Effect of Oxidative Stress on Protein Tyrosine Phosphatase-1B in Scleroderma Dermal Fibroblasts

Scleroderma (SSc) is characterized by Raynaud’s phenomenon, proliferative vascular lesions, and fibrosis of the skin and internal organs. The exact mechanism of tissue scarring is still unknown, however, the abnormal phenotype has been suggested to be due to excessive production of extracellular matrixes, such as collagen I (Col I), by fibroblasts (1). These cells proliferate rapidly, stain positive for α-smooth muscle actin, and therefore possess a myofibroblast phenotype (2). Platelet-derived growth factor (PDGF) and its receptor (PDGFR) have been suggested as key elements in fibrosis. Increased PDGF activity was observed in plasma-derived serum from patients with progressive SSc (3). Immunohistological study revealed increased expression of PDGF and PDGFR in SSc skin biopsies (4, 5). Cytokines such as transforming growth factor β (TGFβ) and interleukin-1α stimulate PDGFR expression and PDGF production in SSc fibroblasts, and appear to be critical for promoting fibrosis in SSc fibroblasts (6, 7). PDGF stimulates the Ras- ERK1/2- reactive oxygen species (ROS) signaling pathway and results in stimulation of Col gene expression (8). All these studies point to the importance of PDGFR signaling in the development of skin lesions in SSc. Reactive oxygen species such as superoxide (O2•−) act as messengers to maintain cellular functions through oxidation-reduction reaction (redox) signaling (9). However, when the production of ROS exceeds the cellular antioxidant capacity and disturbs the balance of the normal redox state, this can lead to oxidative damage of proteins, lipids, and DNA. Increased oxidative damage by ROS has been suggested in the pathogenesis of SSc (8, 10–14). The imbalance of the redox state can cause endothelial dysfunction and injury (15), fibroblast proliferation (16), increased Col synthesis (11), and favoring autoantibody production through a Th2 profile (17); all major characteristics of SSc. Free radical generators such as bleomycin can result in skin fibrosis similar to SSc (18), and in fact, bleomycin injected into mouse skin serves as an animal model of SSc (19). Recently an animal model for SSc has been established by injecting different forms of ROS subcutaneously (20). N-acetylcysteine (NAC), a thiol antioxidant, decreases O2•− production in vitro (11), and shows promising results in alleviating SSc symptoms (14, 21–23). NAC is also used as a treatment for idiopathic pulmonary fibrosis (24). These studies imply that there can be a thiol-oxidation mechanism involved in SSc oxidative stress, since NAC not only acts as a free radical scavenger (25), but also acts specifically on protein thiols to support glutathione synthesis (26) and generate free sulfhydryl groups (27). The protein tyrosine phosphatase (PTP) family is a critical regulator of a variety of cellular signaling pathways including the PDGFR. These PTPs act by dephosphorylating the activation signals elicited by protein tyrosine kinases. PTPs are characterized by their signature motif, HC(X)5R, which contains a cysteine (Cys) residue that is essential for their catalytic activity. The low pKa of this Cys residue allows it to function as a nucleophile, but also makes it susceptible to oxidation. Several studies have shown that the Cys residue is oxidized in the presence of ROS for various PTPs (28, 29). These observations imply that the increased ROS in SSc can oxidize PTPs that are involved in the PDGFR signaling pathway. In this study, we examined the effect of excessive oxidative stress on PTP1B, a phosphatase that has been shown to dephosphorylate the PDGFR (30, 31). The effect of the thiol antioxidant, NAC, on cellular ROS production, PDGFR phosphorylation, tyrosine-phosphorylated proteins, and PTP1B activity was also investigated.