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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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Expression ofα2 integrin constructs in HRA-19, human colorectal cancer cells. A, wild-type α2 and chimeric α2α1 integrin constructs transfected into HRA-19 cells. B, α2 integrin expression in α2 and α2α1 transfectants and HRA-19 cells. The experiment was performed twice. C, α2α1 integrin expression. α2α1 integrin was immunoprecipitated using an α1 cytoplasmic domain antibody, then detected using Western blot with an α2 extracellular domain antibody. The chimeric protein band was found only in α2α1-transfected colonies, α2α1β and α2α1E. The experiment was performed five times. D, α2 integrin localization was examined by immunofluorescence in α2F, HRA-19, and α2α1E cells. Bar, 100 μm.
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fig5: Expression ofα2 integrin constructs in HRA-19, human colorectal cancer cells. A, wild-type α2 and chimeric α2α1 integrin constructs transfected into HRA-19 cells. B, α2 integrin expression in α2 and α2α1 transfectants and HRA-19 cells. The experiment was performed twice. C, α2α1 integrin expression. α2α1 integrin was immunoprecipitated using an α1 cytoplasmic domain antibody, then detected using Western blot with an α2 extracellular domain antibody. The chimeric protein band was found only in α2α1-transfected colonies, α2α1β and α2α1E. The experiment was performed five times. D, α2 integrin localization was examined by immunofluorescence in α2F, HRA-19, and α2α1E cells. Bar, 100 μm.

Mentions: Integrin α2 Cytoplasmic Tail Is Required for Endocrine and Mucous Lineage Commitment—To support a role for the α2 integrin chain in cell fate regulation, we generated HRA-19 transfectants overexpressing either wild-type α2 integrin or a non-signaling chimeric protein composed of the extracellular and transmembrane domain of α2 integrin and the cytoplasmic domain of α1 integrin (Fig. 5A). Cell colonies were analyzed for their expression of α2 integrin (Fig. 5B) and α2α1 integrin (Fig. 5C). Two colonies, α2B and α2F, were chosen for further study as they showed markedly higher α2 integrin expression than the parent cell line (Fig. 5B). The chimeric protein was immunoprecipitated using an antibody to the cytoplasmic tail of α1 integrin and then detected on Western blots using an Ab to the extracellular region of the α2 chain (Fig. 5C). The α2 band was not observed in α1 immunoprecipitates of parent cells or α2 transfectants (α2B or α2F) but was present in chimeric transfectants α2α1B and α2α1E cells (Fig. 5C), which were selected for use in subsequent experiments. In the parent HRA-19 cells, α2 integrin is primarily localized at cell-cell contacts (Fig. 5D), as shown previously in the intestine (38), a localization retained by cells transfected with either wild-type α2 integrin ((α2F) or chimeric α2α1 integrin (α2α1E) (Fig. 5D).


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

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

Expression ofα2 integrin constructs in HRA-19, human colorectal cancer cells. A, wild-type α2 and chimeric α2α1 integrin constructs transfected into HRA-19 cells. B, α2 integrin expression in α2 and α2α1 transfectants and HRA-19 cells. The experiment was performed twice. C, α2α1 integrin expression. α2α1 integrin was immunoprecipitated using an α1 cytoplasmic domain antibody, then detected using Western blot with an α2 extracellular domain antibody. The chimeric protein band was found only in α2α1-transfected colonies, α2α1β and α2α1E. The experiment was performed five times. D, α2 integrin localization was examined by immunofluorescence in α2F, HRA-19, and α2α1E cells. Bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Expression ofα2 integrin constructs in HRA-19, human colorectal cancer cells. A, wild-type α2 and chimeric α2α1 integrin constructs transfected into HRA-19 cells. B, α2 integrin expression in α2 and α2α1 transfectants and HRA-19 cells. The experiment was performed twice. C, α2α1 integrin expression. α2α1 integrin was immunoprecipitated using an α1 cytoplasmic domain antibody, then detected using Western blot with an α2 extracellular domain antibody. The chimeric protein band was found only in α2α1-transfected colonies, α2α1β and α2α1E. The experiment was performed five times. D, α2 integrin localization was examined by immunofluorescence in α2F, HRA-19, and α2α1E cells. Bar, 100 μm.
Mentions: Integrin α2 Cytoplasmic Tail Is Required for Endocrine and Mucous Lineage Commitment—To support a role for the α2 integrin chain in cell fate regulation, we generated HRA-19 transfectants overexpressing either wild-type α2 integrin or a non-signaling chimeric protein composed of the extracellular and transmembrane domain of α2 integrin and the cytoplasmic domain of α1 integrin (Fig. 5A). Cell colonies were analyzed for their expression of α2 integrin (Fig. 5B) and α2α1 integrin (Fig. 5C). Two colonies, α2B and α2F, were chosen for further study as they showed markedly higher α2 integrin expression than the parent cell line (Fig. 5B). The chimeric protein was immunoprecipitated using an antibody to the cytoplasmic tail of α1 integrin and then detected on Western blots using an Ab to the extracellular region of the α2 chain (Fig. 5C). The α2 band was not observed in α1 immunoprecipitates of parent cells or α2 transfectants (α2B or α2F) but was present in chimeric transfectants α2α1B and α2α1E cells (Fig. 5C), which were selected for use in subsequent experiments. In the parent HRA-19 cells, α2 integrin is primarily localized at cell-cell contacts (Fig. 5D), as shown previously in the intestine (38), a localization retained by cells transfected with either wild-type α2 integrin ((α2F) or chimeric α2α1 integrin (α2α1E) (Fig. 5D).

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