Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement
Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.
Oxidation of Protein Tyrosine Phosphatase-1B Leads to Pronounced PDGFR Activation in Scleroderma Fibroblasts.
Tsou1, Pei-Suen, Pinney1, Adam J., Talia1, Nadin N., Piera-Velazquez2, Sonsoles, Jimenez3, Sergio A., Seibold4, James R., Phillips1, Kristine
University of Michigan Medical School, Ann Arbor, MI
Thomas Jefferson University, Philadelphia, PA
Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA
Scleroderma Research Consultants LLC, Avon, CT
Department of Veteran's Affairs and University of Michigan, Ann Arbor, MI
Skin fibrosis is a main characteristic of systemic sclerosis (SSc). Platelet-derived growth factor (PDGF) and its receptor (PDGFR) have been shown to play key roles in promoting fibrosis in SSc. Upon PDGF stimulation, the PDGFR is phosphorylated (p-PDGFR), and its downstream signaling pathways, such as ERK1/2, are activated. The PDGFR is dephosphorylated by phosphatases, including protein tyrosine phosphatase 1B (PTP1B), and the signaling cascade is hence terminated. We previously following PDGF stimulation, ERK1/2 was phosphorylated (p-ERK1/2) to a greater extent in SSc dermal fibroblasts. Moreover, increased oxidative stress was observed in SSc cells compared to normal (NL) fibroblasts, and PTP1B was inactivated. In this study we hypothesize that the p-PDGFR profile in SSc fibroblasts is affected by the inactive PTP1B, and the mechanism of PTP1B inactivation is due to oxidation of its active site. The effect of the thiol antioxidant n-acetylcysteine (NAC) on p-PDGFR, p-ERK1/2, tyrosine-phosphorylated proteins, collagen I (Col I) production, and PTP1B mRNA expression was investigated.
SSc and NL dermal fibroblasts were isolated from skin biopsies. Cellular thiol content was measured by using 5,5'-dithiobis (2-nitrobenzoic acid). Tyrosine phosphorylated proteins were immunoprecipitated, and p-PDGFR was probed with rabbit anti-human PDGFR antibodies. Oxidized PTPs and p-ERK1/2 were measured by Western blotting. Col I was detected by immunofluorescence and quantitative PCR.
Col I expression was significantly higher in SSc fibroblasts, accompanied by significantly lower amounts of free thiol compared to NL fibroblasts (464 ± 18 in NL vs. 344 ± 15 mM/mg protein in SSc cells, n=3, p<0.01). After PDGF stimulation, multiple proteins including the PDGFR, were phosphorylated to a greater extent in SSc fibroblasts. Moreover, the significantly lower PTP1B activity in SSc fibroblasts resulted from cysteine oxidation at its active site by higher levels of ROS, since oxidation of multiple PTPs, including PTP1B, was observed. NAC improved the profile of p-PDGFR and p-ERK1/2, decreased the number of tyrosine-phosphorylated proteins, and decreased both Col I protein and mRNA expression. The PTP1B mRNA level did not change in the presence of NAC in NL fibroblasts when stimulated with PDGF, but PTP1B mRNA significantly increased in SSc fibroblasts after 2 hr of PDGF stimulation (0.0023 ± 0.0005 vs. 0.0036 ± 0.0002 arbitrary unit in the absence or presence of NAC, p<0.05, n>=5).
The inactivation of PTP1B was caused by oxidation of its active site due to the excess oxidative stress in SSc fibroblasts, as indicated by the lower cellular free thiol levels. This led to pronounced and prolonged activation of the PDGFR and ERK1/2 pathways and therefore the increase in Col I production. NAC treatment, by reducing oxidative stress and restoring the low PTP1B activity and expression, decreased Col I production in SSc dermal fibroblasts. We introduce a new mechanism by which ROS promote a profibrotic phenotype in SSc fibroblasts through oxidative inactivation of PTP1B leading to pronounced PDGFR activation and thus increased Col I production.
To cite this abstract, please use the following information:
Tsou, Pei-Suen, Pinney, Adam J., Talia, Nadin N., Piera-Velazquez, Sonsoles, Jimenez, Sergio A., Seibold, James R., et al; Oxidation of Protein Tyrosine Phosphatase-1B Leads to Pronounced PDGFR Activation in Scleroderma Fibroblasts. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :2317