Thomas Jefferson University Jefferson Digital Commons Cardeza Foundation for Hematologic Research Sidney Kimmel Medical College 5-2001 Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal- regulated kinase signaling pathway. Douglas Woods Cancer Research Institute and Department of Cellular and Molecular Pharmacology Holly Cherwinski DNAX Research Institute Eleni Venetsanakos Cancer Research Institute and Department of Cellular and Molecular Pharmacology Arun Bhat FCancerollow thisResear andch additional Institute worksand Depar at: https:/tment/jdc.jeff of Cellularerson.edu/car and Moleculardeza_foundation Pharmacology Stephan Part of Gysin the Hemat ology Commons LetCancer us Resear knowch Institute how and Deparaccesstment of t Cellularo this and document Molecular Pharmacology benefits ouy RecommendedSee next page for Citation additional authors Woods, Douglas; Cherwinski, Holly; Venetsanakos, Eleni; Bhat, Arun; Gysin, Stephan; Humbert, Martine; Bray, Paul F.; Saylor, Vicki L.; and McMahon, Martin, "Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway." (2001). Cardeza Foundation for Hematologic Research. Paper 24. https://jdc.jefferson.edu/cardeza_foundation/24 This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in Cardeza Foundation for Hematologic Research by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: [email protected]. Authors Douglas Woods, Holly Cherwinski, Eleni Venetsanakos, Arun Bhat, Stephan Gysin, Martine Humbert, Paul F. Bray, Vicki L. Saylor, and Martin McMahon This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/cardeza_foundation/24 MOLECULAR AND CELLULAR BIOLOGY, May 2001, p. 3192–3205 Vol. 21, No. 9 0270-7306/01/$04.00ϩ0 DOI: 10.1128/MCB.21.9.3192–3205.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Induction of ␤3-Integrin Gene Expression by Sustained Activation of the Ras-Regulated Raf–MEK–Extracellular Signal-Regulated Kinase Signaling Pathway DOUGLAS WOODS,1† HOLLY CHERWINSKI,2 ELENI VENETSANAKOS,1 ARUN BHAT,1 STEPHAN GYSIN,1 MARTINE HUMBERT,2 PAUL F. BRAY,3 1 1 VICKI L. SAYLOR AND MARTIN MCMAHON * Downloaded from Cancer Research Institute and Department of Cellular and Molecular Pharmacology, San Francisco, California 941151; Department of Cell Signaling, DNAX Research Institute, Palo Alto, California 943042; and Section of Thrombosis, Baylor College of Medicine, Houston, Texas 770303 Received 30 October 2000/Returned for modification 13 December 2000/Accepted 7 February 2001 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 http://mcb.asm.org/ 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 ␣6- and ␤3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led toa5to20-fold induction of cell surface ␣6- and ␤3-integrin expression. Induced ␤3-integrin was expressed on the cell surface as a heterodimer with ␣v-integrin; however, the overall level of ␣v-integrin expression was not altered by Ras or Raf. Raf- induced ␤3-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 ␤3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells on February 4, 2015 by Thomas Jefferson Univ led to elevated ␤3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, ␤3-integrin mRNA was induced by Raf in a manner that was cyclohex- imide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on ␤3-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 ␣v␤3-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. Adhesion of cells to extracellular matrix (ECM) is mediated transduction events that participate in the control of cell cycle by a family of transmembrane proteins known as integrins that progression and apoptosis in a process known as “outside-in” are expressed on the cell surface as ␣/␤-heterodimers (21, 30, signaling. For example, integrin engagement can elicit activa- 40). Different combinations of ␣ and ␤ subunits give rise to a tion of members of the Src and FAK family of protein tyrosine multiplicity of ECM receptors, the expression of which shows kinases (26, 49). These initial signaling events promote the considerable cell type specificity (40). Moreover, the intracel- activation of Ras and Rho family GTPases that in turn influ- lular regions of integrin subunits are believed to mediate the ence the activation of a number of intracellular signaling path- assembly of components of the focal adhesion complex, which ways (16, 47). A second mode of integrin regulation known as in turn participate in marshalling the actin cytoskeleton (14, 21, “inside-out” signaling has also been described. For example, 72). Regulation of integrin function is believed to be essential the Ras-activated Raf–MEK–extracellular signal-regulated ki- in promoting stable cell adhesion as well as being required for nase (ERK) signaling pathway can influence the activation cell migration. state of the ␣IIb␤3 (also known as gpIIb/IIIa) integrin as In addition to their role in cell adhesion and migration, measured by the binding of a monoclonal antibody that rec- engagement and clustering of integrins elicits a series of signal ognizes the activated form of this integrin. Interestingly, the mechanism of alteration of the ␣IIb␤3 integrin affinity state is * Corresponding author. Mailing address: Cancer Research Insti- independent of de novo RNA and protein synthesis and may tute and Department of Cellular and Molecular Pharmacology, UCSF/ be due to the direct modification of preexisting integrin sub- Mt. Zion Comprehensive Cancer Center, 2340 Sutter St., Box 0128, units on the surface of the cell (39). San Francisco, CA 94115. Phone: (415) 502 5829. Fax: (415) 502 3179. In addition to their role in normal cell physiology, there is an E-mail: [email protected]. † Present address: NCI-Frederick Cancer Research and Develop- extensive body of literature indicating that alterations in the ment Center, National Cancer Institute, Frederick, MD 21702. expression of specific integrin subunits on the surface of cancer 3192 VOL. 21, 2001 REGULATION OF ␤3-INTEGRIN EXPRESSION BY Ras AND Raf 3193 cells contributes to the invasive and metastatic properties of to 4-hydroxytamoxifen (4-HT) and ICI compounds but not to 17-␤-estradiol or ⌬ the cells (43, 67, 79). For example, reduced expression of phenol red as described previously (51). DNA sequences encoding Raf:ER* ␣ ␤ ␣ ␤ and ⌬MEK1:ER* were inserted into the replication-defective retrovirus vector 5 1-integrin in K-562, CHO, and HT-29 cells and of 2 1in pBabepuro3 (pBP3) for expression in mammalian cells (60). Retrovirus con- breast cancer cells correlates with increased tumorigenicity. structs expressing EGFP⌬Raf-1:ER, c-Myc:ERTM, and Akt:ER* have been de- Moreover, in certain circumstances, elevated expression of ␣6-, scribed previously (48, 51, 88). Retrovirus vectors (pZAS4) encoding v-Myc or ␣3- or ␤3-integrins appears to be closely associated with on- v-Ha-Ras and resistance to mycophenolic acid were provided by J. Kaplan (45). cogenic transformation and tumor progression. Indeed, there Additional details of retrovirus expression vectors are available on request. Cell culture, virus production, and virus infection. Cells were cultured at 37°C is strong evidence that the expression of ␣v␤3-integrin is tightly in a humidified atmosphere containing 6% (vol/vol) CO2, in phenol red-free correlated with the acquisition of invasive and/or metastatic Dulbecco’s modified Eagle’s medium supplemented with 10% (vol/vol)