Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

Abstract

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4α, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

Cingulin Regulates Claudin-2 Expression and Cell Proliferation through the Small GTPase RhoA

“…The present results confirm the notion that cingulin is not an essential structural component of TJ, because the junctional localizations of claudin-2, ZO-3, ZO-1, and occludin were not significantly altered by cingulin depletion (also see Guillemot et al, 2004). With regard to the barrier function of TJs, it should be noted that exogenous claudin-2 expression decreases the TER in MDCKI cells, which lack claudin-2 (Furuse et al, 2001).…”

“…These results highlight the possible role of cingulin as a junctional protein that contributes to the regulation of cell growth and shape, through its effect on RhoA. The increase in cellular cingulin content in differentiating cells (Bordin et al, 2004;Guillemot et al, 2004) may both fine-tune the expression of claudin-2 (and possibly other TJ proteins), and contribute to the regulation of cell proliferation. This could be one of a number of regulatory mechanisms that control epithelial tissue biogenesis.…”

“…Up-Regulation of the Expression of Claudin-2, ZO-3, and RhoA, and Increased RhoA Activity In mouse EBs, the targeted mutation of cingulin alters the expression of genes coding for TJ proteins, with claudin-2 up-regulation being quantitatively the most striking effect (21-fold; Guillemot et al, 2004). To determine whether cingulindependent control of gene expression is restricted to EBs or has a more general significance, stable MDCK clones in which cingulin expression was knocked down by siRNA were obtained.…”

“…Anti-c-Jun NH 2 -terminal kinase (JNK) (9252) and anti-p-JNK (9255) antibodies were from Cell Signaling Technology (Beverly, MA). Other antibodies were as described in Guillemot et al (2004). The anti-ZO-3 antibody was from K. Matter and M. Balda (University College, London, United Kingdom).…”

“…Inhibitor concentrations were chosen based on manufacturer's directions, current literature (Altan and Fenteany, 2004), and preliminary titrations of three different concentrations of each inhibitor, which showed dose-dependent effects on cell morphology, indicating a biological activity. Lysates (with or without inhibitors) were prepared and analyzed by immunoblotting as described previously (Bordin et al, 2004;Guillemot et al, 2004). Protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA), and gel loadings were normalized by immunoblotting with antibodies against ␣-tubulin.…”