Skip to main content
Log in

Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia

  • Original Paper
  • Published:
Histochemistry and Cell Biology Aims and scope Submit manuscript

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 specific antibodies and RT-qPCR following laser-capture microdissection. The mCLCA5 protein was localized to granular layer keratinocytes of virtually all stratified 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Abdel-Ghany M, Cheng HC, Elble RC, Pauli BU (2001) The breast cancer beta 4 integrin and endothelial human CLCA2 mediate lung metastasis. J Biol Chem 276:25438–25446

    Article  CAS  PubMed  Google Scholar 

  • Abdel-Ghany M, Cheng HC, Elble RC, Pauli BU (2002) Focal adhesion kinase activated by beta(4) integrin ligation to mCLCA1 mediates early metastatic growth. J Biol Chem 277:34391–34400

    Article  CAS  PubMed  Google Scholar 

  • Abdel-Ghany M, Cheng HC, Elble RC, Lin H, DiBiasio J, Pauli BU (2003) The interacting binding domains of the beta(4) integrin and calcium-activated chloride channels (CLCAs) in metastasis. J Biol Chem 278:49406–49416

    Article  CAS  PubMed  Google Scholar 

  • Adams D (1976) Keratinization of the oral epithelium. Ann R Coll Surg Engl 58:351–358

    CAS  PubMed  Google Scholar 

  • Agnel M, Vermat T, Culouscou JM (1999) Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea. FEBS Lett 455:295–301

    Article  CAS  PubMed  Google Scholar 

  • Anton F, Leverkoehne I, Mundhenk L, Thoreson WB, Gruber AD (2005) Overexpression of eCLCA1 in small airways of horses with recurrent airway obstruction. J Histochem Cytochem 53:1011–1021

    Article  CAS  PubMed  Google Scholar 

  • Beckley JR, Pauli BU, Elble RC (2004) Re-expression of detachment-inducible chloride channel mCLCA5 suppresses growth of metastatic breast cancer cells. J Biol Chem 279:41634–41641

    Article  CAS  PubMed  Google Scholar 

  • Bodey B, Bodey B Jr, Siegel SE, Kaiser HE (2000) Novel insights into the function of the thymic Hassall’s bodies. In Vivo 14:407–418

    CAS  PubMed  Google Scholar 

  • Bothe MK, Braun J, Mundhenk L, Gruber AD (2008) Murine mCLCA6 is an integral apical membrane protein of non-goblet cell enterocytes and co-localizes with the cystic fibrosis transmembrane conductance regulator. J Histochem Cytochem 56:495–509

    Article  CAS  PubMed  Google Scholar 

  • Brouillard F, Bensalem N, Hinzpeter A, Tondelier D, Trudel S, Gruber AD, Ollero M, Edelman A (2005) Blue native-SDS PAGE analysis reveals reduced expression of the mClCA3 protein in cystic fibrosis knock-out mice. Mol Cell Proteomics 4:1762–1775

    Article  CAS  PubMed  Google Scholar 

  • Carter WG, Kaur P, Gil SG, Gahr PJ, Wayner EA (1990) Distinct functions for integrins alpha 3 beta 1 in focal adhesions and alpha 6 beta 4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol 111:3141–3154

    Article  CAS  PubMed  Google Scholar 

  • Connon CJ, Yamasaki K, Kawasaki S, Quantock AJ, Koizumi N, Kinoshita S (2004) Calcium-activated chloride channel-2 in human epithelia. J Histochem Cytochem 52:415–418

    CAS  PubMed  Google Scholar 

  • Connon CJ, Kawasaki S, Yamasaki K, Quantock AJ, Kinoshita S (2005) The quantification of hCLCA2 and colocalisation with integrin beta4 in stratified human epithelia. Acta Histochem 106:421–425

    Article  CAS  PubMed  Google Scholar 

  • Cserzo M, Wallin E, Simon I, von Heijne G, Elofsson A (1997) Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10:673–676

    Article  CAS  PubMed  Google Scholar 

  • Denecker G, Ovaere P, Vandenabeele P, Declercq W (2008) Caspase-14 reveals its secrets. J Cell Biol 180:451–458

    Article  CAS  PubMed  Google Scholar 

  • Elble RC, Widom J, Gruber AD, Abdel-Ghany M, Levine R, Goodwin A, Cheng HC, Pauli BU (1997) Cloning and characterization of lung-endothelial cell adhesion molecule-1 suggest it is an endothelial chloride channel. J Biol Chem 272:27853–27861

    Article  CAS  PubMed  Google Scholar 

  • Elble RC, Ji G, Nehrke K, DeBiasio J, Kingsley PD, Kotlikoff MI, Pauli BU (2002) Molecular and functional characterization of a murine calcium-activated chloride channel expressed in smooth muscle. J Biol Chem 277:18586–18591

    Article  CAS  PubMed  Google Scholar 

  • Elble RC, Walia V, Cheng HC, Connon CJ, Mundhenk L, Gruber AD, Pauli BU (2006) The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment. J Biol Chem 281:29448–29454

    Article  CAS  PubMed  Google Scholar 

  • Evans SR, Thoreson WB, Beck CL (2004) Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine. J Biol Chem 279:41792–41800

    Article  CAS  PubMed  Google Scholar 

  • Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana, Totowa

    Google Scholar 

  • Gibson A, Lewis AP, Affleck K, Aitken AJ, Meldrum E, Thompson N (2005) hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore are not ion channels. J Biol Chem 280:27205–27212

    Article  CAS  PubMed  Google Scholar 

  • Gruber AD, Levine RA (1997) In situ assessment of mRNA accessibility in heterogeneous tissue samples using elongation factor-1 alpha (EF-1 alpha). Histochem Cell Biol 107:411–416

    Article  CAS  PubMed  Google Scholar 

  • Gruber AD, Pauli BU (1999) Tumorigenicity of human breast cancer is associated with loss of the Ca2+-activated chloride channel CLCA2. Cancer Res 59:5488–5491

    CAS  PubMed  Google Scholar 

  • Gruber AD, Gandhi R, Pauli BU (1998) The murine calcium-sensitive chloride channel (mCaCC) is widely expressed in secretory epithelia and in other select tissues. Histochem Cell Biol 110:43–49

    Article  CAS  PubMed  Google Scholar 

  • Gruber AD, Schreur KD, Ji HL, Fuller CM, Pauli BU (1999) Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland. Am J Physiol 276:C1261–C1270

    CAS  PubMed  Google Scholar 

  • Gruber AD, Elble RC, Pauli BU (2002) Discovery and cloning of the CLCA gene family. Curr Top Membr 53:367–387

    Article  CAS  Google Scholar 

  • Hale LP, Markert ML (2004) Corticosteroids regulate epithelial cell differentiation and Hassall body formation in the human thymus. J Immunol 172:617–624

    CAS  PubMed  Google Scholar 

  • Hamann M, Gibson A, Davies N, Jowett A, Walhin JP, Partington L, Affleck K, Trezise D, Main M (2009) Human ClCa1 modulates anionic conduction of calcium dependent chloride currents. J Physiol

  • Hirokawa T, Boon-Chieng S, Mitaku S (1998) SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14:378–379

    Article  CAS  PubMed  Google Scholar 

  • Hofmann W, Stoffel W (1993) TMbase—a database of membrane spanning protein segments. Biol Chem Hoppe Seyler 374:166 (Abstr. MFC-135)

    Google Scholar 

  • Hoshino M, Morita S, Iwashita H, Sagiya Y, Nagi T, Nakanishi A, Ashida Y, Nishimura O, Fujisawa Y, Fujino M (2002) Increased expression of the human Ca2+-activated Cl channel 1 (CaCC1) gene in the asthmatic airway. Am J Respir Crit Care Med 165:1132–1136

    PubMed  Google Scholar 

  • Itoh R, Kawamoto S, Miyamoto Y, Kinoshita S, Okubo K (2000) Isolation and characterization of a Ca(2+)-activated chloride channel from human corneal epithelium. Curr Eye Res 21:918–925

    Article  CAS  PubMed  Google Scholar 

  • Kaye GI, Pappas GD (1962) Studies on the cornea. I. The fine structure of the rabbit cornea and the uptake and transport of colloidal particles by the cornea in vivo. J Cell Biol 12:457–479

    Article  CAS  PubMed  Google Scholar 

  • Koster MI, Roop DR (2007) Mechanisms regulating epithelial stratification. Annu Rev Cell Dev Biol 23:93–113

    Article  CAS  PubMed  Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    Article  CAS  PubMed  Google Scholar 

  • Leverkoehne I, Gruber AD (2002) The murine mCLCA3 (alias gob-5) protein is located in the mucin granule membranes of intestinal, respiratory, and uterine goblet cells. J Histochem Cytochem 50:829–838

    CAS  PubMed  Google Scholar 

  • Leverkoehne I, Horstmeier BA, von Samson-Himmelstjerna G, Scholte BJ, Gruber AD (2002) Real-time RT-PCR quantitation of mCLCA1 and mCLCA2 reveals differentially regulated expression in pre- and postnatal murine tissues. Histochem Cell Biol 118:11–17

    CAS  PubMed  Google Scholar 

  • Leverkoehne I, Holle H, Anton F, Gruber AD (2006) Differential expression of calcium-activated chloride channels (CLCA) gene family members in the small intestine of cystic fibrosis mouse models. Histochem Cell Biol 126:239–250

    Article  CAS  PubMed  Google Scholar 

  • Lippens S, Denecker G, Ovaere P, Vandenabeele P, Declercq W (2005) Death penalty for keratinocytes: apoptosis versus cornification. Cell Death Differ 12(Suppl 2):1497–1508

    Article  CAS  PubMed  Google Scholar 

  • Mundhenk L, Alfalah M, Elble RC, Pauli BU, Naim HY, Gruber AD (2006) Both cleavage products of the mCLCA3 protein are secreted soluble proteins. J Biol Chem 281:30072–30080

    Article  CAS  PubMed  Google Scholar 

  • Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36

    Article  CAS  PubMed  Google Scholar 

  • Nakanishi A, Morita S, Iwashita H, Sagiya Y, Ashida Y, Shirafuji H, Fujisawa Y, Nishimura O, Fujino M (2001) Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci USA 98:5175–5180

    Article  CAS  PubMed  Google Scholar 

  • Nakano T, Inoue H, Fukuyama S, Matsumoto K, Matsumura M, Tsuda M, Matsumoto T, Aizawa H, Nakanishi Y (2006) Niflumic acid suppresses interleukin-13-induced asthma phenotypes. Am J Respir Crit Care Med 173:1216–1221

    Article  CAS  PubMed  Google Scholar 

  • Nielson H, Engelbrecht J, Brunack S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Article  Google Scholar 

  • Patel DD, Whichard LP, Radcliff G, Denning SM, Haynes BF (1995) Characterization of human thymic epithelial cell surface antigens: phenotypic similarity of thymic epithelial cells to epidermal keratinocytes. J Clin Immunol 15:80–92

    Article  CAS  PubMed  Google Scholar 

  • Patel AC, Morton JD, Kim EY, Alevy Y, Swanson S, Tucker J, Huang G, Agapov E, Phillips TE, Fuentes ME, Iglesias A, Aud D, Allard JD, Dabbagh K, Peltz G, Holtzman MJ (2006) Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members. Physiol Genomics 25:502–513

    Article  CAS  PubMed  Google Scholar 

  • Pauli BU, Abdel-Ghany M, Cheng HC, Gruber AD, Archibald HA, Elble RC (2000) Molecular characteristics and functional diversity of CLCA family members. Clin Exp Pharmacol Physiol 27:901–905

    Article  CAS  PubMed  Google Scholar 

  • Plog S, Mundhenk L, Klymiuk N, Gruber AD (2009) Genomic, tissue expression and protein characterization of pclca1, a putative modulator of cystic fibrosis in the pig. J Histochem Cytochem

  • Ritzka M, Stanke F, Jansen S, Gruber AD, Pusch L, Woelfl S, Veeze HJ, Halley DJ, Tummler B (2004) The CLCA gene locus as a modulator of the gastrointestinal basic defect in cystic fibrosis. Hum Genet 115:483–491

    Article  CAS  PubMed  Google Scholar 

  • Sandilands A, Sutherland C, Irvine AD, McLean WH (2009) Filaggrin in the frontline: role in skin barrier function and disease. J Cell Sci 122:1285–1294

    Article  CAS  PubMed  Google Scholar 

  • Steinert PM, Marekov LN (1995) The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J Biol Chem 270:17702–17711

    Article  CAS  PubMed  Google Scholar 

  • Toda M, Tulic MK, Levitt RC, Hamid Q (2002) A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma. J Allergy Clin Immunol 109:246–250

    Article  CAS  PubMed  Google Scholar 

  • Tusnady GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850

    Article  CAS  PubMed  Google Scholar 

  • von Smolinski D, Leverkoehne I, von Samson-Himmelstjerna G, Gruber AD (2005) Impact of formalin-fixation and paraffin-embedding on the ratio between mRNA copy numbers of differently expressed genes. Histochem Cell Biol 124:177–188

    Article  CAS  Google Scholar 

  • Yuspa SH, Hennings H, Tucker RW, Jaken S, Kilkenny AE, Roop DR (1988) Signal transduction for proliferation and differentiation in keratinocytes. Ann N Y Acad Sci 548:191–196

    Article  CAS  PubMed  Google Scholar 

Download references

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 mCLCA3neoE7–11 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 help with immune electron microscopy and Astrid Bethe for help with confocal laser scanning microscopy.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Achim D. Gruber.

Additional information

J. Braun and M. K. Bothe contributed equally.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Braun, J., Bothe, M.K., Mundhenk, L. et al. Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia. Histochem Cell Biol 133, 285–299 (2010). https://doi.org/10.1007/s00418-009-0667-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00418-009-0667-0

Keywords

Navigation