Sox2 is required for maintenance and differentiation of bronchiolar Clara, ciliated, and goblet cells

Abstract

The bronchioles of the murine lung are lined by a simple columnar epithelium composed of ciliated, Clara, and goblet cells that together mediate barrier function, mucociliary clearance and innate host defense, vital for pulmonary homeostasis. In the present work, we demonstrate that expression of Sox2 in Clara cells is required for the differentiation of ciliated, Clara, and goblet cells that line the bronchioles of the postnatal lung. The gene was selectively deleted in Clara cells utilizing Scgb1a1-Cre, causing the progressive loss of Sox2 in the bronchioles during perinatal and postnatal development. The rate of bronchiolar cell proliferation was decreased and associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of Clara cells (Scgb1a1), ciliated cells (FoxJ1 and alpha-tubulin), and goblet cells (Spdef and Muc5AC). By adulthood, bronchiolar cell numbers were decreased and Sox2 was absent in extensive regions of the bronchiolar epithelium, at which time residual Sox2 expression was primarily restricted to selective niches of CGRP staining neuroepithelial cells. Allergen-induced goblet cell differentiation and mucus production was absent in the respiratory epithelium lacking Sox2. In vitro, Sox2 activated promoter-luciferase reporter constructs for differentiation markers characteristic of Clara, ciliated, and goblet cells, Scgb1a1, FoxJ1, and Agr2, respectively. Sox2 physically interacted with Smad3 and inhibited TGF-beta1/Smad3-mediated transcriptional activity in vitro, a pathway that negatively regulates proliferation. Sox2 is required for proliferation and differentiation of Clara cells that serve as the progenitor cells from which Clara, ciliated, and goblet cells are derived.

Figure 1. Progressive deletion of Sox2 from the bronchiolar epithelium.

In control mice, strong nuclear staining for Sox2 is observed in all epithelial cells lining the conducting airways (A–D). Sox2 was progressively deleted from the conducting airway epithelium of Sox2^Δ/Δ mice (E–H) from late gestation to adulthood. Arrows indicate regions shown in insets. Scale bar is 50 µm, inset scale bar is 10 µm for all panels. NB, newborn; PN, postnatal.

Figure 2. Lack of ciliated and Clara cell selective markers after deletion of Sox2.

In four week old mice, hematoxylin and eosin staining of control and Sox2^Δ/Δ lung sections demonstrated abnormal low cuboidal-to-squamous epithelium in large bronchioles (A, B) and small bronchioles (C,D). Immunohistochemical staining demonstrated loss of CCSP (Scgb1a1) a Clara cell marker (E,F), ciliated cell markers α-tubulin and FoxJ1 (G–J). Lung epithelial markers Nkx2.1 and FoxA2 were unchanged in control vs Sox2^Δ/Δ mice (K–N). Arrows indicate regions shown in insets. Scale bar is 50 µm, inset scale bar is 10 µm.

Figure 3. Maintenance of Sox2 expression in neuroepithelial cells.

Immunofluorescence staining of six week old Sox2^Δ/Δ mice demonstrated Sox2-positive ciliated and Clara cells in control mice (A,C), the extensive deletion of Sox2, and the presence of rare ciliated and Clara cells lacking Sox2 in Sox2^Δ/Δ mice (B,D). Serial sections from Sox2^Δ/Δ mice (E,F) identified a cluster of more cuboidal, untargeted cells which stained for Sox2 (E) and a distinct subset of Sox2 reactive cells expressing the neuroendocrine cell marker CGRP in which the Scgb1a1 promoter is not active (F). Arrows indicate regions shown in insets. Scale bars: 20 µm (A–D), 40 µm (E,F). Inset scale bars: 5 µm (A–D), 10 µm (E,F).

Figure 4. Sox2 is required for goblet cell differentiation after pulmonary allergen sensitization.

Lung sections from normal four week old mice were co-stained for mucins with Alcian blue and Sox2 demonstrating the presence of goblet cells in the airways of control mice (A) but not in Sox2^Δ/Δ littermates (B). Response to allergen challenge (C–H): In control mice, Alcian blue reactive cells expressed Sox2 (C,D), and the goblet cell associated proteins Spdef (E,F) and Muc5AC (G,H). Extensive goblet cell differentiation was observed in bronchi and larger bronchioles of control (C,E,G) but not Sox2^Δ/Δ mice (D,F,H), in spite of the robust inflammatory response seen in both groups. Asterisk in D indicates a small cluster of Sox2-positive (untargeted) goblet cells. Arrows indicate regions shown in insets. Scale bar is 50 µm, inset scale bar is 10 µm. OVA+, ovalbumin treatment.

Figure 5. Decreased proliferation and decreased cell density after deletion of Sox2.

Bronchiolar cells were co-stained for Sox2 (green) and Ki-67 (red). Average cell height, cell density, and proliferation index was assessed by morphometric analysis (A,B,C). Representative bronchiolar epithelium of control (D) and Sox2^Δ/Δ (E) are shown in side view (upper portion) and plan view (lower portion). Graphs depict mean±standard error of the mean.

Figure 6. Sox2 induced promoter activity for differentiation markers Scgb1a1, FoxJ1, and Agr2.

HBECs were transfected with (A) Scgb1a1-luciferase reporter (0.5 µg), (B) FoxJ1-luciferase reporter (0.5 µg), or (C) Agr2-luciferase reporter (0.5 µg) in the presence of various combinations of Sox2 (0.1 µg), Nkx2.1 (0.1 µg), and FoxA2 (0.1 µg). Sox2 induced modest activation of Scgb1a1, FoxJ1, and Agr2 (A,B,C). Synergistic activation of Scgb1a1 by Nkx2.1 and FoxA2 was observed (A). Sox2 did not synergize with Nkx2.1 or FoxA2 to regulated Scgb1a1, FoxJ1, or Agr2 constructs. Graphs depict representative results of 3 separate experiments performed in triplicate, and are expressed as the mean±standard deviation.

Figure 7. Sox2 inhibited TGF-β1 sensitive promoter activity reporter assay and co-immunoprecipitated with Smad3.

(A) HBECs were transfected with a TGF-β1/Smad-responsive reporter 3TP-luciferase (3TP-Luc) and increasing amounts of pCIG empty vector or pCIG-Sox2 (0.25 µg and 0.5 µg) in the presence or absence of TGF-β1 (2 ng/ml). Sox2 inhibits TGF-β1-mediated reporter activation. (B) Coimmunoprecipitations performed on lysates from HBECs expressing Sox2-V5 and FLAG-Smad3 demonstrated an interaction between Sox2 and Smad3. (C) Sox2 inhibits Smad3-induced activation of 3TP-Luc in the presence or absence of TGF-β1 in HBECS. IP, immunoprecipitation; IB, immunoblot; IgG, immunoglobulin negative control. Graphs depict representative results from three experiments±standard deviation of the mean.

Figure 8. Model in which Sox2 is required for Clara cell self renewal and differentiation of facultative progenitors of the bronchiolar epithelium.

(A) The Clara cell is postulated to be the facultative progenitor cell of the bronchiolar epithelium, possessing differentiated cell characteristics yet capable of self-renewal and differentiation into ciliated or goblet cells. (B) Loss of Sox2 from Clara cells results in an epithelium lacking differentiated cells, consistent with the Clara/facultative stem cell model. Sox2 is required for normal proliferation during perinatal growth and for the postnatal differentiation of Clara, ciliated, and goblet cells in the bronchiolar epithelium.