alpha1 and alpha2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression - PubMed (original) (raw)

alpha1 and alpha2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression

A Lochter et al. Mol Biol Cell. 1999 Feb.

Free PMC article

Abstract

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits alpha6 and beta1, but not against alpha1 and alpha2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against beta1, but not against alpha6 or alpha2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against alpha1 integrins impaired only cell adhesion to type IV collagen. Antibodies against alpha1, alpha2, alpha6, and beta1, but not alpha5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins alpha1 and alpha2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against alpha1 and alpha2, but not alpha6 and beta1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against alpha1 and alpha2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-alpha6 antibodies. Our data indicate that alpha1 and alpha2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas alpha6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

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Figures

Figure 1

Integrin expression by SCg6 cells. Autoradiogram of nonreducing SDS-polyacrylamide gels run on immunoprecipitations performed with antibodies against α1, α2, α5, α6, αv, and β1 integrin subunits on radiolabeled cell lysates of SCg6 cells is shown. Note that, except for αv, all integrin subunits examined could be detected in SCg6 cells and that all antibodies coimmunoprecipitated a band corresponding to the expected molecular mass (130 kDa) of the β1 integrin subunit (◂). Positions (←) of integrin subunits α1 at 180 kDa, α2 at 150 kDa, α5 at 160 kDa, and α6 at 150 kDa are indicated.

Figure 2

Effects of antibodies against α1, α2, α6, and β1 integrin subunits on the invasion of rBM by SCg6 cells. Invasion assays were performed for 24 h in modified Boyden chambers coated with rBM. SCg6 cells were then fixed and stained, and the number of cells that had migrated through rBM was counted by microscopic inspection. Invasion assays were performed in the absence of antibodies (control [C]), in the presence of antibodies against α1, α2, α5, α6, αv, or β1 integrin subunits, or in the presence of rat (r) or hamster (h) IgGs. Results are normalized with the number of cells migrating through rBM in the absence of antibodies set to 100. Means ± SD from three independent experiments are shown. In the absence of IgGs, 169.5 ± 8.89 cells/mm2 migrated through rBM.

Figure 3

Cell number and morphologies of SCg6 cells maintained on rBM gels in the presence of antibodies against integrins. (A) SCg6 cells were maintained for 2 d on rBM, and the number of viable cells was determined by the MTT method. Means ± SD from three independent experiments are expressed as the absorbance (ABS) measured at 570 nm. Note that only minor differences in the number of viable cells were observed for the different antibody treatments. w/o, without antibody. (B–F) Micrographs of toluidine blue-stained SCg6 cells maintained for 2 d on rBM in the absence of antibodies (B) or in the presence of antibodies against α1 (C), α2 (D), α6 (E), and β1 (F) integrins are shown. Bar, 100 μm.

Figure 4

Effect of integrin antibodies on adhesion of SCg6 cells to ECM constituents. Adhesion assays on substrata coated with laminin (A), type IV collagen (B), fibronectin (C), or type I collagen (D) were performed for 1 h in the presence of increasing concentrations of antibodies against α1 (▵), α2 (•), α6 (▪), or β1 (○) integrin subunits. Results are normalized with the number of cells adhering to ECM substrata in the absence of antibodies set to 100. Means ± SD from three independent experiments are shown.

Figure 5

Effects of antibodies against α6 and β1 integrins on SCg6 cell migration along 2D rBM and laminin substrata. Cell migration assays were performed for 6 h in modified Boyden chambers coated with rBM (A), laminin (B), or fibronectin (C). SCg6 cells were then fixed and stained, and the number of cells that had migrated along the ECM was counted by microscopic inspection. Cell migration assays were performed in the absence of antibodies (control [C]), in the presence of antibodies against α1, α2, α5, α6, αv, or β1 integrin subunits, or in the presence of rat (r) or hamster (h) IgGs. Results are normalized with the number of cells that had migrated along the ECM in the absence of antibodies set to 100. Means ± SD from three independent experiments are shown. In the absence of IgGs, the number of cells migrating per square millimeter was 60.1 ± 7.55 on 2D rBM, 90.3 ± 12.25 on laminin, and 102.9 ± 13.27 on fibronectin.

Figure 6

Effect of antibodies against integrins on the production of ECM-degrading proteinases. Medium conditioned by SCg6 cells maintained for 2 d on rBM in the absence of antibodies (w/o) or in the presence of antibodies against α1, α2, α6, or β1 integrin subunits was analyzed by gelatin and casein substrate gel zymography. Negative images of zymograms are shown. Molecular masses of major gelatinolytic and caseinolytic bands are indicated. Left, gelatinases of 72, 66, and 62 kDa represent gelatinase A, and the 102-kDa gelatinase represents gelatinase B. Right, the 57-kDa caseinase represents stromelysin-1, and the 80-kDa caseinase represents an unidentified metalloproteinase secreted by SCg6 cells (Lochter et al., 1997b).

Figure 7

Effect of recombinant stromelysin-1 (SL-1) on invasion and migration of SCg6 cells. (A and B) Invasion assays were performed for 24 h in modified Boyden chambers coated with rBM gels. SCg6 cells were then fixed and stained, and the number of cells that had migrated through rBM was counted by microscopic inspection. Invasion assays were performed in the absence of antibodies (control [C]) or in the presence of antibodies against α1, α2, α6, or β1 integrin subunits with (+ SL-1, black bars) or without (− SL-1, white bars) the addition of recombinant stromelysin-1 to the culture medium. Results are normalized with the num-ber of cells migrating through rBM in the absence of antibodies and stromelysin-1 set to 100. Means ± SD from three independent experiments are shown. In the absence of antibodies, 183.2 ± 16.21 cells/mm2 migrated through rBM. (C) Two-dimensional cell migration assays were performed for 6 h in modified Boyden chambers coated with rBM. SCg6 cells were then fixed and stained, and the number of cells that had migrated along rBM was counted by microscopic inspection. Cell migration assays were performed in the absence of additives (control) or in the presence of recombinant stromelysin-1, the matrix metalloproteinase inhibitor GM6001, or its inactive homologue GM1210. Results are normalized with the number of cells that had migrated along rBM in the absence of additives set to 100. Means ± SD from three independent experiments are shown. Under control conditions, 72.8 ± 9.16 cells/mm2 migrated along rBM.

Figure 8

Effect of antibodies against α1 and α2 integrins on stromelysin-1 promoter activity in SCg6 cells. Luciferase (A, C, and D) and β-galactosidase (B) activity of SCg6 cells cotransfected with SL-1 promoter–luciferase and RSV promoter–β-galactosidase constructs was analyzed 2 d after maintenance of cells on rBM gels (A and B), on tissue culture plastic (C), or in suspension culture (poly-HEMA, D). Cells were cultured in the presence of increasing concentrations of antibodies against α1 (▵), α2 (•), α6 (▪), or β1 (○) integrin subunits (A and C) or in the presence of 10 μg/ml antibodies (B and D). Results are normalized with values obtained for cells maintained in the absence of antibodies (control [C] in B and D) set to 100. Means ± SD from three independent experiments are shown. In the absence of antibodies, the average number of relative light units obtained per well was 22371 on rBM, 6580 on plastic, and 20689 on poly-HEMA for the stromelysin-1 promoter and 9618 on rBM for the RSV promoter.

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alpha1 and alpha2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression - PubMed (original) (raw)