title: Progastrin stimulates colonic cell proliferation via CCK2R- and β-arrestin-dependent suppression of BMP2.
abstract: OBJECTIVE
  Progastrin stimulates colonic mucosal proliferation and carcinogenesis through the cholecystokinin 2 receptor (CCK2R)-partly by increasing the number of colonic progenitor cells. However, little is known about the mechanisms by which progastrin stimulates colonic cell proliferation. We investigated the role of bone morphogenetic proteins (BMPs) in progastrin induction of colonic cell proliferation via CCK2R.
  
  
  METHODS
  We performed microarray analysis to compare changes in gene expression in the colonic mucosa of mice that express a human progastrin transgene, gastrin knockout mice, and C57BL/6 mice (controls); the effects of progastrin were also determined on in vitro colonic crypt cultures from cholecystokinin 2 receptor knockout and wild-type mice. Human colorectal and gastric cancer cells that expressed CCK2R were incubated with progastrin or Bmp2; levels of β-arrestin 1 and 2 were knocked down using small interfering RNAs. Cells were analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division.
  
  
  RESULTS
  The BMP pathway was down-regulated in the colons of human progastrin mice compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by β-arrestin 1 and 2. In mouse colonic epithelial cells, down-regulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of inhibitor of DNA binding 4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these effects were blocked when cells were incubated with recombinant Bmp2. Incubation with progastrin increased the number of CD44(+), bromodeoxyuridine+, and NUMB(+) cells, indicating an increase in symmetric divisions of putative cancer stem cells.
  
  
  CONCLUSIONS
  Progastrin stimulates proliferation in colons of mice and cultured human cells via CCK2R- and β-arrestin 1 and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division.
authors.affiliation: Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, New York.
authors.forename: Guangchun
authors.initials: G
authors.lastname: Jin
authors.forename: C Benedikt
authors.initials: CB
authors.lastname: Westphalen
authors.forename: Yoku
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Worthley
authors.forename: Samuel
authors.initials: S
authors.lastname: Asfaha
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Yang
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Chen
authors.forename: Yiling
authors.initials: Y
authors.lastname: Si
authors.forename: Hongshan
authors.initials: H
authors.lastname: Wang
authors.forename: Yagnesh
authors.initials: Y
authors.lastname: Tailor
authors.forename: Richard A
authors.initials: RA
authors.lastname: Friedman
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Wang
mesh_terms: D000818:Animals; D019390:Arrestins / Q000502:physiology*; D055396:Bone Morphogenetic Protein 2 / Q000037:antagonists & inhibitors* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects*; D003106:Colon / Q000166:cytology / Q000187:drug effects*; D005755:Gastrins / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011498:Protein Precursors / Q000494:pharmacology*; D043887:Receptor, Cholecystokinin B / Q000502:physiology*; D015398:Signal Transduction; D013234:Stem Cells / Q000187:drug effects; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605255:Arrb1 protein, mouse; D019390:Arrestins; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D005755:Gastrins; D011498:Protein Precursors; D043887:Receptor, Cholecystokinin B; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; C013706:big gastrin
keywords: 5-bromo-2′-deoxyuridine; BMP; BrdU; CCK2R; G-17; G-protein−coupled receptor; GAS(−/−); GFP; GPCR; Id4; PCR; Progastrin; WT; amidated gastrin 17; bone morphogenetic protein; cholecystokinin-2 receptor; gastrin knockout; green fluorescent protein; hGAS; human progastrin; inhibitor of DNA binding 4; mRNA; messenger RNA; polymerase chain reaction; wild-type
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