Epithelium-specific deletion of TGF-β receptor type II protects mice from bleomycin-induced pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic fibroproliferative pulmonary disorder for which there are currently no treatments. Although the etiology of IPF is unknown, dysregulated TGF-β signaling has been implicated in its pathogenesis. Recent studies also suggest a central role for abnormal epithelial repair. In this study, we sought to elucidate the function of epithelial TGF-β signaling via TGF-β receptor II (TβRII) and its contribution to fibrosis by generating mice in which TβRII was specifically inactivated in mouse lung epithelium. These mice, which are referred to herein as TβRIINkx2.1-cre mice, were used to determine the impact of TβRII inactivation on (a) embryonic lung morphogenesis in vivo; and (b) the epithelial cell response to TGF-β signaling in vitro and in a bleomycin-induced, TGF-β-mediated mouse model of pulmonary fibrosis. Although postnatally viable with no discernible abnormalities in lung morphogenesis and epithelial cell differentiation, TβRIINkx2.1-cre mice developed emphysema, suggesting a requirement for epithelial TβRII in alveolar homeostasis. Absence of TβRII increased phosphorylation of Smad2 and decreased, but did not entirely block, phosphorylation of Smad3 in response to endogenous/physiologic TGF-β. However, TβRIINkx2.1-cre mice exhibited increased survival and resistance to bleomycin-induced pulmonary fibrosis. To our knowledge, these findings are the first to demonstrate a specific role for TGF-β signaling in the lung epithelium in the pathogenesis of pulmonary fibrosis.

Figures

Figure 1. Epithelial specificity of recombination induced by Nkx2.1-cre in organs of double-transgenic Nkx2.1-cre;Rosa26R mice.

LacZ staining was observed in the brain (arrowheads) and thyroid primordium (arrows) in E9.5 embryos (A). Nkx2.1-cre–mediated recombination in the lung was first visible in E11 lungs (arrow in B and outlined in C). In E13, E15, and E17 embryonic lungs, LacZ staining was restricted to epithelial cells in the lung (D, F, G, I, and J), but was also expressed in the tracheal epithelium (E and H). In Pn7 neonates (K), LacZ staining was localized to the bronchoalveolar duct junction (arrowheads) and alveolar type II cell progenitors (arrows). Scale bar: 2 mm (B and G); 800 μm (A and D); 400 μm (H and J); 200 μm (E); 100 μm (F, I, and K); and 40 μm (C).

Figure 2. Generation and validation of TβRII conditional knockout alleles.

(A) Schematic map of the mouse TβRII floxed locus. Arrowheads designate loxP sequences. Primers a, b and c, used in genotyping the mice, are also shown. (B) PCR genotyping using the 3 primers in A. Heart (lanes 1, 3, and 5) and lung tissue (lanes 2, 4, and 6) from floxed alleles and wild-type alleles can be distinguished by their size using primers a and b. Primers for Cre detection are provided in Methods. Deleted TβRII allele is detected using primers a and b. (C and D) Immunohistochemical analysis for TβRII protein in TβRIIfl/fl and TβRIINkx2.1-cre lungs, respectively. Arrows show alveolar type II–appearing cells in TβRIIfl/fl and TβRIINkx2.1-cre adult lungs. (E and F) Western blot analysis and densitometric quantification of total protein homogenates from TβRIIfl/fl (control) and TβRIINkx2.1-cre (mutant) lungs, respectively. Values were normalized against α-tubulin. (G) p-Smad2 and p-Smad3, normalized against total Smad2 and Smad3, respectively. (H) Densitometric quantification of the Western blot in G. (I and J) p-ERK normalized against total ERK and densitometric quantification. **P < 0.01; *P < 0.05. n = 3. Scale bar: 40 μm.

Figure 3. Characterization of the TGF-β pathway in isolated alveolar epithelial type II cells.

(A) TβRII levels determined by Western blot analysis in TβRIIfl/fl and TβRIINkx2.1-cre type II cells. (C) Western blot analysis of Smads and p-Smads in control and TGF-β–treated type II cells. (E) Western blot analysis of ERK and p-ERK in control and TGF-β–treated type II cells. Quantification of A, C, and E is shown in B, D, and F, respectively. **P < 0.01; *P < 0.05.

Figure 4. Morphological and real-time PCR analysis of TGF-β targets in TβRIIfl/fl and TβRIINkx2.1-cre lungs.

(A–D) Morphological analysis by H&E staining on sections of 8-week-old lungs showing the presence of alveolar enlargement. (E) Distal airway counts in multiple sections of control and mutant lungs at both 3 weeks and 8 weeks of age. (F) Quantification of average size in pixels of airways using Image J. (G) Mean linear intercept measurements (1.0 = 200 μm). n = 3 per group; 5–10 slides from each group. (H) Real-time PCR analysis of ECM components. (I) Real-time PCR analysis of TGF-β targets. n = 3 per group; **P < 0.01; *P < 0.05. Scale bar: 200 μm.

Figure 5. Physiological impact of bleomycin injury in TβRIIfl/fl and TβRIINkx2.1-cre mice.

(A) Survival profile of mice receiving i.t. injection of bleomycin or saline (control). (B) Weight loss in mice receiving i.n. instillation of bleomycin or saline (control). Animals were monitored up to 21 days. (C) Compliance (kPa/ml) in control and mutant lungs on day 14 of bleomycin treatment. (D) Epithelial permeability on day 14 after bleomycin exposure as measured by protein content of BAL. **P < 0.01; *P < 0.05. n = 5 per group.

Figure 6. Relative resistance to bleomycin-induced fibrosis in TβRIINkx2.1-cre mice.

Representative lung histology on day 21 after exposure to bleomycin in TβRIIfl/fl (A) and TβRIINkx2.1-cre (B) mice. Trichrome staining on sections of the same lungs are shown in C and D, respectively. Quantification of collagen in the lungs by Sircol assay (E); **P < 0.005. TUNEL assays are shown in F and H for TβRIIfl/fl and G and I for TβRIINkx2.1-cre lungs. H and I are with DAPI. (J) Quantification of positive (green) cells in the fibrotic foci of TβRIIfl/fl and TβRIINkx2.1-cre lungs. Scale bar: 2 mm (A and B); 200 μm (C and D); 50 μm (F–I). **P < 0.01; n = 3–6 slides from each lung.

Figure 7. Characterization of the fibrogenic markers and TGF-β targets in TβRIIfl/fl and TβRIINkx2.1-cre lungs exposed to bleomycin.

Immunofluorescence for α-SMA in TβRIIfl/fl (A) and TβRIINkx2.1-cre lungs (B) on day 21 after bleomycin injury. (C and D) Immunofluorescence analysis for FSP1 in control and mutant lungs. (E and F) Western blot analysis of α-SMA and FSP1 normalized to lamin A/C in control and mutant lungs. Fold increase in mRNAs (injured/uninjured ratio) for ECM components (G) and Ctgf, Pai1, metalloproteinases, and their inhibitors (H) by qPCR. n = 3 per group. **P < 0.05; *P < 0.01. Scale bar: 100 μm.

Figure 8. Quantitative analysis of mRNA expression of TGF-β targets in isolated TβRIIfl/fl and TβRIINkx2.1-cre alveolar type II (AT2) cells.

(A) Real-time PCR analysis for Ctgf, Pai1, Mmps, and Timps. Values in the TβRIIfl/fl type II cells were adjusted to unity and compared with values obtained in TβRIINkx2.1-cre cells. (B) Response (fold induction) of the same genes as in A to TGF-β treatment of type II cells. **P < 0.05; *P < 0.01.