Quantification of GADD34 Expression in RA

The endoplasmic reticulum (ER) is the subcellular compartment where transmembrane and secreted proteins are produced. In normal conditions biosynthesis rate, folding and trafficking of the proteins are tightly regulated through an efficient 'quality control' system. The ER responds to the accumulation of misfolded proteins in its lumen through different signaling pathways known as the unfolded protein response (UPR) [1]. Upon activation, the ER transmembrane protein PERK phosphorylates the alpha subunit of the eukaryotic initiation factor eIF2 [2]. Under these conditions, the transcription factor ATF4 directs the expression of genes involved in resistance to oxidative stress, in amino acid metabolism, as well as the expression of GADD34 (Growth arrest and DNA damage-inducible gene 34) [3]. GADD34 is a regulatory subunit of PP1 phosphatase which dephosphorylates eIF2alpha [4], representing a negative feedback loop of UPR, essential for protein synthesis recovery and cell survival [5].

The UPR is more than just an adaptive response to unfolded protein accumulation in the ER, and UPR signaling pathways intersect with immune responses at many levels [6]. In B lymphocytes activation of UPR is part of the normal program of cell differentiation, playing an important role in immunoglobulin synthesis [7]. ER stress has also been implicated in the pathogenesis of many human diseases, including metabolic, neurodegenerative diseases, and cancer [8]. A direct link between UPR and inflammation has been demonstrated for the development of autoimmune diseases such as inflammatory bowel disease [9].

Rheumatoid arthritis is a chronic multifactorial inflammatory disease. Proinflammatory cytokines, such as TNF-alpha and IL-6, secreted by macrophages and monocytes have an important pathogenic role in the phases of inflammation, synovial proliferation and cartilage damage [10]. Rheumatoid factor (RF) has been the most widely used antibody to diagnose RA [11]; anti-citrullinated protein antibodies (ACPA) have also been included in the diagnostic criteria of ACR/EULAR in 2010 [12] and they are highly associated with the development of erosive RA [13].

GADD34 has recently been shown to be necessary for the production of proinflammatory cytokines by dendritic cells and fibroblasts exposed to double-strand RNA in a murine model [14]. In fibroblasts, GADD34 expression is dependent on PKR (Protein Kinase RNA-activated) activation, showing a direct link between pathogen detection and the eIF2alphaP/ATF4 pathway of UPR. The importance of this link for anti-viral immunity has been demonstrated by the mortality of GADD34-deficient neonate mice infected by Chikungunya virus, due to a significant reduction of IFN-beta production [15]. It has been proposed that GADD34 could have a qualitative role on the selection of mRNAs being translated in particular conditions, such as viral infections [16]. These results suggest that GADD34 might be a key molecule of inflammatory processes in human pathologies as well; however, the role of GADD34 in cytokine production in humans remains to be elucidated. The aim of our study was to investigate GADD34 expression in RA patients.

1. Hetz, C., et al., The unfolded protein response: integrating stress signals through the stress sensor IRE1alpha. Physiol Rev. 91 (4): p. 1219-43.

2. Harding, H.P., et al., Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell, 2000. 5 (5): p. 897-904.

3. Harding, H.P., et al., An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell, 2003. 11 (3): p. 619-33.

4. Novoa, I., et al., Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha. J Cell Biol, 2001. 153 (5): p. 1011-22.

5. Novoa, I., et al., Stress-induced gene expression requires programmed recovery from translational repression. Embo J, 2003. 22 (5): p. 1180-7.

6. Janssens, S., B. Pulendran, and B.N. Lambrecht, Emerging functions of the unfolded protein response in immunity. Nat Immunol. 15 (10): p. 910-919.

7. Iwakoshi, N.N., et al., Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol, 2003. 4 (4): p. 321-9.

8. Wang, S. and R.J. Kaufman, The impact of the unfolded protein response on human disease. J Cell Biol. 197 (7): p. 857-67.

9. Kaser, A., et al., XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease. Cell, 2008. 134 (5): p. 743-56.

10. McInnes, I.B. and G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med. 365 (23): p. 2205-19.

11. Ingegnoli, F., R. Castelli, and R. Gualtierotti, Rheumatoid factors: clinical applications. Dis Markers. 35 (6): p. 727-34.

12. Aletaha, D., et al., 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Arthritis Rheum. 62 (9): p. 2569-81.

13. Majka, D.S., et al., Duration of preclinical rheumatoid arthritis-related autoantibody positivity increases in subjects with older age at time of disease diagnosis. Ann Rheum Dis, 2008. 67 (6): p. 801-7.

14. Clavarino, G., et al., Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells. Proc Natl Acad Sci U S A. 109 (8): p. 3006-11.

15. Clavarino, G., et al., Induction of GADD34 is necessary for dsRNA-dependent interferon-beta production and participates in the control of Chikungunya virus infection. PLoS Pathog. 8 (5): p. e1002708.

16. Claudio, N., et al., Mapping the crossroads of immune activation and cellular stress response pathways. Embo J. 32 (9): p. 1214-24.