Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.
Anti-U1C Autoantibody Specifically Interferes with the U1RNP-Mediated Splicing of Angiopoietin-1 (Ang1) Gene and Induces Functionally Defective Ang-1/ins Variant in the Patients with MCTD.
Kosugi1, Yuka, Komai1, Koichiro, Funaki1, Shogo, Mashida1, Aya, Shiozawa3, Kazuko, Hashiramoto2, Akira, Shiozawa2, Shunichi
Dep. of Biophysics, Graduate School of Health Sciences, Kobe Univ., Kobe, Japan
Dep. of Biophysics, Graduate School of Health Sciences, Kobe Univ./Dep. of Medicine, Graduate School of Medicine, Kobe Univ./The Center for Rheumatic Diseases, Kobe Univ., Hosp., Kobe, Japan
Rheumatic Diseases Center, Konan Kakogawa Hosp., Kakogawa, Japan
Angiopoietin-1 (Ang1) is the ligand of endothelial tyrosine kinase receptor Tie2 and induces cell proliferation via ERK phosphorylation. Ang1 is strongly up-regulated in the lung of patients with mixed connective tissue disease (MCTD) accompanied by pulmonary hypertension (PH) indicating that Ang1 can play a key role in the pathogenesis of PH associated with MCTD. We previously showed that the frequency of the splicing variant of Ang1 mRNA with nt805GGT insertion (Ang1/ins) encoding 269Gly was significantly increased in the Japanese patients with MCTD and those with PH as compared with Ang1/del with nt805GGT deletion (Arth. & Rheum. 52 (9): S283, '05). We also found that Ang1/ins dose proliferate pulmonary endothelial cells and potentially induce hyperplasia of smooth muscle cell in relation to the pathogenesis of PH as compared with Ang1/del (Arth. & Rheum. 58 (9): S659, '08 and 60 (10): S8, '09). On the other hand, anti-U1RNP antibodies (Abs), elevated in MCTD significantly induced Ang1/ins mRNA in Jurkat cells (Arth. & Rheum. 58 (9): S224, '08). We have now studied the contribution of subclass of Abs against U1A, U1C or U170K to the induction of Ang1/ins variant. We also evaluated the reactivity of anti-U1RNP auto-Abs against each components of U1RNP in sera of patients with MCTD.
Anti-U1A, U1C and U170K Abs (LifeSpan Biosc. and Bio Acad.) were introduced into human pulmonary artery smooth muscle cells (HPASMC) using PULS-in reagent (Polyplus transfection), followed by treatment with IL6 and TNFa (20ng/ml each) for 24 h. Ang1 mRNA was obtained by using RT-PCR and subjected to length analysis by using ABI3130xl sequencer. Expression levels of Ang1 variants were evaluated with calculated peak areas. Anti-U1RNP Ab titer in sera of Japanese patients with MCTD was determined by using Autoimmune EIA Test (BIO-RAD) and the Ang1 mRNA phenotype was determined. We also constructed ELISA by using recombinant U1A, U1C and U170K proteins (DIARECT), as coated antigen and Ab titers in sera of patients were determined.
Anti-U1C and U170K Abs significantly induced Ang1/ins mRNA with IL6 and TNFa (P<0.05). Otherwise, neither anti-U1A, U1C nor U170K Abs did alter the Ang1 splicing without cytokine. We also confirmed that Ang1/ins wasn't induced with cytokine. Frequency of the patients with MCTD having anti-U1RNP Ab titer exceeding 200 was 81.3% (39/48) and 50.0% (6/12) (P<0.05) among those with Ang1/ins dominant and Ang1/del dominant phenotypes, respectively. While comparison between Ang1/del and Ang1/ins dominant phenotypes wasn't possible because of 2 sera specimens of those Ang1/del dominant phenotype, the titer of each subclass of anti-U1RNP Ab in the sera of patients with MCTD among those with Ang1/hetero (n=13) and Ang1/ins dominant phenotypes (n=18) was: 11.8±23.6 and 47±58.9 (P<0.05) for anti-U1A Ab, 13.6±25.7 and 122.1±124.5 (P<0.005); for anti-U1C Ab, 406.2±599.5 and 5512.9±12484.7 (not significant); for anti-U170K Ab.
It is suggested that anti-U1RNP Ab, especially those reactive with U1C affect the splicing of Ang1 with cytokines and induce functionally defective Ang1/ins in the patients with MCTD.
To cite this abstract, please use the following information:
Kosugi, Yuka, Komai, Koichiro, Funaki, Shogo, Mashida, Aya, Shiozawa, Kazuko, Hashiramoto, Akira, et al; Anti-U1C Autoantibody Specifically Interferes with the U1RNP-Mediated Splicing of Angiopoietin-1 (Ang1) Gene and Induces Functionally Defective Ang-1/ins Variant in the Patients with MCTD. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :893