Calcium/calmodulin-dependent protein kinase IV signaling pathway is upregulated in experimental necrotizing enterocolitis

  • Mashriq Alganabi
  • Haitao Zhu
  • Joshua S. O’Connell
  • George Biouss
  • Andrea Zito
  • Bo Li
  • Edoardo Bindi
  • Agostino PierroEmail author
Original Article



Activation of calcium/calmodulin-dependent protein kinase IV (CaMKIV) has been shown to increase intestinal injury and inhibit epithelial cell proliferation in dextran sulfate sodium (DSS)-induced colitis mice. However, the role of CaMKIV in necrotizing enterocolitis (NEC) is unknown. We aimed to study the expression and activation of CaMKIV in experimental NEC.


Following ethical approval, NEC (n = 10) was induced in C57BL/6 mouse pups by hypoxia, gavage hyperosmolar formula feeding and lipopolysaccharide from postnatal days P5 to 9. Breastfed pups served as control (n = 10). Mouse pups were sacrificed on P9 and the terminal ileum was harvested. Gene NEC injury was scored blindly by three independent investigators. CaMKIV, CREM and IL17 gene expression, and CaMKIV and pCaMKIV protein expression were assessed. The data were compared using Mann–Whitney U test. P < 0.05 was considered significant.


Intestinal injury was induced in the NEC mice and confirmed by histological scoring and inflammatory cytokine IL6. CaMKIV and its downstream target genes of CREM and IL17 were significantly elevated in NEC mice relative to control. Similarly, phosphorylated-CaMKIV (pCaMKIV), the active form of CaMKIV, was more notably expressed in the NEC ileal tissue relative to control ileal tissue. Elevated pCaMKIV protein expression was also confirmed by western blot.


CaMKIV expression and activation are upregulated in experimental NEC suggesting a potential contributing factor in the pathogenesis of NEC.


Necrotizing enterocolitis (NEC) Calcium/calmodulin-dependent protein kinase IV (CaMKIV) pCaMKIV IL17 



AP is supported by a Canadian Institutes of Health Research (CIHR) Foundation Grant 353857 and The Hospital for Sick Children. The funding had no impact on study design, data collection, analysis, interpretation, the writing of the report, or the decision to submit the paper for publication. We would also like to thank Maarten Janssen Lok for his contribution to histological scoring and manuscript revision.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Animal experiments received ethical approval by the Animal Care Committee of The Hospital for Sick Children (No. 44032) and were performed in accordance with its guidelines and regulations.


  1. 1.
    Fitzgibbons SC, Ching Y, Yu D, Carpenter J, Kenny M, Weldon C, Lillehei C, Valim C, Horbar JD, Jaksic T (2009) Mortality of necrotizing enterocolitis expressed by birth weight categories. J Pediatr Surg 44(6):1072–1075. CrossRefPubMedGoogle Scholar
  2. 2.
    Wojkowska-Mach J, Rozanska A, Borszewska-Kornacka M, Domanska J, Gadzinowski J, Gulczynska E, Helwich E, Kordek A, Pawlik D, Szczapa J, Heczko PB (2014) Necrotising enterocolitis in preterm infants: epidemiology and antibiotic consumption in the Polish neonatology network neonatal intensive care units in 2009. PLoS ONE 9(3):e92865. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Alganabi M, Lee C, Bindi E, Li B, Pierro A (2019) Recent advances in understanding necrotizing enterocolitis. F1000Research 8:9–10 doi:10.12688/f1000research.17228.1.CrossRefGoogle Scholar
  4. 4.
    Neu J, Walker WA (2011) Necrotizing enterocolitis. New England J Med 364(3):255–264. CrossRefGoogle Scholar
  5. 5.
    Zhang C, Sherman MP, Prince LS, Bader D, Weitkamp JH, Slaughter JC, McElroy SJ (2012) Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice. Dis Model Mech 5(4):522–532. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Molteni M, Gemma S, Rossetti C (2016) The role of toll-like receptor 4 in infectious and noninfectious inflammation. Mediators Inflamm 2016:6978936. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    De Plaen IG (2013) Inflammatory signaling in necrotizing enterocolitis. Clin Perinatol 40(1):109–124. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Lin MY, Zal T, Ch'en IL, Gascoigne NR, Hedrick SM (2005) A pivotal role for the multifunctional calcium/calmodulin-dependent protein kinase II in T cells: from activation to unresponsiveness. J Immunol 174(9):5583–5592CrossRefGoogle Scholar
  9. 9.
    Pan F, Means AR, Liu JO (2005) Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation. EMBO J 24(12):2104–2113. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    McGargill MA, Sharp LL, Bui JD, Hedrick SM, Calbo S (2005) Active Ca2+/calmodulin-dependent protein kinase II gamma B impairs positive selection of T cells by modulating TCR signaling. J Immunol 175(2):656–664CrossRefGoogle Scholar
  11. 11.
    Raman V, Blaeser F, Ho N, Engle DL, Williams CB, Chatila TA (2001) Requirement for Ca2+/calmodulin-dependent kinase type IV/Gr in setting the thymocyte selection threshold. J Immunol 167(11):6270–6278CrossRefGoogle Scholar
  12. 12.
    Ishiguro K, Green T, Rapley J, Wachtel H, Giallourakis C, Landry A, Cao Z, Lu N, Takafumi A, Goto H, Daly MJ, Xavier RJ (2006) Ca2+/calmodulin-dependent protein kinase II is a modulator of CARMA1-mediated NF-kappaB activation. Mol Cell Biol 26(14):5497–5508. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Racioppi L, Means AR (2008) Calcium/calmodulin-dependent kinase IV in immune and inflammatory responses: novel routes for an ancient traveller. Trends Immunol 29(12):600–607. CrossRefPubMedGoogle Scholar
  14. 14.
    Matthews RP, Guthrie CR, Wailes LM, Zhao X, Means AR, McKnight GS (1994) Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-dependent gene expression. Mol Cell Biol 14(9):6107–6116. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Koga T, Ichinose K, Mizui M, Crispin JC, Tsokos GC (2012) Calcium/calmodulin-dependent protein kinase IV suppresses IL2 production and regulatory T cell activity in lupus. J Immunol Baltimore Md 189(7):3490–3496. CrossRefGoogle Scholar
  16. 16.
    Koga T, Hedrich CM, Mizui M, Yoshida N, Otomo K, Lieberman LA, Rauen T, Crispín JC, Tsokos GC (2014) CaMK4-dependent activation of AKT/mTOR and CREM-α underlies autoimmunity-associated Th17 imbalance. J Clin Investig 124(5):2234–2245. CrossRefPubMedGoogle Scholar
  17. 17.
    Maeda K, Otomo K, Yoshida N, Abu-Asab MS, Ichinose K, Nishino T, Kono M, Ferretti A, Bhargava R, Maruyama S, Bickerton S, Fahmy TM, Tsokos MG, Tsokos GC (2018) CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease. J Clin Investig 128(8):3445–3459. CrossRefPubMedGoogle Scholar
  18. 18.
    Cunningham KE, Novak EA, Vincent G, Siow VS, Griffith BD, Ranganathan S, Rosengart MR, Piganelli JD, Mollen KP (2019) Calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation contributes to the pathogenesis of experimental colitis via inhibition of intestinal epithelial cell proliferation. FASEB J 33(1):1330–1346. CrossRefPubMedGoogle Scholar
  19. 19.
    Biouss G, Antounians L, Li B, O'Connell JS, Seo S, Catania VD, Guadagno J, Rahman A, Zani-Ruttenstock E, Svergun N, Pierro A, Zani A (2019) Experimental necrotizing enterocolitis induces neuroinflammation in the neonatal brain. J Neuroinflammat 16(1):97. CrossRefGoogle Scholar
  20. 20.
    Koike Y, Li B, Lee C, Cheng S, Miyake H, Welsh C, Hock A, Belik J, Zani A, Pierro A (2017) Gastric emptying is reduced in experimental NEC and correlates with the severity of intestinal damage. J Pediatr Surg 52(5):744–748. CrossRefPubMedGoogle Scholar
  21. 21.
    Li B, Hock A, Wu RY, Minich A, Botts SR, Lee C, Antounians L, Miyake H, Koike Y, Chen Y, Zani A, Sherman PM, Pierro A (2019) Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis. PLoS ONE ONE 14(1):e0211431. CrossRefGoogle Scholar
  22. 22.
    Miyake H, Chen Y, Koike Y, Hock A, Li B, Lee C, Zani A, Pierro A (2016) Osmolality of enteral formula and severity of experimental necrotizing enterocolitis. Pediatr Surg Int 32(12):1153–1156. CrossRefPubMedGoogle Scholar
  23. 23.
    Miyake H, Li B, Lee C, Koike Y, Chen Y, Seo S, Pierro A (2018) Liver damage, proliferation, and progenitor cell markers in experimental necrotizing enterocolitis. J Pediatr Surg 53(5):909–913. CrossRefPubMedGoogle Scholar
  24. 24.
    Zani A, Cordischi L, Cananzi M, De Coppi P, Smith VV, Eaton S, Pierro A (2008) Assessment of a neonatal rat model of necrotizing enterocolitis. Eur J Pediatr Surg 18(6):423–426. CrossRefPubMedGoogle Scholar
  25. 25.
    Dvorak B, Halpern MD, Holubec H, Williams CS, McWilliam DL, Dominguez JA, Stepankova R, Payne CM, McCuskey RS (2002) Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model. Am J Physiol Gastrointest Liver Physiol 282(1):G156–164. CrossRefPubMedGoogle Scholar
  26. 26.
    Li B, Lee C, Filler T, Hock A, Wu RY, Li Q, Chen S, Koike Y, Ip W, Chi L, Zani-Ruttenstock E, Maattanen P, Gonska T, Delgado-Olguin P, Zani A, Sherman PM, Pierro A (2017) Inhibition of corticotropin-releasing hormone receptor 1 and activation of receptor 2 protect against colonic injury and promote epithelium repair. Sci Rep 7:46616. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Khan D, Ansar Ahmed S (2015) Regulation of IL-17 in autoimmune diseases by transcriptional factors and microRNAs. Front Genet 6:236. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Noack M, Miossec P (2014) Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimmun Rev 13(6):668–677. CrossRefPubMedGoogle Scholar
  29. 29.
    Moote W, Kim H, Ellis AK (2018) Allergen-specific immunotherapy. Allergy Asthma Clin Immunol 14(2):53. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Egan CE, Sodhi CP, Good M, Lin J, Jia H, Yamaguchi Y, Lu P, Ma C, Branca MF, Weyandt S, Fulton WB, Nino DF, Prindle T Jr, Ozolek JA, Hackam DJ (2016) Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis. J Clin Invest 126(2):495–508. CrossRefPubMedGoogle Scholar
  31. 31.
    Lawrence SM, Ruoss JL, Wynn JL (2018) IL-17 in neonatal health and disease. Am J Reprod Immunol 79(5):e12800. CrossRefPubMedGoogle Scholar
  32. 32.
    Beringer A, Noack M, Miossec P (2016) IL-17 in Chronic Inflammation: From Discovery to Targeting. Trends Mol Med 22(3):230–241. CrossRefPubMedGoogle Scholar
  33. 33.
    Onishi RM, Gaffen SL (2010) Interleukin-17 and its target genes: mechanisms of interleukin-17 function in disease. Immunology 129(3):311–321. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Cua DJ, Tato CM (2010) Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol 10(7):479–489. CrossRefPubMedGoogle Scholar
  35. 35.
    Stoll BJ, Hansen NI, Bell EF, Shankaran S, Laptook AR, Walsh MC, Hale EC, Newman NS, Schibler K, Carlo WA, Kennedy KA, Poindexter BB, Finer NN, Ehrenkranz RA, Duara S, Sanchez PJ, O'Shea TM, Goldberg RN, Van Meurs KP, Faix RG, Phelps DL, Frantz ID 3rd, Watterberg KL, Saha S, Das A, Higgins RD (2010) Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics 126(3):443–456. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Bystrom J, Al-Adhoubi N, Al-Bogami M, Jawad AS, Mageed RA (2013) Th17 lymphocytes in respiratory syncytial virus infection. Viruses 5(3):777–791. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Croxford AL, Karbach S, Kurschus FC, Wortge S, Nikolaev A, Yogev N, Klebow S, Schuler R, Reissig S, Piotrowski C, Brylla E, Bechmann I, Scheller J, Rose-John S, Thomas Wunderlich F, Munzel T, von Stebut E, Waisman A (2014) IL-6 regulates neutrophil microabscess formation in IL-17A-driven psoriasiform lesions. J Invest Dermatol 134(3):728–735. CrossRefPubMedGoogle Scholar
  38. 38.
    Sehrawat S, Rouse BT (2017) Interplay of Regulatory T Cell and Th17 Cells during Infectious Diseases in Humans and Animals. Front Immunol 8:341. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Neu J (2005) Neonatal necrotizing enterocolitis: an update. Acta Paediatr Suppl 94(449):100–105. CrossRefPubMedGoogle Scholar
  40. 40.
    Zhang X, Zhivaki D, Lo-Man R (2017) Unique aspects of the perinatal immune system. Nat Rev Immunol 17(8):495–507. CrossRefPubMedGoogle Scholar
  41. 41.
    Koga T, Otomo K, Mizui M, Yoshida N, Umeda M, Ichinose K, Kawakami A, Tsokos GC (2016) Calcium/Calmodulin-Dependent Kinase IV Facilitates the Recruitment of Interleukin-17-Producing Cells to Target Organs Through the CCR6/CCL20 Axis in Th17 Cell-Driven Inflammatory Diseases. Arthrit Rheumatol 68(8):1981–1988. CrossRefGoogle Scholar
  42. 42.
    Tang H, Pang S, Wang M, Xiao X, Rong Y, Wang H, Zang YQ (2010) TLR4 activation is required for IL-17-induced multiple tissue inflammation and wasting in mice. J Immunol 185(4):2563–2569. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Division of General and Thoracic Surgery, Translational Medicine Program, The Hospital for Sick ChildrenUniversity of TorontoTorontoCanada

Personalised recommendations