A Role for cAMP and Protein Kinase A in Experimental Necrotizing Enterocolitis
2016; Elsevier BV; Volume: 187; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2016.10.014
ISSN1525-2191
AutoresBrian P. Blackwood, Douglas R. Wood, Carrie Yuan, Joseph Nicolas, Isabelle G. De Plaen, Kathryn N. Farrow, Pauline M. Chou, Jerrold R. Turner, Catherine J. Hunter,
Tópico(s)Breastfeeding Practices and Influences
ResumoNecrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA in vitro and in vivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC. Necrotizing enterocolitis (NEC) is a devastating intestinal disease that has been associated with Cronobacter sakazakii and typically affects premature infants. Although NEC has been actively investigated, little is known about the mechanisms underlying the pathophysiology of epithelial injury and intestinal barrier damage. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are important mediators and regulators of apoptosis. To test the hypothesis that C. sakazakii increases cAMP and PKA activation in experimental NEC resulting in increased epithelial apoptosis, we investigated the effects of C. sakazakii on cAMP and PKA in vitro and in vivo. Specifically, rat intestinal epithelial cells and a human intestinal epithelial cell line were infected with C. sakazakii, and cAMP levels and phosphorylation of PKA were measured. An increase in cAMP was demonstrated after infection, as well as an increase in phosphorylated PKA. Similarly, increased intestinal cAMP and PKA phosphorylation were demonstrated in a rat pup model of NEC. These increases were correlated with increased intestinal epithelial apoptosis. The additional of a PKA inhibitor (KT5720) significantly ameliorated these effects and decreased the severity of experimental NEC. Findings were compared with results from human tissue samples. Collectively, these observations indicate that cAMP and PKA phosphorylation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC. Necrotizing enterocolitis (NEC) affects 5% of all infants in the neonatal intensive care unit, and surgical NEC survivors incur significantly greater health care costs than age-matched infants without NEC.1Ganapathy V. Hay J.W. Kim J.H. Lee M.L. Rechtman D.J. Long term healthcare costs of infants who survived neonatal necrotizing enterocolitis: a retrospective longitudinal study among infants enrolled in Texas Medicaid.BMC Pediatr. 2013; 13: 127Crossref PubMed Scopus (54) Google Scholar NEC typically affects premature infants (90% of cases) and is fatal in up to 40% of those with the most severe disease.2Hull M.A. Fisher J.G. Gutierrez I.M. Jones B.A. Kang K.H. Kenny M. Zurakowski D. Modi B.P. Horbar J.D. Jaksic T. 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We identified that cAMP and PKA activation are associated with increased apoptosis in NEC and that inhibition of PKA activation protects against apoptosis and experimental NEC. Rat intestinal epithelial cells (IEC-6 passages 19 to 29; Sigma-Aldrich, Milwaukee, WI) were grown in Dulbecco's modified Eagle media supplemented with 10% fetal calf serum, 1 U/mL insulin, 100 U/mL penicillin G, and 100 U/mL streptomycin. Various doses (1 × 103 to 1 × 107 cfu/mL) of C. sakazakii were added to confluent monolayers of IEC-6 cells separately and incubated for 0 to 12 hours. Media were aspirated and replenished every 2 hours to limit bacterial multiplication. Experiments were repeated with 1 × 107 cfu/mL of a second cell line, FHs 74 Int cells (CCL-241; ATCC). FHs 74 Int is a nontransformed human cell line. The cells were grown in Hybri-Care Medium ATCC 46-X supplemented with 30 ng/mL epidermal growth factor, 10% fetal bovine serum. Cells were grown to 90% confluence on Chamber Slides (Nunc Lab-Tek, Naperville, IL). Cells were pretreated with PKA inhibitors (0.1–20 μmol/L) KT5720 (Cayman Chemical, Ann Arbor, MI), cAMP Dependent Protein Kinase Inhibitor (Sigma-Aldrich, St. Louis, MO), Rp-8-cAMPS (Santa Cruz Biotechnology, Dallas, TX) for 30 minutes then subjected to co-culture with C. sakazakii at various concentrations over a time course. These experiments were repeated with IEC-6 cells exposed to LPS 10 μg/mL. In addition, IEC-6 cells were treated with cAMP analogues 8-pCPT-2′-O-Me-cAMP (8C; Sigma-Aldrich), adenosine 3′,5′-cyclophosphate (Sigma-Aldrich), rat tumor necrosis factor α (TNF-α; LifeTech, Elmhurst, IL), and rat IL-6 (LifeTech). Cells were stained for apoptosis using the ApopTag Red in Situ Apoptosis Detection Kit (Chemicon, Billerica, MA). C. sakazakii, clinical strain BAA-894 (ATCC) was grown at 37°C in Luria broth, centrifuged (3000 rpm), and washed twice before being added to cell cultures or formula. C. sakazakii growth was assessed by growing bacteria in broth with KT5720 (1 μmol/L) over a time course and by measuring optical density (Supplemental Figure S1A). C. sakazakii binding to IEC-6 cells were also performed (Supplemental Figure S1B).45Hunter C.J. Petrosyan M. Ford H.R. Prasadarao N.V. Enterobacter sakazakii: an emerging pathogen in infants and neonates.Surg Infect (Larchmt). 2008; 9: 533-539Crossref PubMed Scopus (77) Google Scholar LPS from Escherichia coli clinical strain 0111:B4 (Sigma-Aldrich) was stored at 4°C. LPS was dissolved in sterile 0.9% normal saline (VWR, Radnor, PA) to achieve a stock concentration of 10 mg/mL. IEC-6 cells were seeded in a 12-well plate with 105 cells/well using serum-free media. Cells were transfected with Lipofectamine 2000 (LifeTech). PKA c-α siRNA (Cell Signaling, Danvers, MA), CREB siRNA (Santa Cruz Biotechnology, Dallas, TX), or control siRNA for 6 hours at 37°C and 5% CO2 were transfected. Knockdown was confirmed by Western blot analysis of phosphorylated PKA (pPKA), or quantitative RT-PCR with CREB primers supplied by Santa Cruz Biotechnology and normalized by GAPDH (forward: 5′-ATCACCATCTTCCAGGAGCG-3′; reverse: 5′-TTCTGAGTGGCAGTGAGGGC-3′). Data were analyzed by Bio-Rad CFX Manager (Bio-Rad, Hercules, CA) (Supplemental Figure S1E). After transfection the cells were changed to complete media. Thereafter, the siRNA PKA cells were treated with 1 × 107 cfu/mL C. sakazakii. Cells were harvested at times up to 6 hours after C. sakazakii addition. The siRNA CREB group was treated 24 hours after transfection. Female Sprague-Dawley rats with synchronized pregnancies were purchased from Charles River Laboratories (Roanoke, IL) and induced near-term at embryonic day 21 with a subcutaneous injection of oxytocin 0.1 U. Rat pups were separated from the dams and placed into experimental groups. Pups were gavaged thrice daily with formula 0.2 to 0.3 mL (15 g Similac 60/40 [Ross Pediatrics, Columbus, OH] in 75 mL of Esbilac canine milk replacer [Pet-Ag Inc., Hampshire, IL]). Pups were exposed to hypoxia (5% O2, 95% N2) for 5 minutes twice daily in a modular chamber (Billups-Rothenberg, Del Mar, CA). Experimental groups included formula-fed (FF) controls, a FF group with hypoxia (FF + H group), a FF + H group in which one of the daily feeds contained a known quantity of C. sakazakii (FF + H + C. sakazakii), and FF + H + C. sakazakii or FF + H groups pretreated with KT5720 (5 mL/kg) or vehicle (dimethyl sulfoxide) on the first day of life. Rat pups were euthanized on day 4 after birth or if they displayed symptoms of NEC (abdominal distention and discoloration), respiratory distress, or >20% weight loss. Intestine was harvested for analysis. NEC was graded microscopically by a pediatric pathologist (P.C.) blinded to groups, from grade 0 (normal) to grade 3 (severe with perforation) on the basis of pathologic manifestations, including submucosal edema, epithelial sloughing, hemorrhage, neutrophil infiltration, derangement of intestinal villus architecture, intestinal perforation, and necrosis. Animals were housed in the Northwestern University facilities that are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. They were provided with environmental enrichment. All procedures and protocols were approved by Northwestern University Institutional Animal Care and Use Committee and were conducted in accordance with guidelines set forth by NIH's Guide for the Care and Use of Laboratory Animals.46Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar After institutional review board approval (number 2013-15152), human intestinal tissue samples were obtained from infants undergoing bowel resection. The type of tissue obtained, reason for surgery, positive clinical cultures (blood and urine), and corrected gestational age were recorded. Intestinal specimens were categorized as active NEC (ie, the intestine was resected during surgery for perforation or sepsis) and NEC-free (eg, intestine resected during ostomy takedown or for intestinal atresia). In total 7 patients with NEC and 8 control samples were obtained. Tissue was collected in 10% buffered formalin (Cardinal Health, Dublin, OH) and processed into paraffin blocks, snap-frozen in liquid nitrogen, or preserved in optimal cutting temperature media Sakura Finetek, Torrance, CA) at −80°C. Tissue samples from humans and rats were isolated before either being suspended in Allprotect tissue reagent (Qiagen, Valencia, CA) and stored at −80°C or snap-frozen in liquid nitrogen. The tissue was sectioned and suspended in lysis buffer (20 mmol/L Tris-HCl [pH 7.5]), 150 mmol/L NaCl, 1 mmol/L Na2 EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L glycerophosphate, 1 mmol/L Na3VO4, 1 μg/mL leupeptin; Cell Signaling Tech) and containing 1 mmol/L phenylmethylsulfonyl fluoride, protease inhibitors [1.02 mmol/L 4-(2-Aminomethyl)benzenesulfonyl fluoride hydrochloride, 0.0008 mmol/L aprotinin, 0.02 mmol/L leupeptin, 0.04 mmol/L bestatin, 0.015 mmol/L pepstatin A, 0.014 mmol/L E-64], phosphatase inhibitors (sodium vanadate, sodium molybdate, sodium tartrate, and imidazole; Sigma-Aldrich). Samples were homogenized for 3 minutes on ice using a ground glass tissue grinder. After centrifugation for 2 minutes at 14,000 × g and 4°C, supernatant fluids were collected and stored at −80°C. To isolate proteins, cultured cells were scraped into and centrifuged at 5000 rpm at 4°C for 10 minutes. Supernatant fluids were discarded, and cell pellets were resuspended in lysis buffer, drawn three times through a 27-gauge needle, and mixed on a rotating platform for 30 minutes at 4°C. Insoluble debris was then removed by centrifugation 4°C, 15 minutes, at 10,000 rpm, and supernatant fluids were stored at −80°C. Antibodies were obtained from the following sources: mouse anti–β-actin (dilution 1:50,000; Sigma-Aldrich), rabbit anti-PKA C-a (dilution 1:500; Cell Signaling), rabbit anti–phospho-PKA C (dilut
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