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

Abstract

BackgroundPeptidyl-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-light-chain-enhancer of activated B cells (NF-κB), the p65/RelA subunit, suggesting that CypA may mediate modulation of NF-κB activity to exert its biological effects.MethodologyWestern blotting and qRT-PCR analyses were used to evaluate the association of CypA deficiency with reduced activation of NF-κB/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-κB was assessed by electrophoretic mobility-shift assays (EMSA), luciferase assays, and changes in expression levels of target genes.ResultsGST 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-κB subunit p65 at the N-terminal 170–176 amino acid residues. Significantly, CypA provided stability for NF-κB p65 and promoted NF-κB p65 nuclear translocation, resulting in increased nuclear accumulation and enhanced NF-κB activity.ConclusionsOur findings revealed important mechanisms that regulate NF-κB activation, and offer new insights into the role of CypA in aberrant activation of NF-κB-mediated signaling for altered expression of its target genes, resulting in pathological effects in various diseases.

Novel Role for Cyclophilin A in Regulation of Chondrogenic Commitment and Endochondral Ossification

“…However, the functional role of the closely related PPIase family member CypA (PPIA) in skeletal development remains elusive. In previous reports, we demonstrated a close relationship between CypA and NF-B signaling (22,28). NF-B was found to regulate certain aspects of endochondral ossification during embryogenesis (29,30).…”

“…We had previously demonstrated that CypA knockdown (Kd) results in the downregulation of NF-B/p65 (RelA, a subunit of NF-B) in glioblastoma cell lines and a murine myoblast line, C2C12 (22,28). Decreased NF-B/p65 expression levels were also observed in CypA Kd mouse chondrogenic ATDC5 stable clones, 2T7 and 2T3, as well as in CypA KO mouse proximal tibia growth plates ( Fig.…”

“…CypA functions as a novel NF-B/p65-interacting protein (22). It acts in concert with p65 to mediate BMP-2-induced Sox9 production and hence regulation of chondrogenesis signaling.…”

Proline isomerisation as a novel regulatory mechanism for p38MAPK activation and functions

“…For example, the stability, localisation and activity of the p65 subunit of NF-κB was recently shown to be regulated by association with CypA because its downregulation impaired production of NF-κB-induced cytokines while reducing the proliferation of glioblastoma cells. 32,33 Similar to CypA, genetic ablation of CypB reduces glioma cell survival and proliferation, as shown both in vitro and in vivo. 34,35 We found that multiple cyclophilins -CypA, CypB and CypH -directly interact with a newly identified target, p38MAPK, facilitating isomerisation of Pro224, which in turn is required to regulate phosphorylation of kinase regulatory Thr-Gly-Tyr motif.…”

Cyclophilin A: A Key Factor in Virus Replication and Potential Target for Anti-viral Therapy

“…It can inhibit IL-4 induction by Itk (Colgan et al, 2004) and regulates the RA-induced neuronal differentiation (Song et al, 2004). Moreover, CypA may also facilitate the interleukin-6 (IL-6)-induced signal transducer and activation of transcription 3 (Stat3) tyrosine phosphorylation and nuclear translocation (Bauer et al, 2009), and can strongly associate and activate NF-kB in vitro and in vivo (Obchoei et al, 2009;Sun, et al 2014).…”