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Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells.

Vellón L, Royo F, Matthiesen R, Torres-Fuenzalida J, Lorenti A, Parada LA - BMC Cell Biol. (2010)

Bottom Line: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration.These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus.Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cytogenomics, CIC bioGUNE-CIBEREHD, Par, Tec, Bizkaia Ed, 801 A, 48160 - Derio, Spain.

ABSTRACT

Background: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells.

Results: Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus.

Conclusion: Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

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α5β1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells. (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α5β1-functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α5β1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α5β1, which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α5β1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.
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Figure 2: α5β1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells. (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α5β1-functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α5β1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α5β1, which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α5β1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.

Mentions: Considering that integrin α5β1 is one of the main ECM receptors of hepatocytes, we wondered whether specific disruption of the α5β1-mediated cell-ECM interactions would trigger invasive-like cell growth. In order to substantiate our hypothesis, MLP29 cell cultures were treated with a specific antibody against α5β1 integrin and HGF/SF1 as control, owing to its capacity to induce cell scattering and migration. By microscopic inspection, we observed that untreated MLP29 cells grow in tightly packed patches, mimicking the structure of an epithelial sheet, whereas blocking α5β1 caused the loss of cell-cell junctions and induced cell spreading throughout the culture dish, similar to the cell scattering induced by HGF/SF1. These morphological changes involved reorganization of the cell cytoskeleton, from cortical F-actin structures to parallel bundles disposed along the axis of the cellular prolongations (Figure 2A). Moreover, immunoblot and immunofuorescent detection showed that functional blockade of α5β1 integrin induced down-regulation of E-cadherin, demonstrating that effectively the process involved cell-cell dissociation (Figure 2A).


Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells.

Vellón L, Royo F, Matthiesen R, Torres-Fuenzalida J, Lorenti A, Parada LA - BMC Cell Biol. (2010)

α5β1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells. (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α5β1-functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α5β1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α5β1, which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α5β1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2967514&req=5

Figure 2: α5β1 integrin functional block induced spreading and scattering of MLP29 hepatic progenitor cells. (A) Immunofluorescent detection of F-actin in MLP29 cells showed that the cells grow in packed islands, while after the treatment with a specific α5β1-functional blocking antibody the cells undergo spreading and scattering. Higher magnification (252×) of untreated and treated MLP29 cells shows the characteristic actin microfilaments reorientation associated with cytoskeleton re-organization. Immunofluorescent and immunoblot detection of E-cadherin in MLP29 cells following α5β1 functional blockade showed that the expression levels of E-cadherin decreased at the cell-cell junctions in cells treated with the specific functional blocking antibody against α5β1, which was corroborated by immunoblot analysis. (B) Representative images of the phospho-MAPKs arrays. Activation of the different members of the MAPKs family was identified by means of a key provided with the kit. MLP29 cells were treated with the α5β1 functional blocking antibody or HGF/SF1, in the presence or absence of the specific MEK inhibitor U0126. Cells were lysed and total protein (20 μg) was resolved by SDS-PAGE and analyzed by immunoblot for ERK1/ERK2 MAPK and phospho-ERK1/ERK2 MAPKs. Blots were then reprobed with an antibody for β-actin as a control for protein loading. Results are representative of at least three independent experiments.
Mentions: Considering that integrin α5β1 is one of the main ECM receptors of hepatocytes, we wondered whether specific disruption of the α5β1-mediated cell-ECM interactions would trigger invasive-like cell growth. In order to substantiate our hypothesis, MLP29 cell cultures were treated with a specific antibody against α5β1 integrin and HGF/SF1 as control, owing to its capacity to induce cell scattering and migration. By microscopic inspection, we observed that untreated MLP29 cells grow in tightly packed patches, mimicking the structure of an epithelial sheet, whereas blocking α5β1 caused the loss of cell-cell junctions and induced cell spreading throughout the culture dish, similar to the cell scattering induced by HGF/SF1. These morphological changes involved reorganization of the cell cytoskeleton, from cortical F-actin structures to parallel bundles disposed along the axis of the cellular prolongations (Figure 2A). Moreover, immunoblot and immunofuorescent detection showed that functional blockade of α5β1 integrin induced down-regulation of E-cadherin, demonstrating that effectively the process involved cell-cell dissociation (Figure 2A).

Bottom Line: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration.These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus.Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cytogenomics, CIC bioGUNE-CIBEREHD, Par, Tec, Bizkaia Ed, 801 A, 48160 - Derio, Spain.

ABSTRACT

Background: Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells.

Results: Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus.

Conclusion: Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape.

Show MeSH
Related in: MedlinePlus