Identification of carbonic anhydrase XII as the membrane isozyme ...

Molecular Human Reproduction vol.6 no.1 pp. 68–74, 2000

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium

¨ .Tu¨reci3, A.Waheed4, J.H.Grubb4, G.Shah4, A.-K.Parkkila1, P.Karhumaa1,6, S.Parkkila1,2, O 1 5 K.Kaunisto , J.Tapanainen , W.S.Sly4, and H.Rajaniemi1 1Department

of Anatomy and Cell Biology, University of Oulu, Box 5000, FIN-90401 Oulu, 2Department of Clinical Chemistry, University of Oulu, FIN-90401 Oulu, Finland, 3Department of Internal Medicine, University of Saarland, 66421 Homburg, Germany, 4Edward A.Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St Louis, MO 63104, USA and 5Department of Obstetrics and Gynecology, University of Oulu, FIN-90401 Oulu, Finland

6To

whom correspondence should be addressed

Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergentsolubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle. Key words: bicarbonate/carbonic anhydrase/immunohistochemistry/pH/uterus

Introduction Bicarbonate plays a fundamental role in sperm function. Both male and female reproductive tract epithelia produce bicarbonate ions that are needed for sperm motility, capacitation and the acrosome reaction. Carbonic anhydrase isozyme II (CA II) present in the epithelium of ductus deferens and seminal vesicle equips the ejaculate with bicarbonate. These male-derived bicarbonate ions may participate in maintaining sperm motility in vagina and cervix and also buffer the low pH of vaginal milieu. Thereafter, the endometrial and oviductal epithelia produce bicarbonate that probably accelerates the migration of spermatozoa to the site of fertilization, and facilitates sperm capacitation and the acrosome reaction. CA enzymatic activity in the human endometrium has been demonstrated using biochemical methods (Falk and Hodgen, 1972; Friedley and Rosen, 1975). Friedley and Rosen (1975) showed histochemically that the endometrial epithelial cells contain CA activity, but the isozyme responsible for this activity has remained undefined. The CA gene family includes 10 enzymatically active CAs, which catalyse the reversible hydration of CO2 in the reaction CO2 ⫹ H2O ←→ H⫹ ⫹ HCO3–ı. Three most recently discovered isozymes (CA IX, XII, and XIV) are transmembrane proteins. Two of them (CA IX and XII) are highly expressed in some tumours and may be functionally related to oncogenesis (Za´vada et al., 1993; Pastorek et al., 1994; Ivanov et al., 1998; Tu¨reci et al., 1998). Both isozymes are also expressed in some normal tissues that 68

has been confirmed by immunohistochemistry or Northern blotting (Liao et al., 1994; Pastorekova´ et al., 1997; Ivanov et al., 1998; Tu¨reci et al., 1998). Liao et al. (1994) showed that CA IX is expressed in the reserve cells of the human cervix, while the normal endometrium was negative. Although positive signals for CA XII have been reported in Northern blots of normal human kidney, colon, prostate, pancreas, ovary, testis, lung, and brain (Ivanov et al., 1998; Tu¨reci et al., 1998), this is the first report on the expression and location of CA XII protein in any tissue. The basolateral location of CA XII in the endometrial epithelium suggests that the enzyme participates in the regulation of both the luminal and mural pH homeostasis in the human uterus.

Materials and methods Transfection of Chinese hamster ovary (CHO) cells cDNA sequences expressing either wild-type or truncated human CA XII were ligated into the mammalian expression vector pCXN (Tu¨reci et al., 1998). To produce a secretory form of CA XII, a stop codon was introduced at Q260X. These gene constructs were used to transfect CHO-K1 cells by electroporation. After selection in 400 µg/ml G418 for 10 days, colonies were isolated and cultured. Clones secreting high levels of human CA XII into the medium were identified by CA activity assay (Sundaram et al., 1986). CA XII secreted into the medium was affinity-purified using a sulphonamideagarose resin and used to prepare antibody as previously described (Zhu and Sly, 1990; Waheed et al., 1996). © European Society of Human Reproduction and Embryology

Carbonic anhydrase XII in human endometrium Antibodies The production and characterization of the anti-human CA XII antiserum raised against a histidine-tagged CA XII fusion protein have been described earlier (Tu¨ reci et al., 1998). The polyclonal antibody against the Q260X secretory form of human CA XII was raised as described above. Polyclonal rabbit antisera to human CA I, II, IV, V, and VI have been produced and characterized previously (Parkkila et al., 1991, 1993a,b, 1994, 1996; Saarnio et al., 1999). The cross-reactivities of these antibodies were tested in dot and Western blots and time-resolved immunofluorometric assays in which they showed high isozyme specificity. Purification of CAs from human endometrium Human endometrium specimen (secretory phase) was obtained from a patient with leiomyoma. The sample was homogenized using a Ultra-Turrax homogenizer and sonicated in ice-cold 0.1 mol/l Tris–SO4 buffer, pH 8.7, containing 1 mmol/l phenylmethylsulphonylfluoride (PMSF), 1 mmol/l benzamidine, and 1 mmol/l o-phenanthroline as protease inhibitors. The tissue homogenates were centrifuged at 100 000 g for 30 min. The cytosol and total membrane pellets were recovered, and the membrane pellets were suspended and incubated in the 0.1 mol/l Tris–SO4 buffer containing 1% TritonX-100 for 30 min on ice. The membrane fraction was centrifuged 13 000 g for 10 min and the supernatant was subjected to affinity purification. The inhibitor affinity chromatography was performed using the CM Bio-Gel A coupled to p-aminomethyl benzenesulphonamide as described in detail previously (Parkkila et al., 1990). SDS-polyacrylamide gel electrophoresis (SDS–PAGE) All the reagents for SDS–PAGE were from Bio-Rad Laboratories (Richmond, CA, USA). The electrophoreses were performed in a Mini-Protean electrophoresis unit (Bio-Rad Laboratories) under reducing conditions according to Laemmli (1970), using a 10% acrylamide separating gel and a 4% acrylamide stacking gel. CAs analysed included CA XII (20 µg cell protein from stably transfected CHO-cells), CA IX (5 µl of Nutrient Mixture Ham’s F-12 medium; Life Technologies, Paisley, Scotland) containing truncated CA IX produced in transfected NIH3T3 cells which were a generous gift from Drs Silvia and Jaromir Pastorek, (Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic) and purified CA I, II, IV, and VI (0.5 µg). These aliquots of isozymes show strong immunoreactions in a Western blot analysis when the corresponding antibodies are used (data not shown). Isoelectric focusing (IEF) IEF was carried out using Novex Pre-Cast vertical IEF gels (pH 3–10) (Novex, San Diego, CA, USA) containing 5% polyacrylamide and 2% ampholytes. 15 µl samples of 5⫻ eluate from inhibitor affinity chromatography of endometrial proteins or 20 µg protein from CHO cell homogenate transfected with CA XII cDNA were applied to each lane. The electrophoreses were performed in an Xcell II Mini-Cell unit (Novex) at a constant power of 2 W per gel for 2 h with a voltage limit of 500 V. Western blotting The proteins were transferred electrophoretically from the gel to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Bedford, MA, USA) in a Novex Blot Module (Novex). After the transblotting the membrane was first incubated with Tris-buffered saline/Tween (TBST) buffer (10 mmol/l Tris–HCl, pH 7.5, 150 mmol/ l NaCl, 0.05% Tween-20) containing 10% cow colostral whey (BioTop, Oulu, Finland) for 25 min at room temperature and then with anti-human CA XII antiserum diluted 1:2000 in TBST buffer

for 1 h at room temperature. The sheets were washed four times for 5 min with TBST buffer and incubated for 30 min at room temperature with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG; Bio-Rad Laboratories) diluted 1:3000 in TBST buffer. After washing three times for 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (Bio-Rad Laboratories). Northern blotting The detailed procedure for Northern blot analysis was as previously described (Tu¨ reci et al., 1998). Briefly, RNA was extracted from renal tumour, normal kidney and uterus using guanidium thiocyanate (Chomczynski and Sacchi, 1987). Gels containing 10 µg of RNA per lane were blotted onto nylon membranes (Hybond N, Amersham). After prehybridization, the membranes were hybridized with the specific [32P]-labelled CA XII cDNA probe (Tu¨ reci et al., 1998). The membranes were washed at progressively higher stringency followed by autoradiography. Immunocytochemistry The CHO cells transfected with CA XII cDNA were fixed with 4% neutral-buffered formaldehyde for 25 min. Then they were rinsed with phosphate-buffered saline (PBS) and subjected to immunofluorescence staining that consisted of the following steps: (i) pretreatment of the cells with 0.1% bovine serum albumin (BSA) in PBS for 30 min and rinsing in PBS; (ii) incubation for 1 h with the anti-CA XII antiserum diluted 1:100 in 0.1% BSA–PBS; (iii) incubation with 1:20 diluted fluorescein-conjugated goat anti-rabbit IgG antibodies (Dakopatts, Glostrup, Denmark) in 0.1% BSA–PBS. The cells were washed three times for 2 min after the incubation steps. All incubations and washings were done in the presence of 0.05% saponin. The cells were viewed with a confocal laser-scanning microscope (Leitz CLSM; Leica Laser Technics, Heidelberg, Germany). The specimens were excited with a laser beam at a wavelength of 488 nm and the emission light was focused through a pinhole aperture. The full field was scanned in square image formats of 512⫻512 pixels and built-in software was used to reconstruct the images obtained from the confocal sections. Samples of the human endometrium of proliferative (n ⫽ 2) and secretory (n ⫽ 3) phases were obtained together with routine histopathological specimens taken during surgical operations for leiomyoma of the uterus. Informed consent was obtained from each patient and the research was carried out according to the provisions of the Declaration of Helsinki. Each tissue sample was divided into several small pieces, ~5 mm thick. The specimens were fixed in Carnoy’s fluid (absolute ethanol ⫹ chloroform ⫹ glacial acetic acid 6:3:1) for 6 h at 4°C. The samples were then dehydrated, embedded in paraffin wax in a vacuum oven at 58°C, and sections of 5 µm were placed on gelatin-coated microscope slides. The phase of endometrium was defined using clinical data and histological examination. The CA isozymes were immunostained by the biotin–streptavidin complex method, employing the following steps: (i) pre-treatment of the sections with undiluted cow colostral whey for 40 min and rinsing in PBS; (ii) incubation for 1 h with the primary antiserum diluted 1:100 in 1% BSA–PBS; (iii) treatment with cow colostral whey for 40 min and rinsing in PBS; (iv) incubation for 1 h with biotinylated swine anti-rabbit IgG (Dakopatts) diluted 1:300 in 1% BSA–PBS; (v) incubation for 30 min with peroxidase-conjugated streptavidin (Dakopatts) diluted 1:500 in PBS; (vi) incubation for 2 min in DAB solution containing 9 mg 3,3⬘-diaminobenzidine tetrahydrochloride (Fluka, Buchs, Switzerland) in 15 ml PBS ⫹ 5 µl 30% H2O2. The sections were washed three times for 10 min in PBS after incubation steps ii, iv, and v. All the incubations and washings were carried out

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P.Karhumaa et al. at room temperature, and the sections were finally mounted in Permount (Fisher Scientific, Fair Lawn, NJ, USA). The stained sections were examined and photographed with a Leitz Aristoplan microscope (Wetzlar, Germany). To examine the subcellular localization of CA XII in the endometrial epithelium, tissue sections were stained using an immunofluorescence method and analysed by confocal laser scanning microscopy. The steps in the immunostaining were as follows: (i) pre-treatment of the sections with 1% BSA–PBS for 40 min; (ii) incubation for 1 h with 1:100 diluted (1% BSA–PBS) antibody raised against the secretory form of CA XII. (iii) Washing three times for 5 min in PBS; (iv) incubation for 1 h with 1:30 diluted fluorescein-conjugated goat antirabbit IgG antibodies (Dakopatts) in 1% BSA–PBS; (v) Washing three times for 5 min in PBS. The immunostained sections were examined with a confocal laser-scanning microscope (Leitz CLSM).

Results Characterization of anti-CA XII antibodies Production of the anti-human CA XII antiserum raised against histidine-tagged CA XII fusion protein has been described recently (Tu¨ reci et al., 1998). The second antiserum was raised against the recombinant human CA XII produced in CHOcells transfected with human CA XII cDNA. Figure 1 shows a confocal laser scanning microscopy image of immunofluorescence staining for wild-type CA XII in transfected CHO cells. Both antisera showed positive immunoreaction at the plasma membrane. The less intense intracellular signal probably represents the newly synthesized enzyme in the endoplasmic reticulum. Control immunostaining of these cells using non-immune rabbit serum instead of the anti-CA XII serum showed no positive immunoreaction (data not shown). The specificities of the anti-human CA XII antisera were tested by SDS–PAGE and Western blotting. Both antisera identified a strong 45 kDa and weaker 40 kDa polypeptide bands of CA XII on Western blots of CHO cells transfected with wild-type human CA XII cDNA (Figure 2). The 45 kDa and 40 kDa polypeptides represent the glycosylated and deglycosylated forms of the enzyme respectively (Tu¨ reci et al., 1998). In addition to these polypeptides, the antiserum raised against histidine-tagged CA XII fusion protein recognized a faint, unidentified band of 66 kDa. The anti-CA XII antibodies showed no cross-reactivity with other CA isozymes analysed (CA I, II, IV, VI, and IX). Immunohistochemistry Immunohistochemical staining of the human endometrium for CA XII revealed that both antibodies, raised either for histidinetagged CA XII fusion protein or recombinant CA XII, are useful and give similar staining pattern in these immunostaining conditions. In the body of the uterus, CA XII was localized to both luminal and glandular epithelia, the reaction being strongest in the deep parts of the glands (Figure 3A–C). The confocal microscopic image reveals that the most distinct signal is associated with the basolateral plasma membrane (Figure 3F). The endometrium samples representing the proliferative phase of the menstrual cycle (Figure 3A) showed a somewhat stronger immunoreaction than the samples obtained during the secretory phase (Figure 3B,C). For controls, human 70

Figure 1. A confocal laser scanning microscopy image of CA XII in Chinese hamster ovary cells transfected with CA XII cDNA. Immunostaining was performed using antisera raised against (A) truncated CA XII and (B) histidine-tagged CA XII fusion protein (B). Bar ⫽ 10 µm.

endometrium specimens were also immunostained using antihuman CA I and II sera. These antibodies showed the most distinct immunoreactions in the erythrocytes and the endothelium of the blood vessels (Figure 3D,E), indicating the specificity of the CA XII immunostaining in the epithelium. In the cervix sections, only occasional epithelial cells showed a weak, positive reaction for CA XII (Figure 3G,H). Purification of CA XII from endometrium and IEF To confirm the presence of CA XII in the endometrium, CAs were affinity-purified from both cytosol and membrane fractions of a human endometrium sample, and subsequently identified using IEF followed by Western blotting. In the purified cytosolic fraction, anti-CA I and II antibodies recognized the corresponding enzymes, the pI values being 6.5 and 7.5 respectively (Figure 4A). These were presumably of erythrocyte origin (Figure 3D,E). The pI value of 7.5 for CA II is fairly similar to that (7.6) reported previously (Jones et al., 1982). The pI value 6.5 for CA I was confirmed using human CA I standard purchased from Bio-Rad Laboratories. None of the other CA isozymes tested (CA IV, V, VI, or XII)

Carbonic anhydrase XII in human endometrium

Figure 2. Specificity of antisera raised against (A) truncated CA XII and (B) histidine-tagged CA XII fusion protein tested by Western blotting. An aliquot of 20 µg of Chinese hamster ovary (CHO)-cell homogenate for CA XII, 5 µl of serum-free culture medium containing truncated CA IX, or 0.5 µg of purified CA I, II, IV, or VI was applied to each lane. Both antibodies identified a major 45-kDa and a minor 40 kDa polypeptide bands in the Western blot of CHO-cells transfected with CA XII cDNA, while they showed no cross-reactivity with other CAs.

were detectable in this fraction. Purified membrane proteins were similarly subjected to IEF followed by Western blotting. Figure 4B shows that both anti-CA I and II antibodies recognized the corresponding enzymes, which probably results either from cytoplasmic contamination of the membrane fraction with erythrocyte enzyme, or some membrane-associated CA I and II. Figure 4B also demonstrates that small amounts of CA XII (pI 6.3) have been purified from the solubilized membrane fraction of the endometrium. As a positive control for CA XII in endometrial membranes, CHO cells transfected with wild-type CA XII cDNA showed a protein band of the same isoelectric pH. Northern blotting Figure 5 shows Northern blots of mRNA from human renal carcinoma, normal kidney and uterus. The 4.5 kb transcript previously reported to be present in RNA from kidney and renal cell carcinoma was also seen in uterus. The signal for uterine CA XII mRNA transcript was about half that of the level in kidney.

Discussion CA XII was originally discovered as a cell surface CA isozyme that is over-expressed in renal cell carcinomas and subject to regulation by the von Hippel–Lindau tumour suppressor gene (Ivanov et al., 1998; Tu¨ reci et al., 1998). It is also expressed

in normal kidney, but its functional role there is not yet established. Discovery of CA XII in normal endometrium suggests that it may play a role in the reproductive functions of the uterus by contributing to bicarbonate production at this site. Spermatozoa are exposed to changing microenvironments during their transit through the male and female reproductive tracts. Changes in the bicarbonate concentration in the luminal fluids have been implicated in the regulation of sperm motility, capacitation, and acrosome reaction (Okamura et al., 1985; Boatman, 1997). Bicarbonate ion has been reported to activate sperm motility via a bicarbonate-sensitive adenylate cyclase present in the plasma membrane of the sperm cell (Okamura et al., 1985; Rojas et al., 1992). To transfer bicarbonate ions across the plasma membrane, human spermatozoa express a specific band 3-related carrier protein located in the equatorial segment of the sperm head (Parkkila et al., 1993c). Spermatozoa are stored in a quiescent state in cauda epididymidis and proximal ductus deferens (Carr and Acott, 1984; Robaire and Hermo, 1988; Bedford, 1994). The motility of spermatozoa is increased during ejaculation when they come into contact with the alkaline secretion from accessory sex glands (Mann and Lutwak-Mann, 1981). The bicarbonate present in the ejaculate has been proposed to maintain the sperm motility until the cells enter the lumen of the uterus through the cervical canal (Okamura et al., 1985; Kaunisto et al., 1990). In the female genital tract, the endometrial and oviductal epithelium may produce an alkaline environment for maintaining the sperm motility. This suggestion is in agreement with other observations (Guerin et al., 1997), that the sperm motility is improved by co-culture of human spermatozoa with either endometrial or oviductal epithelial cells. Bicarbonate has been suggested to be the key factor also in sperm capacitation, a process which involves changes in the sperm plasma membrane (Harrison et al., 1996; Boatman, 1997). In fact, bicarbonate has been considered as an essential component for successful in-vitro fertilization (Suzuki et al., 1994). Capacitation is followed by the acrosome reaction which is a Ca2⫹-dependent process leading to the exocytotic release of the contents of acrosome. Previous studies have shown that a rise in intracellular pH triggers the activation and opening of calcium channels in spermatozoa that result in the progesterone-induced acrosome reaction (Fraser, 1995; Sabeur and Meizel, 1995; Benoff, 1998). The present immunohistochemical results show that CA XII is expressed in the human endometrial epithelium. The strongest signal for CA XII was localized to the basolateral plasma membrane of the epithelial cells. CA XII is the second CA isozyme locating at the basolateral plasma membrane of polarized cells. CA IX, the other transmembrane isozyme, has a basolateral localization in the gastrointestinal tract and uterine cervix (Liao et al., 1994; Pastorekova´ et al., 1997; Saarnio et al., 1998). Both CA IX and XII have been reported to be related to von Hippel–Lindau-mediated carcinogenesis in that higher expression levels have been reported in various cancers (Ivanov et al., 1998; Tu¨ reci et al., 1998), and both isozymes have been shown to be down-regulated by expression of the wild-type von Hippel–Lindau tumour suppressor gene (Ivanov 71

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Figure 3. Immunohistochemical staining of CA XII (A–C, F–H), CA I (D) and CA II (E) in human endometrium (A-F) and cervix (G, H). Both antibodies raised either against the truncated CA XII (A, B, F) or histidine-tagged CA XII fusion protein (C) show positive staining in the endometrial epithelial cells. A high magnification view by confocal laser scanning microscopy (F) shows a strong signal in the basolateral plasma membrane (bl), whereas the apical plasma membrane (a) is negative. Immunohistochemical staining of human cervix using antibodies raised against the truncated CA XII (G) and histidine-tagged CA XII fusion protein (H) showed only a weak reaction in occasional epithelial cells (arrows in H). Proliferative phase (A, F, G, H), secretory phase (B–E). A–E, G, H Bar ⫽ 30 µm; (F) bar ⫽ 5 µm.

et al., 1998). In malignant tumours, CA XII has been proposed to acidify the immediate extracellular milieu surrounding the cancer cells (Ivanov et al., 1998). The acidification would create a microenvironment conducive to tumour growth and spread (Warburg, 1926). Very little is known about the extracellular pH milieu required for normal development of the endometrium during the menstrual cycle. It will be of interest to explore the role of CA XII in the regulation of extracellular pH in normal endometrium and to study changes in its expression with hormonal changes that influence the menstrual cycle, and that 72

occur during gestation. The active centre of the enzyme is located on the cell exterior, beneath the basolateral plasma membrane. Hypothetically, at this site, CA XII could regulate the extracellular pH in close proximity to the epithelium as it undergoes morphological changes during the menstrual cycle. What is less clear is whether and how CA XII contributes to alkalization of the uterine cavity which have been reported to be bicarbonate rich (Wishwakarma, 1962). CA XII could be functionally coupled to an unidentified bicarbonate transporter to move bicarbonate across the basolateral membrane into the epithelial cells and to another transporter on the apical surface

Carbonic anhydrase XII in human endometrium grants from Sigrid Juselius Foundation to S.P., from National Institutes of Health (DK 40163) to W.S.S., and from Deutsche Forschungs¨ .T. gemeinschaft to O

References

Figure 4. Analysis of inhibitor affinity-purified proteins from cytosolic (A) and membrane (B) fractions using isoelectric focusing followed by Western blot analysis. Anti-CA I and II sera recognized the corresponding isozymes, the signal being more intense in the cytosolic fraction. The signal present in the third lane of panel B (aCA IV) is due to non-specific binding of antibodies. Anti-CA XII antibody recognized a 6.3-pI protein band in the membrane fraction. Anti-CA XII antibody also recognized a protein band of the same isoelectric pH in Chinese hamster ovary cells transfected with CA XII cDNA.

Figure 5. Northern blots of mRNA from renal carcinoma, normal kidney and uterus. Strong positive signal for CA XII mRNA is seen in all of these specimens.

to effect secretion of the bicarbonate ions into the uterine cavity (Fong et al., 1998).

Acknowledgements We thank Ms Lissu Hukkanen, Mr Mika Kihlstro¨ m, and Mr Eero Oja for skilful technical assistance. The work was supported by the

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