SH2 Domain-Containing Phosphatase-2 Is a Novel Antifibrotic Regulator in Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease with dismal prognosis and no cure. The potential role of the ubiquitously expressed SH2 domain-containing tyrosine phosphatase-2 (SHP2) as a therapeutic target has not been studied in IPF. OBJECTIVES: To determine the expression, mechanistic role, and potential therapeutic usefulness of SHP2 in pulmonary fibrosis. METHODS: The effects of SHP2 overexpression and inhibition on fibroblast response to profibrotic stimuli were analyzed in vitro in primary human and mouse lung fibroblasts. In vivo therapeutic effects were assessed in the bleomycin model of lung fibrosis by SHP2-lentiviral administration and transgenic mice carrying a constitutively active SHP2 mutation. MEASUREMENTS AND MAIN RESULTS: SHP2 was down-regulated in lungs and lung fibroblasts obtained from patients with IPF. Immunolocalization studies revealed that SHP2 was absent within fibroblastic foci. Loss of SHP2 expression or activity was sufficient to induce fibroblast-to-myofibroblast differentiation in primary human lung fibroblasts. Overexpression of constitutively active SHP2 reduced the responsiveness of fibroblasts to profibrotic stimuli, including significant reductions in cell survival and myofibroblast differentiation. SHP2 effects were mediated through deactivation of fibrosis-relevant tyrosine kinase and serine/threonine kinase signaling pathways. Mice carrying the Noonan syndrome-associated gain-of-function SHP2 mutation (SHP2D61G/+) were resistant to bleomycin-induced pulmonary fibrosis. Restoration of SHP2 levels in vivo through lentiviral delivery blunted bleomycin-induced pulmonary fibrosis. CONCLUSIONS: Our data suggest that SHP2 is an important regulator of fibroblast differentiation, and its loss as observed in IPF facilitates profibrotic phenotypic changes. Augmentation of SHP2 activity or expression should be investigated as a novel therapeutic strategy for IPF.

SH2 Domain-Containing Phosphatase-SHP2 Attenuates Fibrotic Responses through Negative Regulation of Mitochondrial Metabolism in Lung Fibroblasts.

BACKGROUND: We have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence. OBJECTIVE: To determine the effect of SHP2 on fibrotic responses. METHODS AND RESULTS: Primary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy. CONCLUSIONS: SHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases.

Monitoring priority substances of directives 76/464/EEC and 2000/60/EC in Greek water bodies.

The priority substances of List I, 76/464/EEC Directive, some of which belong to the new Water Framework Directive 2000/60/EC, have been monitored in the surface waters of Greece through the developed network of 53 sampling stations. The analytical methods used for the determination of these substances included Purge and Trap-Gas chromatography-Mass spectrometry for volatile and semivolatile organic compounds, Gas Chromatography-Electron Capture Detection for organochlorine insecticides, High Performance Liquid Chromatography for pentachlorophenol and Atomic Absorption Spectrometry for metals. The results have shown the presence of several priority substances in Greek surface waters, in most cases at concentrations well below the regulatory limits. However, non-compliance was observed for a limited number of compounds. The monitoring network and the analytical determinations have to be expanded to more water bodies and more priority substances, in order to safeguard the quality of Greek surface waters.