Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

Department of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany.
Journal of Histochemistry and Cytochemistry (Impact Factor: 1.96). 09/2009; 57(12):1169-81. DOI: 10.1369/jhc.2009.954594
Source: PubMed


Recent studies have identified members of the CLCA (chloride channels, calcium-activated) gene family as potential modulators of the cystic fibrosis (CF) phenotype, but differences between the human and murine CLCA genes and proteins may limit the use of murine CF models. Recently established pig models of CF are expected to mimic the human disease more closely than the available mouse models do. Here, we characterized the porcine CLCA gene locus, analyzed the expression pattern and protein processing of pCLCA1, and compared it to its human ortholog, hCLCA1. The porcine CLCA gene family is located on chromosome 4q25, with a broad synteny with the human and murine clca gene loci, except for a pig-specific gene duplication of pCLCA4. Using pCLCA1-specific antibodies, the protein was immunohistochemically localized in mucin-producing cells, including goblet cells and mucinous glands in the respiratory and alimentary tracts. Similar to hCLCA1, biochemical characterization of pCLCA1 identified a secreted soluble protein that could serve as an extracellular signaling molecule or functional constituent of the protective mucous layers. The results suggest that pCLCA1 shares essential characteristics of hCLCA1, supporting the pig model as a promising tool for studying the modulating role of pCLCA1 in the complex pathology of CF.

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Available from: Lars Mundhenk, May 07, 2014
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    • "After incubation with secondary horseradish-peroxidase-conjugated anti-rabbit IgG for 1 h, labeling was visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, USA). To exclude cross-reactivity with other porcine CLCA members, HEK293 cells transiently transfected with the pCLCA1 (Plog et al. 2009), pCLCA4a (Plog et al. 2012), or pCLCA4b (primers: 5′-gaaaagcctcaacaag-3′; 3′-acctaaatatccattctagatt-5′; unpublished results) ORF cloned in pcDNA3.1 were also subjected to immunoblot analyses and processed similarly. The specific antibodies used to confirm the successful protein expression of pCLCA1, pCLCA4a, and pCLCA4b were p1- N-1ab-p (Plog et al. 2009), p4a-N-1b (Plog et al. 2012), and p4b-N-1a (unpublished, generated similarly against the pCLCA4b peptide HFYTTDQSESRGLT). "
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    ABSTRACT: Despite the discovery of the widely expressed CLCA (chloride channel regulators, calcium-activated) proteins more than 15 years ago, their seemingly diverse functions are still poorly understood. With the recent generation of porcine animal models for cystic fibrosis (CF), members of the porcine CLCA family are becoming of interest as possible modulators of the disease in the pig. Here, we characterize pCLCA2, the porcine ortholog of the human hCLCA2 and the murine mCLCA5, which are the only CLCA members expressed in the skin. Immunohistochemical studies with a specific antibody against pCLCA2 have revealed a highly restricted pCLCA2 protein expression in the skin. The protein is strictly co-localized with filaggrin and trichohyalin in the granular layer of the epidermis and the inner root sheath of the hair follicles, respectively. No differences have been observed between the expression patterns of wild-type pigs and CF transmembrane conductance regulator -/- pigs. We speculate that pCLCA2 plays an as yet undefined role in the structural integrity of the skin or, possibly, in specialized functions of the epidermis, including barrier or defense mechanisms.
    Full-text · Article · Sep 2012 · Cell and Tissue Research
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    • "To control for mRNA quality and efficacy of reverse transcription, a 542-bp product of the mRNA coding for the housekeeping factor EF-1a was RT-PCR amplified from each sample using the primers 5′-GAACGGGCAGACCCGTGAGC-3′ (upstream) and 5′-AGCCCACGTTGTCCCCAGGA-3′ (downstream). Primer pairs were designed to specifically discriminate among all known pCLCA homologs (Plog et al. 2009) and to encompass an intron to exclude amplification of contaminating genomic DNA. No crossreactions were detected when cloned cDNA samples of pCLCA1, pCLCA2, or pCLCA4b were used as templates. "
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    ABSTRACT: Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4.
    Full-text · Article · Jan 2012 · Journal of Histochemistry and Cytochemistry
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    • "Species-specific variations in genomic structure may give rise to limited comparability of expression data observed for a specific molecule in different species. For example , entire gene duplications or silencing of genes have been observed in the pig but not in humans for the chloride channels, calcium-activated (CLCA) family of genes, which have been proposed as potential modulators of the CF phenotype (Plog et al. 2009). Our computational genomic analyses revealed no such differences in the pCFTR gene locus, including duplications, premature stop codons within the coding region, or other structural differences that could account for principally different expressions when compared with hCFTR (not shown). "
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    ABSTRACT: Emerging porcine models of cystic fibrosis (CF) are expected to mimic the human disease more closely than current mouse models do. However, little is known of the tissue and cellular expression patterns of the porcine CF transmembrane conductance regulator (pCFTR) and possible differences from human CFTR (hCFTR). Here, the expression pattern of pCFTR was systematically established on the mRNA and protein levels. Using specific anti-pCFTR antibodies, the majority of the protein was immunohistochemically detected on paraffin-embedded sections and on cryostate sections in the apical cytosol of intestinal crypt epithelial cells, nasal, tracheal, and bronchial epithelial cells, and other select, mostly glandular epithelial cells. Confocal laser scanning microscopy with co-localization of the Golgi marker 58K localized the protein in the cytosol between the Golgi apparatus and the apical cell membrane with occasional punctate or diffuse staining of the apical membrane. The tissue and cellular distribution patterns were confirmed by RT-PCR from whole tissue lysates or select cells after laser capture microdissection. Thus, expression of pCFTR was found to largely resemble that of hCFTR except for the kidney, brain, and cutaneous glands, which lack expression in pigs. Species-specific differences between pCFTR and hCFTR may become relevant for future interpretations of the CF phenotype in pig models.
    Full-text · Article · May 2010 · Journal of Histochemistry and Cytochemistry
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