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Aktuelle Rheumatologie 2019; 44(02): 128-135
DOI: 10.1055/a-0576-6409
Original Article
© Georg Thieme Verlag KG Stuttgart · New York

Inhibition of Tubulin β-chain May Play a Regulatory Role in the Development of Rheumatoid Arthritis

Inhibition der Tubulin-β-Kette als möglicher Regulator bei der Entwicklung der rheumatoiden Arthritis
Yan Zhao
1   Shandong Normal University, Biotechnology, Jinan, China
,
Shufeng Li
2   Shandong Qianfoshan Hospital, Laboratory, Jinan, China
,
Pan Jihong
3   Shandong Medicinal and Biotechnology Centre, Biochemistry Laboratory, Jinan, China
,
Rui Zhang
3   Shandong Medicinal and Biotechnology Centre, Biochemistry Laboratory, Jinan, China
,
Zhiyuan Li
1   Shandong Normal University, Biotechnology, Jinan, China
,
Qingsong Meng
4   Shandong Provincial Qianfoshan Hospital, Clinical Laboratory, Jinan, China
› Institutsangaben
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Publikationsverlauf

Publikationsdatum:
13. März 2018 (online)

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Abstract

Pathological characteristics of Rheumatoid arthritis (RA), a systemic autoimmune disease, includes abnormal proliferation of synovial fibroblasts and increased angiogenesis.The study assessed the effect of tubulin β-chain gene (TBB) gene knockdown on the behaviour of rheumatoid arthritis (RA) fibroblast-like synoviocytes (SFs), which is significantly affected by the synthesis of tubulin. Cultured SFs were isolated from the mixed knee-joint synovial tissue of five RA patients and treated with siRNA targeting TBB. Knockdown efficiency and expression of relevant genes were detected by RT-PCR following 24 h and 36 h of transfection. MTT, Transwell, and wound scratch assays and flow cytometric analysis were used to assess cell proliferation and invasive and migratory capacity following TBB knockdown. The expression of TBB was detected after inhibition of the ERK, STAT3 and NF-kB pathways. The expression of IL-17, TNF-α, IL-1α, IL1-β, and IL-6 was quantified following TBB knockdown. The proliferation, invasion, and migration of RASFs were significantly decreased following TBB knockdown. ELISA showed a significant decrease in interleukin 1β (IL-1β) secretion and insignificantly reduced secretion of TNF-α, IL-17, and IL-1α after TBB knockdown. The expression of TBB in RASFs decreased with the increasing concentration of a STAT3 pathway inhibitor while TBB expression increased after down regulation of the ERK pathway. These results support a protective role for TBB knockdown in suppressing RASFs, laying a foundation for further studies on the pathogenic mechanisms of RA.

Zusammenfassung

Zu den pathologischen Charakteristika der rheumatoiden Arthritis (RA), einer systemischen Autoimmunerkrankung, zählen die abnorme Proliferation synovialer Fibroblasten sowie eine verstärkte Angiogenese. In dieser Studie wurde untersucht, wie sich ein Gen-Knockdown beim Tubulin-β-Ketten(TBB)-Gen auf das Verhalten der an der rheumatoiden Arthritis (RA) beteiligten fibroblastenähnlichen Synoviozyten (SF) auswirkt, welches signifikant von der Tubulinsynthese beeinflusst wird. Bei fünf RA-Patienten wurden kultivierte SF aus dem gemischten Synovialgewebe des Kniegelenks isoliert und mit gegen TBB gerichteten siRNA behandelt. Nach 24- und 36-stündiger Transfektion wurden mittels RT-PCR die Effizienz des Knockdowns und die Expression der relevanten Gene bestimmt. Mithilfe von MTT-, Transwell- und Wound-Scratch-Assays sowie einer durchflusszytometrischen Analyse wurden die Zellproliferation und die Migrationskapazität nach dem TBB-Knockdown beurteilt. Außerdem wurden die TBB-Expression nach Inhibition der ERK-, STAT3- und NF-kB-Signalwege ermittelt und die Expression von IL-17, TNF-α, IL-1α, IL1-β und IL-6 nach dem TBB-Knockdown quantifiziert. Die Proliferation, Invasion und Migration von RASF waren nach dem TBB-Knockdown signifikant reduziert. Im ELISA zeigten sich eine signifikante Reduktion der Interleukin-1β(IL-1β)-Sekretion und eine nicht signifikant reduzierte Sekretion von TNF-α, IL-17 und IL-1α nach dem TBB-Knockdown. Die TBB-Expression in RASF nahm mit zunehmender Konzentration eines Inhibitors des STAT3-Signalweges ab, während die TBB-Expression nach Downregulation des ERK-Signalweges zunahm. Diese Ergebnisse sprechen dafür, dass ein TBB-Knockdown eine protektive Rolle bei der Suppression von RASF einnimmt, und bilden daher eine Grundlage für weitere Studien über die pathogenen Mechanismen bei RA.

Key words

tubulin β-chain - siRNA - rheumatoid arthritis - fibroblast-like synoviocytes

Schlüsselwörter

Tubulin-β-Kette - rheumatoide Arthritis - fibroblastenahnliche Synoviozyten - siRNA
 

  • References

  • 1 Ben Hassine H, Zemni R, Bouagina E. et al. Lack of association between PADI4 polymorphisms and rheumatoid arthritis in the Tunisian population. Joint Bone Spine 2012; 79: 329-330
    CrossrefPubMedGoogle Scholar
  • 2 Seeuws S, Jacques P, Van Praet J. et al. A multiparameter approach to monitor disease activity in collagen-induced arthritis. Arthritis Res Ther 2010; 12: R160
    CrossrefPubMedGoogle Scholar
  • 3 Veale DJ, Fearon U. Inhibition of angiogenic pathways in rheumatoid arthritis: potential for therapeutic targeting. Best Pract Res Clin Rheumatol 2006; 20: 941-947
    CrossrefPubMedGoogle Scholar
  • 4 Cheung CH, Wu SY, Lee TR. et al. Cancer cells acquire mitotic drug resistance properties through beta I-tubulin mutations and alterations in the expression of beta-tubulin isotypes. Plos One 2010; 5: e12564
    CrossrefPubMedGoogle Scholar
  • 5 Kavallaris M, Burkhart CA, Horwitz SB. Antisense oligonucleotides to class III beta-tubulin sensitize drug-resistant cells to Taxol. Br J Cancer 1999; 80: 1020-1025
    CrossrefPubMedGoogle Scholar
  • 6 Catrina AI, Lampa J, Ernestam S. et al. Anti-tumour necrosis factor (TNF)-alpha therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis. Rheumatology (Oxford) 2002; 41: 484-489
    CrossrefPubMedGoogle Scholar
  • 7 Dayer JM. The process of identifying and understanding cytokines: from basic studies to treating rheumatic diseases. Best Pract Res Clin Rheumatol 2004; 18: 31-45
    CrossrefPubMedGoogle Scholar
  • 8 Chang XT, Zhao Y, Wang YJ. et al. Screening Citrullinated Proteins in Synovial Tissues of Rheumatoid Arthritis Using 2-dimensional Western Blotting. Journal of Rheumatology 2013; 40: 219-227
    CrossrefPubMedGoogle Scholar
  • 9 Firestein GS. NF-kappaB: Holy Grail for rheumatoid arthritis?. Arthritis Rheum 2004; 50: 2381-2386
    CrossrefPubMedGoogle Scholar
  • 10 Simon LS, Weaver AL, Graham DY. et al. Anti-inflammatory and upper gastrointestinal effects of celecoxib in rheumatoid arthritis: a randomized controlled trial. JAMA 1999; 282: 1921-1928
    CrossrefPubMedGoogle Scholar
  • 11 Wakefield RJ, Gibbon WW, Conaghan PG. et al. The value of sonography in the detection of bone erosions in patients with rheumatoid arthritis: a comparison with conventional radiography. Arthritis Rheum 2000; 43: 2762-2770
    CrossrefPubMedGoogle Scholar
  • 12 Wang Q, Jiang H, Li Y. et al. Targeting NF-kB signaling with polymeric hybrid micelles that co-deliver siRNA and dexamethasone for arthritis therapy. Biomaterials 2017; 122: 10-22
    CrossrefPubMedGoogle Scholar
  • 13 Schett G, Zwerina J, Firestein G. The p38 mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis. Ann Rheum Dis 2008; 67: 909-916
    CrossrefPubMedGoogle Scholar
  • 14 Ohori M. ERK inhibitors as a potential new therapy for rheumatoid arthritis. Drug News Perspect 2008; 21: 245-250
    CrossrefPubMedGoogle Scholar
  • 15 Choi EM, Lee YS. Luteolin suppresses IL-1beta-induced cytokines and MMPs production via p38 MAPK, JNK, NF-kappaB and AP-1 activation in human synovial sarcoma cell line, SW982. Food Chem Toxicol 2010; 48: 2607-2611
    CrossrefPubMedGoogle Scholar
  • 16 Schirmer M, Vallejo AN, Weyand CM. et al. Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28- T cells from rheumatoid arthritis patients. J Immunol 1998; 161: 1018-1025
    PubMedGoogle Scholar
  • 17 Choe JY, Park KY, Park SH. et al. Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6 R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes. Arthritis Res Ther 2013; 15: R26
    CrossrefPubMedGoogle Scholar
  • 18 Lao M, Shi M, Zou Y. et al. Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis. J Immunol 2016; 196: 596-606
    CrossrefPubMedGoogle Scholar
  • 19 Dudley DT, Pang L, Decker SJ. et al. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A 1995; 92: 7686-7689
    CrossrefPubMedGoogle Scholar
  • 20 Cuzzocrea S, Chatterjee PK, Mazzon E. et al. Pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation. Br J Pharmacol 2002; 135: 496-510
    CrossrefPubMedGoogle Scholar
  • 21 Schust J, Sperl B, Hollis A. et al. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol 2006; 13: 1235-1242
    CrossrefPubMedGoogle Scholar
  • 22 McMurray JS. A new small-molecule Stat3 inhibitor. Chem Biol 2006; 13: 1123-1124
    CrossrefPubMedGoogle Scholar
  • 23 Aletaha D, Neogi T, Silman AJ. et al. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. Ann Rheum Dis 2010; 69: 1580-1588
    CrossrefPubMedGoogle Scholar
  • 24 Neogi T, Aletaha D, Silman AJ. et al. The 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for rheumatoid arthritis: Phase 2 methodological report. Arthritis Rheum 2010; 62: 2582-2591
    CrossrefPubMedGoogle Scholar
  • 25 Muller-Ladner U, Kriegsmann J, Franklin BN. et al. Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage when engrafted into SCID mice. Am J Pathol 1996; 149: 1607-1615
    PubMedGoogle Scholar
  • 26 Barksby HE, Milner JM, Patterson AM. et al. Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis. Arthritis Rheum 2006; 54: 3244-3253
    CrossrefPubMedGoogle Scholar
  • 27 Liu M, Sun H, Wang X. et al. Association of increased expression of macrophage elastase (matrix metalloproteinase 12) with rheumatoid arthritis. Arthritis Rheum 2004; 50: 3112-3117
    CrossrefPubMedGoogle Scholar
  • 28 Pap T, Claus A, Ohtsu S. et al. Osteoclast-independent bone resorption by fibroblast-like cells. Arthritis Res Ther 2003; 5: R163-R173
    CrossrefPubMedGoogle Scholar
  • 29 Pettit AR, Ji H, von Stechow D. et al. TRANCE/RANKL knockout mice are protected from bone erosion in a serum transfer model of arthritis. Am J Pathol 2001; 159: 1689-1699
    CrossrefPubMedGoogle Scholar
  • 30 Diarra D, Stolina M, Polzer K. et al. Dickkopf-1 is a master regulator of joint remodeling. Nat Med 2007; 13: 156-163
    CrossrefPubMedGoogle Scholar
  • 31 Frixione E. Recurring views on the structure and function of the cytoskeleton: a 300-year epic. Cell Motil Cytoskeleton 2000; 46: 73-94
    CrossrefPubMedGoogle Scholar
  • 32 Li H, DeRosier DJ, Nicholson WV. et al. Microtubule structure at 8 A resolution. Structure 2002; 10: 1317-1328
    CrossrefPubMedGoogle Scholar
  • 33 Heald R, Nogales E. Microtubule dynamics. J Cell Sci 2002; 115: 3-4
    PubMedGoogle Scholar
  • 34 Howard J, Hyman AA. Dynamics and mechanics of the microtubule plus end. Nature 2003; 422: 753-758
    PubMedGoogle Scholar
  • 35 Giarnieri E, De Francesco GP, Carico E. et al. Alpha- and beta-tubulin expression in rectal cancer development. Anticancer Res 2005; 25: 3237-3241
    PubMedGoogle Scholar
  • 36 Burke D, Gasdaska P, Hartwell L. Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol Cell Biol 1989; 9: 1049-1059
    CrossrefPubMedGoogle Scholar
  • 37 Weinstein B, Solomon F. Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin. Mol Cell Biol 1990; 10: 5295-5304
    CrossrefPubMedGoogle Scholar
  • 38 Tarazona R, Lopez-Lluch G, Galiani MD. et al. HLA-B2702 (77-83/83-77) peptide binds to beta-tubulin on human NK cells and blocks their cytotoxic capacity. J Immunol 2000; 165: 6776-6782
    CrossrefPubMedGoogle Scholar
  • 39 Berrieman HK, Lind MJ, Cawkwell L. Do beta-tubulin mutations have a role in resistance to chemotherapy?. Lancet Oncol 2004; 5: 158-164
    CrossrefPubMedGoogle Scholar
  • 40 Hari M, Loganzo F, Annable T. et al. Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules. Mol Cancer Ther 2006; 5: 270-278
    CrossrefPubMedGoogle Scholar
  • 41 Alghasham A, Rasheed Z. Therapeutic targets for rheumatoid arthritis: Progress and promises. Autoimmunity 2014; 47: 77-94
    CrossrefPubMedGoogle Scholar
  • 42 Shetty A, Hanson R, Korsten P. et al. Tocilizumab in the treatment of rheumatoid arthritis and beyond. Drug Design Development and Therapy 2014; 8: 349-364
    PubMedGoogle Scholar
  • 43 Williams RO, Feldmann M, Maini RN. Cartilage destruction and bone erosion in arthritis: the role of tumour necrosis factor alpha. Ann Rheum Dis 2000; 59 (Suppl. 01) i75-i80
    CrossrefPubMedGoogle Scholar
  • 44 Moelants EA, Mortier A, Van Damme J. et al. Regulation of TNF-alpha with a focus on rheumatoid arthritis. Immunol Cell Biol 2013; 91: 393-401
    CrossrefPubMedGoogle Scholar
  • 45 Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004; 21: 467-476
    CrossrefPubMedGoogle Scholar
  • 46 Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008; 118: 3537-3545
    CrossrefPubMedGoogle Scholar
  • 47 Astry B, Harberts E, Moudgil KD. A cytokine-centric view of the pathogenesis and treatment of autoimmune arthritis. J Interferon Cytokine Res 2011; 31: 927-940
    CrossrefPubMedGoogle Scholar
  • 48 Gaffen SL, Jain R, Garg AV. et al. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol 2014; 14: 585-600
    CrossrefPubMedGoogle Scholar
  • 49 Lin CC, Edelson BT. New Insights into the Role of IL-1beta in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis. J Immunol 2017; 198: 4553-4560
    CrossrefPubMedGoogle Scholar
  • 50 Eastgate JA, Symons JA, Wood NC. et al. Correlation of plasma interleukin 1 levels with disease activity in rheumatoid arthritis. Lancet 1988; 2: 706-709
    PubMedGoogle Scholar
  • 51 Dhimolea E. Canakinumab. MAbs 2010; 2: 3-13
    CrossrefPubMedGoogle Scholar
  • 52 Jiang Y, Genant HK, Watt I. et al. A multicenter, double-blind, dose-ranging, randomized, placebo-controlled study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis: radiologic progression and correlation of Genant and Larsen scores. Arthritis Rheum 2000; 43: 1001-1009
    CrossrefPubMedGoogle Scholar
  • 53 Cohen S, Hurd E, Cush J. et al. Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin-1 receptor antagonist, in combination with methotrexate: results of a twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial. Arthritis Rheum 2002; 46: 614-624
    CrossrefPubMedGoogle Scholar
  • 54 Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol 2014; 6: a016295
    CrossrefPubMedGoogle Scholar
  • 55 Yao X, Huang J, Zhong H. et al. Targeting interleukin-6 in inflammatory autoimmune diseases and cancers. Pharmacol Ther 2014; 141: 125-139
    CrossrefPubMedGoogle Scholar
  • 56 Mimata Y, Kamataki A, Oikawa S. et al. Interleukin-6 upregulates expression of ADAMTS-4 in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Int J Rheum Dis 2012; 15: 36-44
    CrossrefPubMedGoogle Scholar
  • 57 Wang F, Wang L, Jiang H. et al. Inhibition of PCSK6 may play a protective role in the development of rheumatoid arthritis. J Rheumatol 2015; 42: 161-169
    PubMedGoogle Scholar
  • 58 Cantini F, Niccoli L, Goletti D. Tuberculosis risk in patients treated with non-anti-tumor necrosis factor-alpha (TNF-alpha) targeted biologics and recently licensed TNF-alpha inhibitors: data from clinical trials and national registries. J Rheumatol Suppl 2014; 91: 56-64
    CrossrefPubMedGoogle Scholar
  • 59 Krause A, Scaletta N, Ji JD. et al. Rheumatoid arthritis synoviocyte survival is dependent on Stat3. J Immunol 2002; 169: 6610-6616
    CrossrefPubMedGoogle Scholar
  • 60 Fischer SE. RNA Interference and MicroRNA-Mediated Silencing. Curr Protoc Mol Biol 2015; 112 26 21 21-25
    PubMedGoogle Scholar