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

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.

Figure 1

Β1-integrin expression and adhesive properties of MLP29 and Hep16 cells. (A) Quantitative analysis by flow cytometry revealed that most (90%) of cells were positive for β₁ staining and that the intensity levels (Geo Mean fluorescence) of β₁ expression was higher in MLP29 than in Hep16 cells. The experiment was performed three times with similar results. (B) MTT assay was used to assess MLP29 and Hep16 cell adhesion capacity to different components of the extracellular matrix, including FN, VN, LMN and COL I. To prevent nonspecific cell adhesion, plates were blocked with BSA. Cell adhesion to the different substrates was estimated by measuring the optic density at A570 nm. The data presented summarize the mean (±SD) of three independent experiments, each performed in triplicate.

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.

Figure 3

α5β1 integrin functional blockade induced migration of MLP29 hepatic progenitors. α₅β₁ functional blockade induced morphological modifications and triggered cell motility in MLP29 cells, in a manner comparable to HGF/SF1. Cell migration was calculated as the mean of six different measurements along the scratch and is expressed as percentage of the wound width covered by the migrating cells. The data presented summarize the mean (±SD) of three independent experiments.

Figure 4

Gene expression profiling of MLP29 hepatic progenitor cells after α₅β₁ integrin blockade and HGF/SF stimulation. (A) (B) Plots showing the changes in the expression of gene sets involved in cell adhesion and migration, respectively. Data from three independent experiments were pooled and subjected to permutation analyses to assess the expression level changes of gene sets obtained from the Molecular Signatures Database. The resulting data are presented as color-encoded plots in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation.

Figure 5

Changes in the expression level of genes encoding chromatin remodeling and transcription factors in MLP29 cells during migration. (A) Changes in the expression level of genes encoding chromatin remodeling factors were plotted according to their level of significance as a color-encoded map, in which a p-value close to 1 indicates significant up-regulation, and a p-value close to 0 indicates a significant down-regulation. (B) Changes in the expression level of transcription factors following the treatment with α₅β₁ integrin blocking antibody or with HGF/SF1 for 24 hours.

Figure 6

Change of the nuclear organization of MLP29 hepatic progenitor cells. Quantitative FISH was performed with a pan-centromeric probe. Computational analysis of the spatial organization of chromocenters showed marked variations in their average volume, whereas the nuclear volume remained unchanged in MLP29 cells following α₅β₁ blockade or stimulation with HGF/SF1. 3D computational reconstruction of the image stacks and quantitative analysis showed that the average fluorescence intensity of the Me3H3K9 foci remained unaltered. However, the number of foci increased concomitantly with a reduction of their volume after both types of treatments. Immunofluorescent detection of Ac3K9/14 H3, on the other hand, revealed that cell scattering is associated with an increase in the level of acetylation of histone H3 at K9.

Figure 7

Change of the nuclear organization of MLP29 hepatic progenitor cells. (A) Bar graphs summarize the mean (±S.E.) volume of at least 1000 chromocenters analyzed and the average nuclear volume. (B) Bar graphs summarize the number per nuclei and the mean volume (±S.E.) of at least 500 Me3H3K9 foci analyzed. (C) The level of Ac3K9/14H3 expressed as the mean (±S.E.) of the fluorescence intensity of 100 nuclei analyzed for each treatment.

Figure 8

Radial position of integrin genes during migration of MLP29 cells. Three-dimensional FISH analysis was performed with BAC clones for the Itgb1 or Itgb3 loci and the position of fluorescent signals was determined using computational programs for image analysis. Left panels, 3D reconstruction of MLP29 cell nuclei subjected to FISH analysis with a probe for the Itgb1 and Itgb3 genes. Right panel, absolute radial position of these genes in control and treated cells. Scale bar = 3 μm.