Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells

J Clin Immunol. 2013 Apr;33(3):558-66. doi: 10.1007/s10875-012-9841-6. Epub 2012 Nov 28.


In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / immunology
  • Antibodies / metabolism
  • Antibodies / pharmacology*
  • Arthritis, Rheumatoid / immunology
  • Arthritis, Rheumatoid / metabolism
  • Case-Control Studies
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Endoplasmic Reticulum Chaperone BiP
  • Enzyme Activation / drug effects
  • Heat-Shock Proteins / metabolism*
  • Humans
  • I-kappa B Kinase / metabolism
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • Leukocytes, Mononuclear / drug effects*
  • Leukocytes, Mononuclear / metabolism*
  • MAP Kinase Signaling System / drug effects
  • Mitogen-Activated Protein Kinase 1 / metabolism*
  • Mitogen-Activated Protein Kinase 3 / metabolism*
  • Phosphorylation / drug effects
  • Protein Binding
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-akt / metabolism
  • Tumor Necrosis Factor-alpha / biosynthesis
  • U937 Cells


  • Antibodies
  • Endoplasmic Reticulum Chaperone BiP
  • HSPA5 protein, human
  • Heat-Shock Proteins
  • Protein Kinase Inhibitors
  • Tumor Necrosis Factor-alpha
  • Proto-Oncogene Proteins c-akt
  • I-kappa B Kinase
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3