Indeed, we identified a conserved region, which included eleven transcription factor binding sites

Indeed, we identified a conserved region, which included eleven transcription factor binding sites. analyzed for progastrin binding, proliferation, changes in gene expression, and symmetric cell division. Results The BMP pathway was downregulated in the colons of hGAS mice, compared with controls. Progastrin suppressed transcription of Bmp2 through a pathway that required CCK2R and was mediated by ARRB1 and ARRB2. In mouse colonic epithelial cells, downregulation of Bmp2 led to decreased phosphorylation of Smads1/5/8 and suppression of Id4. In human gastric and colorectal cancer cell lines, CCK2R was necessary and sufficient for progastrin binding and induction of proliferation; these MIF Antagonist 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 ARRB1- and 2-dependent suppression of Bmp2 signaling. This process promotes symmetric cell division. = 6 mice/group). Expression MIF Antagonist levels were normalized to GAPDH mRNA. (E) ELISA for Bmp2 protein in the colonic LATS1 mucosa of hGAS and WT mice (= 4 mice/group). All values represent the mean SD. *< 0.05, **< 0.01. (F) Western blot analysis of Bmp2, pSmad1/5/8, Id4 protein levels from murine colonic mucosa (= 3 mice/group). A cluster diagram of the 3 comparisons for select BMP pathway genes is shown in Supplemental Figure 2A. Bmp2 indirectly induces the transcription of inhibitor of DNA binding 4 (Id4),22 a postulated tumor suppressor,21, 23 such that downregulation of Bmp2 would be expected to decrease Id4. Indeed, Id4 was downregulated in hGAS mice in our microarray study confirmed by quantitative RT-PCR (Figure 1D) and by immunohistochemistry (Figure 1A). Thus, downregulation of Bmp2 leads to decreased phosphorylated Smads1/5/8 (pSmads1/5/8) and downregulation of Id4 (Supplemental Figure 2B), changes which could enhance tumorigenesis. Taken together, microarray and PCR analyses strongly suggest that progastrin stimulates colonic proliferation and progenitor expansion at least in part by suppression of the Bmp2 pathway. Progastrin downregulates phosphorylated Smad1/5/8 protein expression in the mouse colon Smads1/5/8 transduce the majority of intracellular signaling by bone morphogenetic proteins.24 Western blot analysis of colonic mucosa revealed significant downregulation of pSmad1/5/8 upon progastrin overexpression (Figure 1F). Immunoreactivity for pSmads1/5/8 was easily detectable in the nuclei of colonic crypt epithelial cells from WT mice, but was markedly lower in hGAS mice (Figure 1A). ID proteins, which have also been linked to the BMP pathway,25 are key regulators in embryonic development where they MIF Antagonist inhibit premature differentiation of stem cells. MIF Antagonist In contrast, inappropriate suppression of ID proteins in differentiated cells can contribute to tumorigenesis.26 Consistent with our findings of decreased Id4 mRNA, we found significantly downregulated Id4 protein level in the hGAS colon (Figure 1A, 1F). Thus, our findings strongly suggest that progastrin stimulates colonic growth in part by suppression of the BMPs, Smads1/5/8 and Id4. Progastrin increases colonic epithelial proliferation through Bmp2 In order to investigate the possibility that progastrin regulates Bmp2 expression through the CCK2R, we isolated colonic crypts27 from WT and CCK2R?/? mice. Noggin, a Bmp2 antagonist, is required for successful colonic crypt culture.27 Progastrin had no effect in noggin-containing media (Supplemental Figure 3A); however, following three to seven days in culture under noggin-free media conditions, progastrin treatment led to increased colonic organoid survival (Figure 2A, 2B, Supplemental Figure 3A) and suppressed Bmp2 mRNA expression in cultured organoids (Figure 2C), suggesting that progastrin restores noggin function through Bmp2 suppression. These effects were clearly mediated through CCK2R, since they were absent in CCK2R?/? organoids. To assess the.