Oncogene (2008) 27, 6930–6938 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc REVIEW Tight junction-based epithelial microenvironment and cell proliferation S Tsukita1, Y Yamazaki, T Katsuno, A Tamura and S Tsukita2 Laboratory of Biological Science, Graduate School of Frontier Biosciences/Graduate School of Medicine, Osaka University, Suita, Osaka, Japan Belt-like tight junctions (TJs), referred to as zonula bodies of multicellular organisms. The sheet-like divi- occludens, have long been regarded as a specialized sions allow diffusion barrier formation and selective differentiation of epithelial cell membranes. They are permeation of substances and ions (permselectivity), required for cell adhesion and paracellular barrier func- both excluding unnecessary or toxic molecules and tions, and are now thought to be partly involved in fence including necessary components to maintain home- functions and in cell polarization. Recently, the molecular ostasis. The combination of the belt-like adherens bases of TJs have gradually been unveiled. TJs are junctions (AJs) and tight junctions (TJs) have important constructed by TJ strands, whose basic frameworks are functions in the formation of epithelial cell sheets and composed of integral membrane proteins with four also in the formation of paracellular permselective transmembrane domains, designated claudins. The claudin barriers through their functions as septa (Mitic and family is supposedly composed of at least 24 members in Anderson, 1998; Tsukita and Furuse, 1999; Hartsock mice and humans. Other types of integral membrane and Nelson, 2008). Paracellular barrier functions with proteins with four transmembrane domains, namely occlu- permselectivity are unique to the epithelial cell system din and tricellulin, as well as the single transmembrane and regulate the internal homeostasis of ions and solutes proteins, JAMs (junctional adhesion molecules)and CAR in the body. As the proteinaceous components of TJs, (coxsackie and adenovirus receptor), are associated with such as claudins, occludin, tricellulin, JAMs and CAR, TJ strands, and the high-level organization of TJ strands is have been characterized (Furuse et al., 1993; Liu et al., likely to be established by membrane-anchored scaffolding 2000; Cohen et al., 2001; Ikenouchi et al., 2005), proteins, such as ZO-1/2. Recent functional analyses of evidence has accumulated that TJs are the main claudins in cell cultures and in mice have suggested that components of paracellular permselective barriers (Tsu- claudin-based TJs may have pivotal functions in the kita et al., 2001; Van Itallie and Anderson, 2006a; regulation of the epithelial microenvironment, which is Angelow et al., 2008). Recently, the epithelial-specific critical for various biological functions such as control of TJ-based permselective barrier system has been sug- cell proliferation. These represent the dawn of ‘Barriology’ gested to be involved in the regulation of cell prolifera- (defined by Shoichiro Tsukita as the science of barriers in tion, possibly by regulating the microenvironment multicellular organisms). Taken together with recent around epithelial cells (Tamura et al., 2008). This notion reports regarding changes in claudin expression levels, has attracted particular attention because over 90% of understanding the regulation of the TJ-based microenvir- cancers are of epithelial origin. This review will present onment system will provide new insights into the regula- some interesting points regarding the TJ-based epithelial tion of polarization in the respect of epithelial microenvironment and cell proliferation. microenvironment system and new viewpoints for devel- oping anticancer strategies. Oncogene (2008) 27, 6930–6938; doi:10.1038/onc.2008.344 Ultrastructural identification of TJ: implications for Keywords: tight junction; cell adhesion; paracellular their regulatory roles in the epithelial microenvironment barrier; paracellular permeability; permselectivity; epithelial cell More than half a century ago, local modification of the apical side of epithelial cells was recognized at the light and electron microscopic levels to affect cell-cell adhesion and paracellular barrier functions. Detailed Introduction electron microscopic analyses in 1963 revealed the morphological bases for this local modification and Epithelial cell sheets cover the internal and external defined them as junctional complexes composed of TJs, surfaces of organs and form compartments within the AJs and desmosomes (Farquhar and Palade, 1963). Among these junctional complexes, TJs are usually Correspondence: Dr S Tsukita, Laboratory of Biological Science, located closest to the lumen of the epithelia. Character- Graduate School of Frontier Biosciences/Graduate School of istically, the TJ-associated membranes of adjoining cells Medicine, Osaka University, Suita, Osaka 565-0871, Japan. E-mail: [email protected] come together, obliterating the intercellular space by 1Sachiko Tsukita in full name. fusion of their outer leaflets at the thin-section electron 2Shoichiro Tsukita in full name. microscopic level (the so-called kissing points of plasma TJ-based epithelial microenvironment and cell proliferation S Tsukita et al 6931 the epithelial physiology, paracellular permeability/ transport constitutes a substantial part of the total permeability/transport through epithelial and endothe- lial cell sheets, the ratios of which vary relative to transcellular transport, depending on different cell types (Van Itallie et al., 2003). Thus, the TJ physiology should be an important contributor to the epithelial and endothelial physiology. As an example, it is well known that paracellular ionic permeability dominates the transcellular ionic permeability in the small intestine (Frizzell and Schultz, 1972; Okada et al., 1977), and has an important function in the intestinal physiology, such as in the absorption of nutrients (Hopfer, 1977; Turk et al., 1991). Similarly, re-absorption of ions through paracellular pathways in the kidney is very important for regulating the ionic homeostasis of the body (Simon et al., 1999). Non-ionic solutes are thought to permeate through paracellular as well as transcellular pathways (Watson et al., 2001) and the molecular mechanisms for the permeabilities for ions and solutes appear to vary depending on the cell type, with the characteristics of the conductance-flux paradox (Yap et al., 1998; Van Itallie and Anderson, 2006a). It appears that the solute flux of Figure 1 Electron microscopic images of tight junctions (TJs), the low-capacity/size-independent pathway, the high- adherens junctions (AJs) and desmosomes. TJs are located at the capacity/size-restrictive pathway and ion-based electri- most apical parts of the plasma membranes of intestinal epithelial cells, whereas AJs and desmosomes are localized in the more basal cal permeability together determine the paracellular parts of the lateral membranes, as revealed by thin-section electron permeability. The proportions of their different con- microscopic images (a and c). Note the kissing points of TJs (c). TJs tributions to the overall permeability define the para- are represented by TJ strands at the apical parts of the plasma cellular permeability of individual tissue types. In any membranes in freeze-fracture replica images (b and d). Micrographs courtesy of H.Sasaki (b and c) (see references, Tamura et al., 2008 case, the molecular bases for TJ strands should be in and Tsukita et al., 2001). Bars, 100 nm. accord with the physiological aspects of the TJ concerned. membranes) (Figure 1). Consistent with such ultra- structural aspects, experiments with mass external tracers have suggested that TJs act as barriers to Molecular bases for TJ diffusion of the tracer by sealing off the lumen from the intercellular spaces. Thus, TJs are considered to have Although it was most likely that the intramembranous critical functions in regulating the microenvironment or particles in the freeze-fracture replica samples reflected circumstances around epithelial cells. the integral membrane proteins, the real nature of the TJ Another key ultrastructural aspect of TJs was strands, lipids and/or proteins remained a mystery until characterized by freeze-fracture replica electron micro- the proteinaceous components constituting the TJ scopy (Staehelin, 1973). Intramembranous TJ strands or strands were identified (Kachar and Reese, 1982; Pinto fibrils appear to underlie the lines of fusion of the outer da Silva and Kachar, 1982; Verkleij, 1984). Hence, when leaflets of the adjoining cell membranes at the epithelial a novel integral membrane protein with four transmem- apical sites. Thus, TJ strands have been suggested to be brane domains of B60 kDa, termed occludin (Furuse responsible for the intercellular sealing of epithelial/ et al., 1993), was identified as a component of TJ strands endothelial cell sheets (Figure 1). from hepatic junctional fractions (Tsukita and Tsukita, 1989), it was regarded as the Holy Grail in this field (Gumbiner, 1993). However, knockout mice for occlu- din still possessed TJ strands, suggesting that occludin TJ as a permselective barrier: implications for the was not necessarily required for TJ strand formation TJ-based epithelial microenvironment system (Saitou et al., 2000) (Figure 2). It was subsequently revealed that the molecular backbones of TJ strands Further evidence for the barrier functions of TJs was were likely to be constituted by the relatively