Protein tyrosine phosphatase α mediates profibrotic signaling in lung fibroblasts through TGF-β responsiveness

Abstract

Fibrotic lung diseases represent a diverse group of progressive and often fatal disorders with limited treatment options. Although the pathogenesis of these conditions remains incompletely understood, receptor type protein tyrosine phosphatase α (PTP-α encoded by PTPRA) has emerged as a key regulator of fibroblast signaling. We previously reported that PTP-α regulates cellular responses to cytokines and growth factors through integrin-mediated signaling and that PTP-α promotes fibroblast expression of matrix metalloproteinase 3, a matrix-degrading proteinase linked to pulmonary fibrosis. Here, we sought to determine more directly the role of PTP-α in pulmonary fibrosis. Mice genetically deficient in PTP-α (Ptpra(-/-)) were protected from pulmonary fibrosis induced by intratracheal bleomycin, with minimal alterations in the early inflammatory response or production of TGF-β. Ptpra(-/-) mice were also protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-β1. In reciprocal bone marrow chimera experiments, the protective phenotype tracked with lung parenchymal cells but not bone marrow-derived cells. Because fibroblasts are key contributors to tissue fibrosis, we compared profibrotic responses in wild-type and Ptpra(-/-) mouse embryonic and lung fibroblasts. Ptpra(-/-) fibroblasts exhibited hyporesponsiveness to TGF-β, manifested by diminished expression of αSMA, EDA-fibronectin, collagen 1A, and CTGF. Ptpra(-/-) fibroblasts exhibited markedly attenuated TGF-β-induced Smad2/3 transcriptional activity. We conclude that PTP-α promotes profibrotic signaling pathways in fibroblasts through control of cellular responsiveness to TGF-β.

Figure 1

Genetic deletion of PTP-α protects mice from bleomycin-induced fibrosis. A: Sircol assay of lung collagen content illustrates that bleomycin-treated Ptpra^−/− mice develop significantly less collagen deposition than bleomycin-treated WT controls at 21 days. B: Representative lung sections from Ptpra^−/− and WT mice at day 21 after administration of bleomycin. Lung mechanics were measured in anesthetized and mechanically ventilated mice at baseline and at 2, 7, and 21 days after administration of saline or 1.5 to 2.5 U/kg bleomycin. C: Static compliance was determined by fitting the Salazar–Knowles equation to pressure–volume curves. Data are expressed as means ± SEM. ^∗∗P < 0.01. Original magnification, ×10. Scale bar = 100 μm. Bleo, bleomycin; +/+, WT; -/-, Ptpra^−/−.

Figure 2

WT and Ptpra^−/− mice exhibit similar recruitment of inflammatory cells and production of cytokines in response to bleomycin. A: BAL fluid was collected at baseline (day 0) and at 2, 7, and 21 days after bleomycin instillation. Total cell counts and neutrophil counts were determined by differential cell counting. B: Meso Scale assay for BAL fluid for proinflammatory cytokines IL-1β and keratinocyte chemokine at 14 and 48 hours after bleomycin administration. C: Total TGF-β concentration in BAL fluid determined by ELISA at 7 days after bleomycin instillation. D: Levels of BAL matrix metalloproteinase MMP-3 as measured by ELISA at baseline and at 2 and 21 days after bleomycin treatment. Data are expressed as means ± SEM. ^∗P < 0.05.

Figure 3

Ptpra^−/− mice are protected from pulmonary fibrosis induced by adenoviral-mediated expression of active TGF-β. A: Representative lung sections from WT and Ptpra^−/− mice treated with active vector (AdTGF-β), empty vector (AdDL70), or saline control stained with trichrome. B: Collagen content in the lungs of WT or Ptpra^−/− mice was determined 21 days after treatment using a Sircol assay. Data are expressed as means ± SEM. ^∗P < 0.05. Original magnification, ×40. Scale bar = 200 μm.

Figure 4

Protection from pulmonary fibrosis is mediated by absence of PTP-α in resident lung parenchymal cells. A: Compliance measurements obtained via FlexiVent ventilation after reciprocal bone marrow transplantation and treatment with 1.5 to 2.5 U/kg bleomycin for 21 days. B: Representative stained lung sections of mice undergoing reciprocal bone marrow transplantation 14 days after administration of bleomycin. C: A Sircol assay was performed to determine collagen content after reciprocal bone marrow transplantation and treatment with bleomycin for 21 days. Data are expressed as means ± SEM. ^∗P < 0.05. Original magnification, ×40. Scale bar = 200 μm.

Figure 5

Ptpra^−/− fibroblasts exhibit diminished responsiveness to profibrotic TGF-β signals. A and B: After stimulation with 2 ng/mL TGF-β or buffer control for 6 hours, WT and Ptpra^−/− mouse embryonic fibroblasts (A) and primary mouse lung fibroblasts (B) were analyzed by qPCR to assess mRNA expression of EDA-fibronectin, αSMA, collagen 1A, and CTGF. Data are expressed as means ± SEM.^∗P < 0.05; ^∗∗P < 0.01.

Figure 6

TGF-β receptor type I and II expression is similar in WT and Ptpra^−/− fibroblasts. Primary mouse lung fibroblasts were isolated and qPCR analysis performed for TGFβRI and TGFβRII expression. Data are expressed as means ± SEM.

Figure 7

Ptpra^−/− fibroblasts exhibit attenuated contractile responses to TGF-β. Mouse lung fibroblasts were cast into collagen gels and allowed to contract over 5 days. Images were captured every 24 hours, and gel area was quantified using ImageJ software. ^∗P < 0.05.

Figure 8

Smad reporter activity is attenuated in Ptpra^−/− fibroblasts. A and B: WT and Ptpra^−/− mouse embryonic fibroblasts were transfected with Smad2 or Smad3 luciferase reporter constructs for 24 hours and then were incubated in the absence or presence of 2 ng/mL TGF-β for 16 hours. Lysates were assayed for luciferase activity. Both Smad2 (A) and Smad3 (B) reporter activity were significantly reduced in Ptpra^−/− fibroblasts, compared with WT. C: NIH 3T3 fibroblasts were reverse-transfected with control or PTP-α siRNA, followed by transfection with Smad3 luciferase reporter construct for 24 hours. Cells were then stimulated with 2 ng/mL TGF-β for 16 hours or with buffer medium. Luciferase activity in lysates was normalized to control siRNA treated with buffer. Data are expressed as means ± SEM. ^∗P < 0.05.