Study of Claudin Function by RNA Interference

Abstract

Claudins are tight junction proteins that play a key selectivity role in the paracellular conductance of ions. Numerous studies of claudin function have been carried out using the overexpression strategy to add new claudin channels to an existing paracellular protein background. Here, we report the systematic knockdown of endogenous claudin gene expression in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells using small interfering RNA against claudins 1-4 and 7. In MDCK cells (showing cation selectivity), claudins 2, 4, and 7 are powerful effectors of paracellular Na ؉ permeation. Removal of claudin-2 depressed the permeation of Na ؉ and resulted in the loss of cation selectivity. Loss of claudin-4 or -7 expression elevated the permeation of Na ؉ and enhanced the proclivity of the tight junction for cations. On the other hand, LLC-PK1 cells express little endogenous claudin-2 and show anion selectivity. In LLC-PK1 cells, claudin-4 and -7 are powerful effectors of paracellular Cl ؊ permeation. Knockdown of claudin-4 or -7 expression depressed the permeation of Cl ؊ and caused the tight junction to lose the anion selectivity. In conclusion, claudin-2 functions as a paracellular channel to Na ؉ to increase the cation selectivity of the tight junction; claudin-4 and -7 function either as paracellular barriers to Na ؉ or as paracellular channels to Cl ؊ , depending upon the cellular background, to decrease the cation selectivity of the tight junction.Tight junctions are cell-cell interactions that provide the primary barrier to the diffusion of solutes through the paracellular pathway, creating an ion-selective boundary between the apical and basolateral extracellular compartments (see reviews in Refs. 1-3). The integral membrane proteins of the tight junction include occludin, a 65-kDa membrane protein bearing four transmembrane domains and two extracellular loops, and claudins, a family with at least 22 homologous proteins of 20 -28 kDa that share a common topology with occludin (4 -7).Claudins have been shown to confer ion selectivity to the paracellular pathway. In MDCK 2 cells, claudin-4, -5, -8, -11, and -14 selectively decrease the permeability of cation through tight junction, whereas the permeation of anion is largely unchanged (8 -12). MDCK cells express five endogenous claudins, claudin-1-4 and -7. LLC-PK1 cells express four endogenous claudins, claudin-1, -3, -4, and -7. In LLC-PK1 cells, claudin-2, -15, -16 selectively increase the permeability of cation through the tight junction with no significant effects on anions (13-14). When exogenous claudins are added to the tight junction, they constitute new charge-selective channels leading to a physiological phenotype that combines the contributions of both endogenous and exogenous claudins in the cell. A biochemical inventory of claudin-claudin interactions is not yet available, although the principle of specificity has been demonstrated in mouse L-fibroblasts (15). In addition, although efforts have been made to demonstrate the oligomerization p...

Tight junction-based epithelial microenvironment and cell proliferation

“…Thus, the proteinaceous TJ strands are dominantly responsible for the paracellular barrier functions, consistent with the dominant contribution of TJs, compared with the minor contribution of the lateral intercellular space, to paracellular resistance that was revealed by physiological measurements (Kottra et al, 1993a; Kottra and Fro¨mter, 1993b; Yap et al, 1998). Second, transfection or knockdown of individual claudins or their combinations in cultured epithelial cells led us to speculate that claudin-2 and -15 contribute to cation channels/pores Van Itallie et al, 2003), whereas claudin-4, -7 and -10a contribute to anion channels/pores or cation barriers (Hou et al, 2006;Van Itallie et al, 2006b) (Figure 3). The plus or minus charges of the first loops of claudin molecules thus appear to have functions in paracellular epithelial permeation (Colegio et al, 2003).…”

The Blood–Brain Barrier

“…Evidence in vitro suggests that claudins are essential for the paracellular barrier formation. Expression of claudins is sufficient to form TJ strands in fibroblasts, and disruption of claudins decreases the paracellular barrier properties of canine kidney cells (Furuse et al 1998(Furuse et al , 2001Van Itallie et al 2001;Amasheh et al 2005;Hou et al 2006). Work with chimeric claudins has shown that amino acid residues in the first extracellular loop define the size and charge selectivity of the pore within the cellular junction, and, thus, the composition of the claudins within a given cell can determine the permeability of the paracellular barrier (Colegio et al 2003).…”

Biology of claudins

“…It is important to recognize that a claudin that acts as a selective pore to one population of ions must, by definition, constitute a relative barrier to all other ions; that this function is manifest may depend on the host cell. For example, Hou et al (43) used small interfering (si) RNA to knock down endogenous claudins in epithelial cells and found that claudin-4 and claudin-7 act as cation barriers in MDCKII cells while forming anion-selective pores in LLC-PK 1 cells (43).…”