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.
The Effect of Oxidative Stress on Protein Tyrosine Phosphatase 1B in Scleroderma Fibroblasts.
Tsou5, Pei-Suen, Talia6, Nadin N., Piera2, Sonsoles, Jimenez1, Sergio A., Seibold3, James R., Phillips4, Kristine, Koch7, Alisa E.
Thomas Jefferson Univ, Philadelphia, PA
Thomas Jefferson University, Philadelphia, PA
University of Connecticut Health Center, Farmington, CT
University of Michigan, Ann Arbor, MI
University of Michigan Medical School, Ann Arbor, MI
University of Michigan Medical School
Unversity 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, and its downstream signaling pathways, including ERK1/2, are activated. The PDGFR is dephosphorylated by phosphatases, including protein tyrosine phosphatase 1B (PTP1B), and the signaling cascade is hence terminated. In addition, increased superoxide production is observed in SSc dermal fibroblasts compared to normal fibroblasts (NL). In this study we sought to determine whether the thiol-sensitive PTP1B is affected by oxidative stress in these cells, enhancing the ERK1/2 signaling pathway of PDGFR. The effect of the thiol antioxidant n-acetylcysteine (NAC) on PTP1B activity was also investigated.
SSc and NL fibroblasts were isolated from skin biopsies. Cells were stimulated with 30 ng/ml PDGF and phosphorylation of ERK1/2 as well as PTP1B levels were measured by Western blotting. A phosphate release assay was used to determine PTP1B activity. Superoxide was measured by dihydroethidium.
Activation of PDGFR by PDGF resulted in phosphorylation of ERK1/2. In NL fibroblasts, ERK1/2 phosphorylation was maximal at 45 min (3 fold increase vs. unstimulated [US], p<0.05, n=5) and returned to baseline at 4 hr. In contrast, in SSc cells, ERK1/2 was significantly phosphorylated at 10 min and remained phosphorylated at 4 hr (2 fold increase vs. US, p<0.05). PDGF increased PTP1B protein expression significantly at 45 min and 2 hr in NL. In contrast, PTP1B expression in SSc fibroblasts remained the same, and at 45 min the level of PTP1B in NLs was significantly higher than that in SSc (p<0.05). PTP1B activities were 20.1 ± 2.7 and 11.9 ± 1.3 nmoles (phosphate released, p<0.05) in NL and SSc fibroblasts, respectively. In the presence of NAC, PTP1B activity was restored in SSc fibroblasts (from 11.9 ± 1.3 nmoles without NAC to 19.2 ± 2.1 nmoles with NAC, p<0.05). In contrast, the activity of PTP1B was unaffected with NAC treatment in NLs (20.1 ± 2.7 nmoles without NAC vs. 16.2 ± 0.4 nmoles with NAC, p>0.05). Superoxide was significantly higher in SSc than in NL dermal fibroblasts, and the level in SSc cells was reduced significantly after incubation with NAC (p<0.05).
The profile of ERK1/2 phosphorylation, which indicated PDGF-induced PDGFR activation, was different in NL and SSc dermal fibroblasts. The inability to dephosphorylate ERK1/2 in SSc fibroblasts suggests that the phosphatases that are responsible for ERK1/2 dephosphorylation are deficient in SSc. The ability to produce PTP1B after PDGF stimulation, and hence terminate PDGFR signaling, was hampered in SSc dermal fibroblasts. PTP1B activity was significantly inactivated in SSc fibroblasts, which may have resulted from higher levels of superoxide than in NL. NAC treatment both restored the low PTP1B activity and decreased superoxide levels in SSc dermal fibroblasts. Thus, we introduce a new class of proteins dysregulated in SSc. Our study also provides a novel molecular mechanism by which NAC therapy may act on these proteins to benefit SSc patients.
To cite this abstract, please use the following information:
Tsou, Pei-Suen, Talia, Nadin N., Piera, Sonsoles, Jimenez, Sergio A., Seibold, James R., Phillips, Kristine, et al; The Effect of Oxidative Stress on Protein Tyrosine Phosphatase 1B in Scleroderma Fibroblasts. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2007