Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1.

Barrier dysfunction is pivotal to the pathogenesis of inflammatory bowel diseases (IBD) and collagenous colitis. Glucocorticoids restore barrier function in Crohn's disease, but whether this reflects attenuated inflammation or an epithelial-specific action has not yet been addressed. Using filter-grown Caco-2 monolayers as an in vitro model of the intestinal epithelial barrier, we observed that glucocorticoids induced a time- and dose-dependent increase in transepithelial electrical resistance (TEER) in a glucocorticoid receptor-dependent manner without altering flux of larger solutes or changing principal tight junction architecture. This was accompanied by reduced paracellular cation flux, reduced expression of the pore-forming tight junction component claudin-2, and upregulation of the sealing tight junction protein claudin-4. In contrast, expression of occludin, claudin-1, -7, or -8 was not altered. Dexamethasone increased expression and activity of MAPK phosphatase-1 and inhibition of this phosphatase prevented the glucocorticoid-induced changes in TEER and claudin expression, whereas inhibiting p38 or MEK1/2 was not sufficient to replicate the glucocorticoid effects. Upon exposure to IFN-γ, TNF-α, or IL-1ß, TEERs declined in dexamethasone-treated cells but remained consistently higher than in cells not receiving glucocorticoids. Treatment with IFN/TNF resulted in an upregulation of claudin-2 that was significantly attenuated by dexamethasone, whereas increased claudin-2 expression upon IL-1ß stimulation was not affected by glucocorticoids. Taken together, barrier augmentation might represent a previously unrecognized mechanism of action, potentially contributing to the therapeutic efficacy of glucocorticoids in IBD and collagenous colitis.

Adalimumab prevents barrier dysfunction and antagonizes distinct effects of TNF-α on tight junction proteins and signaling pathways in intestinal epithelial cells.

Intestinal barrier dysfunction is pivotal in the etiology of inflammatory bowel diseases. Combined clinical and endoscopic remission ("mucosal healing") in patients who received anti-TNF-α therapies suggests restitution of the intestinal barrier, but the mechanisms involved are largely unknown. We therefore investigated the impact of the anti-TNF-α antibody adalimumab on barrier function in two in vitro models. Combined stimulation of Caco-2 and T-84 cells with interferon-γ and TNF-α resulted in a significant decrease of transepithelial electrical resistance (TEER) within 6 h that was prevented by adalimumab in concentrations down to 100 ng/ml. Adalimumab furthermore antagonized the appearance of irregular membrane undulations and prevented internalization of tight junction proteins upon cytokine exposure. In addition, TNF-α induced a downregulation of claudin-1, claudin-2, claudin-4, and occludin as well as activation of phosphatidylinositol 3-kinase signaling in T-84 but not Caco-2 cells, which was reversed by adalimumab. At the signaling level, adalimumab prevented increased phosphorylation of myosin light chain as well as activation of p38 MAPK and NF-κB accompanying the decline in TEER in both model systems. Pharmacological inhibition of NF-κB signaling partially prevented the TNF-α-induced TEER loss, whereas inhibition of p38 worsened barrier dysfunction in Caco-2 but not T-84 cells. Taken together, these data demonstrate that adalimumab prevents barrier dysfunction induced by TNF-α both functionally and structurally as well as at the level of signal transduction. Barrier protection might therefore constitute a novel mechanism how anti-TNF-α therapy contributes to epithelial restitution and tissue repair in inflammatory bowel diseases.

Impaired tight junction sealing and precocious involution in mammary glands of PKN1 transgenic mice.

The mammary gland undergoes a complex set of changes to establish copious milk secretion at parturition. To test the hypothesis that signaling through the Rho pathway plays a role in secretory activation, transgenic mice expressing a constitutively activated form of the Rho effector protein PKN1 in the mammary epithelium were generated. PKN1 activation had no effect in late pregnancy but inhibited milk secretion after parturition, diminishing the ability of transgenic dams to support a litter. Mammary gland morphology as well as increased apoptosis and expression of IFGBP5 and TGFbeta3 suggest precocious involution in these animals. Furthermore, tight junction sealing at parturition was impaired in transgenic mammary glands as demonstrated by intraductal injection of [14C]sucrose. Consistent with this finding, tight junction sealing in response to glucocorticoid stimulation was highly impaired in EpH4 mammary epithelial cells expressing constitutively activated PKN1, whereas expression of a dominant-negative PKN1 mutant resulted in accelerated tight junction sealing in vitro. Tight junction formation was not impaired as demonstrated by the correct localization of occludin and ZO1 at the apical cell borders. Our results provide evidence that PKN1 participates in the regulation of tight junction sealing in the mammary gland by interfering with glucocorticoid signaling.