Caveolin 1 is Associated with Upregulated Claudin 2 in Necrotizing Enterocolitis

Guillermo Ares,Catherine J Hunter,Douglas R Wood,John Sincavage,Christie Buonpane,Carrie Yuan

doi:10.1038/s41598-019-41442-4

Abstract

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal emergency of neonates. Epithelial tight junction (TJ) proteins, such as claudins, are essential for regulation and function of the intestinal barrier. Rho kinase (ROCK) affects cellular permeability and TJ regulation. We hypothesized that TJ protein changes would correlate with increased permeability in experimental NEC, and ROCK inhibitors would be protective against NEC by regulation of key claudin proteins. We tested this hypothesis using an in vivo rat pup model, an in vitro model of experimental NEC, and human intestinal samples from patients with and without NEC. Experimental NEC was induced in rats via hypoxia and bacteria-containing formula, and in Caco-2 cells by media inoculated with LPS. The expression of claudins was measured by gene and protein analysis. Experimental NEC in rat pups and Caco-2 cells had increased permeability compared to controls. Gene and protein expression of claudin 2 was increased in experimental NEC. Sub-cellular fractionation localized increased claudin 2 protein to the cytoskeleton. ROCK inhibition was associated with normalization of these alterations and decreased severity of experimental NEC. Co-immunoprecipitation of caveolin-1 with claudin 2 suggests that caveolin-1 may act as a shuttle for the internalization of claudin 2 seen in experimental NEC. In conclusion, NEC is associated with intestinal permeability and increased expression of claudin 2, increased binding of caveolin-1 and claudin 2, and increased trafficking of claudin 2 to the cytoskeleton.

Highlights

It is well documented that infants with Necrotizing enterocolitis (NEC) have increased intestinal permeability, resulting in increased bacterial translocation, systemic bacteremia, sepsis, and death[3,4]
We have previously identified a role of ROCK in protecting against experimental NEC, and in this manuscript we seek to build upon these findings[13]
Alterations in tight junction (TJ) proteins and epithelial barrier function have been linked to human disease, such as infectious enteritis and inflammatory bowel disease[27]

Summary

Introduction

It is well documented that infants with NEC have increased intestinal permeability, resulting in increased bacterial translocation, systemic bacteremia, sepsis, and death[3,4]. The mechanisms leading to increased permeability are poorly understood and are the focus of this study Gram negative bacteria, such as Cronobackter sakazakii (CS), have been implicated in NEC outbreaks in multiple neonatal intensive care units[5]. Claudin 2 is a pore-forming TJ, selectively increasing permeability to molecules of certain size or charge, whereas claudin 4 contributes to further strengthening the barrier[11]. In addition to their structural functions, TJ are regulators of multiple intracellular signaling pathways, such as protein kinase C, NF kappa B, and the Rho and Rho kinase (ROCK) pathway. We hypothesize that ROCK inhibition mitigates the effects of experimental NEC on the alteration of TJ protein expression

Methods

Results

Conclusion