Article Text
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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- 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
Footnotes
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Published Online First 4 April 2007
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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.
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Competing interests: None.