Studies of intestinal morphology and cathepsin B expression in a transgenic mouse aiming at intestine-specific expression of Cath B-EGFP.

Cathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin B in vivo. Previously we showed that the A33-antigen promoter could be successfully used in vitro in order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chimeric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translated in situ although the transgene was maintained over several generations.

Intestine-specific expression of green fluorescent protein-tagged cathepsin B: proof-of-principle experiments.

We hypothesized that tissue-specific expression of cathepsin B-enhanced green fluorescent protein (CB-EGFP) can be driven by the A33-antigen promoter that contains positive cis-regulatory elements, including caudal-related homeobox (CDX) binding sites. The intestine-specific transcription factor Cdx1 is crucial for A33-antigen promoter activation and could thereby induce expression of CB-EGFP. This concept was tested by construction of the vector pA33-CathB-EGFP encoding CB-EGFP downstream of the A33-antigen promoter. Its Cdx1 dependence, as an indication of its intestine-specific expression, was tested in Cdx1-negative CHO-K1 cells. Cdx1 expression was achieved upon transfection with pCdx1-DsRed-Express and was indicated by red fluorescence of the simultaneously translated reporter protein. Immunolabeling with Cdx1-specific antibodies showed correct targeting of the transcription factor to its point of action in nuclei of transfected cells. Co-transfection experiments with plasmids pA33-CathB-EGFP and pCdx1-DsRed-Express confirmed the hypothesis that Cdx1 indeed activates CB-EGFP expression in a manner dependent on the A33-antigen promoter. Co-localization with compartment-specific markers and subcellular fractionation confirmed CB-EGFP trafficking along the expected route to endolysosomal compartments. Hence, the A33-antigen promoter represents a potent tool for induction of Cdx1-dependent CB-EGFP expression in vitro. Our proof-of-principle studies confirm the suitability of this approach in visualizing protease transport in Cdx1-positive tissues of the gastrointestinal tract.