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Insulin-like Growth Factor-1 Regulates Endogenous RUNX2 Activity in Endothelial Cells through a Phosphatidylinositol 3-Kinase/ERK-dependent and Akt-independent Signaling Pathway*

  • Meng Qiao
    Affiliations
    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
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  • Paul Shapiro
    Affiliations
    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201
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  • Rakesh Kumar
    Affiliations
    Department of Cellular and Molecular Oncology, M. D. Anderson Cancer Center, Houston, Texas 77030, and
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  • Antonino Passaniti
    Correspondence
    To whom correspondence should be addressed: The University of Maryland School of Medicine, Greenebaum Cancer Center, Bressler Research Bldg. 7-021, 655 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-328-5470; Fax: 410-328-6559;
    Affiliations
    Departments of Pathology and Biochemistry & Molecular Biology, Greenebaum Cancer Center Program in Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Author Footnotes
    * This work was supported in part by American Heart Association Grant 0151434U (to A. P.) and National Institutes of Health Grants CA95350 (to A. P.) and CA90970 (to R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:August 09, 2004DOI:https://doi.org/10.1074/jbc.M404480200
      Insulin-like growth factor-1 (IGF-1) is an angiogenic and oncogenic factor that activates signal transduction pathways involved in the expression of transcriptional regulators of tumorigenesis. RUNX2, a member of the Ig-loop family of transcription factors is expressed in vascular endothelial cells (EC) and regulates EC migration, invasion, and proliferation. Here we show that IGF-1 and its receptor regulate post-translational changes in RUNX2 to activate DNA binding in proliferating EC. The phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, reduced both basal and IGF-1-stimulated RUNX2 DNA binding activity in the absence of changes in RUNX2 protein as did the overexpression of the phosphatidylinositol 3-phosphate phosphatase, confirming that PI3K signaling mediates RUNX2 activation. IGF-1 increased ERK1/2 activation, which was abrogated by the inhibition of PI3K, thus linking these two pathways in EC. Treatment with U0126, which inhibits ERK1/2 activation, reduced IGF-1-stimulated RUNX2 DNA binding without affecting RUNX2 protein levels. Overexpression of constitutively active MKK1 increased RUNX2 DNA binding and phosphorylation. No additive effects of PI3K or ERK inhibitors on DNA binding were evident. Surprisingly, these IGF-1-mediated effects on RUNX2 were not regulated by Akt phosphorylation, a common downstream target of PI3K, as determined by pharmacological or genetic inhibition. However, an inhibitor of the p21-activated protein kinase-1, glutathione S-transferase-Pak1-(83–149), inhibited both basal and IGF-1-stimulated RUNX2 DNA binding, suggesting that Pak1 mediates IGF-1 signaling to increase RUNX2 activity. These results indicate that the angiogenic growth factor, IGF-1, can regulate RUNX2 DNA binding through sequential activation of the PI3K/Pak1 and ERK1/2 signaling cascade.
      RUNX2 is a member of the family of transcription factor genes (RUNX) that contains a conserved Runt DNA-binding domain (
      • Stein G.S.
      • Lian J.B.
      • van Wijnen A.J.
      • Stein J.L.
      • Montecino M.
      • Javed A.
      • Zaidi S.K.
      • Young D.W.
      • Choi J.Y.
      • Pockwinse S.M.
      ) and that regulates mammalian developmental events related to hematopoiesis (
      • Miyoshi H.
      • Shimizu K.
      • Kozu T.
      • Maseki N.
      • Kaneko Y.
      • Ohki M.
      ), bone formation (
      • Ducy P.
      • Zhang R.
      • Geoffroy V.
      • Ridall A.L.
      • Karsenty G.
      ), and epithelial development (
      • Li Q.L.
      • Ito K.
      • Sakakura C.
      • Fukamachi H.
      • Inoue K.I.
      • Chi X.Z.
      • Lee K.Y.
      • Nomura S.
      • Lee C.W.
      • Han S.B.
      • Kim H.M.
      • Kim W.J.
      • Yamamoto H.
      • Yamashita N.
      • Yano T.
      • Ikeda T.
      • Itohara S.
      • Inazawa J.
      • Abe T.
      • Hagiwara A.
      • Yamagishi H.
      • Ooe A.
      • Kaneda A.
      • Sugimura T.
      • Ushijima T.
      • Bae S.C.
      • Ito Y.
      ). The RUNX proteins function as strong transcriptional activators or repressors to regulate target gene expression (
      • Wheeler J.C.
      • Shigesada K.
      • Gergen J.P.
      • Ito Y.
      ). The expression of the RUNX2 (Cbfa/PEBP2) gene was originally reported in T cells during thymic development (
      • Satake M.
      • Nomura S.
      • Yamaguchi-Iwai Y.
      • Takahama Y.
      • Hashimoto Y.
      • Niki M.
      • Kitamura Y.
      • Ito Y.
      ), but it was also found to regulate the expression of the osteoblast-specific gene, osteocalcin (
      • Ducy P.
      • Zhang R.
      • Geoffroy V.
      • Ridall A.L.
      • Karsenty G.
      ,
      • Ducy P.
      • Karsenty G.
      ). Many matrix gene promoters in osteoblasts and chondrocytes are activated by RUNX2, including osteocalcin, type I collagen-α1 and α2 chains, bone sialoprotein, osteopontin (
      • Kern B.
      • Shen J.
      • Starbuck M.
      • Karsenty G.
      ), and more recently, the marker of hypertrophic chondrocytes, collagen X (
      • Zheng Q.
      • Zhou G.
      • Morello R.
      • Chen Y.
      • Garcia-Rojas X.
      • Lee B.
      ). RUNX2 has been shown to mediate the expression of vascular endothelial growth factor in hypertrophic chondrocytes that regulates angiogenesis during bone formation (
      • Zelzer E.
      • Glotzer D.J.
      • Hartmann C.
      • Thomas D.
      • Fukai N.
      • Soker S.
      • Olsen B.R.
      ). Targeted inactivation of the Runx2 gene in mice leads to impaired bone formation and failure of vascularization (
      • Ducy P.
      • Zhang R.
      • Geoffroy V.
      • Ridall A.L.
      • Karsenty G.
      ,
      • Zelzer E.
      • Glotzer D.J.
      • Hartmann C.
      • Thomas D.
      • Fukai N.
      • Soker S.
      • Olsen B.R.
      ). RUNX2 was also found to be elevated in mouse (
      • Namba K.
      • Abe M.
      • Saito S.
      • Satake M.
      • Ohmoto T.
      • Watanabe T.
      • Sato Y.
      ) and human (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ,
      • Sun L.
      • Vitolo M.
      • Qiao M.
      • Anglin I.
      • Passaniti A.
      ) models of angiogenesis, suggesting a possible role for RUNX2 in neovascularization of adult tissues. RUNX2 mRNA and protein expression in human bone marrow endothelial cell (EC)
      The abbreviations used are: EC, endothelial cell(s); ECM, extracellular matrix; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FGF-2, fibroblast growth factor-2; IGF-1, insulin-like growth factor-1; IGF-1R, IGF-1 receptor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKK1, MAPK kinase-1; PI3K, phosphatidylinositol 3-kinase; PTEN, PI 3-phosphate phosphatase; OC, osteocalcin; pAkt, phosphorylated Akt; Pak1, p21-activated protein kinase-1; HBME, human bone marrow EC line; GST, glutathione S-transferase; CA, constitutively active; STAT, signal transducers and activators of transcription.
      1The abbreviations used are: EC, endothelial cell(s); ECM, extracellular matrix; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FGF-2, fibroblast growth factor-2; IGF-1, insulin-like growth factor-1; IGF-1R, IGF-1 receptor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MKK1, MAPK kinase-1; PI3K, phosphatidylinositol 3-kinase; PTEN, PI 3-phosphate phosphatase; OC, osteocalcin; pAkt, phosphorylated Akt; Pak1, p21-activated protein kinase-1; HBME, human bone marrow EC line; GST, glutathione S-transferase; CA, constitutively active; STAT, signal transducers and activators of transcription.
      were found to increase in cells that were activated for in vitro angiogenesis (tube formation) when cultured on basement membrane proteins (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ). Although endothelium is a tissue that expresses RUNX genes, it is not clear what the transcriptional targets are in EC. The expression of a dominant-negative Runt DNA-binding domain inhibited EC migration and invasion and expression of the genes encoding the proteolytic enzymes urokinase plasminogen activator and membrane-type matrix metalloproteinase-1, which mediate cell invasion (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ). RUNX2 expression in EC and during angiogenesis suggests that it may be a contributing factor for tumorigenesis. However, RUNX2 may also directly promote the malignant phenotype. RUNX2 cooperates with c-Myc to enhance lymphomagenesis in transgenic mice (
      • Vaillant F.
      • Blyth K.
      • Terry A.
      • Bell M.
      • Cameron E.R.
      • Neil J.
      • Stewart M.
      ). Endogenous RUNX2 in malignant breast cancer cells was found to regulate bone sialoprotein expression (
      • Barnes G.L.
      • Javed A.
      • Waller S.M.
      • Kamal M.H.
      • Hebert K.E.
      • Hassan M.Q.
      • Bellahcene A.
      • Van Wijnen A.J.
      • Young M.F.
      • Lian J.B.
      • Stein G.S.
      • Gerstenfeld L.C.
      ), which has been significantly associated clinically with skeletal metastases (
      • Waltregny D.
      • Bellahcene A.
      • de Leval X.
      • Florkin B.
      • Weidle U.
      • Castronovo V.
      ). RUNX2 mRNA was expressed in two malignant melanoma cell lines in vitro, and RUNX2 protein was detected in higher grade (Clarke's level IV) human melanomas (
      • Riminucci M.
      • Corsi A.
      • Peris K.
      • Fisher L.W.
      • Chimenti S.
      • Bianco P.
      ). Clinical prostate cancer specimens and the metastatic androgen-independent PC-3 cell line (
      • Brubaker K.D.
      • Vessella R.L.
      • Brown L.G.
      • Corey E.
      ,
      • Yeung F.
      • Law W.K.
      • Yeh C.H.
      • Westendorf J.J.
      • Zhang Y.
      • Wang R.
      • Kao C.
      • Chung L.W.
      ) express RUNX2, raising the possibility that it may play a role in hormone-independent prostate tumor growth.
      Extracellular factors, such as transforming growth factor-β, extracellular matrix (ECM), fibroblast growth factor-2 (FGF-2), bone morphogenic protein, retinoids, and vitamin D3 have been shown to activate, whereas tumor necrosis factor-α has been shown to reduce RUNX2 expression and/or activity (
      • Franceschi R.T.
      • Xiao G.
      ,
      • Gilbert L.
      • He X.
      • Farmer P.
      • Rubin J.
      • Drissi H.
      • van Wijnen A.J.
      • Lian J.B.
      • Stein G.S.
      • Nanes M.S.
      ). In addition, RUNX2 itself is known to negatively regulate its own promoter (
      • Drissi H.
      • Luc Q.
      • Shakoori R.
      • Chuva De Sousa Lopes S.
      • Choi J.Y.
      • Terry A.
      • Hu M.
      • Jones S.
      • Neil J.C.
      • Lian J.B.
      • Stein J.L.
      • Van Wijnen A.J.
      • Stein G.S.
      ). RUNX2 DNA binding activity is enhanced by interaction with the α2 integrin (
      • Xiao G.
      • Wang D.
      • Benson M.D.
      • Karsenty G.
      • Franceschi R.T.
      ) or treatment with the potent angiogenic factor, FGF-2 (
      • LaVallee T.M.
      • Prudovsky I.A.
      • McMahon G.A.
      • Hu X.
      • Maciag T.
      ,
      • Folkman J.
      ). FGF-2-stimulated angiogenesis is synergistic with the αVβ3 integrin pathway (
      • Friedlander M.
      • Brooks P.C.
      • Shaffer R.W.
      • Kincaid C.M.
      • Varner J.A.
      • Cheresh D.A.
      ). FGF-2 can also regulate skeletal development (
      • Coffin J.D.
      • Florkiewicz R.Z.
      • Neumann J.
      • Mort-Hopkins T.
      • Dorn G.W.
      • Lightfoot P.
      • German R.
      • Howles P.N.
      • Kier A.
      • O'Toole B.A.
      • et al.
      ,
      • Liang H.
      • Pun S.
      • Wronski T.J.
      ) and increase osteocalcin gene expression in pre-osteoblastic MC3T3-E1 cells (
      • Boudreaux J.M.
      • Towler D.A.
      ). FGF-2 increased while treatment with the MEK/ERK inhibitor, U0126, prevented RUNX2 phosphorylation in pre-osteoblast cells (
      • Xiao G.
      • Jiang D.
      • Gopalakrishnan R.
      • Franceschi R.T.
      ). RUNX2-mediated osteocalcin promoter activation was also increased by FGF-2 but inhibited by U0126, suggesting that an ERK pathway was responsible for transcriptional activity. This FGF-2 activated response was synergistically enhanced by the protein kinase A pathway after forskolin treatment (
      • Selvamurugan N.
      • Pulumati M.R.
      • Tyson D.R.
      • Partridge N.C.
      ).
      Insulin-like growth factor-1 (IGF-1) is part of a family of related growth factors including IGF-2 and insulin that interact with specific receptor tyrosine kinases (IGF-1R and IGF-2R) that transmit intracellular signals to regulate normal development and cellular function (
      • Baker J.
      • Liu J.P.
      • Robertson E.J.
      • Efstratiadis A.
      ). IGF-1 also regulates specific hormone receptor and cytokine genes that are important in postnatal growth, differentiation, and angiogenesis. Elevated IGF-1 levels appear to be associated with prostate cancer and IGF-1 regulates the growth of several breast cancer cells (
      • Manes S.
      • Llorente M.
      • Lacalle R.A.
      • Gomez-Mouton C.
      • Kremer L.
      • Mira E.
      • Martinez A.C.
      ,
      • Dunn S.E.
      • Torres J.V.
      • Nihei N.
      • Barrett J.C.
      ). Signal transduction cross-talk has been described for many receptor-regulated pathways (
      • Chang F.
      • Steelman L.S.
      • Lee J.T.
      • Shelton J.G.
      • Navolanic P.M.
      • Blalock W.L.
      • Franklin R.A.
      • McCubrey J.A.
      ). IGF-1 is a potent growth factor for many cells and an angiogenic factor that can stimulate EC proliferation and differentiation through selective cross-talk between the αVβ5 integrin and protein kinase C signaling pathways (
      • Brooks P.C.
      • Klemke R.L.
      • Schon S.
      • Lewis J.M.
      • Schwartz M.A.
      • Cheresh D.A.
      ). IGF-1 activates cell growth and survival through the phosphatidylinositol 3-kinase (PI3K) and Akt pathway. IGF-1 treatment also leads to mitogen-activated protein kinase (MAPK) kinase (MKK) and ERK1/2 activation (
      • Lopaczynski W.
      ,
      • Shelton J.G.
      • Steelman L.S.
      • White E.R.
      • McCubrey J.A.
      ). The p21-activated kinase-1 (Pak1) in cooperation with Ras/Raf may serve as a link between PI3K and ERK signaling pathways (
      • Jaffer Z.M.
      • Chernoff J.
      ). Pak1 is associated with chromatin during the G2/M phase of the cell cycle (
      • Li F.
      • Adam L.
      • Vadlamudi R.K.
      • Zhou H.
      • Sen S.
      • Chernoff J.
      • Mandal M.
      • Kumar R.
      ) and has a role in transcriptional repression (
      • Barnes C.J.
      • Vadlamudi R.K.
      • Mishra S.K.
      • Jacobson R.H.
      • Li F.
      • Kumar R.
      ). PI3K activates Pak1 (
      • Adam L.
      • Vadlamudi R.
      • Kondapaka S.B.
      • Chernoff J.
      • Mendelsohn J.
      • Kumar R.
      ), and Pak1 phosphorylates MKK1 at position Ser-298 (
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ) or Raf-1 kinase on Ser-338 (
      • Zang M.
      • Hayne C.
      • Luo Z.
      ), contributing to the activation of the ERK pathway (
      • Chang F.
      • Steelman L.S.
      • Lee J.T.
      • Shelton J.G.
      • Navolanic P.M.
      • Blalock W.L.
      • Franklin R.A.
      • McCubrey J.A.
      ,
      • Coles L.C.
      • Shaw P.E.
      ).
      We showed previously that angiogenesis and RUNX2 mRNA and protein expression were stimulated by treatment with the angiogenic factor IGF-1 in a human bone marrow EC line (HBME-1) with an IC50 of 14 pm (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ). RUNX2 expression was associated with a dose-dependent increase in EC tube formation on extracellular matrix, which could be inhibited by incubation with neutralizing IGF-1 receptor antibodies. Although these results show that IGF-1 may increase RUNX2 expression in EC (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ), the specific components of the IGF-1 signaling pathway that regulate RUNX2 DNA binding activity (in the absence of changes in RNA and protein expression) have not been characterized. From the current studies, it now appears that the PI3K signaling pathway may mediate both IGF-1-dependent synthesis of RUNX2 and posttranslational regulation of RUNX2 DNA binding activity through an Akt-independent pathway requiring ERK1/2.

      MATERIALS AND METHODS

      Reagents—Recombinant human IGF-1 and neutralizing antibody for IGF-1 receptor were purchased from R&D Systems (Minneapolis, MN). LY294002 and U0126 were from Calbiochem. Each was prepared as a 20 mm stock solution in dimethyl sulfoxide and stored at –20 °C. The Akt-specific inhibitor (SH5) was from Alexis Biochemicals (San Diego, CA). Actinomycin D and cycloheximide were from Calbiochem. Anti-AML3 (RUNX2) polyclonal antibody was obtained from Oncogene Research Product (Cambridge, MA), and total and phospho-Akt antibodies were from Cell Signaling Technology (Beverly, MA). Total phospho-ERK1/2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Akt dominant-negative (DN) and constitutively active (CA) expression vectors and the CA-MKK1 vector were prepared in our laboratories. PTEN wild-type and mutant vectors were from Dr. Yun Qiu (Department of Pharmacology, University of Maryland). FLAG-M2 monoclonal and γ-tubulin antibodies were from Sigma. Anti-GST antibody was from Zymed Laboratories Inc. (San Francisco, CA).
      Cell Culture and Treatment—HBME cells (
      • Lehr J.E.
      • Pienta K.J.
      ), a gift from Dr. Kenneth Pienta (Comprehensive Cancer Center, University of Michigan), and HEK293 cells (from Dr. Robert Fenton, University of Maryland) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin from Invitrogen at 37 °C in a humidified atmosphere with 5% CO2. HBME cells were treated with either IGF-1 or with inhibitors of the PI3K, Akt, or ERK signaling pathways as indicated in the figure legends. For experiments in which IGF-1 activation of RUNX2 was examined, HBME cells were harvested with trypsin and replated for 16 h in complete medium prior to IGF-1 treatment. Under these conditions, RUNX2 levels were generally low prior to IGF-1 treatment. In some cases, cells were serum-starved for 24 h after replating prior to IGF-1 treatment. For inhibition experiments, HBME cells were cultured for 3 days after replating until 90% confluent (subconfluent cells). Under these conditions, RUNX2 levels were generally high prior to inhibition with LY294002, U0126, or SH5.
      Transient Transfection—All of the cell lines were plated on 35-mm dishes at a density of 5 × 105 cells/cm2. After 24 h, the transfection of HBME or HEK293 cells was carried out in complete medium according to the manufacturer's protocols with Mirus TransIT LT1 reagent from Mirus Corporation (Madison, WI). Two days after transfection, the cells were harvested and analyzed for protein expression by Western blotting using specific antibodies.
      Nuclear Protein Isolation—For electrophoretic mobility shift assay (EMSA) assays, cells were washed with chilled phosphate-buffered saline and centrifuged at 800 × g for 5 min at 4 °C and then treated with hypotonic lysis buffer (10 mm Tris, 10 mm NaCl, 3 mm MgCl2, 0.5% Nonidet P-40) for 30 min at 4 °C. Nuclei were centrifuged at 14,000 rpm for 30 min at 4 °C, and nuclear proteins were extracted with buffer containing 10 mm HEPES, 20% glycerol, 800 mm KCl, 1.5 mm MgCl2, and 0.2 mm EDTA and diluted with hypotonic buffer (1:1, v:v) prior to use. Protein concentrations were determined by the Bradford assay using the Bio-Rad reagent. All of the buffers contained a mixture of protease and phosphatase inhibitors consisting of 2 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotonin, 1 mm EGTA, 10 mm NaF, 1 mm sodium pyrophosphate, and 1 mm sodium orthovanadate. For subcellular fractionation of RUNX2, hypotonic low salt buffer containing 0.5% Nonidet P-40 was used to solubilize RUNX2 from the cytosolic and nucleoplasmic compartments, high salt buffer (0.8 m KCl) was used to remove RUNX2 bound to nucleic acid, and a final resuspension of salt-insoluble nuclear pellet in 2× SDS sample buffer was used to extract RUNX2 associated with the chromatin fraction (
      • Westendorf J.J.
      • Zaidi S.K.
      • Cascino J.E.
      • Kahler R.
      • van Wijnen A.J.
      • Lian J.B.
      • Yoshida M.
      • Stein G.S.
      • Li X.
      ).
      EMSA—5 μg of nuclear extract protein were incubated for 20 min at room temperature with a 32P-labeled oligonucleotide derived from the human osteocalcin promoter (from –141 to –165) and containing the human Runx2 consensus sequence (shown in boldface): 5′-CGTATTAACCACAATACTCG-3′ and 3′-AATTGGTGTTATGAGCATGC-5′.
      The double-stranded RUNX2 probe was end-labeled using [α-32P]-dATP, a dNTP mixture, and Klenow enzyme (New England Biolab number M0210S) and purified according to standard protocols from Amersham Biosciences. DNA-protein complexes were resolved on 6% Tris borate-EDTA polyacrylamide gels (Invitrogen). Gels were dried and exposed to x-ray film at –80 °C with an intensifying screen. For supershift experiments, nuclear extracts were incubated with RUNX2-specific antibody in binding buffer for 45 min at room temperature before 32P-labeled oligonucleotide was added to the binding mixture.
      Western Blotting—Heated denatured nuclear proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane using the Novex-NuPAGE system from Invitrogen. Membranes were blocked with 5% nonfat milk for 1 h at room temperature and subsequently incubated overnight with primary antibodies. Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) detection kit from Amersham Biosciences.
      Data Analysis—The densitometry analysis to quantify RUNX2 DNA binding activity was performed using the NIH Image analysis program. Arbitrary densitometric units were normalized with total protein levels in each nuclear extract. Results shown are representative of at least three experiments with essentially similar results. Student's t test was used to determine significant differences.

      RESULTS

      RUNX2 protein is expressed in several EC including HBME, human dermal microvascular EC, and human lung microvascular EC (
      • Namba K.
      • Abe M.
      • Saito S.
      • Satake M.
      • Ohmoto T.
      • Watanabe T.
      • Sato Y.
      ,
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ,
      • Sun L.
      • Vitolo M.
      • Qiao M.
      • Anglin I.
      • Passaniti A.
      ,
      • Kozikowski A.P.
      • Sun H.
      • Brognard J.
      • Dennis P.A.
      ) and in tumor cell lines including MDA231 breast (
      • Barnes G.L.
      • Javed A.
      • Waller S.M.
      • Kamal M.H.
      • Hebert K.E.
      • Hassan M.Q.
      • Bellahcene A.
      • Van Wijnen A.J.
      • Young M.F.
      • Lian J.B.
      • Stein G.S.
      • Gerstenfeld L.C.
      ) and PC-3 prostate (
      • Yeung F.
      • Law W.K.
      • Yeh C.H.
      • Westendorf J.J.
      • Zhang Y.
      • Wang R.
      • Kao C.
      • Chung L.W.
      ) carcinoma. To determine whether EC RUNX2 protein could actively bind DNA, endogenous RUNX2 DNA binding activity assays (EMSA) were performed. Nuclear extracts from HBME cells were incubated with the cognate RUNX-binding oligonucleotide from the osteocalcin promoter (
      • Ducy P.
      • Zhang R.
      • Geoffroy V.
      • Ridall A.L.
      • Karsenty G.
      ), and the shifted complexes were resolved on Tris borate-EDTA DNA retardation gels (Fig. 1A). A major binding complex (arrow) was evident, which was competed by specific cold oligonucleotide, but not the nonspecific oligonucleotide containing a STAT-binding site. RUNX2 was present in the binding complex as confirmed with RUNX2-specific antibody (Fig. 1B), which induced a supershifted complex. Several control antibodies, including anti-γ-tubulin antibody, did not affect the RUNX2 gel shift, whereas an antibody to corebinding factor β, a RUNX2-binding cofactor, slightly altered the shifted complex with evidence of a supershift.
      Figure thumbnail gr1
      Fig. 1EMSA and RUNX2 DNA binding activity.A, nuclear extracts were isolated from HBME cells that were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 3 days until 90% confluency. Under these conditions, RUNX2 expression is generally high. Nuclear extracts were incubated with a cognate RUNX-binding oligonucleotide from the osteocalcin promoter, and specific RUNX2-binding complexes were resolved on 6% acrylamide Tris borate-EDTA gels as a shifted band (arrow). Free probe is indicated at the bottom of the gel. Specific (cold RUNX2) or nonspecific (STAT-binding site) oligomers were incubated with nuclear extracts as indicated. B, RUNX2-specific antibody, control anti-γ-tubulin antibody, or anti-core-binding factor β (CBFb) antibody was incubated with radiolabeled oligonucleotide and binding complexes (arrow), or supershifted complex (SS) was resolved. C, nuclear extracts from EC (HBME), breast tumor cells (SUM159), or myoblast cells (C2C12) were isolated after replating cells for 16 h and culturing in the presence or absence of 10% fetal bovine serum for an additional 24 h prior to IGF-1 treatment (20 ng/ml for 30 min) as indicated. The RUNX2-shifted complexes were resolved on Tris borate-EDTA gels. These results are representative of three separate experiments.
      We showed previously that IGF-1 increases the expression of RUNX2 mRNA and protein (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ). IGF-1 stimulation of RUNX2 DNA binding was also conserved in several other cells, including the human breast cancer SUM159 and rat myoblast C2C12 cells (Fig. 1C). IGF-1 treatment increased RUNX2 DNA binding activity in HBME cells up to 10-fold relative to untreated cells (Fig. 2A). Maximum IGF-1 activation was observed between 10 and 20 ng/ml. These doses of IGF-1 are comparable to those used previously (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ) with a similar EC50 of 15 pm. Protein levels of RUNX2 also increased with a concomitant increase in phosphorylated Akt (pAkt), which is a downstream component activated by IGF-1 and used here as a positive control for IGF-1 treatment. The presence of a lower molecular weight band of RUNX2 has been described before by our laboratory (
      • Sun L.
      • Vitolo M.
      • Qiao M.
      • Anglin I.
      • Passaniti A.
      ) and is the result of an alternative splicing event (
      • Sun L.
      • Vitolo M.
      • Qiao M.
      • Anglin I.
      • Passaniti A.
      ,
      • Geoffroy V.
      • Corral D.A.
      • Zhou L.
      • Lee B.
      • Karsenty G.
      ,
      • Zhang Y.W.
      • Bae S.C.
      • Takahashi E.
      • Ito Y.
      ). The increase in RUNX2 activity was also time-dependent with up to 12-fold increase evident after 45 min in the presence of 20 ng/ml IGF-1 (Fig. 2B). The induction of RUNX2 activity was not dependent on new transcription or protein synthesis, because the presence of cycloheximide (Fig. 2B, panel c) or actinomycin D (data not shown) did not inhibit IGF-1-induced RUNX2 protein or DNA binding. Interestingly, in the absence of IGF-1, low levels of RUNX2 protein were present in high salt-extracted nuclear fractions (N), whereas increased RUNX2 was evident after IGF-1 treatment (Fig. 2C). Conversely, chromatin-associated RUNX2 (Chr) declined after IGF-1 treatment, suggesting an IGF-1-mediated redistribution of nuclear RUNX2. To test the hypothesis that the IGF-1-stimulated increase in RUNX2 DNA binding was regulated by IGF-1R, EC were treated with neutralizing IGF-1R antibody in the presence or absence of IGF-1 for 30 min. Nuclear extracts were prepared, and DNA binding activity was measured. Results indicate that neutralization of IGF-1R modestly inhibited the RUNX2 DNA binding induced by IGF-1 (Fig. 2C). RUNX2 protein levels were also lower in cells treated with neutralizing IGF-1R antibody. This is consistent with previous data showing that RUNX2 regulation of EC tube formation was inhibited by IGF-1R antibodies (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ).
      Figure thumbnail gr2
      Fig. 2IGF-1 regulates RUNX2 DNA binding.A, HBME cells were harvested and replated for 16 h prior to treatment. Under these conditions, RUNX2 levels are generally low. Cells were treated with the indicated concentrations of IGF-1 for 30 min, and DNA binding activity was analyzed by EMSA (a). RUNX2 DNA binding activity relative to untreated cells is shown below each lane and represents densitometry of the major gel-shifted complex (normalized to equal protein per lane). Western blotting for nuclear RUNX2, active pAkt (p-Akt), or total Akt (T-Akt) was performed with specific antibodies (b). B, HBME cells were treated with IGF-1 (20 ng/ml) for the indicated times, and RUNX2 DNA binding activity was measured by EMSA (a). Densitometric scans of the gel-shifted band are shown. Western blotting for RUNX2 was performed with specific antibody (b). Cycloheximide (CHX)-pretreated cells were incubated with IGF-1 for the indicated times, and RUNX2 DNA binding activity (c) or protein (d) was detected. Loading control was total Akt. C, cell fractionation and Western blotting were used to isolate detergent-soluble (S), DNA-associated (N), or chromatin-associated (Chr) RUNX2 protein as described under “Materials and Methods.” Cells were either treated with IGF-1 for 1 h (+) or were left untreated (–). Total Akt is the loading control. D, neutralizing IGF-1R antibody (1:400) was used to treat HBME cells in the presence or absence of IGF-1 (20 ng/ml; 30 min) prior to measurement of RUNX2 DNA binding activity by EMSA.
      An early event in IGF-1 signaling is the activation of the PI3K pathway. To determine whether PI3K signaling could regulate RUNX2 activity, HBME cells were treated with the PI3K inhibitor LY294002 for 24 h and RUNX2 DNA binding and protein levels were measured (Fig. 3A). Inhibition of PI3K resulted in a dose-dependent inhibition of RUNX2 activity with 30, 50, and 90% inhibition at LY294002 concentrations of 5, 10, and 20 μm, respectively. RUNX2 protein levels were also reduced, as was pAkt. However, total Akt levels were unchanged. Inhibition with LY294002 was time-dependent (Fig. 3B) with ∼40% inhibition observed as early as 4 h with little change in RUNX2 protein levels. Longer treatment times resulted in complete inhibition of RUNX2 protein expression in the nucleus. Treatment for even shorter periods of time resulted in the inhibition of RUNX2 DNA binding within 15 min, conditions under which no change in RUNX2 protein levels was observed (Fig. 3C). To confirm that PI3K activity was necessary for RUNX2 DNA binding, the protein phosphatase, PTEN, was overexpressed in HBME and RUNX2 DNA binding activity was measured. PTEN dephosphorylates phosphatidylinositol 1,4,5-trisphosphate, the product of PI3K phosphorylation, thus inhibiting further downstream signaling events. EC were transfected with PTEN wild-type (WT) or mutant (GE, inactive lipid phosphatase) vectors followed by treatment with IGF-1 for 30 min. Although transfection resulted in the inhibition of RUNX2 activity, PTEN wild type was more effective at inhibiting RUNX2 DNA binding (70% inhibition) than the mutant vector (30% inhibition) (Fig. 3D). Western blotting with specific PTEN antibody confirmed the increased expression of PTEN in transfected cells (Fig. 3D).
      Figure thumbnail gr3
      Fig. 3The PI3K signaling pathway regulates RUNX2 activity.A, HBME cells were cultured until 90% confluency. Under these conditions, RUNX2 levels are generally high. The PI3K inhibitor LY294002 was used to treat EC for 24 h prior to preparation of nuclear extracts and measurement of RUNX2 activity (a). Relative activity was quantitated by densitometry (below each lane). RUNX2, pAkt (p-Akt), and total Akt (T-Akt) levels were measured by Western blotting (b). B and C, time course of RUNX2 activity (a) and protein levels (b) in the presence of PI3K inhibitor. D, HBME cells were transfected with PTEN wild-type (WT) or mutant (GE) vectors for 48 h, and RUNX2 DNA binding activity (a) and nuclear protein levels (b) were determined as described.
      Akt is one of the primary mediators of IGF-1-stimulated PI3K activation. To determine whether Akt could regulate RUNX2 activation, cells were incubated for 24 h with different doses of the Akt-specific inhibitor, SH5 (
      • Kozikowski A.P.
      • Sun H.
      • Brognard J.
      • Dennis P.A.
      ), and basal DNA binding activity and RUNX2 expression were measured (Fig. 4A). Akt phosphorylation was inhibited by SH5 at low (0.1–10 μm) concentrations. However, no inhibition of RUNX2 activity was observed, even at high concentrations (50 μm), which have been reported to inhibit phosphorylation of Akt (
      • Kozikowski A.P.
      • Sun H.
      • Brognard J.
      • Dennis P.A.
      ). To determine whether IGF-1-inducible RUNX2 activity was regulated by Akt, HBME cells were pretreated with SH5 prior to IGF-1 stimulation (Fig. 4B). As expected, IGF-1 increased RUNX2 DNA binding activity and protein as observed previously and this activation was not altered in the presence of Akt inhibition. Similarly, transfection of constitutively active or dominant-negative Akt did not alter RUNX2 DNA binding activity relative to empty vector (Fig. 4C). As expected, total Akt increased after cell transfection, whereas pAkt increased only with transfection of constitutively active Akt (Fig. 4C). These data suggest that a cellular signal transduction pathway that does not depend on Akt might regulate RUNX2 expression and activity.
      Figure thumbnail gr4
      Fig. 4Intracellular signaling through Akt is not involved in RUNX2 activation.A, Subconfluent HBME cells were incubated with the Akt-specific inhibitor SH5 for 24 h prior to measuring RUNX2 DNA binding activity (a) and nuclear protein levels (b). B, HBME cells were pretreated with SH5 at the indicated doses for 4 h prior to IGF-1 treatment (20 ng/ml) for 1 h. DNA binding activity (a) or nuclear protein (b) was measured as in A. C, dominant-negative (DN) or CA Akt vectors were used to transfect HBME cells prior to measuring changes in RUNX2 DNA binding (a). Untransfected cells (Con) and empty vector (ev) were used as controls. Western blotting was used to verify the expression of RUNX2, pAkt (p-Akt), and total Akt (T-Akt) in transfected EC (b). Control protein (actin) was used to verify equal loading.
      It was shown previously that RUNX2 activation is regulated by an ERK-dependent pathway in pre-osteoblast cells (
      • Xiao G.
      • Jiang D.
      • Gopalakrishnan R.
      • Franceschi R.T.
      ). The PI3K pathway has also been shown to interact with the ERK1/2 pathway in some cells (
      • Lopaczynski W.
      ,
      • Shelton J.G.
      • Steelman L.S.
      • White E.R.
      • McCubrey J.A.
      ). To determine whether IGF-1 treatment of EC would activate the ERK pathway through PI3K, HBME cells were treated with IGF-1 in the presence or absence of LY294002 and the levels of active ERK1/2 were measured (Fig. 5A). IGF-1 increased active ERK1/2 within 5 min, a response that was ablated by pretreatment with LY294002. To determine whether the activation of RUNX2 in EC is also mediated through an ERK pathway, EC were treated with the MKK1/2 inhibitor U0126 and RUNX2 DNA binding was measured by EMSA. U0126 inhibited RUNX2 activity in a dose-dependent manner (Fig. 5B) with greater than 80% inhibition observed after 24 h at 10 μm. Although extensive inhibition was observed after 24 and 48 h (Fig. 5C), very little change in RUNX2 protein levels was evident unlike the inhibition of the PI3K pathway (Fig. 3B), suggesting that an ERK-mediated phosphorylation event could alter RUNX2 DNA binding. Because the inhibition of RUNX2 activity was observed after 4 h, shorter treatment times were also examined. Inhibition of RUNX2 activity was observed within 15 min with no change in RUNX2 protein levels (Fig. 5D).
      Figure thumbnail gr5
      Fig. 5Inhibition of the MKK1/2-regulated ERK pathway reduces RUNX2 activity.A, PI3K regulates ERK1/2 activity. HBME cells were treated with IGF-1 (10 ng/ml) in the presence or absence of LY294002 (10 μm). Antibodies specific for phospho-ERK1/2 (ppERK1/2) were used to measure active ERK. Anti-α-tubulin-specific antibodies were used for a loading control. B, HBME cells were incubated with increasing amounts of the MKK1/2 inhibitor U0126 for 24 h prior to measuring RUNX2 DNA binding activity by EMSA. C and D, time course of RUNX2 activity in the presence of MKK1/2 inhibition. Nuclear proteins were detected by Western blotting (b). Shown are representative results from three separate experiments. Scanning densitometry and statistical comparison of results from panel D showed reproducible and statistically significant (p ≤ 0.04) inhibition of DNA binding activity (t = 0 versus t = 120 min). However, there was no significant difference between t = 60 and t = 120 time points.
      To confirm that IGF-1-stimulated RUNX2 activation is mediated by PI3K and MKK1/2 and to determine whether the two pathways act in an additive or synergistic manner, EC were treated with IGF-1 in the presence or absence of LY294002 and/or U0126 (Fig. 6A). IGF-1 treatment increased RUNX2 DNA binding activity, but the increase was completely abrogated by pretreatment with either LY294002 or U0126. No further inhibition was observed when the two inhibitors were used in combination, suggesting that PI3K and ERK1/2 may function in a linear pathway. Alternatively, the two pathways may act independently but both may be required for RUNX2 activity. In agreement with the pharmacological inhibitors, transfection of CA-MKK1 increased RUNX2 DNA binding activity approximately 40% relative to empty vector (Fig. 6B) and induced a shift in migration of RUNX2 on SDS-PAGE, consistent with phosphorylation (Fig. 6C, arrow).
      Figure thumbnail gr6
      Fig. 6MKK1/2 mediates IGF-1-stimulated RUNX2 activity and phosphorylation.A, HBME cells were treated with the indicated inhibitors (10 μm) alone or in combination either in the presence (+) or absence (–) of IGF-1 treatment (20 ng/ml) for 30 min prior to EMSA (a) or Western blotting (b). B, empty vector (ev), wild-type vector (Wt), CA vector, or dominant-negative (DN) MKK1 cDNA vector was used to transfect HBME cells. RUNX2 DNA binding activity was determined by EMSA. C, Western blotting was used to detect changes in gel mobility (SDS-PAGE) of FLAG·RUNX2 expressed in HEK293 transfected with RUNX2 alone or in combination with CA-MKK1. Specific antibodies were used to detect active or total ERK1/2. Arrow indicates slower migrating RUNX2 protein.
      Several possible molecular intermediates have been shown to facilitate the cross-talk between the PI3K and ERK signaling pathways (
      • Chang F.
      • Steelman L.S.
      • Lee J.T.
      • Shelton J.G.
      • Navolanic P.M.
      • Blalock W.L.
      • Franklin R.A.
      • McCubrey J.A.
      ). One possible link has been identified as the p21/Ras-activated protein kinase, Pak1 (
      • Adam L.
      • Vadlamudi R.
      • Kondapaka S.B.
      • Chernoff J.
      • Mendelsohn J.
      • Kumar R.
      ,
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ,
      • Coles L.C.
      • Shaw P.E.
      ,
      • Kumar R.
      • Wang R.A.
      ). To determine the role of Pak1 in activation of RUNX2, HBME cells were transfected with a GST-tagged Pak1 vector, GST·Pak1-(83–149) that inhibits Pak1 kinase activity and blocks Pak1 translocation to the centrosomes during mitosis (
      • Li F.
      • Adam L.
      • Vadlamudi R.K.
      • Zhou H.
      • Sen S.
      • Chernoff J.
      • Mandal M.
      • Kumar R.
      ). The expression of GST·Pak1-(83–149) in EC inhibited basal (control) RUNX2 DNA binding activity relative to empty vector (Fig. 7A). RUNX2 protein was reduced as well during this 48-h period. To determine whether Pak1 mediates IGF-1-stimulated RUNX2 activity, EC were transfected with GST·Pak1-(83–149) and treated with IGF-1 (10 ng/ml) for 1 h prior to isolation of nuclear extracts. IGF-1 increased RUNX2 activity as expected (Fig. 7B). GST·Pak1-(83–149) inhibited IGF-1-inducible RUNX2 DNA binding activity, whereas empty vector did not. Under these conditions, very little change in RUNX2 protein expression was observed by Western blot (Fig. 7B). In related experiments, GST·Pak1-(83–149) inhibited phospho-ERK activation (data not shown), supporting a role for Pak1 upstream of ERK in the activation of RUNX2 (Fig. 8).
      Figure thumbnail gr7
      Fig. 7The Pak1 regulates RUNX2 activity.A, inhibition of endogenous RUNX2 activity in subconfluent cells. HBME nuclear extracts were prepared from untransfected controls (Ct), cells transfected with empty vector (ev), or cells transfected with the Pak1 inhibitor GST·Pak1-(83–149) (GPI) for 48 h, and RUNX2 DNA binding (a) or nuclear protein levels (b) were determined. B, inhibition of IGF-1-activated RUNX2 activity. HBME cells were untransfected (Ct) or transfected with empty vector (ev) or with the GST-Pak1 inhibitor (GPI) for 24 h and harvested and replated for 16 h prior to treatment with (+) or without (–) IGF-1 (10 ng/ml) for 1 h. DNA binding activity was determined by EMSA 24 h after transfection (a). RUNX2-specific or actin-specific antibodies were used to measure RUNX2 protein or actin (control) by Western blotting (b). PIP3, phosphatidylinositol 1,4,5-trisphosphate.
      Figure thumbnail gr8
      Fig. 8EC signal transduction pathways and RUNX2 activation. IGF-1 receptor binding activates the p110 catalytic subunit of PI3K. Several pathways may activate Raf including PI3K/Akt and PI3K/Pak1. Data presented in this paper implicate the PI3K/Pak1 signaling pathway in the activation of RUNX2 rather than the Akt pathway. PI3K/Pak1 signaling may activate MKK, which phosphorylates ERK1/2 to regulate RUNX2 DNA binding (
      • Chang F.
      • Steelman L.S.
      • Lee J.T.
      • Shelton J.G.
      • Navolanic P.M.
      • Blalock W.L.
      • Franklin R.A.
      • McCubrey J.A.
      ).

      DISCUSSION

      This study provides the first mechanistic data that establish the specific signal transduction pathways that mediate activation of the RUNX2 transcription factor in EC. The results support a model in which a common pathway utilizing both PI3K and MKK1, but not Akt, activates RUNX2 DNA binding (Fig. 8). IGF-1-stimulated PI3K may recruit the Pak1 and the ERK pathway to stimulate RUNX2 activity. The data support a role for IGF-1R activation of PI3K. Neutralizing IGF-1R antibodies, the specific PI3K inhibitor LY294002, and the expression of PTEN phosphatase inhibited RUNX2 DNA binding activity. PI3K inhibition also reduced active ERK1/2 levels in cells treated with IGF-1. The MKK1 inhibitor U0126 reduced IGF-1-stimulated RUNX2 activity, whereas constitutively active MKK1 increased the levels of RUNX2 DNA binding and the presence of a slower migrating RUNX2 protein, consistent with increased RUNX2 phosphorylation. However, in contrast to many other IGF-1/PI3K signal transduction events, RUNX2 activity (either basal or IGF-1-inducible) does not appear to be mediated by Akt phosphorylation as determined by the specific inhibition of Akt with pharmacologic agents or with dominant-negative or constitutively active Akt expression vectors. The expression of an inhibitory Pak1 vector inhibited both basal and IGF-1-stimulated RUNX2 DNA binding. The evidence suggests that Pak1 is one of the components that mediates the cross-talk between the PI3K and ERK pathways in the activation of RUNX2.
      Published reports from our laboratory implicate IGF-1 in the regulation of RUNX2 expression in EC (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ,
      • Sun L.
      • Vitolo M.
      • Qiao M.
      • Anglin I.
      • Passaniti A.
      ). IGF-1 has been shown to increase bone sialoprotein and osteocalcin (OC) gene expression in an in vivo rat model of bone formation (
      • Strayhorn C.L.
      • Garrett J.S.
      • Dunn R.L.
      • Benedict J.J.
      • Somerman M.J.
      ). Bone sialoprotein and OC are RUNX2 target genes in the mouse osteoprogenitor cell line, MC3T3-E1 (
      • Xiao G.
      • Jiang D.
      • Thomas P.
      • Benson M.D.
      • Guan K.
      • Karsenty G.
      • Franceschi R.T.
      ), and in breast cancer cells (
      • Barnes G.L.
      • Javed A.
      • Waller S.M.
      • Kamal M.H.
      • Hebert K.E.
      • Hassan M.Q.
      • Bellahcene A.
      • Van Wijnen A.J.
      • Young M.F.
      • Lian J.B.
      • Stein G.S.
      • Gerstenfeld L.C.
      ), suggesting that IGF-1 may be able to regulate the expression of these genes through RUNX2 activation. It is clear that diabetes can interfere with bone formation by altering the expression of transcription factors that regulate osteoblast differentiation (
      • Lu H.
      • Kraut D.
      • Gerstenfeld L.C.
      • Graves D.T.
      ). RUNX2 expression declined in diabetic mice with the reduction of OC and collagen type-I gene expression (RUNX2 target genes), leading to decreased bone formation. Bone loss was restored with insulin treatment, which increased RUNX2 expression and expression of OC and collagen type-I. These studies are consistent with our results in HBME cells and suggest that the IGF-1 and insulin-activated pathways may be important mediators of RUNX2 activity
      It is noteworthy that several pathways leading to RUNX2 activation converge on the MAPK cascade, which is the subject of much therapeutic interest (
      • Chang F.
      • Steelman L.S.
      • Lee J.T.
      • Shelton J.G.
      • Navolanic P.M.
      • Blalock W.L.
      • Franklin R.A.
      • McCubrey J.A.
      ). The ECM plays an important role in cell differentiation, development, and tumorigenesis, and changes in cellular interactions with the ECM lead to alterations in cell signaling and gene expression. The integrins are membrane-bound proteins that interact with the ECM and initiate intracellular signal transduction to the nucleus to activate nuclear factors such as RUNX2. ECM secretion by MC3T3 pre-osteoblasts increased the expression of the OC gene, a RUNX2 target (
      • Xiao G.
      • Cui Y.
      • Ducy P.
      • Karsenty G.
      • Franceschi R.T.
      ), which required the presence of the osteoblast-specific element (OSE2) in the OC promoter. RUNX2 DNA binding activity was enhanced by interaction with the α2 integrin without much change in RUNX2 protein or mRNA expression (
      • Xiao G.
      • Wang D.
      • Benson M.D.
      • Karsenty G.
      • Franceschi R.T.
      ). Interestingly, in these studies, overexpression of constitutively active MEK1, which is an ERK1/2 kinase, increased RUNX2 phosphorylation, demonstrating that a MAPK signaling pathway activates RUNX2. This may explain the increased ECM-induced activation without changes in synthesis of the protein (
      • Xiao G.
      • Jiang D.
      • Thomas P.
      • Benson M.D.
      • Guan K.
      • Karsenty G.
      • Franceschi R.T.
      ). The data suggested that phosphorylation may occur in a Pro/Ser/Thr-rich region in the C-terminal domain of RUNX2. Metabolic labeling of RUNX2 with [32P]orthophosphate also provided evidence for posttranslational phosphorylation of RUNX2 (
      • Wee H.J.
      • Huang G.
      • Shigesada K.
      • Ito Y.
      ), but the kinases responsible for these modifications were not identified. FGF-2 is a potent angiogenic growth factor and activator of endothelial and tumor cell proliferation (
      • LaVallee T.M.
      • Prudovsky I.A.
      • McMahon G.A.
      • Hu X.
      • Maciag T.
      ,
      • Folkman J.
      ) that is synergistic with the αVβ3 integrin pathway (
      • Friedlander M.
      • Brooks P.C.
      • Shaffer R.W.
      • Kincaid C.M.
      • Varner J.A.
      • Cheresh D.A.
      ). FGF-2 can also regulate skeletal development (
      • Coffin J.D.
      • Florkiewicz R.Z.
      • Neumann J.
      • Mort-Hopkins T.
      • Dorn G.W.
      • Lightfoot P.
      • German R.
      • Howles P.N.
      • Kier A.
      • O'Toole B.A.
      • et al.
      ,
      • Liang H.
      • Pun S.
      • Wronski T.J.
      ) and increase OC gene expression in preosteoblastic MC3T3-E1 cells (
      • Boudreaux J.M.
      • Towler D.A.
      ). FGF-2 increased metabolic labeling of RUNX2 in pre-osteoblast cells with [32P]orthophosphate, whereas treatment with the MEK/ERK inhibitor U0126 prevented this increased phosphorylation (
      • Xiao G.
      • Jiang D.
      • Gopalakrishnan R.
      • Franceschi R.T.
      ). FGF-2 also stimulated RUNX2-mediated OC promoter activation, which was inhibited by U0126, suggesting that an ERK-dependent pathway was responsible for transcriptional activity.
      The data presented in our study support the hypothesis that IGF-1 signaling through the PI3K, Pak1, and ERK can regulate RUNX2 activation. Changes in RUNX2 levels could be regulated by the short half-life (3 h) of RUNX2 mRNA (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ) or protein (
      • Huang G.
      • Shigesada K.
      • Ito K.
      • Wee H.J.
      • Yokomizo T.
      • Ito Y.
      ). IGF-1 treatment for 12–24 h resulted in increased RUNX2 protein levels (
      • Sun L.
      • Vitolo M.
      • Passaniti A.
      ), and LY294002 treatment for 24 h (Fig. 3) showed decreases in RUNX2 protein. However, the current data support the hypothesis that IGF-1 signaling may also regulate RUNX2 activity without changes in RUNX2 protein synthesis. Treatment with IGF-1 for 15 min increased RUNX2 protein in the nucleus and DNA binding activity, even in the presence of actinomycin D or cycloheximide. Conversely, the inhibition of PI3K for 15 min resulted in the inhibition of RUNX2 DNA binding. Our subcellular fractionation data (Fig. 2C) suggest that one of the mechanisms by which IGF-1 may increase RUNX2 DNA binding is by altering its distribution in the nucleus. However, PI3K or MKK1/2 inhibition results suggest that RUNX2 DNA binding may also be regulated without changes in RUNX2 protein levels. It is still unclear which kinases are responsible for the phosphorylation of RUNX2 (
      • Franceschi R.T.
      • Xiao G.
      ,
      • Wee H.J.
      • Huang G.
      • Shigesada K.
      • Ito Y.
      ). However, our data suggest that the ERK1/2 kinases appear to act downstream of the PI3K and Pak1 to activate RUNX2. Because active MKK1 catalyzed the appearance of a slow mobility RUNX2 on SDS gels (Fig. 6C), it may be necessary to phosphorylate RUNX2 protein to enhance its DNA binding activity.
      The MEK/MAPK pathway has been shown to be activated by IGF-1, resulting in vascular endothelial growth factor induction (
      • Miele C.
      • Rochford J.J.
      • Filippa N.
      • Giorgetti-Peraldi S.
      • Van Obberghen E.
      ). One of the molecules that may link the PI3K and ERK pathways is the Pak1. Pak1 is a member of a family of kinases with diverse functions in cytoskeletal reorganization during cell migration (
      • Adam L.
      • Vadlamudi R.
      • Kondapaka S.B.
      • Chernoff J.
      • Mendelsohn J.
      • Kumar R.
      ), mitosis, transcriptional repression, and angiogenesis (
      • Kumar R.
      • Vadlamudi R.K.
      ). The recruitment of Pak1 to the nucleus may be necessary for some of these activities (
      • Li F.
      • Adam L.
      • Vadlamudi R.K.
      • Zhou H.
      • Sen S.
      • Chernoff J.
      • Mandal M.
      • Kumar R.
      ). Pak1 may also be a link between cell surface signals and the ERK signal transduction pathway, and it may mediate the cross-talk between the ERK and Rho family of proteins (
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ). MEKs tend to be a focal point for cross-cascade regulation. Rho family proteins utilize Paks to mediate cross-talk. It was found that the expression of active Pak1 can substitute for Rho family G protein activity, whereas the expression of an interfering Pak1 mutant blocked Rho-type protein stimulation of ERKs (
      • Frost J.A.
      • Steen H.
      • Shapiro P.
      • Lewis T.
      • Ahn N.
      • Shaw P.E.
      • Cobb M.H.
      ). Pak1 acts by phosphorylating MEK1 on Ser-298, thus priming MEK1 for phosphorylation by Raf-1, leading to subsequent activation of ERK (
      • Coles L.C.
      • Shaw P.E.
      ). Active Raf-1 can phosphorylate its targets, MEK1/2, and acts downstream of Ras to mediate ERK activation in response to mitogens. Raf-1 can also be phosphorylated by Pak1 on Ser-338, which is important for activation. Heregulin (a mitogen for breast cancer cells) can trigger a rapid stimulation of Pak1 activity and its redistribution to the leading edges of motile cells (
      • Adam L.
      • Vadlamudi R.
      • Kondapaka S.B.
      • Chernoff J.
      • Mendelsohn J.
      • Kumar R.
      ). Pak1 activation by heregulin resulted in increased PI3K activity, whereas inhibition of PI3K blocked the activation of Pak1 and cell migration. The blocking of PI3K also inhibited Pak1 interaction with actin and the HER2 receptor. Thus a PI3K/Pak1-dependent reorganization of cortical actin occurs upon heregulin treatment to increase cell migration. In addition, Pak1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation (
      • Slack-Davis J.K.
      • Eblen S.T.
      • Zecevic M.
      • Boerner S.A.
      • Tarcsafalvi A.
      • Diaz H.B.
      • Marshall M.S.
      • Weber M.J.
      • Parsons J.T.
      • Catling A.D.
      ). However, reports examining the activation of NF-κB-dependent transcription in bovine pulmonary artery EC did not find evidence of cross-talk between these two pathways reported by Liu et al. (
      • Liu W.
      • Liu Y.
      • Lowe Jr., W.L.
      ). Our results also indicate that the GST·Pak1-(83–149) Pak1 inhibitor can reduce phospho-ERK activation (data not shown), supporting a role for Pak1 upstream of ERK in the activation of RUNX2. Several possible mechanisms for Pak1 activation of RUNX2 are suggested by these published reports. The activation of Raf-1 and MEK1 may lead to Pak1 activation of ERK and indirect phosphorylation of RUNX2 to enhance DNA binding. Alternatively, Pak1 may directly phosphorylate RUNX2 or both events may occur. Current experiments are testing these possibilities.
      In conclusion, we have demonstrated that the angiogenic growth factor, IGF-1, can regulate RUNX2 DNA binding through activation of the PI3K and ERK1/2 signaling cascades in EC. This activation is independent of the Akt kinase but may involve the Pak1 signaling intermediate that links cell surface receptor activation to nuclear transcriptional events.

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