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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 effects are mediated via the ERK signaling pathway. A, HRA-19 cells were transferred to ITA medium with or without the α2 integrin antibody P1E6 (25 ng/ml) and the MEK inhibitor PD98059 (10 μm) or DMSO control. Chromogranin-positive cells were detected by immunocytochemistry. The experiment was performed three times. Quadruplicate chambers were used for each condition. Mean ± S.D. is shown *, p < 0.005. The cell number was determined in replicate wells using the WST1 reagent (absorbance 450/620 nm). B, Caco-2 cells were plated in TSG medium onto surfaces coated with either α2 integrin mAb AK7 or MsIgG (10 μg/ml) with or without the MEK inhibitor PD98059 (10 μm). Alkaline phosphatase expression (abs: 405 nm) was normalized for cell number with the WST-1 reagent (abs 450 nm/abs 620 nm). Quadruplicate wells were used for each condition; mean ± S.D. The experiment was performed four times, **, p < 0.001.
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fig8: α2 integrin effects are mediated via the ERK signaling pathway. A, HRA-19 cells were transferred to ITA medium with or without the α2 integrin antibody P1E6 (25 ng/ml) and the MEK inhibitor PD98059 (10 μm) or DMSO control. Chromogranin-positive cells were detected by immunocytochemistry. The experiment was performed three times. Quadruplicate chambers were used for each condition. Mean ± S.D. is shown *, p < 0.005. The cell number was determined in replicate wells using the WST1 reagent (absorbance 450/620 nm). B, Caco-2 cells were plated in TSG medium onto surfaces coated with either α2 integrin mAb AK7 or MsIgG (10 μg/ml) with or without the MEK inhibitor PD98059 (10 μm). Alkaline phosphatase expression (abs: 405 nm) was normalized for cell number with the WST-1 reagent (abs 450 nm/abs 620 nm). Quadruplicate wells were used for each condition; mean ± S.D. The experiment was performed four times, **, p < 0.001.

Mentions: α2 Integrin Regulates Stem Cell Behavior via the ERK Signaling Pathway—The extracellular signal-regulated kinase (ERK MAPK) signaling pathway is important in intestinal epithelial differentiation (39, 40), and its dysregulation in colorectal cancer is thought to play a part in progression of this disease (41). The MEK inhibitor PD98059, which blocks ERK signaling, was used to determine whether α2 integrin regulation of lineage commitment was mediated via this signaling pathway. PD98059 abrogated the α2 integrin-mediated reduction in endocrine lineage commitment in HRA-19 cells (Fig. 8A) and enterocytic differentiation in Caco-2 cells (Fig. 8B) without a change in cell number (Fig. 8A).


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

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

α2 integrin effects are mediated via the ERK signaling pathway. A, HRA-19 cells were transferred to ITA medium with or without the α2 integrin antibody P1E6 (25 ng/ml) and the MEK inhibitor PD98059 (10 μm) or DMSO control. Chromogranin-positive cells were detected by immunocytochemistry. The experiment was performed three times. Quadruplicate chambers were used for each condition. Mean ± S.D. is shown *, p < 0.005. The cell number was determined in replicate wells using the WST1 reagent (absorbance 450/620 nm). B, Caco-2 cells were plated in TSG medium onto surfaces coated with either α2 integrin mAb AK7 or MsIgG (10 μg/ml) with or without the MEK inhibitor PD98059 (10 μm). Alkaline phosphatase expression (abs: 405 nm) was normalized for cell number with the WST-1 reagent (abs 450 nm/abs 620 nm). Quadruplicate wells were used for each condition; mean ± S.D. The experiment was performed four times, **, p < 0.001.
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fig8: α2 integrin effects are mediated via the ERK signaling pathway. A, HRA-19 cells were transferred to ITA medium with or without the α2 integrin antibody P1E6 (25 ng/ml) and the MEK inhibitor PD98059 (10 μm) or DMSO control. Chromogranin-positive cells were detected by immunocytochemistry. The experiment was performed three times. Quadruplicate chambers were used for each condition. Mean ± S.D. is shown *, p < 0.005. The cell number was determined in replicate wells using the WST1 reagent (absorbance 450/620 nm). B, Caco-2 cells were plated in TSG medium onto surfaces coated with either α2 integrin mAb AK7 or MsIgG (10 μg/ml) with or without the MEK inhibitor PD98059 (10 μm). Alkaline phosphatase expression (abs: 405 nm) was normalized for cell number with the WST-1 reagent (abs 450 nm/abs 620 nm). Quadruplicate wells were used for each condition; mean ± S.D. The experiment was performed four times, **, p < 0.001.
Mentions: α2 Integrin Regulates Stem Cell Behavior via the ERK Signaling Pathway—The extracellular signal-regulated kinase (ERK MAPK) signaling pathway is important in intestinal epithelial differentiation (39, 40), and its dysregulation in colorectal cancer is thought to play a part in progression of this disease (41). The MEK inhibitor PD98059, which blocks ERK signaling, was used to determine whether α2 integrin regulation of lineage commitment was mediated via this signaling pathway. PD98059 abrogated the α2 integrin-mediated reduction in endocrine lineage commitment in HRA-19 cells (Fig. 8A) and enterocytic differentiation in Caco-2 cells (Fig. 8B) without a change in cell number (Fig. 8A).

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