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Urocortin II mediates pro-inflammatory effects in human colonocytes via corticotropin-releasing hormone receptor 2α
  1. Alan C Moss1,
  2. Pauline Anton1,
  3. Tor Savidge3,
  4. Paul Newman3,
  5. Adam S Cheifetz2,
  6. Jerome Gay4,
  7. Sophia Paraschos1,
  8. Michael Weinstein Winter1,
  9. Mary P Moyer5,
  10. Katia Karalis3,
  11. Efi Kokkotou3,
  12. Charalabos Pothoulakis1
  1. 1Gastrointestinal Neuropeptide Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA
  2. 2Center for Inflammatory Bowel Disease, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA
  3. 3Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, Harvard Medical School, Boston, USA
  4. 4Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston, USA
  5. 5INCELL Corporation, San Antonio, Texas, USA
  1. Correspondence to:
    Charalabos Pothoulakis
    MD, Beth Israel Deaconess Medical Center, Harvard Medical School, Division of Gastroenterology, Dana 601, 330 Brookline Avenue, Boston, MA 02215, USA; cpothoul{at}bidmc.harvard.edu

Abstract

Background/Aims: Urocortin II (UcnII) is a neuropeptide that binds with high affinity to the corticotropin-releasing hormone receptor 2 (CRHR2) in peripheral tissues. UcnII is synthesised in the intestine, but its role in human intestinal inflammation is largely unknown.

Methods: Responses of human colonic epithelial cells expressing CRHR2 to stimulation by UcnII were measured using ELISA, western blot analysis, real-time reverse transcription-PCR (RT-PCR) and interleukin (IL)8 promoter activity. Expression levels of CRHR2 and UcnII in human colitis were determined by immunofluorescence and real-time RT-PCR in mucosal biopsies from patients with Crohn’s and ulcerative colitis, and in human intestinal xenografts after exposure to Clostridium difficile toxin A.

Results: It is reported here that expression of CRHR2 mRNA and protein in human colonic epithelial cells (HT-29) are increased by exposure to C difficile toxin A or tumour necrosis factor (TNF)α. Stimulation of non-transformed NCM460 colonocytes overexpressing CRHR2α receptor with UcnII resulted in a time- and concentration-dependent increase in IL8 production. UcnII stimulation also led to activation of nuclear factor-κB (NF-κB) and mitogen-acivated protein (MAP) kinase in these cells, as evidenced by degradation of IκBα and phosphorylation of the p65 subunit of NF-κB and extracellularly regulated kinase (ERK) 1/2. Furthermore, expression of UcnII and CRHR2 mRNA was increased in mucosal samples of patients with inflammatory bowel disease, and after exposure of human intestinal xenografts to C difficile toxin A.

Conclusions: These results suggest that UcnII has pro-inflammatory effects in human intestinal cells via the CRHR2α receptor and may play an important role in the pathophysiology of colitis in humans.

  • CRH, corticotropin-releasing hormone
  • CRHR2 corticotropin-releasing hormone receptor 2
  • DMEM, Dulbecco’s modified Eagle’s medium
  • DMSO, dimethylsulphoxide
  • DTT, dithiothreitol
  • ERK, extracellularly regulated kinase
  • FITC, fluorescein isothiocyanate
  • GAPDH, glyceraldehyde phosphate dehydrogenase
  • HUVEC, human umbilical vein epithelial cell
  • IBD, inflammatory bowel disease
  • IL, interleukin
  • KO, knockout
  • LCM, laser capture microdissection
  • MAP, mitogen-activated protein
  • MCP, monocyte chemoattractant protein
  • NF-κB, nerve factor-κB
  • PBS, phosphate-buffered saline
  • RTR-PCR, reverse transcription-PCR
  • TBP, TATA-binding protein
  • TBS, Tris-buffered saline
  • TNBS, trinitrobenzenesulphonic acid
  • TNF, tumour necrosis factor
  • Ucn, urocortin

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Footnotes

  • Published Online First 4 April 2007

  • Funding: Supported by research grant PO-1 DK 33506 from the National Institutes of Health (C Pothoulakis), and the Crohn’s and Colitis Foundation (T Savidge). A C Moss is the recipient of the AGA/Centocor International Research Fellowship in Intestinal Inflammation & Immunity 2005.

  • Competing interests: None.

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