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Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration.

Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR - J. Cell Biol. (2003)

Bottom Line: We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1).Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion.These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

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cdc2 mRNA, protein, and kinase levels are increased in β3-LNCaP cells. (A) Surface expression of ectopic αvβ3 integrin in LNCaP cell stable transfectants. Mock-, β3-1, β3-2, and β3-3 LNCaP cells were incubated with mAb to αvβ3 integrin (AP-3, thick line) or, as negative control, to the HA epitope (12CA5, thin line) followed by staining with FITC-conjugated goat anti–mouse Ig antibody and analysis using a FACScanTM flow cytometer. (B) cDNA expression array analysis. First strand cDNA probes prepared with either β3-1 or mock-LNCaP mRNA were hybridized to Atlas human cancer cDNA expression arrays. Shown are areas of section A of the cDNA array membrane. The arrow indicates the spots corresponding to cdc2 on the array. The brackets indicate other genes on the array that are not up-regulated in β3-1. (C–F) cdc2 protein and kinase levels are increased in β3-LNCaP cells grown in 2D (C and D) and 3D (E and F) cultures. (C) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 2D culture were analyzed by IB with an antibody to cdc2. In C and E, antibody to ERK-1 was used for protein loading control. (D) Immunocomplexes precipitated from 75 μg of β3-1, β3-3, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments. (E) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 3D Matrigel culture for 48 h were analyzed by IB with an antibody to cdc2. (F) Immunocomplexes precipitated from 75 μg of β3-1, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments.
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fig1: cdc2 mRNA, protein, and kinase levels are increased in β3-LNCaP cells. (A) Surface expression of ectopic αvβ3 integrin in LNCaP cell stable transfectants. Mock-, β3-1, β3-2, and β3-3 LNCaP cells were incubated with mAb to αvβ3 integrin (AP-3, thick line) or, as negative control, to the HA epitope (12CA5, thin line) followed by staining with FITC-conjugated goat anti–mouse Ig antibody and analysis using a FACScanTM flow cytometer. (B) cDNA expression array analysis. First strand cDNA probes prepared with either β3-1 or mock-LNCaP mRNA were hybridized to Atlas human cancer cDNA expression arrays. Shown are areas of section A of the cDNA array membrane. The arrow indicates the spots corresponding to cdc2 on the array. The brackets indicate other genes on the array that are not up-regulated in β3-1. (C–F) cdc2 protein and kinase levels are increased in β3-LNCaP cells grown in 2D (C and D) and 3D (E and F) cultures. (C) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 2D culture were analyzed by IB with an antibody to cdc2. In C and E, antibody to ERK-1 was used for protein loading control. (D) Immunocomplexes precipitated from 75 μg of β3-1, β3-3, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments. (E) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 3D Matrigel culture for 48 h were analyzed by IB with an antibody to cdc2. (F) Immunocomplexes precipitated from 75 μg of β3-1, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments.

Mentions: In an attempt to determine genes regulated by the αvβ3 integrin, which contribute to this phenotype in cancer cells, a gene expression analysis was undertaken. As a model system, LNCaP prostate cancer cells were stably transfected with expression vector containing human β3 integrin cDNA, or empty expression vector (mock), or expression vector containing human ICAM-1 cDNA as a transfection control for the effects of ectopically expressing a cell surface protein. Expression of αvβ3 integrin in three different cell populations (β3-1, β3-2, and β3-3), as well as ICAM expression in two different populations, was confirmed by FACS® analysis (Fig. 1 A; unpublished data; Zheng et al., 1999).


Alpha(v)beta3 integrin expression up-regulates cdc2, which modulates cell migration.

Manes T, Zheng DQ, Tognin S, Woodard AS, Marchisio PC, Languino LR - J. Cell Biol. (2003)

cdc2 mRNA, protein, and kinase levels are increased in β3-LNCaP cells. (A) Surface expression of ectopic αvβ3 integrin in LNCaP cell stable transfectants. Mock-, β3-1, β3-2, and β3-3 LNCaP cells were incubated with mAb to αvβ3 integrin (AP-3, thick line) or, as negative control, to the HA epitope (12CA5, thin line) followed by staining with FITC-conjugated goat anti–mouse Ig antibody and analysis using a FACScanTM flow cytometer. (B) cDNA expression array analysis. First strand cDNA probes prepared with either β3-1 or mock-LNCaP mRNA were hybridized to Atlas human cancer cDNA expression arrays. Shown are areas of section A of the cDNA array membrane. The arrow indicates the spots corresponding to cdc2 on the array. The brackets indicate other genes on the array that are not up-regulated in β3-1. (C–F) cdc2 protein and kinase levels are increased in β3-LNCaP cells grown in 2D (C and D) and 3D (E and F) cultures. (C) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 2D culture were analyzed by IB with an antibody to cdc2. In C and E, antibody to ERK-1 was used for protein loading control. (D) Immunocomplexes precipitated from 75 μg of β3-1, β3-3, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments. (E) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 3D Matrigel culture for 48 h were analyzed by IB with an antibody to cdc2. (F) Immunocomplexes precipitated from 75 μg of β3-1, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199360&req=5

fig1: cdc2 mRNA, protein, and kinase levels are increased in β3-LNCaP cells. (A) Surface expression of ectopic αvβ3 integrin in LNCaP cell stable transfectants. Mock-, β3-1, β3-2, and β3-3 LNCaP cells were incubated with mAb to αvβ3 integrin (AP-3, thick line) or, as negative control, to the HA epitope (12CA5, thin line) followed by staining with FITC-conjugated goat anti–mouse Ig antibody and analysis using a FACScanTM flow cytometer. (B) cDNA expression array analysis. First strand cDNA probes prepared with either β3-1 or mock-LNCaP mRNA were hybridized to Atlas human cancer cDNA expression arrays. Shown are areas of section A of the cDNA array membrane. The arrow indicates the spots corresponding to cdc2 on the array. The brackets indicate other genes on the array that are not up-regulated in β3-1. (C–F) cdc2 protein and kinase levels are increased in β3-LNCaP cells grown in 2D (C and D) and 3D (E and F) cultures. (C) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 2D culture were analyzed by IB with an antibody to cdc2. In C and E, antibody to ERK-1 was used for protein loading control. (D) Immunocomplexes precipitated from 75 μg of β3-1, β3-3, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments. (E) cdc2 protein levels in extracts from LNCaP cell transfectants grown in 3D Matrigel culture for 48 h were analyzed by IB with an antibody to cdc2. (F) Immunocomplexes precipitated from 75 μg of β3-1, β3-2, ICAM-, or mock-LNCaP cell extracts using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. Consistent results were obtained in two additional experiments.
Mentions: In an attempt to determine genes regulated by the αvβ3 integrin, which contribute to this phenotype in cancer cells, a gene expression analysis was undertaken. As a model system, LNCaP prostate cancer cells were stably transfected with expression vector containing human β3 integrin cDNA, or empty expression vector (mock), or expression vector containing human ICAM-1 cDNA as a transfection control for the effects of ectopically expressing a cell surface protein. Expression of αvβ3 integrin in three different cell populations (β3-1, β3-2, and β3-3), as well as ICAM expression in two different populations, was confirmed by FACS® analysis (Fig. 1 A; unpublished data; Zheng et al., 1999).

Bottom Line: We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1).Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion.These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
The alphavbeta3 integrin has been shown to promote cell migration through activation of intracellular signaling pathways. We describe here a novel pathway that modulates cell migration and that is activated by alphavbeta3 and, as downstream effector, by cdc2 (cdk1). We report that alphavbeta3 expression in LNCaP (beta3-LNCaP) prostate cancer cells causes increased cdc2 mRNA levels as evaluated by gene expression analysis, and increased cdc2 protein and kinase activity levels. We provide three lines of evidence that increased levels of cdc2 contribute to a motile phenotype on integrin ligands in different cell types. First, increased levels of cdc2 correlate with more motile phenotypes of cancer cells. Second, ectopic expression of cdc2 increases cell migration, whereas expression of dominant-negative cdc2 inhibits migration. Third, cdc2 inhibitors reduce cell migration without affecting cell adhesion. We also show that cdc2 increases cell migration via specific association with cyclin B2, and we unravel a novel pathway of cell motility that involves, downstream of cdc2, caldesmon. cdc2 and caldesmon are shown here to localize in membrane ruffles in motile cells. These results show that cdc2 is a downstream effector of the alphavbeta3 integrin, and that it promotes cell migration.

Show MeSH
Related in: MedlinePlus