Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway

Abstract

Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and beta-catenin were required for expression of Pdgfralpha and Pdgfrbeta and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfralpha and Pdgfrbeta as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfralpha/beta expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.

Figure 1. Loss of Wnt7b expression leads to early defects in SMC development in the lung.

(A–D) SM22α immunostaining showed vascular (A and C) and airway (B and D) smooth muscle development in wild-type (A and B) and Wnt7b^lacZ-null (C and D) mutants. Arrowhead in A indicates a smaller branching vessel in wild-type embryos, and arrows in C point to gaps in SM22α expression in surrounding larger blood vessels of Wnt7b^lacZ-null mutants. (E) Loss of Wnt7b expression in Wnt7b^lacZ-null mutants led to decreased expression of other SMC markers, including SM-MHC and calponin, as shown by Q-PCR. (F) Wnt7b^lacZ-null mutants exhibited decreased axin2 expression. (G and H) SM22α-cre:Ctnnb1^flox/flox mutants at E12.5 exhibited decreased SM22α expression surrounding both airways and blood vessels (arrows) compared with wild-type littermates. (I) SM22α-cre:Ctnnb1^flox/flox mutants had decreased SM-MHC and calponin expression as shown by Q-PCR. *P < 0.01. Scale bars: 100 μm.

Figure 2. Wnt7b and β-catenin signaling regulates Pdgfrα and Pdgfrβ expression in the lung.

(A–D) Immunostaining for Pdgfrβ expression in the developing lungs of wild-type (A and C) and Wnt7b^lacZ-null mutants (B and D) at E10.5 (A and B) and in developing blood vessels (arrows) at E12.5 (C and D). (E) Q-PCR for Pdgfrα and Pdgfrβ expression at E10.5. (F–I) Immunostaining for Pdgfrα expression in the developing lungs of wild-type (F and H) and Wnt7b^lacZ-null mutants (G and I) at E10.5 (F and G) and E12.5 (H and I). (J) BAT-GAL Wnt reporter activity at E10.5 in the mesenchyme of the lung in an overlapping region where Pdgfrβ expression was observed. (K and L) Immunostaining for Pdgfrβ expression in wild-type (K) and SM22α-cre:Ctnnb1^flox/flox mutants (L) at E12.5. (M) Q-PCR for Pdgfrα and Pdgfrβ expression in wild-type and SM22α-cre:Ctnnb1^flox/flox mutants at E10.5. (N) Q-PCR for axin2, Pdgfrα, and Pdgfrβ expression from the lung buds of NaCl- and LiCl-treated embryos at E10.5. (O–R) SM22α immunostaining in lungs of NaCl- and LiCl-treated E15.5 embryos. (S) Q-PCR for SM-MHC and calponin expression from the lung buds of NaCl- and LiCl-treated embryos at E14.5. ai, airway. *P < 0.01. Scale bars: 250 μm (A–D, F, G, and J), 150 μm (H, I, K, and L), 100 μm (O–R).

Figure 3. Wnt7b regulates proliferation of Pdgfrβ-expressing SMC precursors, and β-catenin specifically regulates Pdgfrβ versus Pdgfrα expression in VSMCs.

(A and B) Immunostaining for Pdgfrβ (red) and PO₄-H3 (green) expression in wild-type (A) and Wnt7b^lacZ-null mutants (B) at E10.5. (C and D) The percentage of Pdgfrβ-expressing cells (C) as well as proliferating lung mesenchyme was significantly reduced in Wnt7b^lacZ-null mutants (D). (E and F) Immunostaining for Pdgfrβ (red) and PO₄-H3 (green) expression in wild-type (E) and SM22α-cre:Ctnnb1^flox/flox mutants (F). (G and H) The percentage of Pdgfrβ-expressing cells (G) as well as proliferating lung mesenchyme was significantly reduced in SM22α-cre:Ctnnb1^flox/flox mutants (H) at E10.5. DAPI was used a counterstain to detect cell nuclei. (I) Wnt7b activated the SuperTOPFLASH Wnt reporter in Pac1 cells. (J) Wnt7b activated expression of Pdgfrβ and axin2 but not Pdgfrα in Pac1 cells. (K) siRNA-mediated knockdown of β-catenin in Pac1 cells led to decreased Pdgfrβ but not Pdgfrα expression. *P < 0.001. Scale bars: 250 μm.

Figure 4. Wnt/β-catenin is necessary and sufficient for Tnc expression.

(A) Wnt7b expression activated Prrx1 and Tnc expression in Pac1 cells. (B) siRNA-mediated knockdown of β-catenin expression led to decreased Tnc but not Prrx1 expression. (C) Prrx1 expression was unchanged in Wnt7b^lacZ-null mutants at both E10.5 and E14.5. *P < 0.01.

Figure 5. β-Catenin and Wnt7b signaling regulate Tnc expression in vivo.

(A and B) Immunostaining for Tnc expression in NaCl- and LiCl-treated wild-type embryos at E15.5 showed increased Tnc expression (arrows) surrounding the airways of LiCl-treated embryos. (C) Expression of Tnc was induced approximately 3-fold by LiCl treatment. (D) Loss of β-catenin expression in SMC precursors in SM22α-cre:Ctnnb1^flox/flox mutants resulted in a quantitative loss of Tnc expression in vivo at E10.5. (E–G) Tnc expression was significantly reduced in Wnt7b^lacZ-null mutants as assessed by immunohistochemistry at E14.5 (E and F) and quantitatively by Q-PCR (G). *P < 0.001. Scale bars: 20 μm (A and B), 40 μm (E and F).

Figure 6. Tnc is a direct target of Wnt signaling in the lung.

(A) The proximal Tnc mouse promoter region contains 5 TBEs. (B) The pTnc.luc reporter was activated in a dose-dependent manner by expression of an activated form of β-catenin in Pac1 cells. (C and D) ChIP assays using agarose gel electrophoresis (C) and Q-PCR (D) showed that β-catenin was associated with the TBE1, TBE2, and TBE3–5 regions in the mouse Tnc promoter in the lung.

Figure 7. Tnc expression directly modulates Pdgfrβ expression.

(A–C) Treatment of lung explants from E11.5 embryos with a Tnc-blocking antibody reduced Pdgfrβ expression (arrows) by immunohistochemistry (A and B) and reduced both Pdgfrα and Pdgfrβ expression by Q-PCR (C). The white dotted lines in A and B denote the external outline of lung explant; yellow dotted lines denote developing airways. (D) Antibody-mediated blocking of Tnc function reduced lung mesenchymal proliferation. (E) Tnc protein treatment of Pac-1 cells led to increased Pdgfrβ but not Pdgfrα expression by Q-PCR. (F) Tnc protein treatment of E11.5 mouse lung explants resulted in increased expression of both Pdgfrα and Pdgfrβ as detected by Q-PCR. (G–K) Treatment of lung explants with Wnt3a and either non-immune IgG (control IgG) or Tnc-blocking antibody (Tnc IgG) showed that blocking Tnc decreased Wnt3a-induced Pdgfrα and Pdgfrβ expression, as assessed by Q-PCR (G) and immunohistochemistry (H–K). (H–L) Blocking Tnc also inhibited the Wnt3a-induced increase in proliferation observed in lung explants as noticed by PO₄-H3 immunostaining (green nuclei). *P < 0.001; **P < 0.03. Scale bars: 250 μm.

Figure 8. Increased expression of the Wnt/Tnc/Pdgfr pathway in A. fumigatus–induced asthma in mice.

(A–L) Mice treated with A. fumigatus to induce airway hyperresponsiveness and inflammation showed increased smooth muscle mass as assessed by H&E staining (A and B) and SM22α immunohistochemistry (C and D). Coincident with this reaction, increased expression of axin2 (E and F), Tnc (G and H), Pdgfrα (I and J), and Pdgfrβ (K and L) were observed. (M) Q-PCR was used to quantitate the increase in expression of axin2, Tnc, Pdgfrα, and Pdgfrβ. *P < 0.001. Epi, epithelium; asm, airway smooth muscle. Scale bars: 100 μm.

Figure 9. Increased expression of the Wnt/Tnc/Pdgfr pathway in pulmonary hypertension in humans.

(A–H) Histological sections from human patients with PAH were stained with H&E (A, B, E, and F) or SM22α (C, D, G, and H) to detect vascular smooth muscle overgrowth in pulmonary arteries. (I–P) Expression of axin2 (I and J), Tnc (K and L), Pdgfrα (M and N), and Pdgfrβ (O and P) was increased in patients with PAH compared with normal controls. Vsm, vascular smooth muscle. Scale bars: 400 mm (A–D), 100 μm (E–P).

Figure 10. Model of a Wnt/Tnc/Pdgfr signaling axis regulating lung SMC development.

Airway epithelium (green) expresses Wnt7b, which acts on mesenchymal SMC precursors (yellow). Wnt7b and β-catenin signaling directly activates Tnc expression and in turn promotes airway SMC (blue) and VSMC (red) precursor proliferation and development.