Gastroenterology

Gastroenterology

Volume 120, Issue 2, February 2001, Pages 411-422
Gastroenterology

Alimentary Tract
Heterogeneity in expression and subcellular localization of claudins 2, 3, 4, and 5 in the rat liver, pancreas, and gut,☆☆

https://doi.org/10.1053/gast.2001.21736Get rights and content

Abstract

Background & Aims: Paracellular transport varies widely among epithelia of the gastrointestinal tract. We determined whether members of the claudin family of tight junction proteins are differentially expressed consistent with a potential role in creating these variable properties. Methods: Rabbit polyclonal antibodies were produced against peptides from claudins 2 through 5. The distribution of individual claudins was detected by immunoblotting, and their cell type and subcellular localization were determined by immunofluorescence on cryosections of rat liver, pancreas, stomach, and small and large intestine. Results: All antibodies detected single bands of the expected size on immunoblots and were monospecific based on peptide competition studies. Immunoblotting detected strong differences among tissues in the expression level of each claudin. Immunolocalization confirmed these differences and revealed striking variations in expression patterns. In the liver, claudin 2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, claudin 3 is uniformly expressed, claudin 4 is absent, and claudin 5 is only expressed in endothelial junctions. In the pancreas, claudin 2 is only detected in junctions of the duct epithelia, claudin 5 only in junctions of acinar cells, whereas claudin 3 and 4 are in both. Among differences in the gut are a crypt-to-villus decrease in claudin 2, a highly restricted expression of claudin 4 to colonic surface cells, and the finding that some claudins can be junctional, lateral, or show a gradient in junctional vs. lateral localization along the crypt-to-villus surface axis. Conclusions: Claudins have very different expression patterns among and within gastrointestinal tissues. We propose these patterns underlie differences in paracellular permeability properties, such as electrical resistance and ion selectivity that would complement known differences in transcellular transport.

GASTROENTEROLOGY 2001;120:411-422

Section snippets

Tissue preparation

Male Sprague–Dawley rats (200–250 g) were obtained through approved protocols of the Yale Liver Center from the Yale Animal Resources Center. Anesthesia was administered by intraperitoneal injection of pentobarbital (5 mg/100 g body wt; Abbott Laboratories, North Chicago, IL). Rats were anesthetized with pentobarbital and underwent laparotomy, and the distal abdominal aorta was exposed and cannulated with a polyethylene-90 catheter. Organs were systemically rinsed free of blood with ice-cold

Specificity of anticlaudin antibodies

To investigate the distribution and localization of claudins in the gastrointestinal tract, we generated rabbit polyclonal antibodies against peptides corresponding to the C-terminal sequences of claudins 1 through 5. Either rat, mouse, or human sequences were used depending on which were available in the database (see Materials and Methods). Subsequent availability of all mouse and human sequences shows them to be conserved and suggests that antibodies raised against these sequences will also

Discussion

In this study, we have documented the tissue distribution and subcellular localization of claudins 2–5 in the gastrointestinal tract. Earlier Northern analysis had indicated the heterogeneity and possible tissue and cell type specificity in expression of some claudins.3, 13, 14, 15, 16 Two claudins had already been shown to have extremely restricted expression patterns: claudin 16 in the thick ascending loop of Henle16 and claudin 11 in Sertoli cells and oligodendrocytes.14, 15 We observed

Acknowledgements

The authors thank Drs. Christina M. Van Itallie, Alan Fanning, and Zenta Walther for helpful discussions and Keith Choate and Dr. Richard Lifton for providing a paracellin-GST fusion protein.

References (32)

  • K Morita et al.

    Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands

    Proc Natl Acad Sci U S A

    (1999)
  • P. Claude

    Morphological factors influencing transepithelial per-meability: a model for the resistance of the zonula occludens

    J Membr Biol

    (1978)
  • L Mitic et al.

    Molecular architecture of tight junctions

    Annu Rev Physiol

    (1998)
  • K. Fujimoto

    Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intra-cellular junctional complexes

    J Cell Sci

    (1995)
  • CM Van Itallie et al.

    Occludin confers adhesiveness when expressed in fibroblasts

    J Cell Sci

    (1997)
  • K Kubota et al.

    Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions

    Curr Biol

    (1999)
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    Address requests for reprints to: Christoph Rahner, M.D., Section of Digestive Diseases, 1080 LMP, Department of Internal Medicine, Yale School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8019. e-mail: [email protected]; fax: (203) 785-7273.

    ☆☆

    Supported by National Institutes of Health grants (DK45134 to J.M.A.) and the Core Facilities of the Yale Liver Center (DK34989).

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