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. 2001 May;21(9):3192-205.
doi: 10.1128/MCB.21.9.3192-3205.2001.

Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway

D Woods  1 , H Cherwinski, E Venetsanakos, A Bhat, S Gysin, M Humbert, P F Bray, V L Saylor, M McMahon
Affiliations

Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway

D Woods et al. Mol Cell Biol. 2001 May.

Abstract

Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of alpha6- and beta3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface alpha6- and beta3-integrin expression. Induced beta3-integrin was expressed on the cell surface as a heterodimer with alphav-integrin; however, the overall level of alphav-integrin expression was not altered by Ras or Raf. Raf-induced beta3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce beta3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated beta3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, beta3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on beta3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface alphavbeta3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

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Figures

FIG. 1
FIG. 1
Expression of β3- and α6-integrins in human cancer cell lines. CFPAC pancreatic cancer cells (A to D) or WM793 melanoma cells (E to H) were treated with either UO126, SB203,580, or DMSO as a solvent control for 48 h as indicated and then stained for the cell surface expression of β3- or α6-integrins by flow cytometry as described in Materials and Methods.
FIG. 2
FIG. 2
Expression of integrin subunits following Raf activation in NIH 3T3 cells. NIH 3T3 cells expressing ΔB-Raf:ER* were either untreated (dotted line) or treated with 100 nM 4-HT for 24 h (solid line) at which time the cells were harvested and either left unstained or stained for the cell surface expression of particular α- and β-integrin subunits as indicated. The expression of integrin subunits was detected by flow cytometry as described in Materials and Methods.
FIG. 3
FIG. 3
Induction of β3-integrin by Raf and MEK1. NIH 3T3 cells expressing ΔA-Raf:ER*, ΔRaf-1:ER*, ΔB-Raf:ER*, or ΔMEK1:ER* were either left untreated or treated with 100 nM 4-HT for 48 h, at which time the cells were harvested and stained for the cell surface expression of β3-integrin as described in Materials and Methods. The expression of β3-integrin subunits was detected by flow cytometry.
FIG. 4
FIG. 4
Induction of β3-integrin by Ras. (A) NIH 3T3 cells were infected with retroviruses encoding either v-Myc or v-Ha-Ras, and virus-infected cells were selected over 14 days in mycophenolic acid as described in Materials and Methods. Parental (dotted line), v-Myc-expressing (solid line), and v-Ha-Ras-expressing (filled curve), cells were stained for the cell surface expression of β3-integrin. (B) Human microvascular endothelial cells immortalized by the expression of the catalytic subunit of telomerase and expressing EGFPΔRaf-1:ER were derived by the use of the appropriate amphotropic retroviruses. Cells were either left untreated (thin line) or treated with 1 μM 4-HT (thick line), at which time they were stained either with an isotype control (dotted line) or with an antiserum that recognizes human β3-integrin.
FIG. 5
FIG. 5
Elevated αvβ3-integrin expression on the surface of Raf-transformed cells. NIH 3T3 cells expressing ΔA-Raf:ER* (A), ΔB-Raf:ER* (B), ΔRaf-1:ER* (C), or ΔMEK1:ER* (D) were either left untreated (0 h) or treated with 100nM 4-HT for 4, 8, 24, or 48 h as indicated. Cells were then subjected to biotinylation using the membrane-impermeant reagent NHS-LC-biotin. Cell extracts were prepared, and β3-integrin and its associated proteins were immunoprecipitated using a polyclonal anti-β3-integrin antiserum. Immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots were prepared. Biotinylated proteins in the immunoprecipitates were detected by probing the Western blots with streptavidin-horseradish peroxidase.
FIG. 6
FIG. 6
Delayed-early induction of β3-integrin mRNA by Raf. NIH 3T3 cells expressing ΔB-Raf:ER* in the absence or presence of FCS were either left untreated (−CHX) or treated with 10 μg of cycloheximide per ml (+CHX) for 1 h. At this time, the cells were either left untreated (0 h) or treated with 1 μM 4-HT for 3, 5, or 7 h as indicated. At this time, total cellular RNA was prepared and probed for the expression of β3-integrin (A), HB-EGF (B), and GAPDH (C) mRNAs using a simultaneous RPA as described in Materials and Methods.
FIG. 7
FIG. 7
Induction of β3-integrin by sustained ERK MAP kinase activation. (A to C) NIH 3T3 cells expressing EGFPΔRaf-1:ER were cultured in serum-free medium for 36 h prior to the addition of 10 ng of EGF per ml, 20% (vol/vol) FCS, 1 μM 4-HT, 0.1% (vol/vol) ethanol (EtOH), 50 ng of phorbol esters per ml (PMA), or 10 ng of PDGF per ml for 12 or 24 h. Cell extracts were prepared and probed for the expression of β3-integrin (A), the activation of the ERK MAP kinases (B), and the overall expression of the ERK MAP kinases as a loading control (C) using the appropriate antisera as described in Materials and Methods. (D) NIH 3T3 cells expressing the NGF receptor (65) were either left untreated or treated with 10 ng of NGF per ml for 24 or 48 h, at which time the expression of β3-integrin and p21Cip1 was assessed by Western blotting with the appropriate antisera.
FIG. 7
FIG. 7
Induction of β3-integrin by sustained ERK MAP kinase activation. (A to C) NIH 3T3 cells expressing EGFPΔRaf-1:ER were cultured in serum-free medium for 36 h prior to the addition of 10 ng of EGF per ml, 20% (vol/vol) FCS, 1 μM 4-HT, 0.1% (vol/vol) ethanol (EtOH), 50 ng of phorbol esters per ml (PMA), or 10 ng of PDGF per ml for 12 or 24 h. Cell extracts were prepared and probed for the expression of β3-integrin (A), the activation of the ERK MAP kinases (B), and the overall expression of the ERK MAP kinases as a loading control (C) using the appropriate antisera as described in Materials and Methods. (D) NIH 3T3 cells expressing the NGF receptor (65) were either left untreated or treated with 10 ng of NGF per ml for 24 or 48 h, at which time the expression of β3-integrin and p21Cip1 was assessed by Western blotting with the appropriate antisera.
FIG. 8
FIG. 8
Raf-transformed fibroblasts display alterations in intracellular architecture and cell morphology. (A and B) NIH 3T3 cells expressing ΔB-Raf:ER* were either left untreated (A) or treated with 1 μM 4-HT for 24 h (B), at which time they were photographed using a Polaroid charge-coupled device camera. Total magnification, ca. ×80. (C and D) NIH 3T3 cells expressing ΔB-Raf:ER* were plated on vitronectin-coated glass microscope slides and either left untreated (C) or treated with 1 μM 4-HT for 24 h (D). At this time, the cells were fixed and costained with phalloidin-FITC to visualize polymerized actin (green) and antivinculin antisera to visualize focal adhesions (red). Areas of colocalization are false colored in yellow. Total magnification, ca. ×400.
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