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, 287 (29), 24103-12

Specific β-Containing Integrins Exert Differential Control on Proliferation and Two-Dimensional Collective Cell Migration in Mammary Epithelial Cells

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Specific β-Containing Integrins Exert Differential Control on Proliferation and Two-Dimensional Collective Cell Migration in Mammary Epithelial Cells

Alexa I Jeanes et al. J Biol Chem.

Abstract

Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration.

Figures

FIGURE 1.
FIGURE 1.
β1-Integrin-null MECs display a proliferation block. a, genomic DNA was isolated from control and 4OHT-treated primary MECs over a time course of 24 h. PCR analysis was carried out to show the Cre-mediated recombination on genomic DNA and deletion of the β1-integrin gene. The 2.1-kb product is the full-length floxed allele, and the 1.3-kb product is the recombined allele. b, control (C) and β1-integrin-null (4OHT-treated at time of isolation) primary β1fx/fx;CreERTM MECs were fixed and stained for β1-integrin (red) and Cre-recombinase (green) to show loss of β1-integrin and nuclear localization of Cre-recombinase. Bar: 20 μm. c, immunoblotting confirmed β1-integrin deletion in 4OHT-treated MECs. D2 and D3, day 2 and day 3. d and e, untreated control (C) and 4OHT-treated β1fx/fx;CreERTM MECs were incubated with EdU 2, 3, or 4 days after isolation, fixed, and stained using EdU-Click reaction buffer and β1-integrin antibody. d, proliferation was quantified by counting the percentage of EdU-positive nuclei when compared with total number of cells. ∼1000 cells were counted per condition. The error bars are ± S.E. **, p = 0.04; ***, p = 0.003. e, representative images of day 4 samples. Bar: 38 μm. f, control and 4OHT-treated MECs from CreERTM-only mice were analyzed for EdU incorporation 2, 3, and 4 days after isolation. g and h, cells as in d were labeled with EdU 2 days after isolation and stained for EdU, β1-integrin, and phospho-histone H3. g, the graph shows the average of two independent experiments. **, p = 0.04; ***, p = 0.003. h, representative images. Arrows show phospho-histone H3 (pH3) staining in the EdU-labeled cells. Note that the sensitivity of the phospho-histone H3 stain was less than EdU, but the relative reduction in proliferation after integrin deletion was the same. Bar: 38 μm. i and j, FSK-7 cells were transfected with pshβ1 or control pLVTHM (LV) and replated for proliferation analysis with EdU. i, percentage of EdU incorporation in the transfected (GFP-positive) and nontransfected (GFP-negative) cells within the same dishes. Note that proliferation is only suppressed in the cells transfected with pshβ1. j, parallel culture with the transfected cells sorted by FACS and lysates immunoblotted to show β1-integrin knockdown.
FIGURE 2.
FIGURE 2.
β1-Integrin-null MECs contain functional β3-containing integrins. a, β1fx/fx;CreERTM MECs treated with 4OHT or without (C) were cultured on collagen-I coated coverslips, and the cell morphology was observed by phase microscopy after 3 days. Bar: 50 μm. b, similar cultures were stained for β1-integrin (green), and actin or tubulin (red). Bar: 20 μm. c, untreated control (C) and 4OHT-treated β1fx/fx;CreERTM MECs were used for an adhesion assay in serum-free medium on collagen I or FN, in the presence of β1-integrin and β3-integrin function blocking antibodies (10 μg/ml). The error bars represent S.D. of triplicate samples within a representative experiment (n = 3). No a/b, no antibodies, d, single cell suspensions of untreated control (C) and β1-integrin-deleted (4OHT) MECs were labeled with control or Alexa Fluor 488-conjugated anti-β3-integrin antibodies and analyzed by FACS. β3-Integrin was expressed on the surface of MECs, and its levels were similar following β1-integrin gene deletion. control ab, control antibody. e, β1-integrin and β3-integrin RNA levels were compared in control and 4OHT-treated β1fx/fx;CreERTM MECs 3 days after isolation from mice, by quantitative PCR. **, p < 0.01.
FIGURE 3.
FIGURE 3.
β1-Integrin-null MECs contain functional adhesomes and show collective two-dimensional cell migration. a, β1fx/fx;CreERTM MECs treated with 4OHT or untreated controls (C) were cultured on collagen-I coated coverslips, fixed 3 days after isolation, and stained for β1-integrin (red) and talin, vinculin, or Fak (green). Bar: 20 μm. b, immunoblotting of lysates from untreated and β1-integrin-deleted (4OHT) MECs cultured for 3 days and probed for the indicated antigens. Mitochondrial Hsp70 (mtHsp70) was used a loading control. The intensity of the bands was quantified using the Odyssey system, and the level of signal in the 4OHT-treated samples is plotted relative to untreated. Error bars = S.E. c, immunoblotting of lysates from CreERTM-only MECs replated onto collagen I. d, immunofluorescence staining of paxillin-Tyr(P)-31 (p-pax) in control and 4OHT-treated MECs. Bar: 20 μm. e, β1-null MECs stained with β1- and β3-integrin antibodies. Bar: 20 μm. f, control (C) and β1-null (4OHT) acini were plated onto two-dimensional collagen I-coated plates. The acini were allowed to attach, and the cells migrated onto the culture surface. At the end of the experiment, the cells were stained for EdU and β1-integrin. Note the absence of EdU incorporation in the β1-null cells. Asterisks indicate location of acini from which the cells emigrated. White dotted line indicates migration front. Bar: 40 μm. g, acini were treated with 10 μm mitomycin C for 30 min and prior to cell migration onto the dishes. The area spread was calculated using ImageJ and was not significantly different (ns) in the controls (C) and β1-integrin-null (4OHT) MECs. h, MECs as in g were imaged by time-lapse cinematography, and the tracks of individual cells were followed using MTrackJ. i, the average speed (μm/min) and directional persistence of cell movements shown in h. **, p < 0.01.
FIGURE 4.
FIGURE 4.
β1-Integrin is required for nuclear translocation of pErk. a, WT MECs were isolated and cultured for 24 h and then treated with Mek inhibitor U0126 for 24 h before assessing the percentage of EdU-positive cells. The equivalent volume of DMSO was used as a control. b and c, cell lysates were harvested from untreated and 4OHT-treated β1fx/fx;CreERTM MECs that had been cultured in steady-state conditions with serum, 2 and 3 days after isolation from mice. b, they were analyzed by immunoblotting for β1-integrin, pErk, and total Erk. c, untreated and 4OHT-treated β1fx/fx;CreERTM MECs were serum-starved for 12 h and subsequently stimulated with full medium for 30 min before analyzing the protein levels of β1-integrin, pErk, total Erk, and pElk1. d and e, control (C) and β1-integrin-deleted (4OHT) MECs were cultured on collagen-coated coverslips, treated as in c, and immunostained for pErk. d, the percentage of nuclear Erk was quantified. Error bars = S.E. SS + S, serum-starved plus serum. e, representative images of cells in serum for 2 days, stained for β1-integrin (red) and pErk (green). The white arrow highlights the localization of pErk inside the nucleus of control cells (C) and outside the nucleus of β1-integrin-null (4OHT) cells. Scale bar: 30 μm.
FIGURE 5.
FIGURE 5.
Rac1 links β1-integrins with proliferation in MECs. a and b, control and 4OHT-treated β1fx/fx;CreERTM MECs were analyzed for Rac activity. Levels of β1-integrin and total Rac were assessed in the same lysates to confirm β1-integrin knockdown and correct loading. a, immunoblots. b, band quantification using the LI-COR Odyssey system. c, lysates of day 2 untreated (−) and β1-integrin-null (+4OHT) MECs were assessed by immunoblotting for β1-integrin, total Rac, and phospho-Pak1. Calnexin was used as a loading control. d, WT MECs were cultured for 24 h and then treated for 20 h with Rac inhibitor NSC23766 before assessing the percentage of EdU-positive cells. The equivalent volume of DMSO was used as a control. Error bars = S.E. e, WT MECs were cultured for 2 days, serum-starved for 12 h, treated with the Mek (24 h) or Rac (20 h) inhibitor, and then treated with serum for 1 h. Lysates were immunoblotted for pErk and total Erk. f, WT MECs were cultured with Rac inhibitor as in e, immunostained for pErk, and assessed for the presence of nuclear pErk. SS + serum, serum-starved plus serum. g and h, FSK7 cells were transfected with the empty pVenus vector (pV), pshβ1, or pshβ1-Rac and then replated onto glass coverslips for 24 h. g, lysates showing expression of Rac-GFP. IB, immunoblot. h, immunostain to show simultaneous loss of β1-integrin and expression of GFP in the transfected cells. i and j, cells as in g were immunostained for phospho-histone H3 (pH3). i, graph showing the percentage of phospho-histone H3-positive cells in nontransfected and transfected cells. **, p = 0.02. j, representative images. Green arrows indicate GFP-positive cells. White arrowheads point to cells in S-phase. Note that in the shβ1 cultures, the transfected cells (green) are phospho-histone H3-negative, whereas the neighboring untransfected cells (red) are phospho-histone H3-positive. In contrast, shβ1-Rac transfected cells were both β1-negative and phospho-histone H3-positive. Bar: 20 μm.

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