Professor Nelson Fernández Department of Biological Sciences, University of Essex

1. Study of glucocorticoid receptors in human synovial fibroblast cells: response to dexamethasone and cytokines. Professor Nelson Fernández Department of Biological Sciences, University of Essex

2. This is an example of an interdisciplinary study between Southend Hospital and the University of Essex. The subject is autoimmunity.

3. This study was undertaken to gain an insight into the role of glucocorticoid receptor isoforms α and β in glucocorticoid sensitivity as seen in rheumatoid arthritis

4. RHEUMATOID ARTHRITIS “IS A CHRONIC, SYSTEMIC, INFLAMMATORY AUTOIMMUNE DISORDER”

5. AETIOLOGY CAUSE – UNKNOWN GENETIC FACTORS HLA-DR4 and HLA-DR1 HLA-DR4 subtypes Dw4 & Dw14 HLA-DR1 subtype Dw1 NEGATIVELY ASSOCIATED: HLA-DR4 subtypes Dw10 & Dw13 HLA Molecule 90% of patients

6. …PATHOGENESIS T-CELL MEDIATED DISEASE en.sanofi-aventis.com/.../im/p_im_arthritis.asp

7. Although rheumatic diseases respond to glucocorticoids (GC), different inflammatory rheumatic diseases vary in their degree of responsiveness. • We hypothesise that this may be due to changes in the expression of human glucocorticoid receptor (hGR) isoforms.

8. Two isoforms of the hGR gene are produced: hGRα which has 777 amino acids and hGRβ which has 742 amino acids and these differ at their carboxyl termini (Gieguere et al., 1986). hGRβ is found in the nucleus and thought not to bind ligand and evidence implies that hGRβ may act as a dominant negative to repress the transcriptional activity of hGRα (Oakley et al., 1996).

9. Activation initiates a pleiotropic effect of the GC, which include the transactivation or transrepression of many target genes, for example those encoding IL-1 ( and ), NF-B, IL-6, IL-8 and TNF-.

10. Aims of the study • We set out to establish the relative levels of hGR- and hGR- expressed in fibroblast-like synovial cells derived from an RA patient (RAMAK-1 cells). • We also developed transfectant cells using plasmid clones encoding either the  or the  genes. • We used a selected panel of isoform-specific antibodies. • We then analysed the effects of the exogenous dexamethasone (DEX) and pro-inflammatory cytokine, TNF- on the relative expression of hGR- and hGR- on these cells. • We also investigated the effect of DEX on TNF-α-induced IL-6 and IL-8 cytokine secretion in untransfected and transfected RAMAK-1 cells.

11. Expression analysis of hGR isoforms The hGR isoforms, α and β where detected in the RAMAK-1, CEM-C7 and THP-1 cell protein extracts and subjected to Western blot analysis. First immunoblotted with polyclonal anti-hGR antibody E-20, which is raised against epitopes common to both α and β isoforms

12. Intracellular presence of glucocorticoid receptors. A B C Red and green: Triton X-100 Black and blue: Saponin FIGURE 4

13. Absence of cell-surface glucocorticoid receptors. A B C RAMAK-1, CEM-C7 and THP-1 obtained on staining with the mouse monoclonal anti-hGR antibody FIGURE 3

14. Typical Western Blot Results A B C D E F 170- 130- 95- 72- 56- 43- 34- hGRα 94 kDa hGRβ 90 kDa Band δ ~60 kDa hGRε ~56 kDa Figure 7 Western Blot analysis for (A) rhGR used as control (B) transfected COS-1 cells with hGRα (C) transfected COS-1 cells with hGRβ (D) RAMAK-1 cells (E) CEM-C7 cells (F) THP-1 cells. Arrows indicate band expression of hGRα at 94 kDa; hGRβ at 90 kDa; Band δ at ~60 kDa and hGRε at ~56 kDa.

15. Western Blot analysis of COS-1 cell. Lanes shows transfection of a cell with an alpha and beta plasmidusing GR (E-20) antibody that recognizes hGRα and hGβ. • Molecular Weight • hGRα = 94kDa • hGRβ = 90 kDa.

16. A B C 170- 130- 95- 72- 56- 43- 34- hGRα 94 kDa hGRβ 90 kDa Band δ ~60 kDa Figure 6 Western Blot analysis for (A) untransfected COS-1 cells (B) transfected COS-1 cells with hGRα (C) transfected COS-1 cells with hGRβ. Arrows indicate band expression of hGRα at 94 kDa; hGR β at 90 kDa and Band δ at ~60 kDa.

17. Comparison of transfected COS-1 alpha and beta hGRs with different human cell lines that express receptors endogenously. COS cells do not naturally express human glucocorticoid receptors α and β We compared expression with 3 human cell lines that do express hGRs

18. 205 116 97 84 66 55 45 36 kDa B Antibody E-20 was raised to recognize both isoforms. We also used the hGRα-specific P-20 antibody and the hGR-β-specific PA3-514 antibody. The antibody detected only a faint hGR-α signal in THP-1 cell extracts. As expected, no bands were detected in extracts from pRShGRβ-transfected COS-1 cells indicating that the antibody did not cross-react with the hGR-β isoform. The additional bands recognised by antibody P-20 in the cell lines might be degradation products of the hGR protein.

19. Lanes 1 2 3 4 5 116 97 84 66 55 45 36 Analysis of sub-cellular localisation of hGR on RAMAK kDa pRShGRα (lane 1) pRShGRβ (lane 2) whole cell (lane 3) cytoplasmic (lane 4) nuclear (lane 5) hGR-α could be detected in both whole cell fraction and the cytoplasmic fraction, No signals were detected that co-migrated with the hGR-β (90 kDa) band from pRShGRβ-transfected COS-1 cell extracts in any of the fractions, pointing out the absence of the isoform expression. FIGURE 2

20. We then determine if synthetic steroids exert an auto-regulatory effect in regulating the alpha and beta isoforms of GCR. We treated cells with increasing concentrations of Dex followed by Western blot analysis.

21. Lanes 1 2 3 4 5 6 7 Analysis of hGR after DEX treatment in RAMAK-1 cells hGR Actin A * * * FIGURE 9 B

22. Cytokines are heavily involved in the pathogenesis of RA. • Using the RAMAK-1 model system we designed experiments to test whether modulation of cytokines and hGC receptor isoforms are related.

23. TNF-α-induced IL-6 cytokine secretion in RAMAK-1 cells FIGURE 10

24. TNF-α-induced IL-8 cytokine secretion in RAMAK-1 cells. FIGURE 13

25. Overall conclusion The emerging picture on hGR expression as a determining factor in steroid insensitivity, might explain the reason for differential individual responsiveness to steroids.

26. Conclusions • The synovial RAMAK-1 cells express hGR-α but no hGR-β. • hGR-α receptors are localised in the cytoplasm and translocate to the nucleus in response to DEX. • RAMAK-1, CEM-C7 and THP-1 cells do not express cell-surface hGR. • TNF-α positively regulates hGR-α in THP-1 but not in CEM-C7 or RAMAK-1 cells. • DEX downregulates hGR-α in RAMAK-1 cells. • DEX suppresses IL-6 and IL-8 production in RAMAK-1 cells. • Effects of GC in RAMAK-1 cells are hGR-α-dependent and are directly proportional to the hGR-α levels. • hGR-β does not inhibit hGR-α mediated glucocorticoid action in RAMAK-1 cells.

27. What are the clinical implications? • Can we measure the GCR alpha and beta ratios? • Do we have ‘good’ monoclonal antibodies to probe the 2 isoforms? • Can a patient’s steroid responsiveness be predicted without the need to undergo a steroid 'trial‘ and the side effects that go with this? • Are GR receptors recruited in active synovitis which increases the efficacy of steroids in the acute setting?

28. Acknowledgements Srividya Madhaviah Sukadev (PhD on this project) Prof Bhaskar Dasgupta Jenny List Dr Manos Mavrakos Dr Ian Morrison Prof Richard Cherry Southend Hospital