Carbonic anhydrase gene expression in CA II-deficient (Car2-/-) and CA IX-deficient (Car9-/-) mice

Abstract

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient (Car2-/-), and CA IX deficient (Car9-/-) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2-/- mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2-/- mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2-/- mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2-/- mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.

Figure 1. Expression of Car1, 2, 3, 4, 9, 12, 13 and 14 mRNAs in the wild-type mice detected by real-time RT-PCR

Note: Car1 mRNA in colon is expressed as copies/100 copies of actin.

Figure 2. Expression of Car1, 2, 3, 4, 9, 12, 13 and 14 mRNAs in the wild-type, Car2^−/− and Car9^−/− mouse tissues

Figure 3. Alignment of the wild-type and mutant mouse CA II sequences

WT, wild-type mice; M, CA II-deficient mice. Asterisks indicate untranslated amino acids.

Figure 4. Expression of Car2 and Car9 mRNAs in the stomach of the wild-type, Car2^−/− and Car9^−/− mice

Car9 mRNA expression was increased in the stomach of the Car2^−/− mice. Also the signal for Car2 mRNA was slightly elevated in the Car9^−/− mice.

Figure 5. Immunohistochemical staining of CA IX protein in the stomach of the wild-type and CA II-deficient mice

In the wild-type mouse, the staining is mainly located in the mucus-producing epithelial cells of the outer part of the mucosa (A). In the Car2^−/− mouse, the signal was widely spread, covering intensively the whole thickness of the gastric mucosa (B). Original magnifications × 200. C shows digital image analysis results, comparing the staining extent of CA IX protein in the stomach of the wild-type and Car2^−/− mice (P = 0.0022). Scale bar, 100 µm, and applies to A and B.