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. 2019 Nov 1;317(5):L678-L689.
doi: 10.1152/ajplung.00264.2018. Epub 2019 Sep 4.

Role of dual-specificity protein phosphatase DUSP10/MKP-5 in pulmonary fibrosis

Nikos Xylourgidis  1 , Kisuk Min  2 , Farida Ahangari  1 , Guoying Yu  1 , Jose D Herazo-Maya  1 , Theodoros Karampitsakos  3 , Vassilis Aidinis  4 , Leonhard Binzenhöfer  1 , Demosthenes Bouros  3 , Anton M Bennett  5 , Naftali Kaminski  1 , Argyrios Tzouvelekis  1   4
Affiliations

Role of dual-specificity protein phosphatase DUSP10/MKP-5 in pulmonary fibrosis

Nikos Xylourgidis et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP-5) is a member of the dual-specificity family of protein tyrosine phosphatases that negatively regulates p38 MAPK and the JNK. MKP-5-deficient mice exhibit improved muscle repair and reduced fibrosis in an animal model of muscular dystrophy. Here, we asked whether the effects of MKP-5 on muscle fibrosis extend to other tissues. Using a bleomycin-induced model of pulmonary fibrosis, we found that MKP-5-deficient mice were protected from the development of lung fibrosis, expressed reduced levels of hydroxyproline and fibrogenic genes, and displayed marked polarization towards an M1-macrophage phenotype. We showed that the profibrogenic effects of the transforming growth factor-β1 (TGF-β1) were inhibited in MKP-5-deficient lung fibroblasts. MKP-5-deficient fibroblasts exhibited enhanced p38 MAPK activity, impaired Smad3 phosphorylation, increased Smad7 levels, and decreased expression of fibrogenic genes. Myofibroblast differentiation was attenuated in MKP-5-deficient fibroblasts. Finally, we found that MKP-5 expression was increased in idiopathic pulmonary fibrosis (IPF)-derived lung fibroblasts but not in whole IPF lungs. These data suggest that MKP-5 plays an essential role in promoting lung fibrosis. Our results couple MKP-5 with the TGF-β1 signaling machinery and imply that MKP-5 inhibition may serve as a therapeutic target for human lung fibrosis.

Keywords: MKP-5; fibroblast; homeostasis; kinase; phosphatase; pulmonary fibrosis.

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Conflict of interest statement

A. Tzouvelekis and N. Kaminski are inventors on a pending patent on use of the thyroid hormone as an antifibrotic agent entitled “Novel Methods of Treating or Preventing Fibrotic Lung Diseases” [OCR 6368-047162-7029P1 (00219)]. A. Tzouvelekis consulted for Boehringer Ingelheim and Hoffmann LaRoche. N. Kaminski consulted for Biogen Idec, Boehringer Ingelheim, Numedii, MMI, and Pliant and has an ongoing collaboration with MiRagen, all outside the submitted work. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

Figures

Fig. 1.
Fig. 1.
MAPK phosphatase 5 (MKP-5) knockout (MKP-5−/−) mice display attenuated fibrotic responses to bleomycin (Bleo) challenge. A: decreased collagen deposition, assessed by hydroxyproline content, following bleomycin challenge at day 14 in MKP-5−/− mice compared with wild-type littermates (MKP-5+/+; 116.14 ± 27.99 vs. 202.25 ± 45.98). Data (μg/mg right lung) are presented as box and whisker plots, with horizontal bars representing means ± SD, n = 8–15 mice from each group-treated animals. One-way ANOVA with Sidak correction for stepwise comparison, ****P < 0.0001. B: relative changes following bleomycin challenge at day 14 in dual-specificity protein tyrosine phosphatase 10 (DUSP10)/MKP-5, α-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), collagen 3A1 (COL3A1), and fibronectin (FN1) mRNA expression levels. Each bar represents mean ± SD expression of n = 5/10/7/9 mice from each group (biological replicates). Bars are shown for the relative changes (fold) by setting the indicated control level to 1.0. One-way ANOVA with Sidak correction for stepwise comparison, *P < 0.05, **P < 0.005, ***P < 0.0002, ****P < 0.0001. C: decreased collagen deposition, assessed by hydroxyproline content, following bleomycin challenge at day 21 in MKP-5−/− mice compared with MKP-5+/+ (93.23 ± 10.38 vs. 126.67 ± 29.73). Data (micrograms/milligrams right lung) are presented as box and whisker plots, with bars representing means ± SD, n = 7 for MKP-5+/+ and n = 4 for MKP-5−/− mice group-treated animals. One-way ANOVA with Sidak correction for stepwise comparison, *P < 0.03. D: relative changes following bleomycin challenge at day 21 in DUSP10/MKP-5, α-SMA, COL1A1, COL3A1, and FN1 mRNA expression levels. Each bar represents mean ± SD expression of n = 3 from each group sample (biological replicates). Bars are shown for the relative changes (fold) by setting the indicated control level to 1.0. One-way ANOVA with Sidak correction for stepwise comparison, *P < 0.05, **P < 0.005, ***P < 0.0008. E: Masson’s trichrome (top) and α-SMA (bottom) staining of representative lung sections (n = 4) from each group of treated mice. Decreased collagen deposition and α-SMA staining are observed in the interstitium, peribronchiolar or perivascular space, in MKP-5−/− mice compared with MKP-5+/+ mice. Scale bars, 100 μΜ. F: relative changes following bleomycin challenge at day 14 of M1/M2 macrophage markers. mRNA expression of nitric oxidase synthase-2 (Nos2) and resistin-like molecule-α (Fizz1), arginase 1 (Arg1), c-type mannose receptor 1 (Mrc1), and chitinase-like 3 protein 1 (Chil1), following challenge with bleomycin. Each bar represents mean ± SD expression of n = 3 from each group samples (biological replicates). Bars are shown for the relative changes (fold) by setting the indicated control level to 1.0. One-way ANOVA with Sidak correction for stepwise comparison, *P < 0.05, **P < 0.002, ***P < 0.001.
Fig. 2.
Fig. 2.
MAPK phosphatase 5 (MKP-5) inhibition attenuates fibroblast-to-myofibroblast differentiation and migration and increases proliferation of mouse lung fibroblasts. Primary mouse lung fibroblasts were isolated from C57BL/6, 9- to 12-week-old mice. MKP-5 knockout mice (MKP-5−/−) or wild-type mice (MKP-5+/+) were stimulated with transforming growth factor β1 (TGF-β1; 10 ng/mL) and then harvested for RNA and protein extraction. Normal human lung fibroblasts were transfected at 70% confluency with a specific dual-specificity protein tyrosine phosphatase 10/MKP-5 siRNA (10 nM) and a nontargeting siRNA (10 nM) as a control for 24 h. A: relative change in α-smooth muscle actin (α-SMA), collagen 1A1 (COL1A1), collagen 3A1 (COL3A1), and fibronectin (FN1) mRNA levels following stimulation with TGF-β1 or PBS. Each bar represents mean expression of 3 samples (in duplicates). Bars (means ± SD) are shown for the relative changes (fold) by setting the indicated control level to 1.0. One-way ANOVA with Sidak correction for stepwise comparison, *P < 0.02, **P < 0.004, ****P < 0.0001. B: immunoblot analyses of α-SMA and β-actin. Each lane represents an individual mouse lung fibroblast preparation (biological replicates). Blots were stripped and reblotted using an anti-β-actin antibody as loading control. Ratio indicates protein densitometry ratio of the target/control protein. C: MKP-5−/− mouse lung fibroblasts exhibited increased proliferation rates following stimulation with PDGF-BB (25 ng/mL) for 6 h compared with wild type (MKP-5+/+). Data analyzed at 24 h post-treatment (ratio of 24 h/0 hr absorbance) are presented as box–whisker plots with bars representing means ± SD, n = 20. One-way ANOVA with Sidak correction for stepwise comparison, **P < 0.085, ****P < 0.0001. D: scratch wound assay was used to quantify the migration capacity of MKP-5+/+ and MKP-5−/− fibroblasts at different time points: baseline (t0h) and at 24 h (t24h). The areas between the dotted lines define the migratory boundary. Bottom: bar plot depicts quantitation of percentage of actual distance covered. E: double immunofluorescence analysis showing colocalization (merged/yellow) of α-SMA (red) with F-actin (green) in MKP-5+/+ and MKP-5−/− mouse lung fibroblasts following stimulation with TGF-β1 or PBS. MKP-5−/− mouse lung fibroblasts exhibited reduced TGF-β1-induced differentiation to myofibroblasts, as indicated by reduced formation of stress fibers and α-SMA expression compared with MKP-5+/+. Scale bars, 50 μΜ.
Fig. 3.
Fig. 3.
MAPK phosphatase 5 (MKP-5) inhibition negatively regulates transforming growth factor β1 (TGF-β1)-induced Smad3 fibrotic pathway. Primary mouse lung fibroblasts were isolated from C57BL/6, 9- to 12-week-old female mice in MKP-5 knockout (MKP-5−/−) or wild-type mice (MKP-5+/+), which were then stimulated with TGF-β1 (10 ng/mL) for 5, 30, and 60 min and then harvested for protein extraction and immunoblot analysis. Normal human lung fibroblasts (NHLFs) were transfected at 70% confluency with dual-specificity protein tyrosine phosphatase 10 (DUSP10)/MKP-5 siRNA (10 nM) for 24 h or a control nontargeting siRNA (10 nM). Cells were stimulated with TGF-β1 (10 ng/mL) for 5, 30, and 60 min. A: immunoblot analyses for phosphorylated (p-)MAPKs (p-JNK, p-ERK1/2, and p-p38 MAPK), along with their respective MAPK totals at the indicated time points of primary mouse lung fibroblast preparation from MKP-5+/+ and MKP-5−/− mice. B: immunoblot analyses for MKP-5 expression following transfection with DUSP10/MKP-5 siRNA (siMKP-5; 10 nM) or a nontargeting siRNA for 24 h (10 nM). Each lane represents an individual NHLF preparation. C: the same cell lysates were immunoblotted and analyzed for p-Smad3 and total Smad3 following TGF-β1 stimulation. D: immunoblot analysis for p-Smad3 and total Smad3 following TGF-β1 stimulation in primary lung fibroblasts from MKP-5+/+ and MKP-5−/− mice. E: immunoblot analysis for Smad7 in primary lung fibroblasts from MKP-5+/+ and MKP-5−/− fibroblasts. Ratio indicates protein densitometry ratio of the target/control protein.
Fig. 4.
Fig. 4.
MAPK phosphatase 5 (MKP-5) expression is increased in idiopathic pulmonary fibrosis (IPF) lung fibroblasts but not in whole IPF lungs. A: relative change in MKP-5 mRNA levels in whole lung IPF. Each bar represents the mean expression of 3 samples (in triplicates). Bars (means ± SD) are shown for the relative fold changes by setting the indicated control (CTRL) level to 1.0. Mann-Whitney U-test for independent samples (left). B: immunoblot analysis showing MKP-5 protein expression in IPF lungs compared with controls vs. β-actin protein levels and corresponding relative densitometry. Ratio indicates ratio values of the target/control protein (left); box plots (right) summarize all 4 experimental samples of each group. NS, not significant. C: relative change in MKP-5 mRNA levels in IPF lung fibroblasts compared with normal human lung fibroblasts (NHLFs). Each bar represents mean expression of 3 samples (in triplicates). Bars (means ± SD) are shown for the relative changes (fold) by setting the indicated control level to 1.0. Mann-Whitney U-test for independent samples, *P < 0.05 (left). D: immunoblot analysis showing protein levels of MKP-5 in IPF lung fibroblasts compared with NHLF and corresponding relative densitometry. β-Actin protein levels serve as loading control. Ratio indicates ratio values of the target/control protein (left); box plots (right) summarize all 4 experimental samples of each group. *P < 0.05. E: immunohistochemistry analysis in representative lung samples (n = 5) showing increased MKP-5 expression within the fibrotic interstitium, as well as the surrounding alveolar epithelium, in IPF lungs compared with normal alveolar epithelium in control lung samples. Boxed regions are enlarged at right and as insets on the left. A nonimmune immunoglobulin of the same isotype and concentration as the primary antibody was used as negative control. Arrows indicate alveolar epithelial cells with minimal expression of MKP-5 (blue color). Arrowheads indicate spindle-shaped, fibroblast-like cells within fibroblastic foci in the fibrotic lung stained positive for MKP-5 (brown color).
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