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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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β1 integrins regulate endocrine and mucous lineage commitment by HRA-19 cells. A, HRA-19 cells were seeded into 8-chamber plastic slides in serum-free medium with varying dilutions of β1 integrin mAbs JB1A or LM534. On Day 5, cells were fixed and stained for the endocrine lineage marker chromogranin using immunocytochemistry. Data shown are mean ± S.D. (n = 7). **, p < 0.001. Results are representative of three independent experiments. B, HRA-19 cells were grown in 8-chamber slides for 3 days and then transferred to serum-free medium. On Day 7, monolayers were stained with the colonic mucous antibody, PR4D4 using immunocytochemistry. Data shown are mean ± S.D. (n = 4) **, p < 0.001. The cell number was determined in replicate wells using the WST1 reagent (absorbance: 450/620 nm) (n = 4). Results are representative of three independent experiments.
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fig1: β1 integrins regulate endocrine and mucous lineage commitment by HRA-19 cells. A, HRA-19 cells were seeded into 8-chamber plastic slides in serum-free medium with varying dilutions of β1 integrin mAbs JB1A or LM534. On Day 5, cells were fixed and stained for the endocrine lineage marker chromogranin using immunocytochemistry. Data shown are mean ± S.D. (n = 7). **, p < 0.001. Results are representative of three independent experiments. B, HRA-19 cells were grown in 8-chamber slides for 3 days and then transferred to serum-free medium. On Day 7, monolayers were stained with the colonic mucous antibody, PR4D4 using immunocytochemistry. Data shown are mean ± S.D. (n = 4) **, p < 0.001. The cell number was determined in replicate wells using the WST1 reagent (absorbance: 450/620 nm) (n = 4). Results are representative of three independent experiments.

Mentions: Endocrine Lineage Commitment Is Regulated by α2β1 Integrin—To determine whether β1 integrins were involved in lineage commitment, HRA-19 cells were transferred to serum-free medium to induce endocrine lineage commitment in the presence of a β1 antibody (JB1A), which blocks cell adhesion (26) and signaling (34). JB1A reduced endocrine lineage commitment to 2% of control values (Fig. 1A). Furthermore LM534, another β1 antibody that binds to the extracellular domain of β1 integrin, also reduced endocrine lineage commitment significantly (Fig. 1A). Function-blocking β1 integrin antibody, JB1A, also blocked mucous lineage commitment in HRA-19 cells (Fig. 1B) while addition of equivalent amounts of isotype control antibody did not affect mucous cell numbers. In addition, total cell number was unaffected by treatment with JB1A or isotype control, indicating that the antibodies had not affected attachment, proliferation, or survival (Fig. 1C). These results indicate a role for the β1 integrins in regulating endocrine and mucous lineage commitment in HRA-19 cells. However, integrins are heterodimers and modulation of β1 chain function could potentially be affecting all members of the β1 integrin family. Therefore, we sought to identify which β1 integrin heterodimer(s) was involved in blocking endocrine/mucous lineage commitment.


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

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

β1 integrins regulate endocrine and mucous lineage commitment by HRA-19 cells. A, HRA-19 cells were seeded into 8-chamber plastic slides in serum-free medium with varying dilutions of β1 integrin mAbs JB1A or LM534. On Day 5, cells were fixed and stained for the endocrine lineage marker chromogranin using immunocytochemistry. Data shown are mean ± S.D. (n = 7). **, p < 0.001. Results are representative of three independent experiments. B, HRA-19 cells were grown in 8-chamber slides for 3 days and then transferred to serum-free medium. On Day 7, monolayers were stained with the colonic mucous antibody, PR4D4 using immunocytochemistry. Data shown are mean ± S.D. (n = 4) **, p < 0.001. The cell number was determined in replicate wells using the WST1 reagent (absorbance: 450/620 nm) (n = 4). Results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: β1 integrins regulate endocrine and mucous lineage commitment by HRA-19 cells. A, HRA-19 cells were seeded into 8-chamber plastic slides in serum-free medium with varying dilutions of β1 integrin mAbs JB1A or LM534. On Day 5, cells were fixed and stained for the endocrine lineage marker chromogranin using immunocytochemistry. Data shown are mean ± S.D. (n = 7). **, p < 0.001. Results are representative of three independent experiments. B, HRA-19 cells were grown in 8-chamber slides for 3 days and then transferred to serum-free medium. On Day 7, monolayers were stained with the colonic mucous antibody, PR4D4 using immunocytochemistry. Data shown are mean ± S.D. (n = 4) **, p < 0.001. The cell number was determined in replicate wells using the WST1 reagent (absorbance: 450/620 nm) (n = 4). Results are representative of three independent experiments.
Mentions: Endocrine Lineage Commitment Is Regulated by α2β1 Integrin—To determine whether β1 integrins were involved in lineage commitment, HRA-19 cells were transferred to serum-free medium to induce endocrine lineage commitment in the presence of a β1 antibody (JB1A), which blocks cell adhesion (26) and signaling (34). JB1A reduced endocrine lineage commitment to 2% of control values (Fig. 1A). Furthermore LM534, another β1 antibody that binds to the extracellular domain of β1 integrin, also reduced endocrine lineage commitment significantly (Fig. 1A). Function-blocking β1 integrin antibody, JB1A, also blocked mucous lineage commitment in HRA-19 cells (Fig. 1B) while addition of equivalent amounts of isotype control antibody did not affect mucous cell numbers. In addition, total cell number was unaffected by treatment with JB1A or isotype control, indicating that the antibodies had not affected attachment, proliferation, or survival (Fig. 1C). These results indicate a role for the β1 integrins in regulating endocrine and mucous lineage commitment in HRA-19 cells. However, integrins are heterodimers and modulation of β1 chain function could potentially be affecting all members of the β1 integrin family. Therefore, we sought to identify which β1 integrin heterodimer(s) was involved in blocking endocrine/mucous lineage commitment.

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