Caveolin-1 and its Effects on Tight Junction and Apoptosis Regulation in Necrotizing Enterocolitis

Author(s):
Cody Dalton; Tyler Leiva; Katherine Snyder; Grant Gershner; Catherine Hunter

Background:

Necrotizing enterocolitis (NEC) is a common gastrointestinal disease in premature infants. It is a multifactorial disease process involving dysregulated cellular mechanisms in tight junction processing and in cell death. Caveolin-1 is a crucial protein in tight junction transportation as well as in cell death signaling cascades. Its expression is upregulated in NEC.

Hypothesis:

We hypothesize that Caveolin-1 deficiency will result in decreased tight junction expression and increased apoptosis markers compared to wild type in the setting of NEC. We aim to further delineate the role of Caveolin-1 in tight junction regulation and apoptotic pathways and how it contributes to necrotizing enterocolitis.

Methods:

Enteroids were generated from intestinal tissue collected from wild type and Caveolin-1 knockout mice. The enteroids were treated with 100ug/mL lipopolysaccharide (LPS), subjected to 24 hours of hypoxia inducing experimental NEC, then compared with untreated controls. Gene expression of pro-apoptotic markers caspase-3, caspase-9, and BCL2-Associated X (BAX), anti-apoptotic markers B-cell lymphoma 2 (BCL-2), nuclear factor kappa B (NF-kB), as well as occludin and claudin-2 were evaluated using RT-qPCR.  ANOVA was used to determine statistical significance (p<0.05).

Results:

NEC-derived enteroids had a significant decrease in occludin expression which was significantly lower in NEC compared to wild type (p=0.023). Claudin-2 expression decreased in NEC, however, this was only significant in the knockout strains (p=0.01). In NEC, expression of pro-apoptotic markers increased and anti-apoptotic markers decreased without a significant difference between wild type and caveolin-1 knockout strains.

Conclusions:

Expression of tight junctions occludin and claudin-2 significantly decreased in enteroids exposed to NEC. The decrease in occludin was significant in the caveolin-1 knockout group compared to wild type. There was no significant difference in expression of pro and anti-apoptotic markers between the two groups. This may suggest a relationship between caveolin-1 and tight junction regulation in the setting of necrotizing enterocolitis while cell death pathways may be independent of caveolin-1.