A role for NRAGE in NF-kappaB activation through the non-canonical BMP pathway

Abstract

Background: Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-kappaB has yet to be explored.

Results: Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -alpha/beta and subsequent transcriptional activation of the p65 subunit of NF-kappaB. Ablation of endogenous NRAGE by siRNA inhibited NF-kappaB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-kappaB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor.

Conclusion: Modulation of NRAGE expression revealed novel roles in regulating NF-kappaB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.

Figure 1

Activation of the NF-κB pathway requires NRAGE in the BMP-4 pathway. A: A total of 293 cells were transfected with NRAGE:EGFP, NRAGE siRNA or a control vector, stimulated with 10 ng/ml BMP-4 for 15, 30 and 60 minutes, and western blotted for NRAGE, phospho-IKK α/β and total IKK α. B: A total of 293 cells were transfected with NRAGE siRNA or a control siRNA, stimulated with 10 ng/nl BMP-4, and activation assessed by luciferase assay. Luciferase assays were performed in triplicate and presented as fold increase over renilla. C: Immunofluorescence of phospho-p65 in the NRAGE:EGFP stable line. Magnification at 40×. * P < 0.05; siRNA control by the two-tailed unpaired Student's t test.

Figure 2

Activation of NF-κB through NRAGE is specific to the non-canonical BMP pathway. A: 293 cells were transfected with full length NRAGE:EGFP or stimulated with BMP-4 and incubated with or without IKK VII inhibitor, and NF-κB activation assessed by luciferase assay. B: 293 cells were transfected with full length NRAGE:EGFP or stimulated with BMP-4 and incubated with or without Tak1 or Tab1 morpholinos, and NF-κB activation assessed by luciferase assay. C: Endogenous phosphorylation of IKK-α/β in 293 cells transfected with Tak1 or Tab1 morpholinos prior to 10 ng/ml BMP-4 stimulation for one hour. D: 293 cells were transfected with full length NRAGE:EGFP, incubated with or without Tak1 or Tab1 morpholinos immunoprecipitated with rabbit anti-GFP and western blotted for IKK α/β-phosphorylation and total IKK α. E: Western blot of Tak1 and Tab1 proteins in 293 cells with and without morpholinos. Luciferase assays were performed in triplicate and presented as fold increase over renilla. * P < 0.05; vs NRAGE construct or BMP-4 treatment by the two-tailed unpaired Student's t-test.

Figure 3

BMP-4 induces the expression of MIF. A: Dot blot array for the secretion of cytokines and chemokines induced by NRAGE overexpression in 293HEK cells. Squared area indicates duplicate MIF blotting. B: Qualification of macrophage migrating inhibitory factor expression by western blot in 293HEK cells. MIF expression is induced after 24 hours stimulation with 10 ng/ml BMP-4 or NRAGE overexpression, and is inhibited by Tak morpholino and NF-κB inhibitor.

Figure 4

In vivo overexpression of NRAGE in the mouse kidney. A: Control staining of secondary antibodies in the NRAGE:Cherry transgenic mouse. B: Merged picture of NRAGE:Cherry and IKKα/β-phosphorylation. C: Merged picture of NRAGE:Cherry and NF-κB activation. Note the translocation of NF-κB to the nucleus denoted by teal colored nuclei. D: Merged picture of NRAGE:Cherry and MIF. E: Hematoxylin and Eosin staining of NRAGE:Cherry transgenic kidney. F: Control staining for secondary antibodies in the wild type mouse. G: Staining of IKKα/β-phosphorylation in the wild type mouse. H: Staining of NF-κB activation in the wild type mouse. I: Merged picture of NRAGE:cherry and MIF in the wild type mouse. J: Hematoxylin and Eosin staining of Wild-Type kidney. K-O: 63× magnification of C, red arrows point to nuclear localization of p65 as depicted by teal coloring.

Figure 5

NRAGE is required for NF-KB activation in P19 cells. A: P19 cells were transfected with NRAGE siRNA or a control siRNA stimulated with 10 ng/ml BMP-4 for 24 hours, and western blotted for IKK α/β-phosphorylation and total IKK α. B: P19 cells were transfected with NRAGE, NRAGE siRNA or a control siRNA, stimulated with 10 ng/nl BMP-4 for 24 hours, and NF-KB transcriptional activity assessed by luciferase assay. Luciferase assays were performed in triplicate and presented as fold increase over renilla. C: P19 cells were transfected with NRAGE or stimulated with 10 ng/nl BMP-4 for 24 hours and western blotted for MIF expression. * P < 0.05; vs NRAGE construct or BMP-4 treatment by the two-tailed unpaired Student's t- test.