O11 – Cyclic adenosine monophosphate activation of protein kinase A may play a role in the pathogenesis of necrotizing enterocolitis
Author(s):
Background: Necrotizing enterocolitis (NEC) is a deadly intestinal disease that affects newborn infants. Cronobacter sakazakii (CS) has been associated with outbreaks of NEC. Protein kinase A (PKA) is an abundant protein that is typically activated by cyclic adenosine monophosphate (cAMP). PKA has been associated both with cell survival and cell death pathways.
Hypothesis: We hypothesized that cAMP and PKA mediated signaling may contribute to epithelial cell death in NEC.
Methods: Three systems are used in this project: an in vitro cell line, a rat pup model of NEC and human tissue. After IRB approval, human intestinal segments were obtained from infants undergoing bowel resection for NEC (September 2012-November 2013), NEC strictures, or ostomy closures (controls). Protein was extracted for western blot analysis with antibodies for PKA, activated PKA and markers of apoptosis. Rat pups delivered near-term were subject to hypoxia (5%) and feeding with CS-inoculated formula, or dam fed. Intestinal samples were collected and processed for histology and protein extraction. Rat intestinal epithelial cells (IEC-6) were grown to 90% confluence and exposed to CS in vitro. PKA inhibitors (KT-5720 & SC-3010) were added at doses of 0.1uM, 1uM and 10uM prior to CS infection. Cell and tissue lysates were assayed by western blot analysis of PKA, activated PKA and caspase-3. cAMP tissue levels were measured in all model systems by Elisa analysis.
Results: Activated PKA was identified by Western blot analysis in human specimens with NEC (n=4), as compared with controls (n=6) p<0.005. Increased caspase 3 activation was seen in patients with NEC as compared with controls. CS induces caspase 3 activation after 4 hours of co-culture in IEC-6 cells (P<0.005). PKA is present in IEC-6 and is activated by 4 hours of infection with CS. Furthermore, cAMP was increased in experimental NEC compared with breast milk fed controls (p<0.005), and also in vitro after CS exposure. The addition of a PKA inhibitor prior to IEC-6 infection with CS prevents CS-induced apoptosis p<0.005.
Conclusions: We conclude that cAMP-PKA mediated signaling may play an important role in CS-induced intestinal epithelial apoptosis. The prospect of PKA inhibitors presents an interesting potential therapeutic line of investigation.