Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul 4;12(1):19.
doi: 10.1186/1475-2867-12-19.

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

Karim Bahmed  1 , Curtis Henry, Michael Holliday, Jasmina Redzic, Madalina Ciobanu, Fengli Zhang, Colin Weekes, Robert Sclafani, James Degregori, Elan Eisenmesser
Affiliations

Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B

Karim Bahmed et al. Cancer Cell Int. .

Abstract

Background: Although the peptidyl-prolyl isomerase, cyclophilin-A (peptidyl-prolyl isomerase, PPIA), has been studied for decades in the context of its intracellular functions, its extracellular roles as a major contributor to both inflammation and multiple cancers have more recently emerged. A wide range of activities have been ascribed to extracellular PPIA that include induction of cytokine and matrix metalloproteinase (MMP) secretion, which potentially underlie its roles in inflammation and tumorigenesis. However, there have been conflicting reports as to which particular signaling events are under extracellular PPIA regulation, which may be due to either cell-dependent responses and/or the use of commercial preparations recently shown to be highly impure.

Methods: We have produced and validated the purity of recombinant PPIA in order to subject it to a comparative analysis between different cell types. Specifically, we have used a combination of multiple methods such as luciferase reporter screens, translocation assays, phosphorylation assays, and nuclear magnetic resonance to compare extracellular PPIA activities in several different cell lines that included epithelial and monocytic cells.

Results: Our findings have revealed that extracellular PPIA activity is cell type-dependent and that PPIA signals via multiple cellular receptors beyond the single transmembrane receptor previously identified, Extracellular Matrix MetalloPRoteinase Inducer (EMMPRIN). Finally, while our studies provide important insight into the cell-specific responses, they also indicate that there are consistent responses such as nuclear factor kappa B (NFκB) signaling induced in all cell lines tested.

Conclusions: We conclude that although extracellular PPIA activates several common pathways, it also targets different receptors in different cell types, resulting in a complex, integrated signaling network that is cell type-specific.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Extracellular PPIA activity monitored through luciferase reporters assays. Sixteen luciferase reporters were used to monitor the response to extracellular PPIA in several cell lines, which included the following: A) HEK293T cells, B) MOLM13 cells, C) PANC-1 cells, and D) L3.6PL. Cells were transiently transfected with the indicated reporters and then stimulated with recombinant 25 μM PPIA as described in Materials and Methods. Luciferase activity is shown as a fold-increase over the buffer control (Luc Fold-Increase). Dashed line (grey) indicate no Luc Fold-Increase (i.e., unity). *Beta-Catenin (βCate) and *HRE refer to plasmids that comprise the transcription target sites for HIF and βCate transcription factors, respectively. The remaining plasmids comprise the indicated promoters for each protein. Bars indicate the standard error of two replicates.
Figure 2
Figure 2
Probing the involvement of the PPIA active site for both its biological activity and catalytic activity. A) For PPIA biologically activity, dose-dependent luciferase assays were conducted as in Figure 1 for the most highly induced luciferase reporter in each cell line using both wild type PPIA (white) and the active site point mutation PPIA R55A (black). These include IL-6 in HEK293T cells, IL-8 in MOLM13 cells, IL-5 in PANC-1 cells, and IL-8 in L3.6pL cells. B) The 15N-HSQC spectrum of 1 mM 15N-labeled model peptide substrate, GSFGPLRAGD, is shown alone with the associated assignments for each amide (top). Note, two resonances are observed corresponding to the slowly interconverting cis and trans resonances. Catalysis of both the wild type PPIA (middle) and mutant PPIA R55A (bottom) were assessed through ZZ-exchange spectroscopy in the presence of 20 μM of each. ZZ-exchange spectra are shown for the same 240 ms delay. Arrows denote the appearance of exchange peaks due to PPIA-mediated enhancement of the rate of cis/trans interconversion of the model peptide substrate. We note that no exchange peaks were observed for longer mixing times of PPIA R55A indicating no detectable catalytic rate enhancement. All spectra were collected at 720 MHz at 10 C.
Figure 3
Figure 3
Characterizing extracellular PPIA-mediated activity of NFκB within multiple cell lines. A) An NFκB translocation assay was conducted in several adherent cell lines, which included HEK293T cells (top), PANC-1 cells (middle), and L3.6pL cells (bottom) with the addition of either buffer control or PPIA. Cells were transiently transfected with the p65-GFP plasmid (green, left panels) as well as DAPI stained (blue, middle panels) and the merge is shown (green, right panels). B) PPIA-induced luciferase reporter activity was monitored for the wild type IL-6 reporter (WT) and two mutations to the NFκB binding element (Mut1 and Mut2) for HEK293T and L3.6pL cells. C) PPIA-induced luciferase reporter activity was monitored for the wild type IL-8 luciferase reporter (WT) and two mutations within the NFκB binding element (also called Mut1 and Mut2) for MOLM13, PANC-1, and L3.6pL cells. PPIA-induced luciferase reporter activity was monitored as in Figure 1.
Figure 4
Figure 4
Characterizing extracellular PPIA-mediated activation of ERK1/2 phosphorylation within multiple cell lines. A) ERK1/2 phosphorylation in PPIA stimulated HEK293T, MOLM13, PANC-1, and L3.6pL cells was determined by immunoblotting. Cells were treated with either buffer alone (−) or 25 μM PPIA (+) for 10 min and stored in SDS loading buffer for subsequent immunoblotting analysis of pERK1/2 and total ERK1/2 as a control. B) PPIA-induced time-dependent induction of ERK1/2 phosphorylation as monitored by flow cytometry is shown for both MOLM13 and PANC-1 cells for the indicated time points also stimulated with 25 μM PPIA. The data shown represents the mean of fluorescence intensity (MFI) for each time point measured in triplicate and normalized to the average initial MFI.
Figure 5
Figure 5
Assessing the importance of cellular BSG for extracellular PPIA activity on monocytes. A) Top: ERK1/2 phosphorylation was monitored in MOLM13 cells using a flow cytometric assay (as in Figure 4) for both sh0 cells (solid line) and shBSG cells (dashed line). Bottom: Western blot analysis using the BSG antibody shows that shBSG cells exhibit a significant knockdown when compared to sh0 cells. No significant increase in cellular BSG expression was observed in sh0 cells within this 30 min timeframe as assessed by western blot analysis. B) Top: In MOLM13 cells, IL-8 luciferase reporter activity was monitored after stimulation with recombinant PPIA. Bottom: Cellular BSG was monitored via western blot analysis both before and after 24 h stimulation with recombinant PPIA. C) In U937 cells, a similar analysis was conducted as in (B). For all stimulations, 25 μM recombinant PPIA was used and β-actin was used as a loading control. Arrows denote both highly and lowly glycosylated BSG as well as the β-actin loading control.
Figure 6
Figure 6
Quantifying the PPIA interaction with heparin at atomic resolution. A) 15N-HSQC spectra of 0.5 mM 15N-PPIA alone (black) and in complex with 3 mM heparin (red). B) Residues that exhibit chemical shifts greater than 0.1 ppm upon engaging heparin in either 15N or 1H dimensions are highlighted (red) and are all located within the PPIA active site. C) Based on the measured 15N-PPIA chemical shift changes upon titration with heparin, the binding isotherms were used to estimate a KD of 2.5 ± 0.3 mM. Shown are the binding isotherms for the 15N chemical shift changes of K82 and T152. All experiments were conducted at 600 MHz at 25°C.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Saphire ACS, Bobardt MD, Gallay PA. Host cyclophilin A mediates HIV-1 attachment to target cells via heparans. EMBO J. 1999;18(23):6771–6785. doi: 10.1093/emboj/18.23.6771. - DOI - PMC - PubMed
    1. Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, Shimotohno K. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell. 2005;19(1):111–122. doi: 10.1016/j.molcel.2005.05.014. - DOI - PubMed
    1. Satoh K, Nigro P, Zeidan A, Soe NN, Jaffre F, Oikawa M, O'Dell MR, Cui ZQ, Menon P, Lu Y. et al.Cyclophilin A Promotes Cardiac Hypertrophy in Apolipoprotein E-Deficient Mice. Arterioscler Thromb Vasc Biol. 2011;31(5):1116–U1458. doi: 10.1161/ATVBAHA.110.214601. - DOI - PMC - PubMed
    1. Ralhan R, Masui O, Desouza LV, Matta A, Macha M. Michael Siu KW: Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: Strategy for discovery of candidate serological biomarkers. Proteomics. 2011;11(12):2363–2376. doi: 10.1002/pmic.201000186. - DOI - PubMed
    1. Yao QZ, Li M, Yang H, Chai H, Fisher W, Chen CY. Roles of cyclophilins in cancers and other organ systems. World J Surg. 2005;29(3):276–280. doi: 10.1007/s00268-004-7812-7. - DOI - PubMed

LinkOut - more resources