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First published online 19 February 2003
doi: 10.1242/jcs.00338

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Chronic acid exposure leads to activation of the cdx2 intestinal homeobox gene in a long-term culture of mouse esophageal keratinocytes

Marta Marchetti^*, Elise Caliot and Eric Pringault

Laboratory of Lympho-Epithelial Interactions, Department of Cell Biology and Infection, Pasteur Institute, 28, Rue du Dr Roux, 75015 Paris, France
^* Present address: Laboratory of Traffic and Signaling, UMR 144 Curie/CNRS, Curie Institute, 26, rue d'Ulm, 75005 Paris Cedex, France

View larger version (74K): [in a new window]   Fig. 1. Culture of mouse esophageal keratinocyte cells. (A) Phase contrast microscopy view of epithelial cells in primary culture. The dark area represents the explant tissue. (B) Phase contrast microscopy view of esophageal epithelioid cells at confluency (day 11). Scale bar, 10 µm. (C) Growth curve of long-term culture of normal mouse esophageal cells. Cell number is given as mean ± s.e.m. of three independent cultures.

View larger version (134K): [in a new window]   Fig. 2. Characterization of long-term cultures of esophageal cells grown on filters. (A-C) Confocal analysis of immunocytochemistry labeling. (A) CK14 was strongly expressed in the basal cell layer, whereas (B) CK4 was expressed in suprabasal cells. (C) xz confocal section after labeling of nuclei (red) and CK4 (green) showing the cell stratification and the suprabasal epithelioid distribution of CK4 positive cells. Scale bar, 20 µm. (D) Transmission electron microcopy view of pluristratified mouse esophageal epithelioid cells grown on filter. Insert: enlarged view of a desmosome (f: filter). Scale bar, 2 µm.

View larger version (25K): [in a new window]   Fig. 3. Effect of pH on esophageal epithelial cell growth and proliferation on a transwell filter device. Cells were grown by maintaining neutral pH into the basolateral chamber and adding acid pH or neutral pH into the apical chamber. (A) Cell number is given as the mean ± s.e.m. of three independent cultures. (B) Cell lysates of cells at D+1 were used for immunblotting using anti-PCNA antibody. Equal amount of protein per lane (10 µg) were analyzed.

View larger version (99K): [in a new window]   Fig. 4. GFP expression under the control of the cdx2 promoter in long-term cultured mouse esophageal epithelioid cells maintained at pH 3.5. (A) Pluristratified `Malpighien-like' cells exposed for 18 days at pH 3.5 stained with propidium iodide (nuclear staining) showing GFP-positive cells (B) Western blot analysis of GFP expression on cell lysates of cells exposed for 18 days at pH 3.5, revealed a 27 kDa band corresponding to the GFP protein that is absent in cells grown at pH 7.4. (C) Same field as in (A) stained for CK14 antibody (red). Note that GFP-positive cells remained green indicating that they were CK14 negative. Scale bar, 20 µm. (D) The same field as in (A) stained for CK4; the dotted line deliniates the GFP-positive cells that also stain for CK4.

View larger version (88K): [in a new window]   Fig. 5. CDX2 expression analysis in esophageal cells grown on filters and in cell lysates. (A) Caco2 cells, used as control, were all CDX2 positive. (B) Esophageal cells at D+18 grown at pH 7.4 exhibited no CDX2 staining, whereas (C) positive nuclear staining was observed in some cells grown at pH 3.5. Scale bar, 20 µm. (D) Western blot analysis of CDX2 protein expression performed on lysates of cells at D+18 at pH 7.4 (lane 2), pH 5 (lane 3), and pH 3.5 (line 4). Lane 1 represents the positive control for Cdx2 protein in Caco2 cell lysate (C+).