Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation - PubMed (original) (raw)

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

E L Habib Dakir et al. Carcinogenesis. 2008 Dec.

Abstract

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells.

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Figures

Fig. 1.

Fig. 1.

Generation of CC10-hK14 TG mice. (A) hK14 complementary DNA was cloned into a plasmid that contains a 2.1 kb mouse CC10 (mCC10) promoter, β-globin intron as enhancer and bovine growth hormone (bGH) polyA. Sequencing was done using T7 and T3 primers. (B) Southern blot analysis of CC10-hK14 TG mouse founders. Lanes J7, E10, E9, F8 and F7 revealed the expected band size (2.1 kb) for hK14 transgene. The positive control (lane C) included a mixture of plasmid DNA (CC10-hK14) of transgene and WT tail DNA digested with BamHI. Results composed of three separate gels (1–3). (C) Genotyping of CC10-hK14 TG mice by PCR. Two sets of primers were used as indicated in a schematic illustration (top). Amplification of both fragments of tail DNA in lanes B, C, E–G, J–O and R indicated the contiguity of the incorporated transgene. First and last lanes (M) correspond to molecular markers.

Fig. 2.

Fig. 2.

Characterization of CC10-hK14 TG mice. Photomicrographs of normal bronchiolar epithelium from a WT mouse (A) (hematoxylin and eosin stain; bar = 200 μm) and focal epithelial hyperplasia (arrow) from TG mouse (B) (hematoxylin and eosin stain; bar = 100 μm). Normal bronchiolar epithelium from a young TG mouse (C) and squamous metaplasia in a TG mouse (D) (hematoxylin and eosin stain; bar = 400 μm). Inset shows a mitotic figure and intercellular bridges. Bronchiolar epithelium from a WT mouse (E) negative for K14 (immunoperoxidase stain; bar = 200 μm) and K14 immunoreactivity in basal cells (long arrows) and focal squamous metaplasia (short arrows) in TG bronchiolar epithelium (F) (immunoperoxidase stain; bar = 100 μm). CC10 immunorectivity in bronchiolar epithelium from a WT mouse (G) and in hyperplastic, thickened epithelium from a TG mouse (H) (immunoperoxidase stain; bar = 200 μm). LU = lumen. (I) A graph of body weights. Hatched line = WT mice; solid line = TG mice; mo = months. (J) Expression of hK14 mRNA expression by Q-RT-PCR with ribosomal S18 as housekeeping gene. Controls included lungs from two lines of WT mice (WT1 and WT2) and BEAS-2B cells (BE). TG tissues included trachea (TRA) and lungs from independent lines I9, E9, E10 and D1 of mice. M = DNA markers. The primers used in this experiment detected only hK14 gene with a 278 bp product. (K) A bar graph of relative hK14 mRNA expression by Q-RT–PCR in a trachea and lungs from TG lines I9, E9, E10, D1, J7 and a WT mouse. (L) Immunoprecipitation analysis of hK14 protein in human BEAS-2B cells and WT lung (control) and in lungs from TG mouse lines J7, A9 and E9.

Fig. 3.

Fig. 3.

Increased expression of K14 and basal cell hyperplasia in TG trachea. (A) Relative expression of K14 mRNA by Q-RT–PCR in the lung and tracheas from WT littermates of mouse lines D1-1, D1-2 and J7-1 and from TG animals of mouse lines F8-1, D10-1 and D1-3. The primers used in this experiment detected both mouse and human K14 mRNA. Ribosomal S15 and S18 were used as housekeeping genes. Photomicrographs of (B) basal cell hyperplasia (arrows) in tracheal epithelium from a TG mouse (I) and scattered basal cells (arrows) in the trachea of a WT mouse (K14 immunoperoxidase stain; bar = 200 μm). Lu = lumen.

Fig. 4.

Fig. 4.

Expression of CCE precursor mRNAs in the lungs and trachea of TG mice. Relative expression of involucrin, loricrin, transglutaminase 1 (TGase 1), small-proline rich protein 1A (SPRR 1A) and cholesterol sulfotransferase 2B1 (SULT2B1) mRNAs was analyzed by Q-RT–PCR in the lung tissue of WT mouse and in the trachea (TRA) and lungs from CC10-hK14 TG mouse lines I9, E9, E10, D1 and J7. Ribosomal S15 and S18 were used as housekeeping genes.

Fig. 5.

Fig. 5.

Tumor histology and immunohistochemical analysis of differentiation in the lungs from CC10-hK14 TG mice. (A) Photomicrograph of a normal airway from a WT mice (hematoxylin and eosin stain; bar = 200 μm). (B) An adenoma adjacent to a bronchiolus with mild hyperplasia and atypia from a TG mouse (hematoxylin and eosin stain; bar = 400 μm). (C) Histology of a spontaneous adenoma in the lung of a 48-week-old WT mouse (hematoxylin and eosin stain; bar = 400 μm). (D) A poorly differentiated spontaneous carcinoma with marked atypia, increased nuclear:cytoplasmic ratio (arrows) and prominent nucleoli in tumor cells from a TG mouse (hematoxylin and eosin stain; bar = 400 μm). Panels (E–R) show immunoperoxidase stain as follows: bronchiolar epithelium of a WT mouse negative for p63 (E) and K14 (F) (bar = 200 μm). Bronchiolar epithelium from a TG mouse positive for p63 (G) and K14 (H) in basal cells (arrows; bar = 200 μm). (I) A spontaneous tumor from a WT mouse negative for K14 (bar = 200 μm). (J) Scattered K14-positive tumor cells in a TG mouse (arrows; bar = 400 μm). (K) A tumor from WT lung negative for CC10 (bar = 200 μm). (L) A tumor with intense CC10 immunoreactivity (white arrows) from a TG mouse. The epithelial cells lining an airway lumen (LU) are also positive (bar = 200 μm). (M) Characteristic immunoreactivity for proSP-C (arrows) in a pulmonary adenoma from a WT mouse (bar = 200 μm). (N) The same tumor as in (L) from a TG mouse is also positive for proSP-C (arrows), whereas the cells lining an airway lumen (LU) are negative (stain; bar = 200 μm). (O) A tumor from a WT mouse negative for p63 (bar = 200 μm). (P) Focal immunoreactivity for p63 (arrows) in a tumor from a TG mouse (bar = 200 μm). (Q) A tumor (TU) from a WT mouse negative for loricrin (bar = 200 μm). (R) A tumor from a TG mouse with strong immunoreactivity for loricrin (bar = 200 μm).

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