Expression of hypoxia-inducible membrane-bound carbonic anhydrase isozyme XII in mouse tissues.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 277A:171–177 (2004) Expression of Hypoxia-Inducible, Membrane-Bound Carbonic Anhydrase Isozyme XII in Mouse Tissues PIIA HALMI,1 JONNA LEHTONEN,1 ABDUL WAHEED,2 WILLIAM S. SLY,2 1,3 AND SEPPO PARKKILA * 1 Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland 2 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 3 Department of Clinical Chemistry, University of Oulu, Oulu, Finland ABSTRACT Carbonic anhydrase (CA) XII is a membrane-associated enzyme that has been demonstrated to be normally expressed in some human tissues, to be upregulated in some cancers, and to be a hypoxia-inducible gene product. In mouse, CA XII has been recently localized in the kidney. In the present study, we investigated CA XII gene and protein expression in other mouse tissues, with the kidney serving as a positive control for the reagents. The expression of CA XII mRNA was examined using polymerase chain reaction (PCR) ampliﬁcation of commercial cDNAs produced from selected mouse tissues. A strong positive signal for CA XII mRNA was detected in the kidney, and weak signals were obtained in the testis and lung. Heart, spleen, liver, and skeletal muscle were negative. Immunohistochemical staining was performed using a mouse CA XII-speciﬁc antibody and biotin-streptavidin complex method. The results showed high expression of CA XII in the kidney, as expected. It was also highly expressed in the surface epithelial cells of the colon, whereas it was absent in the stomach, proximal small intestine, pancreas, liver, heart, and skeletal muscle. The maturing sperm cells showed a weak staining in a pattern that most probably indicates expression in the developing acrosomal membrane. The high expression in the kidney and colon suggests a role for CA XII in the maintenance of body ion and pH homeostasis in the mouse. However, the present ﬁndings demonstrated that CA XII has a very limited distribution in mouse tissues outside these two organs. Anat Rec Part A 277A:171–177, 2004. © 2004 Wiley-Liss, Inc. Key words: carbonic anhydrase; colon; gastrointestinal; kidney; plasma membrane Carbonic anhydrases (CAs) form a family of enzymes that catalyze the reversible hydration of carbon dioxide in the reaction CO2 ⫹ H2O 7 H2CO3 7 HCO3– ⫹ H⫹. All enzymatically active isoforms contain a zinc ion (Zn2⫹), which is considered critical for the catalytic activity. Different CA isozymes have been implicated in a number of physiological processes such as CO2 transport, regulation of pH balance, production of biological ﬂuids, fertilization, gluconeogenesis, ureagenesis, cell proliferation, and adhesion (Sly and Hu, 1995; Henry, 1996; Parkkila et al., 2003). Recent studies have also suggested that CA activity may be involved in long-term synaptic transformation and gating of memory storage (Sun and Alkon, 2002). CAs are divided into three distinct classes (␣, ␤, and ␥) that evolved independently and have no sequence homol© 2004 WILEY-LISS, INC. ogy (Hewett-Emmet and Tashian, 1996). The ␣-CA gene family includes at least 11 active CA isozymes, which have Grant sponsor: Academy of Finland; Grant number: 200969; Grant sponsor: National Institutes of Health; Grant number: GM34182, DK40163; Grant sponsor: Sigrid Juselius Foundation. *Correspondence to: Professor Seppo Parkkila, MD, PhD, Institute of Medical Technology, University of Tampere, Lenkkeilijänkatu 6, 33520 Tampere, Finland. Fax: ⫹358-3-2158597. E-mail: Seppo.Parkkila@uta.ﬁ Received 9 October 2003; Accepted 12 November 2003 DOI 10.1002/ar.a.20001 172 HALMI ET AL. Fig. 1. PCR analysis of mouse CA XII mRNA expression. The strongest 700-bp signal is seen in the kidney, followed by the testis, embryos, and lung. In embryos, the signal becomes more prominent at stages of 15 and 17 days. been characterized in the animal kingdom. Four of these are cytoplasmic (CA I, CA II, CA III, and CA VII) (Sly and Hu, 1995; Earnhardt et al., 1998), two are mitochondrial (CA VA and CA VB) (Fujikawa-Adachi et al., 1999), one is secreted (CA VI) (Murakami and Sly, 1987), and four are membrane associated (CA IV, CA IX, CA XII, and CA XIV) (Zhu and Sly, 1990; Pastorek et al., 1994; Ivanov et al., 1998; Türeci et al., 1998; Mori et al., 1999). Some isozymes such as CA II are expressed in a number of different tissues, whereas others (e.g., CA VI, IX, and XIV) show a more limited distribution. It is notable, however, that all active isozymes are expressed in the alimentary tract, although the cellular localization is unique for each isozyme (Parkkila et al., 1994; Fleming et al., 1995; Sly and Hu, 1995; Parkkila and Parkkila, 1996, Pastoreková et al., 1997; Kivelä et al., 2000; Parkkila et al., 2003). Human CA XII was cloned and characterized by two groups independently (Ivanov et al., 1998; Türeci et al., 1998), in both cases as a gene whose mRNA is greatly upregulated in renal cell carcinomas. The cDNA sequence predicted a 354-amino acid polypeptide with a molecular mass of 39,448 Da. CA XII is a one-pass, Type I transmembrane protein with an intact catalytic domain in the extracellular CA module (Ivanov et al., 1998). Recently, Whittington et al. (2001) described the crystal structure of a secretory form of human CA XII at 1.55-Å resolution. CA XII was found to exist as a dimer in both solution and the crystal. Interestingly, the CA12 gene has been identiﬁed as a von Hippel-Lindau target gene, suggesting a potential role for CA XII in von Hippel-Lindau carcinogenesis (Ivanov et al., 1998). More recently, CA XII has been found to be a hypoxia-inducible gene (Ivanov et al., 2001; Wykoff et al., 2001; Watson et al., 2003), possibly explaining its upregulation in certain tumors. These recent reports suggest that CA XII may be an excellent marker for hypoxia in tumors. CA XII mRNA is expressed in several normal human tissues such as kidney, colon, prostate, pancreas, ovary, testis, lung, and brain (Ivanov et al., 1998; Türeci et al., 1998). Immunohistochemical studies showed that CA XII protein is expressed in the human reproductive tissues, colon, and kidney (Karhumaa et al., 2000, 2001; Kivelä et al., 2000; Parkkila et al., 2000). Kyllönen et al. (2003) have recently studied the localization of CA XII in the mouse kidney and reported its localization in the proximal tubules and intercalated cells of the collecting ducts. This study was designed to investigate the expression of CA XII in other mouse tissues. MATERIALS AND METHODS Tissue Preparation Samples of stomach, duodenum, colon, jejunum, liver, ileum, rectum, psoas muscle, heart, and testis were obtained from two adult mice. The specimens were ﬁxed in Carnoy’s ﬂuid (absolute ethanol ⫹ chloroform ⫹ glacial acetic acid, 6:3:1) for 18 hr. Then they were dehydrated, embedded in parafﬁn in a vacuum oven at 58°C, and 4-m sections were placed on gelatin-coated microscope slides. Antibody and Immunohistochemical Method The production of polyclonal rabbit antibody raised against the recombinant mouse CA XII has recently been described by Kyllönen et al. (2003). The speciﬁcity of the antibody was conﬁrmed by Western blotting. The tissue sections were immunostained by the biotinstreptavidin complex method, employing the following steps: 1) pretreatment of the sections with undiluted cow colostral whey (Biotop Oy, Oulu, Finland) for 30 min and rinsing in phosphate-buffered saline (PBS), 2) incubation for 1 hr with anti-mouse CA XII serum or normal rabbit serum (1:100) in 1% bovine serum albumin (BSA)-PBS, 3) incubation for 1 hr with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Zymed Laboratories, South San Francisco, CA) diluted 1:300 in 1% BSA-PBS, 4) incubation for 30 min with peroxidase-conjugated streptavidin CARBONIC ANHYDRASE XII IN MOUSE TISSUES 173 Two primers for amplifying CA XII cDNA were chosen based on the published mouse CA XII sequence (accession numbers BC033432 and AK052639): forward 5⬘TGGTGATCCTTAAGAAGCA-3⬘ and reverse 5⬘-GCACAGGGTTTCGGAAAACT-3⬘, which generated a 692-bp ampliﬁcation product. The primers were produced by Sigma Genosys (Cambridgeshire, UK). Primers for glyceraldehyde 3-phosphate dehydrogenase (G3PDH, BD Biosciences) were used to monitor the quality of the cDNA samples. Five nanograms of total cDNAs were used as templates for PCRs. The PCR cycling protocol consisted of denaturation at 94°C for 1 min, followed by 33 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1.5 min, followed by ﬁnal extension at 72°C for 3 min. The PCR products were analyzed by electrophoresis on 1.2% agarose gel containing 0.1 g/ml ethidium bromide. DNA standard (100-bp DNA ladder) was purchased from New England Biolabs (Beverly, MA). RESULTS CA12 Gene Expression in Mouse Tissues The expression of the CA12 gene was investigated by PCR ampliﬁcation of a commercially available set of cDNAs produced for selected mouse tissues, including heart, spleen, lung, liver, skeletal muscle, kidney, testis, 7-day embryo, 11-day embryo, 15-day embryo, and 17-day embryo. Figure 1 shows a strong 700-bp band in kidney and a weak band in the testis. A very faint signal was also seen in the lung. Heart, spleen, liver, and skeletal muscle were negative. Embryos showed positive bands and the signal became stronger with increasing age of the embryo, indicating developmental regulation. Distribution of CA XII Protein Fig. 2. Positive control staining of CA XII in the mouse kidney. The enzyme is located in the proximal convoluted tubule (PCT) and collecting ducts (CD) (A). Immunostaining using normal rabbit serum instead of the anti-CA XII serum is negative (B). GL, glomerulus. Original magniﬁcations, ⫻200 (A and B), ⫻630 (insert in A). (Zymed Laboratories), and 5) incubation for 1 min in DAB solution containing 9 mg of 3,3⬘-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in 15 ml of PBS ⫹ 5 l of 30% H2O2. The sections were washed three times for 10 min in PBS after incubation in steps 2 and 3, and four times for 5 min in PBS after step 4. All the incubations and washings were carried out at room temperature. The stained sections were examined and photographed with Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany). Polymerase Chain Reaction (PCR) Method The expression of mouse CA XII mRNA was examined using a cDNA kit (MTC™ panel I) purchased from BD Biosciences (Palo Alto, CA). The cDNAs included in the MTC™ panel were used as templates for PCR using CA12 gene-speciﬁc primers. The mouse MTC™ panel I contained ﬁrst-strand cDNA preparations produced from total poly-A RNAs isolated from a number of different tissues. Immunohistochemical staining of CA XII revealed a distribution pattern that was limited to only a few mouse tissues. Kidney served as a positive control for the reagents, which were used to demonstrate CA XII by immunoﬂuorescence in the proximal tubules and in the intercalated cells of the collecting ducts (Kyllönen et al., 2003). This result is conﬁrmed in Figure 2. In the gastrointestinal tract, CA XII was not expressed in the stomach, duodenum, and jejunum (Fig. 3). The enterocytes in the ileum showed a faint positive staining, and the reaction became much stronger in the colon and rectum. In the large intestine, the staining was most intense in the surface epithelial cuff region as described previously for human CA XII. The highest expression was seen on the basolateral surfaces. No speciﬁc staining for CA XII was found in the mouse liver and pancreas (Fig. 4). Weak staining was detected in the developing sperm cells, in which the signal showed a crescentic or point-form pattern, suggesting CA XII expression in the acrosomal membrane (Fig. 5). No speciﬁc reaction was found in the psoas and heart muscle specimens (Fig. 6). DISCUSSION Both immunohistochemical and PCR analyses indicated that CA XII is expressed only in a few mouse organs. Based on the present and previous (Kyllönen et al., 2003) results, kidney appears to contain the highest amount of CA XII. This is in line with the ﬁndings by Türeci et al. (1998) and Ivanov et al. (1998), who showed high CA XII 174 HALMI ET AL. Fig. 3. Immunohistochemical staining of CA XII in the mouse gastrointestinal canal. Stomach (A), duodenum (B), and jejunum (C) are negative. Faint staining is seen in the ileal enterocytes (D). The strongest reaction is present in the surface epithelial cuff region of the colon (E) and rectum (F) (arrows). Insert in panel F demonstrates that the most prominent immunoreaction is conﬁned to the basolateral surfaces of the enterocytes. Original magniﬁcations, ⫻200 (A–F), ⫻630 (insert in F). mRNA expression in the human kidney and renal tumors. A more extensive transcription analysis conﬁrmed that the strongest signal for human CA XII mRNA was in the kidney and the second strongest was in the colon (Ivanov et al., 2001). Immunohistochemical staining revealed that CA XII protein is located at the basolateral plasma membrane of certain epithelial cells in both the human colon and kidney (Kivelä et al., 2000; Parkkila et al., 2000). Although CA XII has a similar pattern of expression in the mouse colon to that previously reported in the human colon (Kivelä et al., 2000), i.e., in the most superﬁcial part of the mucosa called the epithelial cuff region, the localization of CA XII in mouse kidney was clearly different from that reported for human kidney. In the human kid- ney, CA XII was localized to the proximal and distal tubules and principal cells of the collecting ducts (Parkkila et al., 2000), whereas in mouse, it was found in the proximal tubules and intercalated cells of the collecting ducts (Kyllönen et al., 2003). The physiological implications of these differences are not yet clear, nor is the role of CA XII in gut obvious. However, conservation of CA XII expression in the colonic enterocytes in both species suggests that the enzyme serves a similar role in human and mouse gut. It is well known that colonic enterocytes are equipped with a number of ion channels, carriers, and pumps, allowing highly efﬁcient transport of salt and water. The enterocytes located in the epithelial cuff region are differ- CARBONIC ANHYDRASE XII IN MOUSE TISSUES Fig. 4. Immunostaining of CA XII in the mouse liver (A) and pancreas (B). No reaction is seen. Original magniﬁcations, ⫻200 (A and B). Fig. 5. Immunohistochemical staining of CA XII in mouse testis. Weak staining is located to the developing sperm cells. Higher magniﬁcation reveals that the reaction is probably located at the site of the developing acrosome (arrows). Original magniﬁcations, ⫻200, ⫻630 (insert). 175 entiated from the colonic crypt cells (Lipkin, 1985). The base crypt enterocytes show the highest proliferative activity, demonstrate limited expression of differentiation markers, and have a high chloride secretory activity (Kunzelmann and Mall, 2002). The most luminal epithelial cells, representing the mature enterocytes, have a lower tendency to proliferate, express several differentiation marker proteins and certain lectins, and have a primarily absorptive function. The colonic absorption of NaCl can be electrogenic via epithelial Na⫹ channels or is electroneutral via parallel Na⫹/H⫹ and Cl–/HCO3– exchange (Kunzelmann and Mall, 2002). From these two pathways, electroneutral NaCl absorption has been shown to take place in both crypts and surface epithelial cuff region. In contrast, electrogenic absorption via Na⫹ channels is mainly located at the epithelial cuff region that is also the site of CA XII expression. Unfortunately, the role of CAs in electrogenic NaCl absorption is largely unknown. On the other hand, the participation of the colonic CA in the electroneutral NaCl absorption has been well established by CA inhibitors (Charney et al., 1986). Here, CA has been shown to be involved in the absorption of NaCl via the synchronous operation of apical Na⫹/H⫹ and Cl⫺/HCO3⫺ exchange processes. CAs also participate in the alkalization of the luminal contents by generating HCO3⫺ for apical Cl⫺/HCO3⫺ exchange (Feldman, 1994). Interestingly, recent results have indicated that CAs can be physically and functionally associated with Cl⫺/HCO3⫺ (AE1) and Na⫹/H⫹ exchangers (Sterling et al., 2001, 2002; Li et al., 2002). So far, CA IV is the only membrane-bound CA isozyme that has been shown to interact with AE1 protein (Sterling et al., 2002). It is quite possible that CA XII also could drive ion exchange processes across the colonic plasma membranes via a direct link with an ion transport protein. To date, there have been two major techniques to deﬁne the physiological role of CA isozymes. One is to use a membrane-permeant or -impermeant CA inhibitor, even though these studies are often hampered by lack of isozyme speciﬁcity. Another approach is to develop and analyze knockout mouse models deﬁcient in one CA isozyme. To date, two such mouse colonies with CA deﬁciency have been reported: CA II-deﬁcient mice produced by chemical mutagenesis (Lewis et al., 1988) and CA IXdeﬁcient mice developed by targeted mutagenesis and homologous recombination (Ortova Gut et al., 2002). Extrapolating from the present ﬁndings, physiological studies of renal and colonic function in CA XII-deﬁcient mice should be quite interesting. One might predict that disruption of CA XII function would result in abnormal regulation of ion and pH homeostasis, because both kidney and colon play key roles in the maintenance of body electrolyte and pH balance. 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