Cyclophilin a (CypA) interacts with NF-κB subunit, p65/RelA, and contributes to NF-κB activation signaling

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Abstract

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-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. Methodology: Western 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. 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-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. Conclusions: Our 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. © 2014 Sun et al.

Figures

  • Figure 1. CypA deficiency reduces activated NF-kB/p65 (RelA) expression. (A) Western blot analysis of CypA, NF-kB subunits and their inhibitory protein, IkBa. Cytoplasmic extracts isolated from MEFs and C2C12 cells were immunoblotted for IkBa, and GDPH was used as internal control. Nuclear extracts prepared from MEFs and C2C12 cells were subjected to Western blot analysis to detect p65/RelA, p50, RelB, and p52 expression levels. p84 served as an internal control. Ppia+/+ and Ppia2/2 represent CypA WT and CypA KO, respectively. SC is a scrambled clone, and 1C2 and 2B6 are C2C12 CypA Kd clones. (B) Semi-quantitative analysis of relative expression levels of p65/RelA in MEFs (Ppia+/+ vs Ppia2/2) and C2C12 cells (WT, SC vs 1C2 and 2B6). (C) qRT-PCR analysis of relative mRNA expression levels of p65/RelA and p50 between Ppia+/+ and Ppia2/2 MEFs. (D) p65/RelA expression levels in MEF cytoplasmic extract (CE) and nuclear extracts (NE) upon treatment cells with CsA and Sfa. doi:10.1371/journal.pone.0096211.g001
  • Figure 2. Interaction of CypA with the NF-kB subunit, p65/RelA. (A) GST pull-down assays. GST-CypA was immobilized on GSH-sepharose 4B beads, then incubated with glioblastoma cell line U87vIII whole-cell lysates. The complexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes, followed by probing with anti-p65/RelA antibody. (B) Co-immunoprecipitation of myc-tagged CypA (myc-CypA) with HAtagged p65/RelA. Cell extracts prepared from myc-CypA and HA-p65/RelA co-transfected cells were subjected to immunoprecipitation by anti-HA antibody, or with anti-myc antibody, followed by immunoblotting with anti-myc or anti-HA antibodies. (C) Mapping the region of p65/RelA required for interaction with CypA. The HA-tagged various truncations were co-trasfected with myc-CypA into 293T cells. Cell lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with anti-HA antibody. As shown in lane 6, the truncation of 2–221 retains binding activity. Furthermore, site-directed mutagenesis of p65’s amino acid residues G170A/P172A/P176A prevented binding and interaction between NF-kB/p65 with CypA (compare lane 26 with lane 23; and compare lane 29 with lane 18). (D) Schematic representation of p65/RelA, its various truncations and site-directed mutants, and their binding activity. doi:10.1371/journal.pone.0096211.g002
  • Figure 3. p65/RelA protein stability. (A) Comparison of p65/RelA and its mutant (p65 G170A/P172A/P176A) (p65/RelAm) stability in 293T cells. (B) Comparison of p65/RelAm stability in the presence or absence of proteasome inhibitor MG-132 in 293T cells. (C) Comparison of p65/RelA and its mutant stability in CypA-deficient MEF. (D) Comparison of p65/RelAm stability in the presence or absence of MG132 in CypA-deficient MEF. Relative protein stabilities were semi-quantified with ImageJ. doi:10.1371/journal.pone.0096211.g003
  • Figure 4. CypA enhances NF-kB/p65 nuclear translocation. (A) Nuclear translocation of p65/RelA but not the CypA-binding site mutant (p65/ RelAm) induced by endogenous CypA. HeLa cells were transfected with pRed-p65/ReA and its mutant, and p65 location was determined by fluorescence microscopy. (B) HeLa cells were co-transfected with pRed-p65/RelA or pRed-p65/RelAm and GFP-CypA, and the locations of p65 and CypA were determined by fluorescence microscopy. (C) Comparison of the TNF-a-induced endogenous p65/RelA nuclear translocation in MEFs isolated from WT (Ppia+/+) and CypA KO (Ppia2/2). Relative intensity of p65/RelA in the nucleus was determined by fluorescence microscopy. (D) p65/ RelA expression levels in CypA-deficient MEF were restored by ectopically expressed CypA. doi:10.1371/journal.pone.0096211.g004
  • Figure 5. CypA is associated with NF-kB binding and transcriptional activities. (A) CypA activates kB site-mediated luciferase activity in a dose-dependent manner. Hela cells were transfected with indicated amounts of CypA, together with pGL4 nf-kb-Luc reporter constructs for 24 hrs, followed by luciferase activity assays. CypA (R55A) was used as negative control. (B) TNF-a-induced nuclear p65 levels degraded more quickly in MEFs isolated from Ppia2/2 mice. MEFs were treated with TNF-a for the indicated times, and nuclear extracts were prepared for Western blot analysis for p65 expression levels. Histone H3 protein served as an internal control. (C) Reduced NF-kB binding activity associated with CypA KO MEFs. EMSAs were performed with nuclear extracts from MEFs isolated from WT (Ppia+/+) and CypA KO (Ppia2/2) mice. NF-kB oligos were used as a probe. For competition experiments, nuclear extracts were pre-incubated with 100-fold molar excess of unlabeled oligos at 25uC for 10 min before addition of labeled probes. P84 was used as an internal loading control. (D) CypA is required for cytokine-induced full activation of kB-Luc activity. C2C12 cells and CypA Kd clones 1C2 and 2B6 were transfected with wild-type (WT) or mutant (MUT) pGL4 nf-kb-Luc reporter constructs, followed by treatment with IL-1a or TNF-a. Cells were harvested for luciferase assays. doi:10.1371/journal.pone.0096211.g005
  • Figure 6. CypA Kd-mediated down-regulation of IL-8, IL-6, and IL-1b expression. IL-8, IL-6, and IL-1b expression levels were determined by a quantitative real-time PCR (qRT-PCR) analysis. One microgram of total RNA was used for cDNA synthesis with Superscript III (Invitrogen) and the oligo(dT)15 primer. qRT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) detected by the 7300 Real Time PCR System (Applied Biosystems). The relative expression levels of IL-8, IL-6, and IL-1b mRNA were normalized to the internal reference 18S rRNA. doi:10.1371/journal.pone.0096211.g006

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Sun, S., Guo, M., Zhang, J. B., Ha, A., Yokoyama, K. K., & Chiu, R. H. (2014). Cyclophilin a (CypA) interacts with NF-κB subunit, p65/RelA, and contributes to NF-κB activation signaling. PLoS ONE, 9(8). https://doi.org/10.1371/journal.pone.0096211

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