Volume 126, Issue 1, Pages 111-121 (January 2004) Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon  James C.H. Hardwick, Gijs R. Van Den Brink, Sylvia A. Bleuming, Isabel Ballester, Jan.M.H. Van Den Brande, Josbert J. Keller, G.Johan A. Offerhaus, Sander J.H. Van Deventer, Maikel P. Peppelenbosch  Gastroenterology  Volume 126, Issue 1, Pages 111-121 (January 2004) DOI: 10.1053/j.gastro.2003.10.067

Figure 1 RT-PCR for BMP receptors Ia, Ib, and II in 5 colon cancer cell lines. A product of the expected length (BMPRIa, 300 bp; BMPRIb, 200 bp; BMPRII, 400 bp) was seen in all cell lines for all 3 receptors. The negative controls in which RNA was omitted showed no product. The osteosarcoma cell line SAOS-2 was used as a positive control. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 2 Immunoblot for BMP receptors Ia, Ib, and II in 6 colon cancer cell lines. Seventy-five micrograms of total protein from cell lysates were loaded per lane. SAOS-2 osteosarcoma cells were used as a positive control. The calculated apparent molecular weights are shown. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 3 MTT assay in 4 colon cancer cell lines treated for 72 hours with various concentrations of BMP2. Values obtained with no BMP2 have been set at 100. Error bars represent the standard error of the mean. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 4 Immunoblots of HT29 cells treated with BMP2 (100 ng/mL) for various times (shown in hours). Fifty micrograms of total protein from cell lysates were loaded per lane and blotted for PCNA, cleaved caspase 3, and β-catenin. Equal loading was confirmed by showing equal β-actin levels. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 5 Immunohistochemistry for BMP2 in mouse (B and C) and human (D) colon. BMP2 is expressed predominantly in colonocytes at the epithelial surface. Strong staining is seen localized to the cytoplasm. Control stainings performed by omitting the primary antibody (not shown) and using a control IgG2b (A) showed no staining. Double staining with PCNA (brown) and BMP2 (red) illustrates that BMP2 is expressed away from the proliferative compartment (E). TUNEL staining in normal mouse colon shows that BMP2 expression overlaps with that of a marker of apoptotis (F). Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 6 Immunohistochemistry for BMP receptors 1a, 1b, and II and phosphorylated Smad1 and Smad4 in normal human and mouse colon. All are expressed predominantly in colonocytes at the epithelial surface. Strong staining is seen localized to the cytoplasm and cell membrane in the receptor stainings, whereas both the Smad stainings show cytoplasmic and clear nuclear staining. Controls were performed in human and mouse colon using isotype antibody controls (not shown) and human (not shown) and mouse controls omitting the primary antibody (shown above); all showed no staining. BMPRIa in human colon is not shown because extremely high signal in the stroma made the slides difficult to interpret. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 7 Graph shows the mean number of TUNEL-labeled cells per crypt in the colons of noggin, saline, or Rituximab (anti-CD20)-treated mice. Statistical analysis was performed with the Student t test. Error bars represent the standard error of the mean. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 9 Immunoblot analysis of lysates of mouse colon from 7 C57/BL6 mice treated with noggin/Fc chimera for 2 weeks and 7 vehicle controls. Fifty micrograms of total protein was loaded per lane. Noggin treatment significantly decreased phosphorylated Smad1 expression, suggesting that BMP signaling was blocked. PCNA expression was significantly decreased as was cleaved caspase 3. β-Catenin expression was also significantly reduced, whereas BMP2 expression rose in treated animals. Equal loading was confirmed by assessing β-actin. Statistical analysis of the relative mean expression of the various molecules using the Student t test gave the following P values: actin = 0.17, BMP2 = 0.0024, β-catenin = 0.018, caspase 3 = 0.0006, PCNA = 0.004, pSmad1 = 0.0003. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 8 Graph shows the mean number of BrdU-labelled cells per crypt in the colons of noggin, saline, or Rituximab (anti-CD20)-treated mice. Statistical analysis was performed with the Student t test. Error bars represent the standard error of the mean. Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 10 Immunohistochemistry for β-catenin in mouse colon from C57/BL6 mice treated with noggin/Fc chimera for 2 weeks (A) and with a control chimeric antibody Rituximab (B). The reduction in β-catenin seen in the immunoblots can be clearly seen in the immunohistochemistry. There is no nuclear staining in either group. The change appears to be largely due to a decrease in membrane β-catenin staining, especially in the cells of the lower half of the crypts. (Original magnification 400×.) Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)

Figure 11 Immunohistochemistry for BMP2 in human colon from a patient with familial adenomatous polyposis (A). The strong staining seen in normal tissue is lost in the epithelial cells of the microadenoma. B and C are higher magnifications of the areas marked with a solid and dotted line, respectively. B shows the microadenoma at high magnification (×200), and C shows nearby normal epithelium (×200). (Original magnification of A 80×.) Gastroenterology 2004 126, 111-121DOI: (10.1053/j.gastro.2003.10.067)