Insulin-like Growth Factor-1 Regulates Endogenous RUNX2 Activity in Endothelial Cells through a Phosphatidylinositol 3-Kinase/ERK-dependent and Akt-independent Signaling Pathway
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m404480200
ISSN1083-351X
AutoresMeng Qiao, Paul Shapiro, Rakesh Kumar, Antonino Passaniti,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoInsulin-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. 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 (1Stein 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. Oncogene. 2004; 23: 4315-4329Crossref PubMed Scopus (428) Google Scholar) and that regulates mammalian developmental events related to hematopoiesis (2Miyoshi H. Shimizu K. Kozu T. Maseki N. Kaneko Y. Ohki M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10431-10434Crossref PubMed Scopus (804) Google Scholar), bone formation (3Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar), and epithelial development (4Li 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. Cell. 2002; 109: 113-124Abstract Full Text Full Text PDF PubMed Scopus (960) Google Scholar). The RUNX proteins function as strong transcriptional activators or repressors to regulate target gene expression (5Wheeler J.C. Shigesada K. Gergen J.P. Ito Y. Semin. Cell Dev. Biol. 2000; 11: 369-375Crossref PubMed Scopus (74) Google Scholar). The expression of the RUNX2 (Cbfa/PEBP2) gene was originally reported in T cells during thymic development (6Satake M. Nomura S. Yamaguchi-Iwai Y. Takahama Y. Hashimoto Y. Niki M. Kitamura Y. Ito Y. Mol. Cell. Biol. 1995; 15: 1662-1670Crossref PubMed Google Scholar), but it was also found to regulate the expression of the osteoblast-specific gene, osteocalcin (3Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 7Ducy P. Karsenty G. Mol. Cell. Biol. 1995; 15: 1858-1869Crossref PubMed Scopus (529) Google Scholar). Many matrix gene promoters in osteoblasts and chondrocytes are activated by RUNX2, including osteocalcin, type I collagen-α1 and α2 chains, bone sialoprotein, osteopontin (8Kern B. Shen J. Starbuck M. Karsenty G. J. Biol. Chem. 2001; 276: 7101-7107Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar), and more recently, the marker of hypertrophic chondrocytes, collagen X (9Zheng Q. Zhou G. Morello R. Chen Y. Garcia-Rojas X. Lee B. J. Cell Biol. 2003; 162: 833-842Crossref PubMed Scopus (257) Google Scholar). RUNX2 has been shown to mediate the expression of vascular endothelial growth factor in hypertrophic chondrocytes that regulates angiogenesis during bone formation (10Zelzer E. Glotzer D.J. Hartmann C. Thomas D. Fukai N. Soker S. Olsen B.R. Mech. Dev. 2001; 106: 97-106Crossref PubMed Scopus (301) Google Scholar). Targeted inactivation of the Runx2 gene in mice leads to impaired bone formation and failure of vascularization (3Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 10Zelzer E. Glotzer D.J. Hartmann C. Thomas D. Fukai N. Soker S. Olsen B.R. Mech. Dev. 2001; 106: 97-106Crossref PubMed Scopus (301) Google Scholar). RUNX2 was also found to be elevated in mouse (11Namba K. Abe M. Saito S. Satake M. Ohmoto T. Watanabe T. Sato Y. Oncogene. 2000; 19: 106-114Crossref PubMed Scopus (52) Google Scholar) and human (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar, 13Sun L. Vitolo M. Qiao M. Anglin I. Passaniti A. Oncogene. 2004; 23: 4722-4734Crossref PubMed Scopus (43) Google Scholar) 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) 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar). 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar). 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 (14Vaillant F. Blyth K. Terry A. Bell M. Cameron E.R. Neil J. Stewart M. Oncogene. 1999; 18: 7124-7134Crossref PubMed Scopus (80) Google Scholar). Endogenous RUNX2 in malignant breast cancer cells was found to regulate bone sialoprotein expression (15Barnes 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. Cancer Res. 2003; 63: 2631-2637PubMed Google Scholar), which has been significantly associated clinically with skeletal metastases (16Waltregny D. Bellahcene A. de Leval X. Florkin B. Weidle U. Castronovo V. J. Bone Miner. Res. 2000; 15: 834-843Crossref PubMed Scopus (112) Google Scholar). 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 (17Riminucci M. Corsi A. Peris K. Fisher L.W. Chimenti S. Bianco P. Calcif. Tissue Int. 2003; 73: 281-289Crossref PubMed Scopus (38) Google Scholar). Clinical prostate cancer specimens and the metastatic androgen-independent PC-3 cell line (18Brubaker K.D. Vessella R.L. Brown L.G. Corey E. Prostate. 2003; 56: 13-22Crossref PubMed Scopus (98) Google Scholar, 19Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 (20Franceschi R.T. Xiao G. J. Cell. Biochem. 2003; 88: 446-454Crossref PubMed Scopus (463) Google Scholar, 21Gilbert L. He X. Farmer P. Rubin J. Drissi H. van Wijnen A.J. Lian J.B. Stein G.S. Nanes M.S. J. Biol. Chem. 2002; 277: 2695-2701Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). In addition, RUNX2 itself is known to negatively regulate its own promoter (22Drissi 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. J. Cell. Physiol. 2000; 184: 341-350Crossref PubMed Scopus (238) Google Scholar). RUNX2 DNA binding activity is enhanced by interaction with the α2 integrin (23Xiao G. Wang D. Benson M.D. Karsenty G. Franceschi R.T. J. Biol. Chem. 1998; 273: 32988-32994Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) or treatment with the potent angiogenic factor, FGF-2 (24LaVallee T.M. Prudovsky I.A. McMahon G.A. Hu X. Maciag T. J. Cell Biol. 1998; 141: 1647-1658Crossref PubMed Scopus (116) Google Scholar, 25Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar). FGF-2-stimulated angiogenesis is synergistic with the αVβ3 integrin pathway (26Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar). FGF-2 can also regulate skeletal development (27Coffin 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.Mol. Biol. Cell. 1995; 6: 1861-1873Crossref PubMed Scopus (260) Google Scholar, 28Liang H. Pun S. Wronski T.J. Endocrinology. 1999; 140: 5780-5788Crossref PubMed Google Scholar) and increase osteocalcin gene expression in pre-osteoblastic MC3T3-E1 cells (29Boudreaux J.M. Towler D.A. J. Biol. Chem. 1996; 271: 7508-7515Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). FGF-2 increased while treatment with the MEK/ERK inhibitor, U0126, prevented RUNX2 phosphorylation in pre-osteoblast cells (30Xiao G. Jiang D. Gopalakrishnan R. Franceschi R.T. J. Biol. Chem. 2002; 277: 36181-36187Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). 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 (31Selvamurugan N. Pulumati M.R. Tyson D.R. Partridge N.C. J. Biol. Chem. 2000; 275: 5037-5042Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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 (32Baker J. Liu J.P. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 73-82Abstract Full Text PDF PubMed Scopus (2072) Google Scholar). 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 (33Manes S. Llorente M. Lacalle R.A. Gomez-Mouton C. Kremer L. Mira E. Martinez A.C. J. Biol. Chem. 1999; 274: 6935-6945Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 34Dunn S.E. Torres J.V. Nihei N. Barrett J.C. Mol. Carcinog. 2000; 27: 10-17Crossref PubMed Scopus (47) Google Scholar). Signal transduction cross-talk has been described for many receptor-regulated pathways (35Chang F. Steelman L.S. Lee J.T. Shelton J.G. Navolanic P.M. Blalock W.L. Franklin R.A. McCubrey J.A. Leukemia. 2003; 17: 1263-1293Crossref PubMed Scopus (603) Google Scholar). 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Chem. 2002; 277: 4395-4405Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), contributing to the activation of the ERK pathway (35Chang F. Steelman L.S. Lee J.T. Shelton J.G. Navolanic P.M. Blalock W.L. Franklin R.A. McCubrey J.A. Leukemia. 2003; 17: 1263-1293Crossref PubMed Scopus (603) Google Scholar, 45Coles L.C. Shaw P.E. Oncogene. 2002; 21: 2236-2244Crossref PubMed Scopus (99) Google Scholar). 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar). 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar), 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. 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 (46Lehr J.E. Pienta K.J. J. Natl. Cancer Inst. 1998; 90: 118-123Crossref PubMed Scopus (190) Google Scholar), 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 (47Westendorf J.J. Zaidi S.K. Cascino J.E. Kahler R. van Wijnen A.J. Lian J.B. Yoshida M. Stein G.S. Li X. Mol. Cell. Biol. 2002; 22: 7982-7992Crossref PubMed Scopus (282) Google Scholar). 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. RUNX2 protein is expressed in several EC including HBME, human dermal microvascular EC, and human lung microvascular EC (11Namba K. Abe M. Saito S. Satake M. Ohmoto T. Watanabe T. Sato Y. Oncogene. 2000; 19: 106-114Crossref PubMed Scopus (52) Google Scholar, 12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar, 13Sun L. Vitolo M. Qiao M. Anglin I. Passaniti A. Oncogene. 2004; 23: 4722-4734Crossref PubMed Scopus (43) Google Scholar, 48Kozikowski A.P. Sun H. Brognard J. Dennis P.A. J. Am. Chem. Soc. 2003; 125: 1144-1145Crossref PubMed Scopus (197) Google Scholar) and in tumor cell lines including MDA231 breast (15Barnes 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. Cancer Res. 2003; 63: 2631-2637PubMed Google Scholar) and PC-3 prostate (19Yeung F. Law W.K. Yeh C.H. Westendorf J.J. Zhang Y. Wang R. Kao C. Chung L.W. J. Biol. Chem. 2002; 277: 2468-2476Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) 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 (3Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar), 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. We showed previously that IGF-1 increases the expression of RUNX2 mRNA and protein (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar). 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar) 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 (13Sun L. Vitolo M. Qiao M. Anglin I. Passaniti A. Oncogene. 2004; 23: 4722-4734Crossref PubMed Scopus (43) Google Scholar) and is the result of an alternative splicing event (13Sun L. Vitolo M. Qiao M. Anglin I. Passaniti A. Oncogene. 2004; 23: 4722-4734Crossref PubMed Scopus (43) Google Scholar, 49Geoffroy V. Corral D.A. Zhou L. Lee B. Karsenty G. Mamm. Genome. 1998; 9: 54-57Crossref PubMed Scopus (84) Google Scholar, 50Zhang Y.W. Bae S.C. Takahashi E. Ito Y. Oncogene. 1997; 15: 367-371Crossref PubMed Scopus (30) Google Scholar). 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 (12Sun L. Vitolo M. Passaniti A. Cancer Res. 2001; 61: 4994-5001PubMed Google Scholar). 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
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