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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 regulates migration of HeLa cells. (A) Expression of cdc2dn or cdc2wt affects migration of HeLa cells. HeLa cells cotransfected with pCMVβgal and pcDNA-3, pCMVcdc2dn, or pCMVcdc2wt were trypsinized at two different time points (24 and 48 h) after transfection and cells were seeded in medium containing 10% FBS on 10 μg/ml FN-coated plates. After 16 h, cells were fixed and stained for βgal activity. The mean and SEM of 10 random fields is shown. (B) Expression of cdc2wt increases cdc2 kinase activity. Immunocomplexes precipitated from 60 μg cell extracts of HeLa cells transfected with pcDNA-3 or cdc2wt using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. (C) Purvalanol A inhibits HeLa cell migration without affecting cell adhesion. Migration assays (top): HeLa cells were incubated for 2 h at 37°C in the presence or absence of purvalanol A and 90,000 HeLa cells were seeded in medium containing purvalanol A at the indicated concentrations on FN-coated transwell insert filters. After 16 h, cells were fixed and stained with crystal violet and the cells on the bottom of the filter were counted. The mean and SEM of 10 random fields is shown. Adhesion assays (bottom): 100,000 HeLa cells grown in the presence or absence of purvalanol A for 2 h were seeded in medium containing purvalanol A at the indicated concentrations in a 96-well plate coated with FN. After 2 h, plates were washed two times with PBS, fixed, and stained with crystal violet. The results show the mean and standard deviation of duplicate observations. Purv A, purvalanol A. (D) PMA increases HeLa cell migration. HeLa cells were allowed to attach for 1 h to filters coated with 10 μg/ml FN and then were incubated in the presence or absence of 100 nM PMA for 3 h and processed as described above. The mean and SEM of 10 random fields is shown. (E) cdc2 is enriched in peripheral areas in motile cells. HeLa cells plated on 10 μg/ml FN-coated coverslips and incubated in the presence of 100 nM PMA were stained with cdc2 mAb (red) and ezrin mAb (green); image-merging analysis of ezrin and cdc2 is shown. (Arrows) Peripheral areas of the cell where ezrin and cdc2 colocalize.
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fig4: cdc2 regulates migration of HeLa cells. (A) Expression of cdc2dn or cdc2wt affects migration of HeLa cells. HeLa cells cotransfected with pCMVβgal and pcDNA-3, pCMVcdc2dn, or pCMVcdc2wt were trypsinized at two different time points (24 and 48 h) after transfection and cells were seeded in medium containing 10% FBS on 10 μg/ml FN-coated plates. After 16 h, cells were fixed and stained for βgal activity. The mean and SEM of 10 random fields is shown. (B) Expression of cdc2wt increases cdc2 kinase activity. Immunocomplexes precipitated from 60 μg cell extracts of HeLa cells transfected with pcDNA-3 or cdc2wt using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. (C) Purvalanol A inhibits HeLa cell migration without affecting cell adhesion. Migration assays (top): HeLa cells were incubated for 2 h at 37°C in the presence or absence of purvalanol A and 90,000 HeLa cells were seeded in medium containing purvalanol A at the indicated concentrations on FN-coated transwell insert filters. After 16 h, cells were fixed and stained with crystal violet and the cells on the bottom of the filter were counted. The mean and SEM of 10 random fields is shown. Adhesion assays (bottom): 100,000 HeLa cells grown in the presence or absence of purvalanol A for 2 h were seeded in medium containing purvalanol A at the indicated concentrations in a 96-well plate coated with FN. After 2 h, plates were washed two times with PBS, fixed, and stained with crystal violet. The results show the mean and standard deviation of duplicate observations. Purv A, purvalanol A. (D) PMA increases HeLa cell migration. HeLa cells were allowed to attach for 1 h to filters coated with 10 μg/ml FN and then were incubated in the presence or absence of 100 nM PMA for 3 h and processed as described above. The mean and SEM of 10 random fields is shown. (E) cdc2 is enriched in peripheral areas in motile cells. HeLa cells plated on 10 μg/ml FN-coated coverslips and incubated in the presence of 100 nM PMA were stained with cdc2 mAb (red) and ezrin mAb (green); image-merging analysis of ezrin and cdc2 is shown. (Arrows) Peripheral areas of the cell where ezrin and cdc2 colocalize.

Mentions: To investigate whether cdc2 regulates migration in cells other than LNCaP, cdc2dn and cdc2wt were ectopically expressed in HeLa cells and their effects on migration and cell cycle were determined over time. At 24 h, there is a modest reduction of HeLa cell migration on FN by cdc2dn and a twofold increase in migration by cdc2wt (Fig. 4 A). Because cdc2 activity is regulated by cyclin levels, we tested whether increased levels of cdc2 would be sufficient to increase kinase levels. Immunoprecipitation of cdc2 from cells transfected with vector alone or cdc2wt determined that ectopic expression of cdc2wt increased cdc2 kinase activity (Fig. 4 B). At 48 h, cdc2dn reduces HeLa cell migration more than threefold, and cdc2wt increases migration modestly (Fig. 4 A); neither adhesion nor cell cycle profile was affected by either cdc2dn or by cdc2wt at these time points (unpublished data). HeLa cells cultured in the presence of micromolar concentrations of purvalanol A for 2 h show a dose-dependent reduction of migration on FN with a negligible effect on adhesion at 8 μM (Fig. 4 C). The results show that expression of cdc2wt and cdc2dn affects HeLa cell migration before a significant effect on cell cycle can be observed and that treatment with cdc2 inhibitors blocks HeLa cell migration.


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 regulates migration of HeLa cells. (A) Expression of cdc2dn or cdc2wt affects migration of HeLa cells. HeLa cells cotransfected with pCMVβgal and pcDNA-3, pCMVcdc2dn, or pCMVcdc2wt were trypsinized at two different time points (24 and 48 h) after transfection and cells were seeded in medium containing 10% FBS on 10 μg/ml FN-coated plates. After 16 h, cells were fixed and stained for βgal activity. The mean and SEM of 10 random fields is shown. (B) Expression of cdc2wt increases cdc2 kinase activity. Immunocomplexes precipitated from 60 μg cell extracts of HeLa cells transfected with pcDNA-3 or cdc2wt using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. (C) Purvalanol A inhibits HeLa cell migration without affecting cell adhesion. Migration assays (top): HeLa cells were incubated for 2 h at 37°C in the presence or absence of purvalanol A and 90,000 HeLa cells were seeded in medium containing purvalanol A at the indicated concentrations on FN-coated transwell insert filters. After 16 h, cells were fixed and stained with crystal violet and the cells on the bottom of the filter were counted. The mean and SEM of 10 random fields is shown. Adhesion assays (bottom): 100,000 HeLa cells grown in the presence or absence of purvalanol A for 2 h were seeded in medium containing purvalanol A at the indicated concentrations in a 96-well plate coated with FN. After 2 h, plates were washed two times with PBS, fixed, and stained with crystal violet. The results show the mean and standard deviation of duplicate observations. Purv A, purvalanol A. (D) PMA increases HeLa cell migration. HeLa cells were allowed to attach for 1 h to filters coated with 10 μg/ml FN and then were incubated in the presence or absence of 100 nM PMA for 3 h and processed as described above. The mean and SEM of 10 random fields is shown. (E) cdc2 is enriched in peripheral areas in motile cells. HeLa cells plated on 10 μg/ml FN-coated coverslips and incubated in the presence of 100 nM PMA were stained with cdc2 mAb (red) and ezrin mAb (green); image-merging analysis of ezrin and cdc2 is shown. (Arrows) Peripheral areas of the cell where ezrin and cdc2 colocalize.
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fig4: cdc2 regulates migration of HeLa cells. (A) Expression of cdc2dn or cdc2wt affects migration of HeLa cells. HeLa cells cotransfected with pCMVβgal and pcDNA-3, pCMVcdc2dn, or pCMVcdc2wt were trypsinized at two different time points (24 and 48 h) after transfection and cells were seeded in medium containing 10% FBS on 10 μg/ml FN-coated plates. After 16 h, cells were fixed and stained for βgal activity. The mean and SEM of 10 random fields is shown. (B) Expression of cdc2wt increases cdc2 kinase activity. Immunocomplexes precipitated from 60 μg cell extracts of HeLa cells transfected with pcDNA-3 or cdc2wt using an mAb to cdc2 were used in a kinase reaction using histone H1 as a substrate. (C) Purvalanol A inhibits HeLa cell migration without affecting cell adhesion. Migration assays (top): HeLa cells were incubated for 2 h at 37°C in the presence or absence of purvalanol A and 90,000 HeLa cells were seeded in medium containing purvalanol A at the indicated concentrations on FN-coated transwell insert filters. After 16 h, cells were fixed and stained with crystal violet and the cells on the bottom of the filter were counted. The mean and SEM of 10 random fields is shown. Adhesion assays (bottom): 100,000 HeLa cells grown in the presence or absence of purvalanol A for 2 h were seeded in medium containing purvalanol A at the indicated concentrations in a 96-well plate coated with FN. After 2 h, plates were washed two times with PBS, fixed, and stained with crystal violet. The results show the mean and standard deviation of duplicate observations. Purv A, purvalanol A. (D) PMA increases HeLa cell migration. HeLa cells were allowed to attach for 1 h to filters coated with 10 μg/ml FN and then were incubated in the presence or absence of 100 nM PMA for 3 h and processed as described above. The mean and SEM of 10 random fields is shown. (E) cdc2 is enriched in peripheral areas in motile cells. HeLa cells plated on 10 μg/ml FN-coated coverslips and incubated in the presence of 100 nM PMA were stained with cdc2 mAb (red) and ezrin mAb (green); image-merging analysis of ezrin and cdc2 is shown. (Arrows) Peripheral areas of the cell where ezrin and cdc2 colocalize.
Mentions: To investigate whether cdc2 regulates migration in cells other than LNCaP, cdc2dn and cdc2wt were ectopically expressed in HeLa cells and their effects on migration and cell cycle were determined over time. At 24 h, there is a modest reduction of HeLa cell migration on FN by cdc2dn and a twofold increase in migration by cdc2wt (Fig. 4 A). Because cdc2 activity is regulated by cyclin levels, we tested whether increased levels of cdc2 would be sufficient to increase kinase levels. Immunoprecipitation of cdc2 from cells transfected with vector alone or cdc2wt determined that ectopic expression of cdc2wt increased cdc2 kinase activity (Fig. 4 B). At 48 h, cdc2dn reduces HeLa cell migration more than threefold, and cdc2wt increases migration modestly (Fig. 4 A); neither adhesion nor cell cycle profile was affected by either cdc2dn or by cdc2wt at these time points (unpublished data). HeLa cells cultured in the presence of micromolar concentrations of purvalanol A for 2 h show a dose-dependent reduction of migration on FN with a negligible effect on adhesion at 8 μM (Fig. 4 C). The results show that expression of cdc2wt and cdc2dn affects HeLa cell migration before a significant effect on cell cycle can be observed and that treatment with cdc2 inhibitors blocks HeLa cell migration.

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