Expression of Sox2 In Vivo
To conditionally express Sox2 in the respiratory epithelium, mice containing a (tetO
transgene were bred to transgenic mice expressing the tetracycline transactivator (rtTA) under control of the rat Scgb1a1
promoter (line 2). Previous studies demonstrated that rtTA expression was highly selective and efficient in targeting most nonciliated epithelial cells in the conducting airways and in a subset of alveolar type II cells (25
). To induce Sox2 expression, 4-week-old double transgenic mice and single transgenic littermate control mice were treated with doxycycline for specified lengths of time and then killed. Consistent with previous findings in normal mice, immunohistochemical staining of Sox2 was observed exclusively in all cells of the conducting airways of single transgenic and untreated double transgenic mice (). Compared with control mice, robust transgenic Sox2 expression was observed in bronchiolar and alveolar epithelial cells of double transgenic (rSox2) mice after 3 days, 1 week, and 6 months of doxycycline treatment (–). Quantitative PCR demonstrated an increase in Sox2
Figure 1. Conditional expression of Sox2 in the respiratory epithelium. Four-week-old mice were treated with doxycyline for the times shown and then killed. Immunohistochemical staining for Sox2 was performed on adult control (A, C, E, G) and double transgenic (more ...)
Figure 3. Exogenous Sox2 induced cell cycle proteins. Immunohistochemical staining and quantification of lung sections from mice treated with doxycycline for the time periods shown demonstrated increased protein expression for cyclin D1 (A–D, I) and FoxM1 (more ...)
Sox2 Induced Cell Proliferation and Caused Alveolar Cell Hyperplasia
Hematoxylin-eosin staining of lungs from mice treated with doxycycline for 6 months demonstrated normal bronchial and alveolar morphology in single transgenic mice. Abnormal clusters of epithelial cells were observed in the alveoli of double transgenic littermates treated with doxycyline. The lesions consisted of clusters of cuboidal cells that were present throughout the alveoli (). Alveolar clusters containing 10 or more cells were rare in control animals (mean, 0.3 clusters per 200× microscopic field; SD, 0.6) and common in rSox2 animals (mean, 6.7 clusters per 200× microscopic field; SD, 1.4).
Figure 2. Exogenous Sox2 caused cell proliferation and epithelial hyperplasia in the respiratory epithelium. Four-week-old mice were treated with doxycyline for the times shown and then killed. (A, B) Hematoxylin-eosin staining of lung sections from mice treated (more ...)
Compared with control mice, increased numbers of Ki-67 reactive cells were observed in the conducting airways and alveoli of double transgenic mice after 3 days or 1 week of doxycyline treatment but not after 6 months of treatment (, , and ). At 3 and 7 days, dual immunofluorescence staining for Sox2 and Ki-67 demonstrated that most of the increased proliferation was due to cells coexpressing Ki-67 and high levels of Sox2 (). Staining for the cell cycle initiator (cyclin D1) and the late cell cycle regulator (FoxM1) was increased in conducting airway (data not shown) and alveolar epithelial cells of the rSox2 mice (), consistent with the sites and extent of proliferation induced by the transgene.
Sox2 Induced Expression of mRNAs Associated with Cell Proliferation
The levels of mRNAs encoding selected cell cycle genes were assessed by qRT-PCR of whole lung homogenates. Messenger RNA for the transcription factor FoxM1 and the cyclins Ccna2 and Ccnb2 were significantly increased after expression of Sox2 (). Similarly, Ccnd1 mRNA was significantly increased after 7 days of doxycycline. Messenger RNA levels of the cell cycle inhibitors Cdkn1a, Cdkn2a, and Cdkn2c (cyclin-dependent kinase inhibitors) were not altered.
Figure 4. Sox2 induced mRNAs associated with cell proliferation and activated Foxm1 gene expression. Messenger RNAs encoding selected genes associated with cell proliferation were assessed by quantitative RT-PCR in total lung from adult control or rSox2 mice after (more ...)
Sox2 Activated the Foxm1 Promoter
To identify possible mechanisms by which Sox2 influenced cell proliferation in the lung, we sought to determine if FoxM1, a transcription factor that promotes proliferation, is a downstream target of Sox2. A Foxm1-luciferase reporter construct containing a regulatory sequence from the first intron, 780 bp upstream of the start codon, was cotransfected with a vector expressing Sox2 in human bronchial epithelial cells. Transcriptional activity of the −0.78 kb Foxm1 was significantly induced by cotransfection with Sox2 (). These data support the concept that Sox2 induces FoxM1 expression, providing insight into a potential mechanism by which increased levels of Sox2 promote cell proliferation.
Sox2 Inhibited Scgb1a1 Expression in Conducting Airway Epithelial Cells
Sox2 is required for the expression of Scgb1a1 and for the differentiation of Clara and ciliated cells in conducting airways (11
). Staining for Scgb1a1 in conducting airways was inhibited by the increased expression of Sox2 (). In contrast, FoxJ1 and α-tubulin staining was maintained in ciliated cells in the conducting airways (see
Figures E1A–E1F in the online supplement). Staining for the neuroendocrine marker, CGRP, was distributed normally in rSox2 mice (data not shown). Staining for proSP-C was readily detected in the alveolar epithelium in control and rSox2 mice (Figures E1G and E1H), whereas staining in the bronchioles for proSP-C was negative (not shown).
Figure 5. Sox2 inhibited expression of the Clara cell marker Scgb1a1 in conducting airways and induced abnormal alveolar clusters expressing markers of conducting airway epithelium. (A–H) Immunohistochemical staining for the Clara cell marker protein Scgb1a1 (more ...)
Sox2 Induced Conducting Airway Cell Differentiation in the Alveoli
Increased Sox2 expression caused widespread focal alveolar hyperplasia, inducing formation of abnormal clusters of highly differentiated cells with characteristics of airway epithelia in the peripheral lung. The hyperplastic alveolar epithelial lesions expressed TTF-1, consistent with maintenance of a respiratory epithelial cell phenotype (). Immunohistochemical staining of the lesions for the lymphocytic marker CD3 was negative (data not shown), indicating that clusters are not composed of inflammatory infiltrates. Hyperplastic lesions consisted of distinct subsets of cuboidal cells that expressed FoxJ1 and α-tubulin () or the Clara cell marker Scgb1a1 (). Ciliated cells were present throughout the alveoli and within the hyperplastic alveolar lesions (). Some alveolar cells expressed proSP-C, consistent with maintenance of a subset of alveolar type II cells (Figures E1E and E1F). Cells staining for proSP-C (an alveolar type II cell marker) or T1-α (an alveolar type I cell marker) were located primarily outside the lesions (Figures E2A–E2D) and did not colocalize with Sox2.
Cellular Heterogeneity of Alveolar Lesions Induced by Sox2
To test the differentiation status of diverse epithelial cells located within the hyperplastic lesions induced by Sox2, triple immunofluorescence staining was performed on mice treated for 6 months with doxycycline. In conducting airways, epithelial cells of control mice coexpressed Sox2 and Scgb1a1 or α-tubulin (). In the bronchioles of double transgenic mice, epithelial cells expressing high levels of Sox2 lacked Scgb1a1 (the Clara cell marker) and α-tubulin (). High levels of exogenous Sox2 observed in conducting airway epithelial cells of double transgenic mice precluded observation of lower endogenous Sox2 levels in Scgb1a1-positive and α-tubulin–positive epithelial cells (). In alveolar epithelium, control mice were negative for Sox2, Scgb1a1, and α-tubulin (). Ectopic alveolar clusters in double transgenic mice contained heterogeneous cell types, including many cells expressing Sox2 and Scgb1a1 () and less frequently Sox2+, α-tubulin+, Scgb1a1− cells. The weaker Scgb1a1 intensity () was due to the weaker expression of Scgb1a1 in the alveolar compartment compared with the bronchiolar compartment of the double transgenic mice.
Figure 6. Sox2-expressing cells in conducting airway lack differentiation markers. Expression of airway differentiation markers was determined in bronchiolar (A, B) and alveolar epithelium (C, D) from control (A, C) and rSox2 mice (B, D) after 6 months of doxycycline (more ...)
Ultrastructural Changes Induced by Sox2
The effects of Sox2 on the ultrastructure of the alveolar epithelium were assessed in control and rSox2 mice after 5 months of doxycycline exposure (). The abnormal epithelial cell clusters induced in the alveoli consisted primarily of nonciliated cells with one or two visible ciliated cells and, more rarely, one or two mature type II cells with multiple lamellar bodies (LBs) (), as well as transitional cells containing one or two residual LBs and type II cells with fusing and/or abnormally enlarged LBs (). The nonciliated cells contained numerous mitochondria, smooth endoplasmic reticulum, and glycogen but no rough endoplasmic reticulum or secretory granules. Ciliated cells, however, were well differentiated and readily detected along the alveolar walls as single cells () or in pairs containing one ciliated and one nonciliated cell (). Cilia consisted of the usual nine pairs of microtubules, arranged concentrically around the periphery, with two central microtubules (); basal bodies exhibited the normal arrangement of nine sets of three microtubules. These abnormal epithelial cell clusters had features of alveolar and bronchiolar epithelial cells, consistent with partial reprogramming of alveolar cells in response to increased Sox2 expression.
Figure 7. Sox2 induced structural features and expression of markers of the conducting airway epithelium in the alveolar epithelium. Ultrastructural features of alveolar epithelial cells were assessed by transmission electron microscopy in rSox2 mice treated with (more ...)
Sox2 Induced the Expression of Genes Expressed in Conducting Airways
The abundance of mRNAs of genes related to bronchiolar (Scgb1a1, Cyp2f2, Tubb3, Calca, Car3, Aqp3, Foxj1, Trp63, Elf3, Hes1) and alveolar epithelia (Sftpc, Abca3, Erm) was assessed by qRT-PCR in whole lung homogenates (). Sox2 increased expression of mRNAs for Clara (Scgb1a1, Elf3) and ciliated (Foxj1) cell markers after 3 days of exposure to doxycycline (). Expression of the alveolar cell marker Sftpc was decreased 3 days after induction with doxycycline (). After 7 days of doxycycline, Clara cell (Scgb1a1, Cyp2f2, Car3) and ciliated cell (Foxj1, Tubb3) markers were significantly increased, likely indicating the differentiation of proliferative cells induced earlier by Sox2 (). Trp63 mRNA, a marker for basal cells, and Calca mRNA, a marker for neuroendocrine cells, were modestly increased after 7 days of doxycycline ().
Lack of Pulmonary Tumors after Expression of Sox2
Pulmonary tumors expressing Sox2 were not detected after 6 months of Sox2 expression (n
= 6 control and n
= 6 rSox2 mice). Single pulmonary adenomatous tumors were seen in one control and in one rSox2 mouse. Each tumor expressed proSP-C, lacked Sox2, and had morphologic features typical of the spontaneous tumors seen with relatively high frequency in the FVB/N strain (26