Barrier function of the tight junction protein tricellulin

Abstract

Tricellulin is a tight junction protein localized in tricellular junctions (tTJ), the meeting points of three cells, but also in bicellular junctions (bTJ). The barrier function of tricellulin in bTJ and tTJ has been characterized by overexpression experiments in kidney tubule MDCK II cells. When expressed in the tTJ alone, tricellulin impeded the permability for macromolecules, while after expression in both, tTJ and bTJ, tricellulin impeded the permeability for macromolecules and also for ions. The surprising finding that in tTJ no changes in ion permeability occured is explained by the fact that tTJ central tubes are wide enough for macromolecular passage but too rare to contribute significantly to ion permeability. In conclusion, tricellulin forms a barrier against macromolecular passage in the tTJ and, if present there, also in the bTJ.

Zusammenfassung

Tricellulin ist ein Tight Junction-Protein, das in trizellulären Tight Junctions (tTJ), dem Verbindungspunkt dreier Zellen, aber auch in bizellulären Tight Junctions (bTJ) lokalisiert ist. Die Barrierefunktion von Tricellulin wurde durch Überexpressionsstudien in MDCK II-Zellen, einer etablierten Nierenzelllinie, charakterisiert. Expression von Tricellulin in tTJ verminderte die Permeabilität für Makromoleküle, während eine Expression sowohl in tTJ als auch bTJ nicht nur zu einer weiteren Permeabilitätsabnahme für Makromoleküle sondern auch einer solchen für Ionen führte. Der überraschende Befund, dass die Ionenpermeabilität in tTJ unverändert blieb, ist dadurch erklärbar, dass das zentrale Lumen der tTJ zwar weit genug für die Passage von Makromolekülen ist, aber zu selten vorkommt, um signifikant zur Ionenpermeabilität beizutragen. Tricellulin bildet somit eine Permeationsbarriere für Makromoleküle in der tTJ und, wenn dort exprimiert, auch in der bTJ.

The tight junction and tricellulin

Paracellular barrier properties of endothelia and epithelia are determined by tight junctions. There are two different types of TJs, the bicellular TJ (bTJ), forming cell-cell contacts between two neighbouring cells, and the tricellular TJ (tTJ) at the meeting point of three cells. The main proteins incorporated in TJ strands are the family of claudins, occludin, and tricellulin. Presently 27 different claudins are described of whom most have tightening but some have channel functions. Occludin is thought to be a regulatory component of the tight junction meshwork, while its barrier function is controversial.

Tricellulin was described in 2005 by Ikenouchi et al. [1] to be localized mainly in tTJs and to a lesser extent also in bTJ. Four different isoforms of tricellulin are described in humans [2]. TRIC-a is the longest one comprising 558 amino acids and is meant in this as well as in other studies when simply termed “tricellulin”. It shares a 32% sequence identity in the C-terminal domain with occludin. Other tricellulin isoforms, TRIC-a1, TRIC-b, and TRIC-c are splice variants lacking exon 2, the occludin C-terminal ELL, or exon 3, respectively.

In knockdown experiments with siRNA against tricellulin changes in TJ morphology and organization could be observed. Bicellular TJs appeared to be thinner and discontinuous, at the meeting point of three cells TJ did not meet, and gaps disturbed TJ strand continuity [1]. These findings support a role of tricellulin in organization of the tTJ and also the bTJ.

In occludin-deficient mice tricellulin was spread out from the tTJ in the bTJ and from this observation it was implied that occludin supports the predominant localization of tricellulin in the tTJ by dislodging it from bTJ [3].

Barrier function of tricellulin

Ikenouchi et. al. (2005, [1]) showed in their knockdown model that absence of tricellulin prevented the development of transepithelial electrical resistance (TER) and caused an elevated permeability for the paracellular tracers FITC-dextran 4 kDa and 250 kDa. However, as tricellulin knockdown affected overall TJ integrity further studies were performed to elucidate tricellulin barrier properties. Krug et al. used a different approach to analyse the function of tricellulin in TJ [4]. By overexpression of TRIC-a in low-resistance MDCK II cells, stable clones were generated that express TRIC-a at different levels and also differed in localization: In subclones with moderate overexpression of TRIC-a, the protein was found in tTJs only (“tTJ clone”, Fig. 7.1). In subclones expressing tricellulin at high levels, protein localization was no longer restricted to tTJ, but tricellulin was also incorporated in bTJ (“tTJ+bTJ clone”, Fig. 7.1).

Fig. 7.1 Graded overexpression of tricellulin in a tTJ clone and a tTJ+bTJ clone. Immunofluorescence confocal microscopy of vector controls exhibited a localization of endogenous tricellulin (green) mainly at tricellular contacts in colocalization (yellow) with occludin (red). In the tTJ clone tricellulin was more strongly expressed in tTJs (as quantified by Western blotting). In contrast, the tTJ+bTJ clone revealed equally strong tricellulin signals in tTJs but also signals in bTJs. Bar = 20 µm (modified from [4]).

In a first set of experiments using two-path impedance spectroscopy [5] it was shown that overexpression in tTJ alone did not lead to changes in paracellular resistance (R^para) or ion permeability. In contrast, R^para was 14-fold increased when tricellulin was incorporated in tTJ and bTJ (Fig. 7.2) and the permeability for Na⁺ and Cl⁻ was equally reduced. The explanation for this puzzling observation is as follows. As tTJs contribute only 1% of overall paracellular conductance, overexpression of tricellulin only in tTJ does not lead to a measurable effect on ion permeability. The bTJ plays the main role in paracellular ion transport and here the tightening effect of tricellulin, if incorporated in this TJ area, becomes apparent [1].

Fig. 7.2 Effects of tricellulin on resistance and macromolecule permeabilty. A As measured by two-path impedance spectroscopy, tricellular overexpression of tricellulin (tTJ clone) did not change epithelial resistance (R^epi) in comparison to controls, while tri- and bicellular overexpression (tTJ+bTJ clone) induced a threefold increase of R^epi. This increase was based on a 14-fold rise of paracellular resistance (R^para) which is represents the reciprocal ion permeability of the TJ. Transcellular resistance (R^trans) was not significantly changed. B Permeability for FITC-dextran 4 kDa was strongly decreased already in the tTJ clone and even more in the tTJ+bTJ clone (modified from [4]).

Passage of macromolecules

The permeability for paracellular tracer molecules FITC-dextran 4 kDa and 10 kDa was measured in Ussing chamber experiments. The permeability for both macromolecules was strongly reduced already in the tTJ clone and even more in the tTJ+bTJ clone (Fig. 7.2B). In the latter, also the permeability for mid-sized molecules like fluorescein (332 Da) or PEG400 (400 Da) was decreased. Large molecules like FITC-dextran 20 kDa did hardly pass the TJ and tricellulin overexpression had no effect on either clone.

From dilution potential measurements it was derived that tricellulin equally affected passage of cations and anions, indicating that tricellulin alters permeability of the bTJ in a size-selective but not charge selective manner.

Paracellular barrier and channel properties of the TJ are mainly determined by claudins, so that passage can be selective e.g. for cations (by claudin-2 [6]), anions (by claudin-10a [7]) or water (by claudin-2 [8]). Here, tricellulin can be added as a component of the TJ that sets a barrier against macromolecular passage.

Inflammatory bowel disease

Starting from a cell type which was selected because of its low average endogenous tricellulin expression, tricellulin overexpression in the tTJ proves to be essential for preventing macromolecular passage across that structure. This effect was enhanced after including tricellulin overexpression also in bTJ. This points to the tTJ as a weak point for macromolecular passage, if under pathological conditions tricellulin would be reduced [4].

Epithelial integrity is diminished in inflammatory bowel diseases (IBD) like Crohn's disease and ulcerative colitis. Caused by inflammatory cytokines, the channel-forming protein claudin-2 was increased [9,10] and barrier-forming claudins like claudin-5 and -8 [10] were decreased. It will be a target of future research which role tricellulin and the tTJ plays for these pathologies.

References

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[10] Zeissig S, Bürgel N, Günzel D et al. Changes in expression and distribution of claudin-2, -5 and -8 lead to discontinuous tight junctions and barrier dysfunction in active Crohn's disease. Gut 2007; 56 (1): 61–72

Correspondence
Dr. Susanne M. Krug
[e-mail]