Disruption of occludin function in polarized epithelial cells activates the extrinsic pathway of apoptosis leading to cell extrusion without loss of transepithelial resistance.

BACKGROUND: Occludin is a tetraspanin protein normally localized to tight junctions. The protein interacts with a variety of pathogens including viruses and bacteria, an interaction that sometimes leads to its extrajunctional localization. RESULTS: Here we report that treatment of mammary epithelial monolayers with a circularized peptide containing a four amino acid sequence found in the second extracellular loop of occludin, LHYH, leads to the appearance of extrajunctional occludin and activation of the extrinsic apoptotic pathway. At early times after peptide treatment endogenous occludin and the LYHY peptide were co-localized in extrajunctional patches, which were also shown to contain components of the death inducing signaling complex (DISC), caspases 8 and 3, the death receptor FAS and the adaptor molecule FADD. After this treatment occludin could be immunoprecipitated with FADD, confirming its interaction with the DISC. Extrusion after LYHY treatment was accomplished with no loss of epithelial resistance. CONCLUSION: These observations provide strong evidence that, following disruption, occludin forms a complex with the extrinsic death receptor leading to extrusion of apoptotic cells from the epithelial monolayer. They suggest that occludin has a protective as well as a barrier forming role in epithelia; pathogenic agents which utilize this protein as an entry point into the cell might set off an apoptotic reaction allowing extrusion of the infected cell before the pathogen can gain entry to the interstitial space.

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.

Transduction of the mammary epithelium with adenovirus vectors in vivo.

Because the mammary parenchyma is accessible from the exterior of an animal through the mammary duct, adenovirus transduction holds promise for the short-term delivery of genes to the mammary epithelium for both research and therapeutic purposes. To optimize the procedure and evaluate its efficacy, an adenovirus vector (human adenovirus type 5) encoding a green fluorescent protein (GFP) reporter and deleted of E1 and E3 was injected intraductally into the mouse mammary gland. We evaluated induction of inflammation (by intraductal injection of [(14)C]sucrose and histological examination), efficiency of transduction, and maintenance of normal function in transduced cells. We found that transduction of the total epithelium in the proximal portion of the third mammary gland varied from 7% to 25% at a dose of 2 x 10(6) PFU of adenovirus injected into day 17 pregnant mice. Transduction was maintained for at least 7 days with minimal inflammatory response; however, significant mastitis was observed 12 days after transduction. Adenovirus transduction could also be used in the virgin animal with little mastitis 3 days after transduction. Transduced mammary epithelial cells maintained normal morphology and function. Our results demonstrate that intraductal injection of adenovirus vectors provides a versatile and noninvasive method of investigating genes of interest in mouse mammary epithelial cells.