Abstract
Human MN/CA IX protein localized on the surface of epithelial cells is a member of the carbonic anhydrase family isozymes. Its multidomain composition implies rather broad functionality. Under normal conditions MN/CA IX is expressed in several tissues of the alimentary tract, notably in the gastric mucosa. Furthermore, its aberrant expression has been connected to carcinogenesis. Recently MN/CA IX protein was linked to the regulation of cell proliferation and identified as a molecule involved in the cell adhesion. Despite the increasing knowledge about MN/CA IX protein, its physiological relevance in the gastrointestinal tract and its role in oncogenesis has not been understood.
In order to assess the function of the MN/CA IX protein in vivo, mice lacking the MN/CA IX protein were generated by gene targeting. In order to fulfil this approach, cDNA and genomic DNA, coding for the murine MN/CA IX protein, had to be identified, isolated and characterized. The murine counterpart of human MN/CA IX was identified on the basis of its sequence homology and expression pattern. The full length of the murine MN/CA9 cDNA is 1982bp. The open reading frame of 1311bp has a coding capacity for a 437 amino acid protein with a theoretical molecular mass of 47,3kDa. The alignment of the predicted amino acid sequence of the murine and human MN/CA9 cDNAs showed only 69,5% sequence identity. However, all the extracellular and intracellular domains were conserved in both. Expression of the murine MN/CA9 mRNA is highest in mouse stomach, then in the proximal intestine and distal colon. The MN/CA9 cDNA was also detected in several tumor cell lines (TS/A; GR/3; and MM5) and one tumor sample. This observation hints at a possible implication of the MN/CA IX protein in oncogenesis also in mouse.
For further investigation the murine MN/CA IX protein was isolated, purified, and the identity validated by MALDI-MS. Since the used purification method is applicable only for enzymatically active carbonic anhydrases, this result at the same time sustains the conservation of at least some carbonic anhydrase activity of the murine MN/CA IX protein. The apparent molecular mass of the murine MN/CA IX was estimated at 54kDa. This result was confirmed by Western blot analysis with prepared polyclonal rabbit antiserum which was raised against an oligopeptide from the proteoglycan-like domain.
The murine MN/CA9 gene was isolated from a mouse genomic library. The gene is divided into 11 exons and 10 introns, spanning 6,7kb of the mouse genomic DNA. A comparison of exon/intron boundaries of the mouse and human genes showed identical gene structure suggesting their high evolutionary conservation.
A replacement type vector with a targeted interruption of the first exon of the murine MN/CA9 gene was introduced into the mouse embryonic stem cells by homologous recombination. The mice, homozygous in the MN/CA9 gene mutation, were normal in terms of size, activity, behavior, ability to reproduce and life span. The most remarkable changes were found in the gastric mucosa of MN/CA9-/- mice. Severe hyperplasia in adult mutant mice concerned all major glandular and superficial epithelial cells. In addition to hyperplastic changes, large pathological cysts were observed. The first mild changes in the thickness of the gastric epithelium were detected already in newborn MN/CA9-/- mice.
Immunohistochemical staining for PCNA indicated a massive enlargement and disorganization of the proliferative zone of the stomach epithelium. A similarly disorganized pattern was observed by E-cadherin immunostaining of the gastric mucosa of mutant mice. The ratio of the number of proliferative cells stained by PCNA and total number of cells in the proliferative area was approximately the same as in the MN/CA9+/+ mice. Since a defect in the cellular death was excluded, the alterations in the gastric epithelium are most likely caused by deregulated cell proliferation. Whether E-cadherin mediated cell adhesion is affected by the absence of MN/CA IX remains still to be determined.
Although the precise physiological role of the MN/CA IX protein in the gastrointestinal tract is still not clear, significant insight has been gained. We propose that the null mutation of MN/CA IX protein perturbs the process of proper mediation and/or processing of the extracellular signaling that normally defines the level of the proliferation and maintenance of the architecture of gastric epithelial cells.
Among the carbonic anhydrase isozymes MN/CA9-/- mice are the first animal model for carbonic anhydrase deficiency constructed by gene targeting. Moreover, MN/CA IX is the first targeted adhesion-related molecule expressed in the gastrointestinal epithelium that revealed involvement in morphogenesis of the mucosa. The MN/CA9-/- deficient mouse provide an excellent system to elucidate the role of this molecule in the morphogenesis of the gastric epithelium and its involvement in carcinogenesis. |
