 0 1 Robert H. Chiu 0 1 Philippe A. Gallay, Scripps Research Institute, United States of America 0 Current address: School of Life Science, Tsinghua University , Beijing , China 1 1 Dental and Craniofacial Research Institute and School of Dentistry, University of California , Los Angeles, CA , United States of America, 2 Department of Neurosurgery, the 2nd Affiliated Hospital of Harbin Medical University , Harbin, Heilonjiang , China , 3 Graduate Institute of Medicine, Kaohsiung Medical University , Kaohsiung, Taiwan , 4 Surgical Oncology & Jonsson Comprehensive Cancer Center, University of California , Los Angeles, CA , United States of America Background: Peptidyl-prolyl isomerase cyclophilin A (CypA) plays important roles in signaling, protein translocation, inflammation, and cancer formation. However, little is known about the mechanisms by which CypA exerts its effects. C57BL/6 Ppia (encoding CypA)-deficient embryonic fibroblasts show reduced activation of the nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-kB activity to exert its biological effects. Methodology: Western blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-kB/p65 at the protein level. GST pull-down and co-immunoprecipitation were used to examine interactions between CypA and p65/RelA. Truncation mutants and site-directed mutagenesis were used to determine the sequences of p65/RelA required for interactions with CypA. Enhancement of p65/RelA nuclear translocation by CypA was assessed by co-transfection and immunofluorescent imaging. Treatment of cells with cycloheximide that were harvested at various time points for Western blot analyses was carried out to evaluate p65/RelA protein stability. The functional activity of NF-kB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes. Results: GST pull-down assays in vitro and co-immunoprecipitation analyses in vivo provided evidence for protein-protein interactions. These interactions were further supported by identification of a CypA-binding consensus-like sequence within NF-kB subunit p65 at the N-terminal 170-176 amino acid residues. Significantly, CypA provided stability for NF-kB p65 and promoted NF-kB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-kB activity. Conclusions: Our findings revealed important mechanisms that regulate NF-kB activation, and offer new insights into the role of CypA in aberrant activation of NF-kB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases. - Funding: This work was supported in part by a research fund from the UCLA Dental and Craniofacial Research Institute. No additional funding received for this study. The funder had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Nuclear factor kB (NF-kB) is a set of multifunctional transcription factors that regulate expression of genes involved in numerous normal cellular activities [1,2]. In addition to the wellestablished role of NF-kB in both immunity and inflammation, deregulation of NF-kB signaling is associated with cancer malignancies, diabetes, and atherosclerosis, further emphasizing the wide spectrum of roles it plays in control of normal growth, tissue homeostasis, and diseases [3,4,5]. A heterodimeric complex of p65/RelA and p50 is the most common form of NF-kB in mammalian cells, and is commonly referred to as NF-kB. Given the protein functions of NF-kB to positively regulate gene expression, there is an inhibitory kB (IkB) family of NF-kB inhibitory proteins called IkBs, which retain NF-kB dimers in the cytoplasm of unstimulated cells [6]. A myriad of endogenous and exogenous stimuli, such as tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b) are capable of inducing activation of the IkB kinase (IKK) complex, which leads to ubiquitin-dependent degradation of IkBa. NF-kB is then free to shuttle into the nucleus and bind to specific sequences in the promoter or enhancer regions of its target genes [7]. Although the molecular events that control translocation of NFkB from the cytoplasm to the nucleus are well characterized [7], knowledge concerning the regulation of NF-kBs activity inside the nucleus still remains largely unclear. NF-kB is believed to recruit co-factors to form a much higher order transcription complex than previously expected [8]. Among the well-characterized NF-kB coactivators, p300/CREB-binding protein (CBP) appears to be a basal component of a functional NF-kB transcription complex [9,10]. A recent report demonstrated that LRP16 integrates into the NF-kB transcriptional complex by associating with its p65 component, and is required for its functional activation [11]. CypA is a member of the peptidyl-prolyl isomerase (PPIase) family, a group of proteins that catalyze cis-trans isomerization of peptidyl-prolyl bonds during protein folding and/or conformational changes [12,13]. CypA was first identified as the primary intracellular target of the immunosuppressive drug, CsA [14]. The immunosuppressive activity of CsA is thought to result from engagement of calcineurin by the CsA-CypA complex [15], an observation supported by the finding that CypA knockout mice are resistant to immunosuppression by CsA [16]. One of the other inhibitory effects of CsA on T cell proliferation is to inhibit the early phase of NF-kB/RelA activation by CD28 costimulatory signaling to reduce the IL-2 expression [17]. In addition, CsA affected not only immune cells but also on non-lymphoid lineages and induced insensitiveness to inflammatory cytokines, especially to TNF-a. It has been demonstrated that CsA inhibited TNF-atriggered MCP-1 induction via unfolded protein response (UPR)mediated suppression of NF-kB. This suppression of NF-kB by UPR was, at least in part, via induction of C/EBP family members [18]. Several lines of research have revealed that PPIase such as CypA may function as molecular signaling switches that can act as novel molecular timers to help control the amplitude and duration of a cellular process [19]. Moreover, the role of CypA nuclear translocation, CypAs participation in activation of other factors or their nuclear translocation also impacts various cellular functions [2023]. One recent report demonstrated that kno (...truncated)