Effect of oxidative stress on protein tyrosine phosphatase 1B in scleroderma dermal fibroblasts

Abstract

Objective: Platelet-derived growth factor (PDGF) and its receptor, PDGFR, promote fibrosis in systemic sclerosis (SSc; scleroderma) dermal fibroblasts, and such cells in scleroderma skin lesions produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and is dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. This study was undertaken to determine whether the thiol-sensitive PTP1B is affected by ROS in SSc dermal fibroblasts, thereby enhancing the phosphorylation of PDGFR and synthesis of type I collagen. This study also sought to investigate the effect of a thiol antioxidant, N-acetylcysteine (NAC), in SSc.

Methods: Fibroblasts were isolated from the skin of patients with diffuse SSc and normal healthy donors for cell culture experiments and immunofluorescence analyses. A phosphate release assay was used to determine the activity of PTP1B.

Results: Levels of ROS and type I collagen were significantly higher and amounts of free thiol were significantly lower in SSc fibroblasts compared to normal fibroblasts. After stimulation with PDGF, not only were PDGFR and ERK-1/2 phosphorylated to a greater extent, but also the ability to produce PTP1B was hampered in SSc fibroblasts. The activity of PTP1B was significantly inactivated in SSc fibroblasts as a result of cysteine oxidation by the raised levels of ROS, which was confirmed by the oxidation of multiple PTPs, including PTP1B, in SSc fibroblasts. Decreased expression of PTP1B in normal fibroblasts led to increased expression of type I collagen. Treatment of the cells with NAC restored the activity of PTP1B, improved the profile of PDGFR phosphorylation, decreased the numbers of tyrosine-phosphorylated proteins and levels of type I collagen, and scavenged ROS in SSc fibroblasts.

Conclusion: This study describes a new mechanism by which ROS may promote a profibrotic phenotype in SSc fibroblasts through the oxidative inactivation of PTP1B, leading to pronounced activation of PDGFR. The study also presents a novel molecular mechanism by which NAC may act on ROS and PTP1B to provide therapeutic benefit in SSc.

Figure 1

Production of O₂•⁻ in NL and SSc dermal fibroblasts by fluorescence microscopy. NL (top panels) and SSc dermal fibroblasts (bottom panels) were treated with DHE, a O₂•⁻ trap that releases red fluorescence when it encounters O₂•⁻ . Nuclei were counterstained with DAPI. Staining was detected using a fluorescence microscope and images were taken at 400x. A significant amount of O₂•⁻ was detected in SSc fibroblasts compared to NL (left panels) while in the presence of NAC, O₂•⁻ in SSc fibroblasts decreased (right panels). These representative images were from 3 NL and 3 SSc lines.

Figure 2

PDGF-stimulated PDGFR and protein phosphorylation in NL and SSc dermal fibroblasts. A. In NL cells, PDGF significantly stimulated PDGFR phosphorylation (p-PDGFR) at 10 and 30 min (p<0.05 vs. NL not-stimulated [NS]). In SSc fibroblasts, p-PDGFR increased significantly until 1 hr (p<0.05 vs. SSc NS). At 30min, the level of p-PDGFR in SSc dermal fibroblasts was significantly higher than in NL cells. B. In the presence of NAC, p-PDGFR occurred at 10 and 30 min in both NL and SSc dermal fibroblasts. PDGF significantly stimulated p-PDGFR at 10 and 30 min in SSc dermal fibroblasts (p<0.05 vs. SSc NS). The intensity of p-PDGFR was significantly lower in the presence of NAC in both NL and SSc dermal fibroblasts. C. In the absence of NAC, multiple proteins were phosphorylated in both NL and SSc dermal fibroblasts. At 1 hour phosphorylated proteins were still visible. PDGF induced more phosphorylated proteins in SSc than in NL cells. D. In the presence of NAC, PDGF stimulated protein tyrosine phosphorylation in both NL and SSc dermal fibroblasts, but with less intensity compared to those in Figure 2C. Results are expressed as mean ± S.E. and p<0.05 is considered significant. n≥3 NL subjects and patients.

Figure 3

ERK_1/2 phosphorylation patterns and Col I in NL and SSc dermal fibroblasts. A. In NL cells, PDGF significantly stimulated ERK_1/2 phosphorylation (p-ERK_1/2) at 45 min and 2 hr (p<0.05 vs. NL not-stimulated [NS]). In SSc fibroblasts, p-ERK_1/2 increased significantly from 10 min until 4 hr (p<0.05 vs. SSc NS). At 4 hr, the level of p-ERK_1/2 in SSc dermal fibroblasts was significantly higher than in NL cells. B. In the presence of NAC, although the basal level of p-ERK_1/2 increased slightly in both NL and SSc cells (p>0.05), PDGF stimulation did not result in significant p-ERK_1/2. C. Col I mRNA levels were significantly higher in SSc fibroblasts compared to NLs. NAC significantly decreased Col I mRNA levels in SSc cells, but had no significant effect in NL dermal fibroblasts. D. A significant amount of Col I was detected in SSc fibroblasts compared to NL. In the presence of NAC, Col I in SSc fibroblasts decreased. These representative images were from 3 NL and SSc lines.

Figure 4

Expression and enzymatic activity of PTP1B in NL and SSc dermal fibroblasts. A. PDGF-stimulated mRNA expression of PTP1B in NL and SSc dermal fibroblasts was not significantly different. B. At the protein level, PDGF stimulated PTP1B production was significantly higher at 45 min and 2 hr (p<0.05 vs. NS) in NL cells. However, in SSc cells, PDGF incubation did not alter PTP1B expression, and at 45 min there was a significant difference between NL and SSc (p<0.05). C. In SSc dermal fibroblasts, the PTP1B activity was significantly lower compared to NL (p<0.05). In the presence of NAC, the activity in SSc cells was restored. D. When PTP1B was significantly knocked down in NL dermal fibroblasts, Col I mRNA significantly increased (p<0.05), mimicking what was observed in SSc dermal fibroblasts. Results are expressed as mean ± S.E. and p<0.05 is considered significant. n≥4 NL subjects and patients.

Figure 5

Oxidation of PTPs in NL and SSc dermal fibroblasts. Oxidized PTPs in NL and SSc dermal fibroblasts were visualized using mouse anti-oxidized human PTP antibody after Western blotting. Equal amounts of protein were subjected to immunoprecipitation using rabbit anti-human PTP1B antibody. The blot was then probed with mouse anti-oxidized human PTP antibody. A. Dermal fibroblasts from 3 NL subjects and 5 SSc patients were used for this study. Overall, more PTPs were oxidized in SSc dermal fibroblasts compared to NLs. In NL cells, 2 bands were observed in all subjects though at different molecular weight. In SSc dermal fibroblasts, at least 3 PTPs were oxidized. The 50 kD protein, which we postulate as PTP1B, was oxidized in all SSc dermal fibroblasts. B. We confirmed that the 50 kD protein was indeed PTP1B by immunoprecipitation. There was significantly more oxidized PTP1B in SSc dermal fibroblasts compared to NL. Results are expressed as mean ± S.E. and p<0.05 is considered significant. n ≥ 3 NL subjects and patients.

Figure 6

Summary of our work. In NL dermal fibroblasts, adequate amounts of ROS perform their role in redox signaling and are not harmful in oxidizing cellular proteins. When PDGF binds to the PDGFR, the receptor is phosphorylated and downstream signaling pathways such as ERK_1/2 are activated, leading to a response such as collagen I production. PTP1B is active and controls the extent of phosphorylation on PDGFR. In SSc dermal fibroblasts, the increased production of ROS inactivates PTP1B through oxidation of the Cys residue in its active site. When PDGF binds to its receptor, due to PTP1B inactivation, prolonged PDGFR phosphorylation results in an increased downstream response. In the presence of the thiol antioxidant NAC, ROS decrease, the oxidized Cys residue on PTP1B is reduced, the activity is restored, and the response returns to baseline. P: tyrosine phosphorylation; -SH: free thiols; -SOxH: oxidized thiols; ROS: reactive oxygen species; NAC: n-acetylcysteine; : oxidation; : less oxidation; : dephosphorylation; : unable to dephosphorylate