The online version of this article (doi:10.1186/1475-2867-12-19) contains supplementary material, which is available to authorized users.
There are no competing interests.
KB designed and acquired all of the cell-based assays, MH and FZ produced and collected the nuclear magnetic resonance experiments. CH, JD, and RAS helped design and acquire the ERK1/2 phosphorylation experiments and provided expertise in culturing monocytic cell lines. JD and JR helped produce stable knockdown cell lines. MC and CW provided expertise in culturing pancreatic cancer cell lines. EZE helped design all of the experiments. All authors read and approved the final manuscript.
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.
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.
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.
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.
Additional file 1: Figure S1. Assessing the purity of recombinant PPIA. A) UV spectrum of recombinantly purified PPIA. The atypical UV spectrum of PPIA has been shown to be an important confirmation of its purity [ 15]. B) 15N-HSQC spectrum of purified PPIA collected at 900 MHz at 25°C along with the three-dimensional structure (inset). (PDF 6 MB)
Additional file 2: Figure S2. The effect of extracellular PPIA on cellular proliferation. Cell cycle was monitored 24 h post incubation using FACS analysis with either buffer alone or recombinant PPIA in (A) HEK293T cells, (B) MOLM13 cells, (C) PANC-1 cells, and (D) L3.6pL cells. No apparent effect was observed. (JPEG 329 KB)12935_2012_338_MOESM2_ESM.jpeg
Additional file 3: Figure S3. Probing the cell-specific responses to in-house purified recombinant proteins. A) MOLM13 cells were used for a comparative analysis of luciferase reporter assays stimulated with various recombinantly purified proteins, which include recombinant PPIA and recombinant IL-6. B) Luciferase reporter activity of PPIA was monitored for both IL-6 (left) and IL-8 (right). All luciferase reporter activities were conducted as in Figure 1. (JPEG 303 KB)
Additional file 4: Figure S4. Further characterization of PPIA-mediated activation of NFκB. A) An NFκB translocation assay was conducted as in Figure 3A, but using the PPIA active site point mutation, PPIA R55A. B) IκBα degradation is shown after treatment with recombinant PPIA. (JPEG 271 KB)
Additional file 5: Luciferase Reporter Plasmid Sources. (DOC 26 KB)12935_2012_338_MOESM5_ESM.doc
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- Extracellular cyclophilin-A stimulates ERK1/2 phosphorylation in a cell-dependent manner but broadly stimulates nuclear factor kappa B
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