Histochem Cell Biol (2010) 133:285299 DOI 10.

1007/s00418-009-0667-0

ORIGINAL PAPER

Murine mCLCA5 is expressed in granular layer keratinocytes of stratied epithelia

Josephine Braun Melanie K. Bothe Lars Mundhenk Carol L. Beck Achim D. Gruber

Accepted: 19 November 2009 / Published online: 11 December 2009 Springer-Verlag 2009

Abstract CLCA proteins represent a large family of proteins widely expressed in mammalian tissues with a unique expression pattern for each family member analyzed so far. However, their functions in normal and diseased tissues are poorly understood. Here, we present the cellular expression pattern of mCLCA5 in murine tissues using immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy with specic antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratied squamous epithelia of the body. Biochemical protein characterizations revealed that the amino-terminal cleavage product is fully secreted by the cell, while the carboxy-terminal cleavage product remains associated with the cell. The results imply that mCLCA5 may play a role in maturation and keratinization of squamous epithelial cells. Keywords Calcium-activated chloride channel Squamous epithelium Mouse Skin Stomach

Introduction The mCLCA5 protein belongs to a multi-gene family of proteins of unknown function. They were originally named chloride channels, calcium-activated due to their putative function as mediators of calcium-activated chloride currents. Recent studies, however, on protein structure and electrophysiology of transfected HEK293 cells suggest a more indirect action via mediating an as yet unidentied calcium-activated chloride conductance (Gibson et al. 2005; Hamann et al. 2009; Mundhenk et al. 2006). For several members, a role has been proposed in the pathogenesis of highly relevant human diseases, including asthma (Anton et al. 2005; Hoshino et al. 2002; Nakanishi et al. 2001; Nakano et al. 2006; Toda et al. 2002), cystic brosis (CF, mucoviscidosis) (Brouillard et al. 2005; Leverkoehne et al. 2006; Ritzka et al. 2004), and tumor metastasis (Abdel-Ghany et al. 2002; Elble et al. 1997). The expression patterns of these proteins are strongly indicative of cell type-specic functions. For example, mCLCA3, its human ortholog hCLCA1, the equine ortholog eCLCA1 and the porcine ortholog pCLCA1 are expressed in goblet cells only (Anton et al. 2005; Gruber et al. 1998; Leverkoehne and Gruber 2002; Plog et al. 2009), whereas mCLCA6 is exclusively expressed in nongoblet cell enterocytes (Bothe et al. 2008). In contrast, more complex and partly contradictory expression patterns have been reported for several other CLCA family members, such as hCLCA2 (Agnel et al. 1999; Connon et al. 2005; Pauli et al. 2000) and its murine ortholog mCLCA5 (Beckley et al. 2004). hCLCA2 has been linked to diseases with secretory dysfunction including cystic brosis (Ritzka et al. 2004) due to its suggested role in transepithelial ion transport (Gruber et al. 1999). It has also been detected in vascular endothelial cells where it is thought to act as an

J. Braun and M. K. Bothe contributed equally. J. Braun M. K. Bothe L. Mundhenk A. D. Gruber (&) Department of Veterinary Pathology, Faculty of Veterinary Medicine, Freie Universitat Berlin, Robert-von-Ostertag Strae 15, 14163 Berlin, Germany e-mail: gruber.achim@vetmed.fu-berlin.de L. Mundhenk e-mail: mundhenk.lars@vetmed.fu-berlin.de C. L. Beck Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, 1020 Locust Street, 366 JAH, Philadelphia, PA 19107, USA

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adhesion molecule for metastatic cancer cells (AbdelGhany et al. 2001, 2003; Pauli et al. 2000). Unique among all other CLCA proteins characterized so far, hCLCA2 has also been found in squamous epithelium (Connon et al. 2004) where it may contribute to chloride conductance (Itoh et al. 2000). However, different groups have reported partly contradictory results about the expression of hCLCA2 (Abdel-Ghany et al. 2001, 2003; Connon et al. 2004; Gruber et al. 1999). The cellular expression pattern of mCLCA5, the murine ortholog to hCLCA2, is unknown. Crude organ extracts contained mCLCA5 mRNA in a variety of tissues including the esophagus, gastrointestinal tract, pancreas, kidney, lung, uterus, spleen, heart, aorta, mammary gland and eye (Beckley et al. 2004; Evans et al. 2004) with no further information as to the expressing cell type. This apparently wide expression pattern and rst functional observations have led to speculations about a role in growth arrest, apoptosis, or anoikis (Beckley et al. 2004; Gruber and Pauli 1999; Patel et al. 2006). Beckley and coworkers hypothesized that mCLCA5 may antagonize mammary tumor progression by inhibiting detachment-sensitive growth (Beckley et al. 2004). It has also been suggested that it may drive mucous cell metaplasia in airway epithelium, possibly compensating for a lack of mCLCA3 in mCLCA3decient mice (Patel et al. 2006). In this study, we systematically characterized the cellular expression pattern of mCLCA5 on protein and mRNA levels using specic anti-mCLCA5 antibodies and realtime RT-qPCR analysis following laser-assisted microdissection. To gain more information on possible functions, we also analyzed the cellular protein processing.

urinary bladder, ovaries, uterus, cervix, vagina, mammary glands, adrenal glands, thyroid glands, eyes, brain (cortex, cerebellum, brainstem, medulla), abdominal wall (skeletal muscle, serosa), skeletal muscle, bone including bone marrow, lymph nodes, and adipose tissue. Unlike the human stomach, the murine stomach is only partially lined by glandular mucosa, whereas the rest is lined by nonglandular, keratinized mucosa. Both types of epithelia were investigated separately and revealed different results. Sequence analyses and generation of antibodies The mCLCA5 amino acid sequence (GenBank accession number NP 848812) was analyzed using the SignalP 3.0 software (Nielson et al. 1997). The software ProtParam was used to predict the molecular mass of the full-length primary translation product and the software NetNGly to identify potential N-linked glycosylation sites (Gasteiger et al. 2005). To identify possible transmembrane regions, hydrophobicity analyses were carried out using the KyteDoolittle (Kyte and Doolittle 1982), SOSUI (Hirokawa et al. 1998), HMMtop (Tusnady and Simon 2001), TMPRED (Hofmann and Stoffel 1993), DAS (Cserzo et al. 1997) and PSORT II (Nakai and Horton 1999) algorithms. Peptide sequences of high predicted immunogenicity were selected from the mCLCA5 protein sequence using computer-aided antigenicity analyses. Seven oligopeptides were synthesized, ve located in the predicted amino-terminal cleavage product of mCLCA5 (am5-N1, corresponding to amino acids 92105, TWTDHNYSRVRQES; am5-N2, corresponding to amino acids 110123, NVIVAEQSEEHGDD; am5-N3, corresponding to amino acids 172185, GVFDEYNNDKPFYV; am5-N4, corresponding to amino acids 363376, SLQQIYSDDDRKLL; am5-N5, corresponding to amino acids 399413, FEVVEERNGRADGS) and two in the predicted carboxy-terminal cleavage product (am5-C1, corresponding to amino acids 784797, WTAPGEDFDQGQTT; am5-C2, corresponding to amino acids 925937, IFNRKKRPSRKENE). The seven oligopeptides were coupled to keyhole limpet hemocyanin (KLH) and used for standard immunization of two rabbits each. Preimmune sera were collected before immunization and used as controls in all immunodetection experiments. The 14 antisera were designated am5-N1-a/am5-N1-b and so forth. Following initial testing of antiserum reactivities, after which non-reactive and poorly reactive antisera were excluded, the anti-amino-terminal antiserum am5-N5-b and the anti-carboxy-terminal antiserum am5-C1-a were afnity-immunopuried using the corresponding peptides coupled to an EAH-sepharose column and designated am5-N5-bp and am5-C1-ap. am5-N5-bp, am5-C1-ap and am5-C1-a were used for all further experiments.

Materials and methods Animals and tissue processing Three female 10-week-old C57BL/6J mice and three mCLCA3neoE711 mice on C57BL/6J background with exons 711 of the mCLCA3 gene replaced by a neomycin cassette were sacriced by cervical dislocation in accordance with national guidelines. Samples from the following tissues were either stored at -80C, immersion xed in neutral-buffered 4% formalin for 24 h or lysed on ice using a precellys 24 homogenizer (Peqlab biotechnology GmbH, Erlangen, Germany) and used for protein and mRNA tissue expression analyses: haired skin (from tail), pinna, nasal cavity, larynx, trachea, lung, heart, aorta, parotid and sublingual salivary glands, tongue, soft palate, esophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, pancreas, gall bladder, liver, spleen, thymus, kidneys,

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Immunohistochemistry and confocal laser scanning microscopy Immunohistochemical (IHC) analyses were performed as described previously (Leverkoehne and Gruber 2002). Briey, xed tissue samples were embedded in parafn, cut at 4-lm thickness and mounted on adhesive glass slides. Consecutive tissue sections were routinely stained with hematoxylin and eosin (HE) for histological examination. For IHC, the avidinbiotin-peroxidase complex (ABC) method (Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA) was applied. After dewaxing of the mounted tissue sections in xylene and rehydration in increasing concentrations of ethanol, the following antigen retrieval (AR) methods were tested: (a) 15-min microwave heating (700 W) in 10 mM citric acid, pH 6.0, (b) 15-min treatment with 0.1% pronase E (Applichem, Darmstadt, Germany) in PBS at 37C, or (c) microwaving after pronase E digestion. Based on these trials, the pronase E pre-treatment method (b) was used for systematic tissue analyses. In addition, cryo-sections were used as templates and treated similarly after xation in either 100% ice cold acetone, 100% methanol, 4% formalin, or equal amounts of ethanol and acetic acid. Slides were incubated with the puried antibodies am5-N-bp or am5-C-ap in PBS containing 1% BSA, with dilutions ranging from 1:100 to 1:40,000, or the respective pre-immune serum, or the unrelated immunopuried rabbit antibodies am6-N-1ap and am6-C-1 bp (Bothe et al. 2008). To further test for specicity, primary antibodies were pre-adsorbed with the peptides (10 lg/ml) used for immunizations, respectively, and the results compared with reactions from non-adsorbed antibodies. Finally, the slides were incubated with 1:200 dilutions of biotinylated secondary goat-anti-rabbit antibodies, and HRP-coupled streptavidin. diaminobenzidine (DAB) was used as substrate for color development. The slides were counterstained with hematoxylin, dehydrated through increasing concentrations of ethanol, cleared in xylene, and coverslipped. For confocal laser scanning microscopy, 10 lm parafn tissue sections were processed as described above. The rst antibody, am5-C1-ap, was diluted 1:100. The secondary DyLight-488 labeled antibody (Dianova; Hamburg, Germany) was diluted 1:200. Sections were analyzed using a confocal microscope (TCS SP2; Leica, Wetzlar, Germany). DyLight-488 was excited at 493 nm, and emission of green uorescence was detected at 517 nm. Single optical scans were repeated several times and recorded separately. Immune electron microscopy Immune electron microscopy was performed as described earlier (Leverkoehne and Gruber 2002). In brief, samples

of the non-glandular mucosa of the stomach were xed in a xative containing 4% paraformaldehyde, 0.25% glutaraldehyde and 3.7% saccharose and embedded in gelatine capsules lled with LR White resin containing LR White accelerator (Plano; Wetzlar, Germany). Resin was allowed to polymerize at room temperature overnight. Ultrathin sections cut at 6090 nm were collected on uncoated 180mesh nickel grids (Pelco International; Clovis, CA, USA). For immunolabeling, grids were immersed upside down in drops of the respective solution. After blocking of aldehydes with PBS containing 50 nM glycine, sections were blocked in PBS containing 0.5% BSA and 0.1% liquid gelatin (washing buffer) and 5% heat-inactivated normal goat serum. Puried serum am5-C1-ap was incubated in washing buffer at 4C overnight in a humid chamber (dilution 1:5,000) followed by repeated washes in washing buffer and by incubation for 1 h with 10-nm gold particleconjugated goat anti-rabbit immunoglobulins (Sigma Aldrich; Munich, Germany) diluted at 1:40 in washing buffer. Sections were postxed, contrasted for 15 min in aqueous uranyl acetate (Merck; Darmstadt, Germany), and washed in distilled water. Grids were air dried and examined with a Zeiss EM 10 CR transmission electron microscope (Zeiss; Oberkochen, Germany). Negative controls were included using puried immune sera from rabbit against irrelevant antigens as primary antibodies (pCFTR-c-1/2-p) that failed to yield any signals in the relevant cell structures at dilutions of 1:200 or higher. Laser-capture microdissection, RNA isolation and reverse transcription For isolation of total RNA for real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), representative tissue samples were snap frozen in liquid nitrogen and stored at -80C until further use. Total RNA was extracted from whole-tissue samples using the Trizol method (Invitrogen) and puried using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol and including a digestion with RNasefree DNase I (Qiagen). Approximately 500 ng total RNA was reverse transcribed into cDNA using random hexamer primers in the presence of RNase Out according to the manufacturers protocol (SuperScript III First-Strand Synthesis System; Invitrogen). For laser-capture microdissection (LCM), frozen stomach, eye, brain and spleen samples from two C57BL/6J mice were cut into multiple consecutive sections of 8 lm thickness and mounted onto glass slides coated with a polyethylene naphthalate membrane (PALM membrane slides; PALM Microlaser Technologies, Martinsried, Germany). Sections were xed for 1.5 min in 95% ethanol at -20C, stained with hematoxylin dissolved in diethylpyrocarbonate

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(DEPC)-treated water for 45 s, rinsed in DEPC-treated water for 45 s, and then stained with 1% eosin dissolved in DEPC-treated water at room temperature. Sections were dehydrated in 70 and 100% ethanol, air dried at room temperature for about 1 min, and stored at -80C. Total RNA was isolated from the following locations. Stomach: nonglandular mucosa as a whole, luminal and basal half of the stratied squamous epithelium; glandular epithelium; submucosa and smooth muscle cells; eye: cornea, lens, retina, sclera and choroidea (posterior uvea); brain: granular and Purkinje cell layers of the cerebellum, hippocampus, choroid plexus of the cerebrum, granular and pyramidal layers of the cerebrum and white matter of the cerebellum; and spleen: capsule, red pulp and white pulp. A total area of up to 3.0 9 106 lm2 cells from each region of interest were separated by laser microdissection and laser-pressure catapulted into 0.5-ml Eppendorf tubes. Total RNA from LCM samples was extracted using the RNeasy Mini Kit (Qiagen) as described above. Real-time quantitative polymerase chain reaction Primers and probe used for real-time qPCR were designed using the Beacon Designer 3.0 software (Premier Biosoft International, Palo Alto, CA, USA). Primer sequences for mCLCA5 were 50 -ACGATGACCGGAAGCTGCTG-30 (forward) and 50 -ACCACCTCAAAGCCTTTCTTAACC30 (reverse) with a predicted amplicon size of 100 bp. The corresponding TaqMan probe sequence was 50 -FAMCCTGCCGACCGCCGTGTCCACT-TAMRA-30 . The primer pair and probe were designed based on sequence comparisons to specically discriminate amongst all known mCLCA homologs (mCLCA 1 to -6) and to encompass an intron to exclude amplication of contaminating genomic DNA. No cross-reactions were detected when linearized cloned full cDNA samples of mCLCA1, mCLCA2, mCLCA3 and mCLCA6, or PCR-derived relevant fragments of mCLCA4 were used as templates containing 102106 copies. To be able to correct for variations in RNA amounts and cDNA synthesis efcacy, a fragment of the cDNA encoding murine elongation factor 1a (EF1a) was amplied in parallel in all experiments as an internal housekeeping gene standard (Gruber and Levine 1997). Primer sequences for EF1a were 50 -AAAAACGACC CACCAATGG-30 (forward) and 50 -GGCCTGGATGGTT CAGGATA-30 (reverse) with a predicted amplicon size of 68 bp. The corresponding TaqMan probe sequence was 50 -FAM-AGCAGCTGGCTTCACGCTCAGGTGTAMRA-30 . GenBank accession numbers for mCLCA5 and EF1a were NM_178697 and NM_178697, respectively. RT-qPCR and data analyses were conducted using the MX3000P Quantitative PCR System and MX Pro Software (Stratagene, La Jolla, CA, USA). PCR reactions

were carried out in 96-well High Prole plates covered with optical-thin wall-caps for real-time PCR (Kisker, Steinfurt, Germany). The plates contained triplicates of each cDNA sample, duplicates of no-template controls and duplicates of positive controls. The optimized 25-ll reaction mix contained 12.5 ll qPCR MasterMix Plus Low ROX (Eurogentec, Seraing, Belgium), 300 nM of each primer, 200-nM probe, 1 ll cDNA, 5.5 mM MgCl2 (mCLCA5 only) and RNase free water (Roth, Karlsruhe, Germany) to a nal volume of 25 ll. The thermal prole used was 10 min at 95C followed by 39 cycles (wholetissue samples) or 38 cycles (LCM samples) of 15 s at 95C and 1 min at 60C. cDNA samples were used with primers and probes for both mCLCA5 and EF1a on the same plate to ensure equal amplication conditions. RT-qPCR protocols were initially established and optimized using tenfold serial dilutions (ranging from 102 to 106 copies of mRNA) of puried mCLCA5-pcDNA3.1 (Evans et al. 2004) or puried (QIAquick PCR Purication Kit; Qiagen) PCR-derived fragments (1,114 bp) generated from cDNA templates from mouse colon for EF1a. Highly reproducible amplication efciencies of between 95 and 105% were observed in all experiments. Quantication of target gene expression For each sample analyzed, the mean cycle threshold (Ct) value and its corresponding standard deviation (SD) were given by the MX Pro Stratagene analysis software. Ct value was calculated based on the normalized baseline corrected uorescence (DRN). Relative gene expressions were calculated as ratios representing the copy numbers of the gene of interest (GOI) relative to the internal reference EF1a and were thus normalized to the housekeeping gene EF1a as internal standard (DCt-method; ratio = 2Ct EF1a - Ct GOI = 2DCt; von Smolinski et al. 2005). Statistical analyses For each cDNA sample analyzed the SD of the triplicates was calculated. Differences of expression levels between the samples of the two genotypes (C57BL/6J mice and mCLCA3neoE711 mice) were considered statistically signicant for p values B 0.05 in the MannWhitney U test. Biochemical protein analyses HEK293 cells were grown on 6-well plates in Dulbeccos modied Eagles medium (DMEM) containing 10% heat-inactivated fetal calf serum, 2% glutamate, and 1% penicillin/streptomycin (culture medium) at 37C in the presence of 5% CO2. Cells were washed between medium changes with prewarmed phosphate-buffered saline (PBS).

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HEK293 cells were transfected with pcDNA3.1 plasmid containing the mCLCA5 open reading frame (Evans et al. 2004) using LipofectamineTM 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturers protocol. Cells transfected with pcDNA3.1-vector alone served as negative controls (mock transfected). HEK293 cells were washed three times with PBS 48 h after transfection, followed by incubation in DMEM supplemented with 2% glutamate for 6 h. Cells and medium were analyzed separately. The medium was kept on ice and cells were lysed in standard lysis buffer. The medium samples were spun at 1,000 9 g for 5 min at 4C. The pellet was discarded and the remaining supernatant spun at 10,000 9 g for 15 min at 4C. This second supernatant was ethanol precipitated overnight at -20C and pellets were resuspended in 40 ll of lysis buffer. Initial experiments using whole-tissue lysates resulted in weak bands on the gel. These lysates were therefore ethanol precipitated and resuspended in 40 ll of lysis buffer prior to loading onto the gel. Medium and cell and tissue lysates were boiled in threefold SDS-PAGE Laemmli buffer containing 150 mM DTT, split in half and separated by SDS-PAGE. Samples for analysis with am5-N5-bp were separated by 7.5% SDSPAGE, while samples for analysis with antibody am5-C1-a were separated by 15% SDS-PAGE. Immunoblot analyses were performed as described (Leverkoehne and Gruber 2002) with am5-N5-bp and am5-C1-a as primary antibodies, respectively, diluted 1:500. Cell lysate and supernatant from mCLCA5 transfected cells were incubated with 500 U/ll endo H or PNGase F (New England Biolabs, Frankfurt, Germany) prior to SDSPAGE analysis, with one sample of each set untreated as a negative control. Gradient gel electrophoresis using Novex 420% Trisglycine gel 1.0 mm (Invitrogen) was performed to better differentiate between similarly sized bands but failed to improve the resolution on the blots (not shown). Immunoprecipitation of mCLCA5 transfected HEK293 cells was performed as described previously (Plog et al. 2009). Unfortunately, all antibodies failed to detect any specic band in the immunoblot after immunoprecipitation (data not shown). Of the mCLCA5 protein epitopes used to generate the antibodies, the carboxy-terminal epitope had partial sequence identity to the human hCLCA2 (92% amino acid similarity) and two of its murine CLCA family members (72% amino acid similarity to mCLCA3 and 75% amino acid similarity to mCLCA6). The amino-terminal epitope had partial sequence identity to hCLCA2 only (72% amino acid similarity). Therefore, to exclude cross-reactivity with the antibodies generated, equal amounts of cell lysates of HEK293 transfected with a myc-tagged hCLCA2-construct (Elble et al. 2006), mCLCA5 (Evans et al. 2004), mCLCA3

(Leverkoehne and Gruber 2002), mCLCA6 (Evans et al. 2004), or vector alone transfected cells (pcDNA3.1) were analyzed. All immunoblot experiments were carried out at least thrice independently. The tissue screening shown in Fig. 3 was performed at least twice for each tissue shown.

Results Sequence analyses and generation of antibodies CLCA proteins can be divided in two structural subgroups, with some CLCA proteins possessing a transmembrane domain in the carboxy-terminal cleavage product (Bothe et al. 2008; Elble et al. 2006) and others without (Gibson et al. 2005; Mundhenk et al. 2006). To examine which of these models best describes mCLCA5, we performed computer-aided sequence analyses followed by biochemical analyses using heterologously expressed mCLCA5 in transiently transfected HEK293 cells. The SignalP 3.0 algorithm predicted a signal sequence at the amino-terminus, with a most likely cleavage site between amino acids 32 and 33. Furthermore, SignalP 3.0 predicted mCLCA5 to be a nonsecretory protein. One potential transmembrane domain between amino acids (aa) 901926 was predicted by all algorithms tested (HMMTOP aa 904926, KyteDoolittle around aa 900, PSORT II aa 905921, SOSUI aa 901923, DAS aa 905923 and TMPred aa 905926), suggesting a carboxy-terminal cytoplasmic tail of no less than 16 hydrophilic amino acids. SOSUI predicted one additional transmembrane domain close to the carboxy-terminus between aa 874 and 895. Only the TMPred algorithm predicted two additional potential transmembrane regions, one at aa 235253 and one at aa 426445. Thus, computer-aided predictions of potential transmembrane domains for mCLCA5 consistently predicted a transmembrane region close to the carboxy-terminus (Fig. 1a, panels 27). All CLCA proteins discovered so far undergo posttranslational cleavage of the primary translation product, resulting in a larger amino-terminal and a smaller carboxyterminal cleavage product of approximately 90 and 35 kDa, respectively (Gruber et al. 2002). To study both cleavage products separately, without the risk of artifacts potentially introduced by epitope tagging, we generated specic antibodies against the predicted amino- and carboxy-terminal cleavage products of the mCLCA5 protein (Fig. 1a, top panel). Antibody am5-N5-b, raised against the predicted amino-terminal cleavage product, detected an approximately 100 kDa protein in mCLCA5 overexpressing HEK293 cells, corresponding to the size of the predicted glycosylated amino-terminal cleavage product (Fig. 1b, lane 1). The full-length primary translation

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290 Fig. 1 Generation of antibodies against mCLCA5. a Antisera were c raised against synthetic peptides corresponding to amino acid (aa) sequences within the predicted amino-(N)-terminal cleavage product (am5-N5-b) and the carboxy-(C)-terminal cleavage product (am5-C1-a; top panel). Panels 27 show the locations (solid bars) of the predicted transmembrane domains using several computer algorithms. All hydrophobicity analyses used predicted a transmembrane domain close to the carboxy-terminus around aa 900926. SOSUI predicted an additional transmembrane domain around aa 874895. Only TMPred predicted two additional transmembrane domains closer to the amino-terminus. Dots in the top panel indicate consensus sites for N-linked glycosylation, bolts show the putative cleavage site around aa 650700. b When lysates of heterologously mCLCA5 transfected HEK293 cells were immunoblotted with unpuried am5-N5-b, a specic protein of 100 kDa in size was detected (lane 1, asterisk), besides several unspecic protein bindings. The immunopuried antibodies detected less unspecic proteins (lane 3). Pre-immune serum from the same rabbit failed to detect any specic protein in the same lysate (lane 5). c Anti-carboxy-terminal antibody am5-C1-a detected two specic proteins, one at 130 and one at 37 kDa in size (lane 1, asterisks). The various protein species of *37 kDa likely represent different glycovariants of the carboxy-terminal cleavage product as shown later by deglycosylation in Fig. 7c. The immunopuried antibody am5-C1-ap reacted only poorly on immunoblots (lane 3). Vector-alone transfected cells served as negative controls (mock). All antibodies were diluted at 1:500 except for am5-C1-ap which was diluted 1:100. Asterisks label specic protein bands. d Antibodies am5-N5-bp and am5-C1a failed to detect the hCLCA2 protein (lane 2), or mCLCA3 (lane 3) or mCLCA6 (lane 4) in transfected HEK293 cells. No sequence homologies were found with other CLCA or non-CLCA proteins. Experiments with hCLCA2-myc, mCLCA3 and mCLCA6 were each positively controlled by the respective specic antibodies (Bothe et al. 2008; Elble et al. 2006; Leverkoehne and Gruber 2002). Vector-alone transfected HEK293 cells showed no specic bands at 100 and 37 kDa, respectively. Asterisks label specic protein bands. Antibodies were diluted 1:500

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product with a predicted size of 124134 kDa was only weakly and inconsistently detected by this antibody in lysates of mCLCA5 overexpressing HEK293 cells (not shown). The immunopuried antibody detected less unspecic bands and completely failed to detect the primary translation product (Fig. 1b, lane 3). Antibody am5-C1-a, raised against the predicted carboxy-terminal cleavage product, detected several poorly dened protein species of between 35 and 39 kDa, most likely representing different glycovariants of the carboxy-terminal cleavage product (Fig. 1c, lane 1). The presence of different glycovariants was conrmed by reduction of the various low molecular weight bands by PNGase F treatment to a single band of approximately 30 kDa in size shown later with experiments on cellular processing (Fig. 7c, lane 3). Additionally, this antibody detected a faint protein of 130 kDa in size, probably representing the full-length primary translation product (Fig. 1c, lane 1). Immunopurication of the am5-C1-a antiserum resulted in a reduction of its reactivity on immunoblots, therefore the non-immunopuried antiserum am5-C1-a was used for further immunoblotting experiments. Gradient gel electrophoresis failed to improve the resolution between bands of interest

and prominent yet non-specic bands running in close proximity (not shown). Sequence comparisons of the mCLCA5 epitopes used for immunization using an NCBI protein BLAST search revealed amino acid identities of 92% (carboxy-terminal epitope) and 72% (amino-terminal epitope) to hCLCA2 and 72 and 75% (carboxy-terminal epitopes) with the corresponding regions of mCLCA3 and mCLCA6,

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Fig. 2 The mCLCA5 protein is expressed in tissues containing stratied epithelium. Ethanol precipitated lysates of different tissues were analyzed by immunoblot. Lysates of transiently mCLCA5overexpressing HEK293 cells served as positive controls (lane 1), glandular mucosa of the stomach served as negative control (lane 2) as backed by RT-qPCR results shown in Fig. 5b. On immunoblots the anti-carboxy-terminal antibody, diluted 1:500, detected an approximately 37 kDa protein in the non-glandular mucosa of the stomach (lane 3), soft palate (lane 4), esophagus (lane 5), cervix (lane 6), and thymus (lane 7). Additional proteins of around 40 kDa in size were detected in lysates of non-glandular mucosa of the stomach, esophagus and cervix, likely representing glycovariants of the protein (see Fig. 7c). All other organs tested were negative (not shown). Incubation with the pre-immune serum as primary antibody instead of the anti-mCLCA5-carboxy-terminal antibody failed to detect any proteins of that size (not shown)

respectively. However, immunoblot analyses using lysates of HEK293 cells transfected with hCLCA2-myc, mCLCA3 or mCLCA6 failed to detect any cross-reactivity of the anti-mCLCA5 antibodies am5-N5-bp or am5-C1-a with the respective proteins (Fig. 1d, lanes 24). No sequence homologies were found with other CLCA or non-CLCA proteins. Experiments of hCLCA2-myc, mCLCA3 and mCLCA6 were each positively controlled by the respective specic antibodies (Bothe et al. 2008; Elble et al. 2006; Leverkoehne and Gruber 2002). Tissue expression pattern All tissues were further analyzed by immunoblots of wholetissue extracts. Antibody am5-C1-a detected a specic protein band at 37 kDa in size in lysates of the non-glandular mucosa of the stomach, soft palate, esophagus, cervix, and thymus. Unlike the human stomach, the murine stomach is only partially lined by glandular mucosa whereas the rest is lined by non-glandular, keratinized mucosa. Both types of epithelia were investigated separately and revealed different results. The specic protein band was not detected in lysates of the glandular mucosa of the stomach (Fig. 2), or any other organ tested (not shown). No other specic protein bands were detected. Pre-immune serum served as negative control. Antibodies am5-N5-b and am5-N5-bp failed to identify any specic protein in any of the tissue lysates. A systematic immunohistochemical analysis of C57BL/ 6J mice using antibody am5-C1-ap detected the mCLCA5

protein in keratinocytes of the stratied epithelium of the oral cavity (soft palate, tongue, epiglottis), esophagus, nonglandular mucosa of the stomach, skin, pinnae, cervix and vagina (Fig. 3). Additional signals were observed in the cornifying Hassalls bodies of the thymus (Fig. 3h). Higher magnications revealed that the mCLCA5 protein was located in cytoplasmic granules of keratinocytes of the granular layer (Fig. 3a, inset). Especially in the stomach, the granules seemed to accumulate to form larger granular bodies, similarly as described for keratohyalin granules (Adams 1976). Signals were additionally observed in the spinous layer of the non-glandular mucosa of the stomach, tongue, and soft palate. In the non-glandular mucosa of the stomach, skin, vagina, and pinnae faint, spotted signals were also observed in the cornied layer (Fig. 3a). Goblet cells were unstained. No staining was observed in vascular endothelial cells or vessel walls of all arteries, veins and lymphatics examined, including the aorta. Unfortunately, no tissue staining was observed in immunohistochemical studies with antibodies am5-N5-b or am5-N5-bp with any of the methods tested and on any of the tissues screened. Lastly, no staining was observed with the respective preimmune serum or an unrelated puried antibody or when the am5-C1-ap antibody was pre-adsorbed with the respective oligopeptide used for immunization. Thus, the expression patterns obtained with the antimCLCA5 antibodies were inconsistent with the expression patterns reported for all other murine CLCA proteins (Bothe et al. 2008; Elble et al. 2002; Leverkoehne and Gruber 2002; Leverkoehne et al. 2002), which further argues against cross-reactivity of the antibodies with other CLCA proteins. Confocal laser scanning microscopy of the stomach conrmed strong expression of mCLCA5 in variably sized cytoplasmic granules of granular layer keratinocytes only (Fig. 4b). Ultrastructural localization Immune electron microscopic detection of the mCLCA5 protein using 10-nm gold-particle-labeled secondary antibodies identied the carboxy-terminal cleavage product of mCLCA5 to be associated most prominently with keratohyalin granules of variable size and shape in granular layer keratinocytes (Fig. 4d). No expression was detected in cell membranes. Quantitation of the mCLCA5 mRNA cellular expression pattern in laser microdissected samples To verify the tissue distribution pattern observed on the protein level, RT-qPCR was carried out. mCLCA5 mRNA was detected in all whole-tissue samples investigated with

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292 Fig. 3 mCLCA5 is only expressed in keratinocytes of stratied c squamous epithelia. Immunohistochemically, the mCLCA5 protein was mainly detected in cytoplasmic granules of keratinocytes of the granular layer of the non-glandular mucosa of the stomach (a, inset), haired skin including hair follicles (b), vagina (d), tongue (e), soft palate (f), pinna (g), cervix, esophagus and epiglottis (not shown). To a lesser extent signals were detected in the spinous layer of the nonglandular mucosa of the stomach, tongue and soft palate (a, d, f). In the non-glandular mucosa of the stomach, haired skin including hair follicles, vagina and pinna (a, b, d, g), faint signals were also detected in the cornied layer (a, arrows). Additionally, mCLCA5 was detected in the cornifying Hassalls bodies of the thymus (h). Tissue sections were alternatively incubated with am5-C1-ap (diluted 1:100 in ah) or, as negative control, the peptide-saturated antibody (c, i). Color was developed using DAB as substrate (brown) with hematoxylin as counterstain (blue). Bars 50 lm (ae), 20 lm (fi), and 10 lm (inset)

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varying copy numbers (Fig. 5a). Tissue samples exceeding an average of 0.3 copies of mCLCA5 per 100 copies of EF1a (relative copies), listed in descending order, were pinnae (8.4), tongue (3.6), soft palate and vagina (both 1.8), larynx and esophagus (both 1.4), cervix (1.3), skin (1.1), nonglandular mucosa of the stomach (0.7) and eye (0.6). All other tissues investigated showed less than 0.3 relative copies of mCLCA5 (not shown). To conrm the distribution along the maturation process of squamous epithelia, RNA levels were quantied following LCM in the stomach and eye (Fig. 5b) as well as in spleen and brain as controls (not shown). mCLCA5 mRNA was detected only in the nonglandular mucosa of the stomach, in both the luminal and basal half of the stratied squamous epithelium, with an average of 1.1 and 2.1 relative copies, respectively. No other components of the stomach including glandular mucosa, submucosa and muscle layer had detectable mCLCA5 mRNA. In the eye, mCLCA5 mRNA was detected mainly in the cornea and to a lesser extent in the sclera (Fig. 5b), in the latter probably due to the covering by conjunctival epithelium. In the brain, mCLCA5 mRNA was detected only in the choroid plexus of the cerebrum (0.09 relative copies). In the spleen, mCLCA5 mRNA was detected close to the lower limit of detection (0.04 relative copies). Differential expression levels of mCLCA5 mRNA in mCLCA3neoE711 versus wild-type C57BL/6J mice It has previously been suggested that mCLCA5 may compensate for a loss in function in mCLCA3 decient mice by driving mucous cell metaplasia (Patel et al. 2006). Mucous cell metaplasia is one of several traits exhibited by complex airway diseases such as asthma. To test this hypothesis, quantitative expression levels of mCLCA5 mRNA in relevant tissues of mCLCA3neoE711 mice were compared to their C57BL/6J wild-type controls. Stomach was included as an internal positive control. mCLCA5 mRNA was detected in very low copy numbers within all tissues tested

and without signicant differences (p C 0.05) between mCLCA3neoE711 mice and their wild-type controls (Fig. 6). Cellular processing and association with the plasma membrane To further characterize the cellular processing of the mCLCA5 protein, the cell lysate and supernatant of

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Fig. 4 Confocal and immune electron microscopic localization of mCLCA5 to keratohyalin granules. Confocal laser scanning microscopy detected mCLCA5 protein in cytoplasmic granules of granular layer keratinocytes of the stomach (b). On the ultrastructural level, the carboxy-terminal cleavage product of the mCLCA5 protein was localized to keratohyalin granules (d) of granular layer keratinocytes of the stomach. Tissue sections were alternatively incubated with am5-C1-ap (diluted 1:100 for confocal, b; and 1:5,000 for immune electron microscopy, (d) or, as negative control a rabbit serum against irrelevant antigen (a, c). Higher magnication of keratohyalin granules are shown in the insets. Bars 10 lm (a, b); 300 nm (c, d); 100 nm (insets)

mCLCA5 transiently transfected HEK293 cells were immunoblotted with antibodies am5-N5-bp and am5-C1-a. Computer programs predicted a full-length non-glycosylated primary translation product of approximately 104 kDa and a glycosylated primary translation product of between 124 and 134 kDa. The full-length primary translation product of approximately 130 kDa was consistently detected in the lysate only when the am5-C1-a antibody was used (Fig. 7a, lane 5). Importantly, the primary translation product was not detected in the supernatant of transfected cells. In contrast, the amino-terminal cleavage product of approximately 100 kDa was present both in the cell lysate and in the supernatant 6 h after replacement of the medium (Fig. 7a; lanes 1, 2). At variance, the carboxy-terminal cleavage product of approximately 37 kDa in size was detected in the cell lysate only (Fig. 7a; lane 5).

Fig. 5 mCLCA5 mRNA is expressed in tissues containing stratied squamous epithelium. a mCLCA5 mRNA was quantied in murine tissues using real-time RT-qPCR. Tissues of C57BL/6J wild-type mice expressing mCLCA5 mRNA with relative copy numbers of above a threshold of 0.3 copies per 100 copies of EF1a (relative copies) were skin, pinna, larynx, tongue, soft palate, esophagus, stomach non-glandular mucosa (Sto ngl. muc.), eye, cervix, and vagina. These results conrmed the immunohistochemical stainings. The thymus, positive in immunohistochemistry, had very low copy numbers (0.13 0.11 relative copies) and is not included in this gure. n = 3 for all tissues. b Laser-capture microdissection was used to isolate different layers of the stomach (Sto) and eye prior to realtime RT-qPCR. The results veried expression in squamous epithelium of the stomach (Sto, sq.ep.). Both the luminal (Sto, sq.ep.lum.) and basal (Sto, sq.ep.basal) half of the squamous epithelium contained mCLCA5 mRNA. The glandular epithelium (Sto, gland.ep.), submucosa (Sto, submucosa) and smooth muscle (Sto, sm.muscle) cells of the stomach were negative for mRNA expression within the sensitivity range. In contrast, both the cornea and, to a lesser extent, the sclera had detectable mRNA at very low levels. n = 3 for all locations; bars standard deviations. Expression levels are given in ratios of mCLCA5 mRNA copy numbers relative to the copy numbers of the housekeeping gene EF1a

We further examined the glycosylation pattern of the mCLCA5 protein to characterize its cellular processing and to nd the cellular compartment where protein cleavage occurs. Cell lysates and supernatants were both treated with endoglycosidases endo H and PNGase F separately to identify the extent and kind of glycosylation. The 130 kDa full-length primary translation product was reduced to

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Fig. 6 mCLCA5 mRNA is not differentially expressed in cystic brosis-relevant tissues of mCLCA3neoE711 C57BL/6J compared to wild-type C57BL/6J mice. No statistically signicant differences (p C 0.05) were detected in mean relative expression levels of mCLCA5 mRNA between mCLCA3neoE711 mice (mCLCA3-/-; grey columns) and wild-type C57BL/6J mice (black columns) using real-time RT-qPCR. Tissues including mucous cells (lung, stomach, duodenum, jejunum, caecum, and colon) as well as tissues normally not expressing mucous cells (pancreas, liver, and kidney) were analyzed. Bars standard deviations. Expression levels are given in ratios of mCLCA5 mRNA copy numbers relative to the copy numbers of the housekeeping gene EF1a. n = 3 for all tissues

110 kDa by both endo H and PNGase F, as detected by antibody am5-C1-a (Fig. 7c; lanes 2, 3). The amino-terminal cleavage product found in the cell lysate and in the supernatant (Fig. 7b; lanes 1, 4) and the carboxy-terminal cleavage product present in the cell lysate only (Fig. 7c; lane 1) were resistant to endo H treatment but sensitive to PNGase F (Fig. 7b; lanes 2, 3, 5, 6; Fig. 7c; lanes 2, 3). The amino-terminal cleavage product was reduced in size to an 80-kDa protein, while the carboxy-terminal cleavage product was reduced to a 30-kDa product (Fig. 7b; lanes 3, 6; Fig. 7c; lane 3). Using antibody am5-N5-b some experiments identied an additional smaller band at approximately 9095 kDa after endo H treatment (not shown). Possibly some glycovariants of the amino-terminal cleavage product were partially sensitive to endo H that could be explained by an oversaturated glycosylation system in the Golgi of overexpressing HEK293 cells. The glycosylation of both cleavage products thus concurs with the glycosylation sites predicted by Evans et al. (2004). In conclusion, the results of our deglycosylation experiments indicated that the cleavage of the full-length primary translation product most likely occurs after its exit from the endoplasmic reticulum but before processing in the Golgi.

Discussion CLCA proteins are widely distributed in mammalian tissues with a specic cellular expression pattern for each

family member analyzed so far. hCLCA2, the human ortholog of mCLCA5, has been described to be expressed in epithelial and endothelial cells (Connon et al. 2004; Pauli et al. 2000) and its function has been the subject of much debate (Connon et al. 2004; Elble et al. 2006; Itoh et al. 2000; Pauli et al. 2000). The cell types expressing the mCLCA5 protein have not been systematically analyzed to date, albeit a variety of functions have been proposed (Beckley et al. 2004; Evans et al. 2004; Patel et al. 2006). Therefore, as a basis for future work on its normal function and signicance in diseased tissues, we characterized the expression of mCLCA5. A broad tissue and cellular screening detected mCLCA5 mRNA and protein virtually exclusively in keratinocytes of all stratied squamous epithelia that undergo cornication, including squamous epithelium of the skin, non-glandular mucosa of the stomach and even in Hassalls bodies of the thymus. Specically, immunohistochemistry, confocal laser scanning microscopy and immune electron microscopy localized the mCLCA5 protein to cytoplasmic granules of granular layer keratinocytes and, to a lesser extent, to spinous and cornied layer keratinocytes. No association with cellular membranes was observed. Relative quantication of mCLCA5 mRNA following LCM agreed with these results. However, the overall expression pattern of mCLCA5 mRNA seemed broader than the immunohistochemical results. Interestingly, all tissues containing stratied squamous epithelium with detectable mCLCA5 protein had a relative gene expression of above an arbitrary threshold of approximately 0.3 relative copies (pinna, tongue, soft palate, vagina, larynx, esophagus, cervix, skin, non-glandular mucosa of the stomach). In contrast, tissues devoid of stratied squamous epithelium and without detectable protein staining merely had minimal mRNA copy numbers, consistently beneath 0.3 relative copies. The only two exceptions were thymus and eye. In the thymus, immunohistochemistry detected mCLCA5 protein in cornied epithelial cells of Hassalls bodies, whereas the mRNA level was 0.1 relative copies. Conceivably, the amounts of keratinized Hassalls bodies are very low in the thymus with little cell turnover leading to low relative mRNA expression at the whole-tissue level. Hassalls bodies are thought to be composed of terminally differentiated medullary thymic epithelial cells that express keratins similar to keratinocytes (Bodey et al. 2000). They are important functional components of the thymus which are involved in intrathymic lymphopoiesis (Bodey et al. 2000). As shown by various authors, Hassalls bodies often react with different antibodies directed against the differentiated upper layers of the epidermis, i.e. CK14, CK5, caspase 14 and laggrin (Denecker et al. 2008; Hale and Markert 2004; Patel et al. 1995).

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Histochem Cell Biol (2010) 133:285299 Fig. 7 The amino-terminal mCLCA5 cleavage product is secreted by c transfected cells, whereas its carboxy-terminal mature glycosylated cleavage product remains associated with the cell. a HEK293 cells transiently transfected with mCLCA5 were analyzed by immunoblotting using antibodies am5-N5-bp, diluted 1:500, or am5-C1-a, diluted 1:500, respectively. For analyses of supernatant (S), a media change was performed 48 h after transfection followed by harvest and ethanol precipitation of the media 6 h later. Vector alone transfected cells served as controls (mock, lanes 3, 4, 7 and 8). Immunoblotting analyses identied both the amino-terminal 100 kDa and the carboxyterminal 37-kDa cleavage products of mCLCA5 in the cell lysate (L, lanes 1 and 5, asterisks), but only the amino-terminal cleavage product in the supernatant (S, lane 2, asterisk). Asterisks label specic protein bands. b The 100 kDa amino-terminal cleavage products in the cell lysate and supernatant of mCLCA5 were resistant to endo H treatment (lanes 2, 5). In contrast, this protein was fully reduced in size to approximately 80 kDa when treated with PNGase F (lanes 3, 6). Thus, deglycosylation experiments identied the amino-terminal cleavage product of mCLCA5 as a complex glycosylated mature protein form that has passed the Golgi apparatus. Aliquots of cell lysate (L) and supernatant (S) of HEK293 cells transiently transfected with mCLCA5 were treated with endo H (H), PNGase F (F) or not treated (-). Asterisks label specic protein bands. c The primary translation product of 130 kDa in the cell lysate was sensitive to both endo H (lane 2) and PNGase F treatment (lane 3) and was reduced in size to approximately 110 kDa, corresponding to the predicted size of the non-glycosylated primary translation product. The 37 kDa carboxy-terminal cleavage product in the cell lysate (L) was also resistant to endo H (lane 2) but reduced in size to 30 kDa when treated with PNGase F (lane 3). Asterisks label specic protein bands

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Conversely, eye had an mRNA expression of 0.6 relative copies although immunohistochemical staining was negative. mCLCA5 mRNA was detected in the cornea and, to a lesser extent, in the sclera. The three layers of the corneal epithelium include a layer of squamous cells on the outer surface without a granular layer typical of stratied, squamous epithelium (Kaye and Pappas 1962) which might account for lack of mCLCA5 protein. The sclera, on the other hand, is chiey formed of brous tissue and attened connective tissue. It is easily conceivable that the mRNA detected in the LCM material collected from the sclera actually stems from the squamous epithelium that lines the conjunctiva and covers the anterior surface of the sclera. The functional role of mCLCA5 in keratinocytes has yet to be established. Its cellular location in granular and spinous epithelial layers suggests a role in the development of the cornied envelope. In stratied squamous epithelia, the granular and spinous layers contain differentiating rather than proliferating keratinocytes (Yuspa et al. 1988), whereas proliferation takes place only in the basal layer. Keratinocyte differentiation in cornifying epithelium ultimately results in the cornied envelope as the primary protective layer of the skin and several mucosal tissues. Immune electron microscopy identied mCLCA5 protein to be located in round to irregular and variably sized keratohyalin granules. At least two types of granules have been described so far (Adams 1976). One type is irregular

in shape and associated with tonolaments, whereas the other type has a more regular shape and is not apparently associated with tonolaments. The latter is believed to play a role in the thickening of the cell membrane of keratinized cells (Adams 1976). Keratohyalin granules in the granular layer also contain the precursor of laggrin. Filaggrin is a keratin-associated matrix protein that is part of the layer of highly insoluble proteins that make up the epidermal cornied cell envelope (Sandilands et al. 2009; Steinert and Marekov 1995). These proteins are situated on the intracellular side of the cell membrane during terminal differentiation (Steinert and Marekov 1995). Cross-reactivity of the immunopuried anti-carboxy-terminal mCLCA5 antibody with laggrin is highly unlikely because of the different sizes of the proteins (Sandilands et al. 2009) and lack of any sequence homologies between laggrin and the epitope used for immunization. Sequence comparison between the mCLCA5 epitope and the laggrin protein

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using the NCBI pairwise protein BLAST search yielded an E value of 1.3 which lies far above the generally accepted limit of 10-4 for possible homology and cross-binding. Localization of mCLCA5 to presumably both types of keratohyalin granules of stratied layer keratinocytes again suggests a role in the development of the cornied envelope. Similar to laggrin, mCLCA5 in granular layer granules may represent a precursor protein of the proteins comprising the cornied cell envelope of stratied squamous epithelia or a keratin-associated protein. As Hassalls bodies represent mature, cornifying cells of the thymic epithelium, expression of mCLCA5 in this cell type might be a further hint for the involvement of mCLCA5 in maturation or differentiation of these cells. An important factor regulating the differentiation of epithelial keratinocytes is a Ca2?-gradient (Yuspa et al. 1988) which activates Ca2?-dependent proteins like protein kinase Ca (PKCa) and transglutaminases (Koster and Roop 2007; Lippens et al. 2005). However, the exact mechanisms resulting in terminal differentiation are unknown and only a few proteins included in this process that are controlled by Ca2? have been identied to date. Evans and coworkers reported that ionomycin-induced calcium inux evokes a novel calcium-dependent chloride conductance in mCLCA5 transiently transfected HEK293 cells (Evans et al. 2004). Possibly, mCLCA5 is a further Ca2?-regulated protein involved in evoking a chloride current during epithelial differentiation (Koster and Roop 2007). Alternatively, a proliferation inhibiting function has been proposed for mCLCA5 in association with growth suppression of metastatic breast cancer cells (Beckley et al. 2004). In accordance with that observation, mCLCA5 could play a role in growth arrest and the maturation process of squamous epithelial cells. To gain more insight into its relevance in keratinocytes, biochemical analyses were performed using mCLCA5transfected HEK293 cells. Our hydrophobicity analyses predicted that the mCLCA5 protein may possess one transmembrane domain in the carboxy-terminal cleavage product. Deglycosylation analyses revealed that, consistent with the general model for CLCA proteins (Gruber et al. 2002), mCLCA5 is processed as follows: the primary translation product is cleaved before being processed in the Golgi. Both cleavage products are complex glycosylated and pass the Golgi. The physiological and functional signicances of this glycosylation pattern, however, remain unknown. The amino-terminal cleavage product is secreted by the cell while the carboxy-terminal cleavage product remains associated with the cell. Lack of any immunohistochemical staining on tissues when the antibodies raised against its amino-terminus were used is likely due to the complete secretion of this part of the protein or due to lower sensitivity of the antibodies. In general, these data

reveal similarities with the proposed processing of hCLCA2 (Elble et al. 2006). Similar to hCLCA2, Evans and coworkers reported a cell membrane staining of mCLCA5 in transfected cells (Evans et al. 2004). Much to our surprise, the carboxy-terminal cleavage product of mCLCA5 was immunohistochemically and immune electron microscopically detected only in cytoplasmic granules of differentiating keratinocytes and was not associated with the cell membrane. If present in the membrane in vivo at all, the copy number of the carboxy-terminal cleavage product in the cell membrane obviously remained below the detection limit. Alternatively, membrane association that has been observed previously (Evans et al. 2004) may have been an artifact due to heterologous overexpression in cultured cells. Also, processing mechanisms, associated proteins and ultimate protein destination may differ between keratinocytes in vivo and the cell lines used in in vitro experiments (Evans et al. 2004). Our investigations of the mCLCA5 protein expression pattern were particularly impeded because attempts to generate specic antibodies against mCLCA5 turned out to be rather unsuccessful. Of seven antibodies generated against predicted immunogenic epitopes of both the aminoand carboxy-terminal cleavage products of mCLCA5, only one anti-carboxy-terminal antibody reacted specically in the immunohistochemical assay. Similar problems with generating antibodies against mCLCA5 in rabbits have been reported previously (Beckley et al. 2004). This may also explain the relatively large number of unspecically recognized proteins in our immunoblots (Fig. 1). Whether these results are coincidental or due to immunological reasons, such as close epitope homologies between mice and rabbits, remains unknown at this point. When compared to its human ortholog hCLCA2, the distribution pattern of mCLCA5 shows both similarities and dissimilarities. In contrast to hCLCA2 which was previously detected in endothelial cells of lung vasculature by both RT-PCR and immunohistochemistry (AbdelGhany et al. 2003), we failed to detect expression of mCLCA5 in endothelial cells of vessel walls in any tissues including lung and aorta by immunohistochemistry and relative mRNA quantication in the aorta remained below the threshold of 0.3 relative copies. Similar to mCLCA5, hCLCA2 has previously been detected in stratied epithelia (Connon et al. 2004, 2005; Gruber et al. 1999). However, unlike mCLCA5, the hCLCA2 protein had been localized immunohistochemically to the basal cells adjacent to the basement membrane (Connon et al. 2004) as opposed to supercial epithelial cells. The close proximity of hCLCA2 to hemidesmosomes had led to the speculation of a role in epithelial stratication and particularly in adhesion mechanisms involving integrin-b4, a component of hemidesmosomes (Carter et al. 1990; Connon et al.

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297 help with immune electron microscopy and Astrid Bethe for help with confocal laser scanning microscopy.

2004). In contrast to hCLCA2, a function in connection with hemidesmosomes is rather implausible for mCLCA5 because of its association with cytoplasmic granules of granular layer keratinocytes, although it has also been reported to possess a b4-binding domain (Abdel-Ghany et al. 2003). However, we cannot exclude the possibility that detection of mCLCA5 mRNA in basal layers may still point towards a functional signicance in that microenvironment with low expression levels, undetectable by immunohistochemistry and immune electron microscopy. In corneal epithelium where chloride secretion is a vital feature to sustain corneal transparency, hCLCA2 has been implicated as the most abundant chloride conductance protein (Itoh et al. 2000). Consistently, mCLCA5 was also found in corneal epithelium in this study, albeit only on the mRNA level and not immunohistochemically. Possibly, mCLCA5 is regulated on the post-transcriptional level in the cornea, for example, by post-transcriptional gene silencing, preventing translation into detectable amounts of protein under normal conditions. Alternatively, this discrepancy could again point towards lower sensitivity of the immunohistochemical assay. A previous study reported that mucous cell metaplasia in airway epithelium of C57BL/6J mice can be linked to increased mCLCA3 expression in goblet cells and mCLCA5 expression in non-goblet cell epithelial cells, respectively (Patel et al. 2006). Specically, a compensatory role has been speculated for mCLCA5 in the absence of mCLCA3 in mCLCA3 knockout mice (Patel et al. 2006). We therefore compared the relative gene expression levels of mCLCA5 in tissues from mCLCA3-decient mice which lack a specic phenotype to their wild-type C57BL/6J controls. Tissues investigated represent tissues associated with diseases involving mucous cells like asthma and CF. However, no statistically signicant differences were found in expression levels in any tissue tested in this study, yielding no evidence of transcriptional up-regulation as a possible mechanism of compensation (Patel et al. 2006). In summary, mCLCA5 appears to be primarily located in the cytoplasmic granules of granular layer keratinocytes in virtually all tissues containing stratied squamous epithelium. Further work is needed to elucidate its function and signicance during stratication of epithelial cells and a possible link to anion conduction across epithelial membranes, cellular adhesion, or growth arrest and differentiation.
Acknowledgments This study was supported by the German Mukoviszidose e.V. The authors acknowledge Stella R. Evans, Ph.D., who made the mCLCA5 constructs as part of her PhD dissertation research. The mCLCA3neoE711 mice were a kind gift from Karim Dabbagh (Roche, Palo Alto, CA, USA). Human hCLCA2-myc was a kind gift from Randolph Elble. We thank Verena Eckert-Funke for

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