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Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells.

Kirkland SC, Ying H - J. Biol. Chem. (2008)

Bottom Line: Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation.To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin.Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Faculty of Medicine, Imperial College London, London W12 ONN, United Kingdom. s.kirkland@imperial.ac.uk

ABSTRACT
The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

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α2 integrin regulates colorectal epithelial stem cell fate. A, endocrine lineage commitment (chromogranin expression) in cells after 48 h in serum-free medium. The mean ± S.D is shown. The experiment was performed three times. Endocrine and mucous cell numbers were normalized to an absorbance of 1 obtained with the WST-1 cell proliferation reagent to eliminate variation in cell number. Cells used were α2-transfected colonies α2B and α2F, chimeric α2α1-transfected colonies α2α1B and α2α1E and the parent non-transfected cell line HRA-19. B, chromogranin expression in α2F, α2α1E cells, and HRA-19 cells after 48 h in serum-free medium. Images obtained using a confocal microscope. The white arrow shows typical endocrine cell with a long process. Phase contrast images of the same fields. Bar, 100 μm. C, mucous lineage commitment in parent and transfected cell colonies detected with mucous antibody PR4D4 after 72 h in serum-free medium. The mean ± S.D. is shown. The experiment was performed three times.
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fig6: α2 integrin regulates colorectal epithelial stem cell fate. A, endocrine lineage commitment (chromogranin expression) in cells after 48 h in serum-free medium. The mean ± S.D is shown. The experiment was performed three times. Endocrine and mucous cell numbers were normalized to an absorbance of 1 obtained with the WST-1 cell proliferation reagent to eliminate variation in cell number. Cells used were α2-transfected colonies α2B and α2F, chimeric α2α1-transfected colonies α2α1B and α2α1E and the parent non-transfected cell line HRA-19. B, chromogranin expression in α2F, α2α1E cells, and HRA-19 cells after 48 h in serum-free medium. Images obtained using a confocal microscope. The white arrow shows typical endocrine cell with a long process. Phase contrast images of the same fields. Bar, 100 μm. C, mucous lineage commitment in parent and transfected cell colonies detected with mucous antibody PR4D4 after 72 h in serum-free medium. The mean ± S.D. is shown. The experiment was performed three times.

Mentions: Parent cells, α2 and α2α1 transfectants were induced to undergo lineage commitment by growth in serum-free (ITA) medium. Endocrine cell numbers were much higher in the wild-type α2 transfectants, α2B and α2F, than in the chimeric transfectants α2α1B and α2α1E, which showed little endocrine lineage commitment (Fig. 6, A and B). α2F and α2α1E cells had the highest expression of α2 and α2α1 proteins, respectively (Fig. 5, B and C) and these colonies showed the most extreme phenotypes with α2F cells showing 10.5-fold higher endocrine lineage commitment than the parent cells while α2α1E cells show only 2% of parent endocrine cell lineage commitment. Immunofluorescence staining of HRA-19 monolayers for chromogranin shows differential endocrine lineage commitment (Fig. 6B) between parent cells and transfectants. Phase contrast images are included to show that cells are present in the α2α1E monolayers, but endocrine lineage commitment is negligible. α2F cells contain many typical chromogranin-positive endocrine cells with long processes (Fig. 6B, white arrow).


Alpha2beta1 integrin regulates lineage commitment in multipotent human colorectal cancer cells.

Kirkland SC, Ying H - J. Biol. Chem. (2008)

α2 integrin regulates colorectal epithelial stem cell fate. A, endocrine lineage commitment (chromogranin expression) in cells after 48 h in serum-free medium. The mean ± S.D is shown. The experiment was performed three times. Endocrine and mucous cell numbers were normalized to an absorbance of 1 obtained with the WST-1 cell proliferation reagent to eliminate variation in cell number. Cells used were α2-transfected colonies α2B and α2F, chimeric α2α1-transfected colonies α2α1B and α2α1E and the parent non-transfected cell line HRA-19. B, chromogranin expression in α2F, α2α1E cells, and HRA-19 cells after 48 h in serum-free medium. Images obtained using a confocal microscope. The white arrow shows typical endocrine cell with a long process. Phase contrast images of the same fields. Bar, 100 μm. C, mucous lineage commitment in parent and transfected cell colonies detected with mucous antibody PR4D4 after 72 h in serum-free medium. The mean ± S.D. is shown. The experiment was performed three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562061&req=5

fig6: α2 integrin regulates colorectal epithelial stem cell fate. A, endocrine lineage commitment (chromogranin expression) in cells after 48 h in serum-free medium. The mean ± S.D is shown. The experiment was performed three times. Endocrine and mucous cell numbers were normalized to an absorbance of 1 obtained with the WST-1 cell proliferation reagent to eliminate variation in cell number. Cells used were α2-transfected colonies α2B and α2F, chimeric α2α1-transfected colonies α2α1B and α2α1E and the parent non-transfected cell line HRA-19. B, chromogranin expression in α2F, α2α1E cells, and HRA-19 cells after 48 h in serum-free medium. Images obtained using a confocal microscope. The white arrow shows typical endocrine cell with a long process. Phase contrast images of the same fields. Bar, 100 μm. C, mucous lineage commitment in parent and transfected cell colonies detected with mucous antibody PR4D4 after 72 h in serum-free medium. The mean ± S.D. is shown. The experiment was performed three times.
Mentions: Parent cells, α2 and α2α1 transfectants were induced to undergo lineage commitment by growth in serum-free (ITA) medium. Endocrine cell numbers were much higher in the wild-type α2 transfectants, α2B and α2F, than in the chimeric transfectants α2α1B and α2α1E, which showed little endocrine lineage commitment (Fig. 6, A and B). α2F and α2α1E cells had the highest expression of α2 and α2α1 proteins, respectively (Fig. 5, B and C) and these colonies showed the most extreme phenotypes with α2F cells showing 10.5-fold higher endocrine lineage commitment than the parent cells while α2α1E cells show only 2% of parent endocrine cell lineage commitment. Immunofluorescence staining of HRA-19 monolayers for chromogranin shows differential endocrine lineage commitment (Fig. 6B) between parent cells and transfectants. Phase contrast images are included to show that cells are present in the α2α1E monolayers, but endocrine lineage commitment is negligible. α2F cells contain many typical chromogranin-positive endocrine cells with long processes (Fig. 6B, white arrow).

Bottom Line: Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation.To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin.Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Faculty of Medicine, Imperial College London, London W12 ONN, United Kingdom. s.kirkland@imperial.ac.uk

ABSTRACT
The human colorectal epithelium is maintained by multipotent stem cells that give rise to absorptive, mucous, and endocrine lineages. Recent evidence suggests that human colorectal cancers are likewise maintained by a minority population of so-called cancer stem cells. We have previously established a human colorectal cancer cell line with multipotent characteristics (HRA-19) and developed a serum-free medium that induces endocrine, mucous and absorptive lineage commitment by HRA-19 cells in vitro. In this study, we investigate the role of the beta1 integrin family of cell surface extracellular matrix receptors in multilineage differentiation by these multipotent human colorectal cancer cells. We show that endocrine and mucous lineage commitment is blocked in the presence of function-blocking antibodies to beta1 integrin. Function-blocking antibodies to alpha2 integrin also blocked both HRA-19 endocrine lineage commitment and enterocytic differentiation by Caco-2 human colon cancer cells; both effects being abrogated by the MEK inhibitor, PD98059, suggesting a role for ERK signaling in alpha2-mediated regulation of colorectal cancer cell differentiation. To further explore the role of alpha2 integrin in multilineage differentiation, we established multipotent cells expressing high levels of wild-type alpha2 integrin or a non-signaling chimeric alpha2 integrin. Overexpression of wild-type alpha2 integrin in HRA-19 cells significantly enhanced endocrine and mucous lineage commitment, while cells expressing the non-signaling chimeric alpha2 integrin had negligible ability for either endocrine or mucous lineage commitment. This study indicates that the collagen receptor alpha2beta1 integrin is a regulator of cell fate in human multipotent colorectal cancer cells.

Show MeSH
Related in: MedlinePlus