Transglutaminase 2 and its role in pulmonary fibrosis

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a deadly progressive disease with few treatment options. Transglutaminase 2 (TG2) is a multifunctional protein, but its function in pulmonary fibrosis is unknown.

Objectives: To determine the role of TG2 in pulmonary fibrosis.

Methods: The fibrotic response to bleomycin was compared between wild-type and TG2 knockout mice. Transglutaminase and transglutaminase-catalyzed isopeptide bond expression was examined in formalin-fixed human lung biopsy sections by immunohistochemistry from patients with IPF. In addition, primary human lung fibroblasts were used to study TG2 function in vitro.

Measurements and main results: TG2 knockout mice developed significantly reduced fibrosis compared with wild-type mice as determined by hydroxyproline content and histologic fibrosis score (P < 0.05). TG2 expression and activity are increased in lung biopsy sections in humans with IPF compared with normal control subjects. In vitro overexpression of TG2 led to increased fibronectin deposition, whereas transglutaminase knockdown led to defects in contraction and adhesion. The profibrotic cytokine transforming growth factor-β causes an increase in membrane-localized TG2, increasing its enzymatic activity.

Conclusions: TG2 is involved in pulmonary fibrosis in a mouse model and in human disease and is important in normal fibroblast function. With continued research on TG2, it may offer a new therapeutic target.

Figure 1.

Transglutaminase 2 (TG2) is increased in the lungs of mice treated with bleomycin (BLEO). C57BL/6 mice were given 2.5 U/kg bleomycin by oropharyngeal aspiration. Twenty-one days after treatment the left lung was inflated and fixed in formalin. Lung sections were stained with an antibody to TG2 and developed with Nova Red. Scale bars = 50 μm. Sections shown are representative of four mice per group. PBS = phosphate-buffered saline.

Figure 2.

Transglutaminase 2 knockout mice (Tgm2^−/−) develop reduced fibrosis after bleomycin (BLEO) challenge. Male 8-week-old Tgm2^−/− mice or C57BL/6 control mice were treated with 2.5 U/kg bleomycin. Twenty-one days after bleomycin administration, the fibrotic response was analyzed. (A) Representative hematoxylin and eosin–stained formalin-fixed paraffin-embedded sections from the left lung; scale bar equals 200 μm. (B) Representative sections were stained with Gomori Trichrome. Collagen appears as blue. Scale bar = 50 μm. PBS = phosphate-buffered saline.

Figure 3.

Transglutaminase 2 knockout mice have reduced histologic fibrosis score, hydroxyproline, and fibrosis-associated genes after bleomycin challenge. Mice were treated as in Figure 2. (A) Fibrosis was scored on hematoxylin and eosin and trichrome sections from 0 to 8 using an Aschroft scoring system. (B) Hydroxyproline content was measured in the right side of the lung. One representative experiment of three performed at different doses of bleomycin is shown. mRNA was isolated from the lungs, analyzed for (C) fibronectin (FN1) and (D) collagen 1 (Col1a1), and normalized to 18s RNA. n = 4 control groups, n = 7 bleomycin groups, error bars represent mean ± SEM; *P < 0.05 by analysis of variance (A, C, D) or Mann Whitney Student t test (B). PBS = phosphate-buffered saline.

Figure 4.

Transglutaminase 2 (TG2) protein and isopeptide bond are expressed in idiopathic pulmonary fibrosis (IPF). Samples of lung biopsies taken from six patients with IPF and four nonfibrotic control subjects. (A) Immunohistochemical staining for TG2; scale bar = 100 μm. (B) High-power serial sections of a fibroblastic focus stained for α-smooth muscle actin (α-SMA) and TG2; scale bar = 100 μm. (C) Immunohistochemical staining for transglutaminase catalyzed Nε(γ-glutamyl) lysine bond; scale bar = 50 μm. Slides were developed with Nova Red. Representative images from three patients with IPF and one control patient are shown.

Figure 5.

Transforming growth factor (TGF)-β drives an increase in cell surface transglutaminase 2 (TG2) expression. Human lung fibroblasts were treated with TGF-β (5 ng/mL). (A) Protein extracts were separated into particulate (memTG2) and cytosolic (cytTG2) fractions and analyzed by Western blot for TG2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) after 72 hours. (B) Surface expression of TG2 was analyzed by flow cytometry 72 hours after TGF-β treatment. (C) Expression of TG2 and collagen I mRNA were measured by quantitative reverse transcriptase–polymerase chain reaction after 24 hours of TGF-β treatment. n = 4 per group ***P < 0.001.

Figure 6.

Transforming growth factor (TGF)-β treatment leads to an increase in extracellular transglutaminase 2 (TG2) and a corresponding increase in activity. (A) Human lung fibroblasts (HLFs) grown in chamber slides were stained with an antibody to TG2 (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) after 72 hours of TGF-β (5 ng/mL) treatment. (B) HLFs were grown in the presence of pentylamine-biotin or fluorescein isothiocyanate (FITC) cadaverine and TGF-β (5 ng/mL) for 72 hours. Cells were stained with strep-Alexa Fluor 594, fixed, and stained with DAPI. (C) HLFs grown in the presence of FITC-cadaverine were collected as whole cell lysates or extracellular matrix proteins. Proteins were then analyzed by Western blot with antibodies against TG2 and against FITC.

Figure 7.

Human lung fibroblasts (HLFs) deficient in transglutaminase 2 (TG2) have functional defects. HLFs were infected with a lentivirus that encodes GFP and either a TG2 (Sh-TG2) or a scrambled (Sh-Scram) Sh-RNA. Cells were then sorted on GFP expression and used at passage 6 to 10. (A) Western blot for TG2, α-smooth muscle actin (α-SMA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression with or without transforming growth factor (TGF)-β. (B) Equal numbers of cells were seeded in a collagen gel, floated in media, and then weighed after 48 hours. Weights were compared with no cell control gels and percent contraction was calculated. (C) Confluent cell layers were wounded with a pipet tip and migration was tracked over 72 hours. n = 3 per group; error bars represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 by analysis of variance.

Figure 8.

Overexpression of transglutaminase 2 (TG2) leads to an increase in fibronectin protein and increased collagen gel contraction. Primary human lung fibroblasts (HLFs) from nonfibrotic patients were infected with an empty control adenovirus (DL-70), an adenovirus containing the wild-type TG2 sequence (wt TG2), or an adenovirus with a transglutaminase activity null mutant (mTG2 C277S). (A) Cells were grown on chamber slides, fixed with methanol, and double stained for fibronectin and TG2. Mounting media containing 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize the nuclei. (B) After infection with adenovirus, cells were serum starved for 24 hours and protein lysates were collected. Lysates were analyzed by Western blot for fibronectin, TG2, α-smooth muscle actin (α-SMA), and GAPDH. (C) Equal numbers of cells were seeded in a collagen gel, floated in media, and then weighed after 48 hours. Weights were compared with no cell control gels and percent contraction was calculated. n = 3 per group; error bars represent mean ± SEM; *P < 0.05, **P < 0.01, by analysis of variance.