Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail

Abstract

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty, Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function. q

Biology of claudins

“…Two studies report direct phosphorylation of claudins by PKC. In the first study, overexpression of claudin-1 on the right side of chick embryos randomized the direction of heart looping, an event in embryonic development that normally departs from the bilateral symmetry in an evolutionarily conserved direction (90). Mutating the predicted PKC phosphorylation target, Thr206, to alanine abolished this randomization, suggesting that appropriately localized claudin-1 expression is important for developing correct heart looping and that PKC phosphorylation of Thr206 is important for this function.…”

Xenopus, an ideal model system to study vertebrate left‐right asymmetry

“…2G,H in Brizuela et al, 2001). Claudin may be a particularly important factor, because it has been involved in laterality determination in the chick upstream of the Nodal cascade as well (Simard et al, 2006).…”

Involucrin–claudin-6 tail deletion mutant (CΔ206) transgenic mice: a model of delayed epidermal permeability barrier formation and repair

“…Although Cldns demonstrate amino acid similarity among family members, the cytoplasmic tails of Cldns are divergent in sequence and possess a number of sites that provide clues about their structure-function relationships in epidermal differentiation, including a PDZ binding sequence (YV) and potential phosphorylation sites (Fujibe et al, 2004;Simard et al, 2006).…”

“…However, it seems likely that as yet unidentified novel molecules interact with Cldns in regulating gene expression and epidermal differentiation. Post-translational modifications within the tail domain, including phosphorylation and palmitoylation, are also thought to regulate Cldn activities, including their targeting to the membrane and their insertion into TJs to regulate paracellular permeability (Simard et al, 2006). Phosphorylation of a number of Cldns has been demonstrated to be required for their assembly into TJs [e.g.…”