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Runx2 Regulates G Protein-coupled Signaling Pathways to Control Growth of Osteoblast Progenitors

2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês

10.1074/jbc.m802453200

ISSN

1083-351X

Autores

Nadiya M. Teplyuk, Mario Galindo, Viktor I. Teplyuk, Jitesh Pratap, Daniel Young, David Lapointe, Amjad Javed, Janet L. Stein, Jane B. Lian, Gary S. Stein, André J. van Wijnen,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-β/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G1. Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2-/- osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals. Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-β/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G1. Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2-/- osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals. Stringent regulation of mesenchymal stem cell renewal and the generation and differentiation of the appropriate number of committed osteoprogenitors are critical for development, remodeling, and repair of bone tissue. Runx2 (Runt-related transcription factor 2) is a critical regulator of bone development by driving osteoblast differentiation and formation of a bone-specific mineralized extracellular matrix (1Lian J.B. Stein G.S. Javed A. van Wijnen A.J. Stein J.L. Montecino M. Hassan M.Q. Gaur T. Lengner C.J. Young D.W. Rev. Endocr. Metab. Disord. 2006; 7: 1-16Crossref PubMed Scopus (375) Google Scholar, 2Komori T. Front. Biosci. 2008; 13: 898-903Crossref PubMed Scopus (197) Google Scholar, 3Blyth K. Cameron E.R. Neil J.C. Nat. Rev. Cancer. 2005; 5: 376-387Crossref PubMed Scopus (379) Google Scholar, 4van Wijnen A.J. Stein G.S. Gergen J.P. Groner Y. Hiebert S.W. Ito Y. Liu P. Neil J.C. Ohki M. Speck N. Oncogene. 2004; 23: 4209-4210Crossref PubMed Scopus (98) Google Scholar). Runx2 regulates osteoblast growth (5Pratap J. Galindo M. Zaidi S.K. Vradii D. Bhat B.M. Robinson J.A. Choi J.-Y. Komori T. Stein J.L. Lian J.B. Stein G.S. van Wijnen A.J. Cancer Res. 2003; 63: 5357-5362PubMed Google Scholar, 6Galindo M. Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Biol. Chem. 2005; 280: 20274-20285Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) in part through epigenetic mechanisms (7Young D.W. Hassan M.Q. Pratap J. Galindo M. Zaidi S.K. Lee S.H. Yang X. Xie R. Javed A. Underwood J.M. Furcinitti P. Imbalzano A.N. Penman S. Nickerson J.A. Montecino M.A. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Nature. 2007; 445: 442-446Crossref PubMed Scopus (195) Google Scholar, 8Young D.W. Hassan M.Q. Yang X.-Q. Galindo M. Javed A. Zaidi S.K. Furcinitti P. Lapointe D. Montecino M. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3189-3194Crossref PubMed Scopus (143) Google Scholar, 9Zaidi S.K. Sullivan A.J. Medina R. Ito Y. van Wijnen A.J. Stein J.L. Lian J.B. Stein G.S. EMBO J. 2004; 23: 790-799Crossref PubMed Scopus (321) Google Scholar) and the ability of Runx2 to promote a non-proliferative state (e.g. senescence or quiescence) (6Galindo M. Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Biol. Chem. 2005; 280: 20274-20285Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 10Zaidi S.K. Pande S. Pratap J. Gaur T. Grigoriu S. Ali S.A. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19861-19866Crossref PubMed Scopus (65) Google Scholar, 11Kilbey A. Blyth K. Wotton S. Terry A. Jenkins A. Bell M. Hanlon L. Cameron E.R. Neil J.C. Cancer Res. 2007; 67: 11263-11271Crossref PubMed Scopus (40) Google Scholar, 12Thomas D.M. Johnson S.A. Sims N.A. Trivett M.K. Slavin J.L. Rubin B.P. Waring P. McArthur G.A. Walkley C.R. Holloway A.J. Diyagama D. Grim J.E. Clurman B.E. Bowtell D.D. Lee J.S. Gutierrez G.M. Piscopo D.M. Carty S.A. Hinds P.W. J. Cell Biol. 2004; 167: 925-934Crossref PubMed Scopus (184) Google Scholar). The first evidence that Runx2 affects bone cell growth was the observation that primary calvarial cells from Runx2 null mice proliferate faster than corresponding wild type osteoblasts, and reintroduction of Runx2 into Runx2 null cells restores normal cell growth (5Pratap J. Galindo M. Zaidi S.K. Vradii D. Bhat B.M. Robinson J.A. Choi J.-Y. Komori T. Stein J.L. Lian J.B. Stein G.S. van Wijnen A.J. Cancer Res. 2003; 63: 5357-5362PubMed Google Scholar). Several studies indicate that Runx2 control of proliferation is cell type-specific. Runx2 inhibits proliferation of osteoprogenitors and committed osteoblasts (5Pratap J. Galindo M. Zaidi S.K. Vradii D. Bhat B.M. Robinson J.A. Choi J.-Y. Komori T. Stein J.L. Lian J.B. Stein G.S. van Wijnen A.J. Cancer Res. 2003; 63: 5357-5362PubMed Google Scholar, 6Galindo M. Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. 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Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Biol. Chem. 2005; 280: 20274-20285Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 12Thomas D.M. Johnson S.A. Sims N.A. Trivett M.K. Slavin J.L. Rubin B.P. Waring P. McArthur G.A. Walkley C.R. Holloway A.J. Diyagama D. Grim J.E. Clurman B.E. Bowtell D.D. Lee J.S. Gutierrez G.M. Piscopo D.M. Carty S.A. Hinds P.W. J. Cell Biol. 2004; 167: 925-934Crossref PubMed Scopus (184) Google Scholar), T cell lymphomas (16Stewart M. Mackay N. Hanlon L. Blyth K. Scobie L. Cameron E. Neil J.C. Cancer Res. 2007; 67: 5126-5133Crossref PubMed Scopus (40) Google Scholar, 17Blyth K. Vaillant F. Hanlon L. Mackay N. Bell M. Jenkins A. Neil J.C. Cameron E.R. Cancer Res. 2006; 66: 2195-2201Crossref PubMed Scopus (89) Google Scholar), and breast cancer (18Pratap J. Lian J.B. Javed A. Barnes G.L. van Wijnen A.J. Stein J.L. Stein G.S. Cancer Metastasis Rev. 2006; 25: 589-600Crossref PubMed Scopus (220) Google Scholar). In non-osseous cells (e.g. T cells), Runx2 has anti-proliferative potential (19Vaillant F. Blyth K. Andrew L. Neil J.C. Cameron E.R. J. Immunol. 2002; 169: 2866-2874Crossref PubMed Scopus (66) Google Scholar) unless c-Myc levels are elevated (17Blyth K. Vaillant F. Hanlon L. Mackay N. Bell M. Jenkins A. Neil J.C. Cameron E.R. Cancer Res. 2006; 66: 2195-2201Crossref PubMed Scopus (89) Google Scholar). The growth regulatory activity of Runx2 is controlled by cell cycle-dependent modulations in its protein levels. Runx2 expression is modulated during the cell cycle at both transcriptional and post-transcriptional levels and maximal in early to mid-G1 (6Galindo M. Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Biol. Chem. 2005; 280: 20274-20285Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 20Galindo M. Kahler R.A. 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Hist. 2007; 38: 501-506Crossref PubMed Scopus (27) Google Scholar, 22Rajgopal A. Young D.W. Mujeeb K.A. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell. Biochem. 2007; 100: 1509-1517Crossref PubMed Scopus (49) Google Scholar, 23Shen R. Wang X. Drissi H. Liu F. O'Keefe R.J. Chen D. J. Biol. Chem. 2006; 281: 16347-16353Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Runx2 expression and activity in osteoblasts are regulated by a large number of growth regulatory factors and external signals, including fibroblast growth factor 2 (24Kim H.J. Kim J.H. Bae S.C. Choi J.Y. Kim H.J. Ryoo H.M. J. Biol. Chem. 2003; 278: 319-326Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 25Guenou H. Kaabeche K. Mee S.L. Marie P.J. Hum. Mol. Genet. 2005; 14: 1429-1439Crossref PubMed Scopus (58) Google Scholar, 26Marie P.J. Gene (Amst.). 2003; 316: 23-32Crossref PubMed Scopus (235) Google Scholar), TGFβ 5The abbreviations used are: TGFβ, transforming growth factor-β; GPCR, G protein-coupled receptor; PTH, parathyroid hormone; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; qRT, quantitative reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IRES, internal ribosome entry site; GFP, green fluorescent protein; EGFP, enhanced GFP; α-MEM, minimum Eagle's α-medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; siRNA, short interfering RNA; FACS, fluorescence-activated cell sorting; MOPS, 4-morpholinepropanesulfonic acid; PMA, phorbol myristate acetate; CREB, cAMP-responsive element-binding protein; BMP, bone morphogenetic protein; Cdk, cyclin-dependent kinase. /BMP2 (27Balint E. Lapointe D. Drissi H. van der Meijden C. Young D.W. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Cell. Biochem. 2003; 89: 401-426Crossref PubMed Scopus (147) Google Scholar, 28Lee M.H. 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Importantly, retention of Runx2 protein on mitotic chromosomes provides an epigenetic memory of its developmental position in the osteogenic lineage (7Young D.W. Hassan M.Q. Pratap J. Galindo M. Zaidi S.K. Lee S.H. Yang X. Xie R. Javed A. Underwood J.M. Furcinitti P. Imbalzano A.N. Penman S. Nickerson J.A. Montecino M.A. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Nature. 2007; 445: 442-446Crossref PubMed Scopus (195) Google Scholar, 8Young D.W. Hassan M.Q. Yang X.-Q. Galindo M. Javed A. Zaidi S.K. Furcinitti P. Lapointe D. Montecino M. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3189-3194Crossref PubMed Scopus (143) Google Scholar) and poises Runx2 to control gene expression in early G1 (21Galindo M. Teplyuk N. Yang X. Young D.W. Javed A. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Bone Miner. Res. 2006; 21 (abstr.): S384Google Scholar). Runx2 is a bifunctional transcription factor that mediates the integration of multiple signaling pathways at nuclear matrix-associated subnuclear foci through interactions with a large cohort of cofactors (>24 partner proteins) that support activation or repression of Runx2 target genes in osteoblasts (reviewed in Ref. 1Lian J.B. Stein G.S. Javed A. van Wijnen A.J. Stein J.L. Montecino M. Hassan M.Q. Gaur T. Lengner C.J. Young D.W. Rev. Endocr. Metab. Disord. 2006; 7: 1-16Crossref PubMed Scopus (375) Google Scholar and see Refs. 33Thomas D.M. Carty S.A. Piscopo D.M. Lee J.S. Wang W.F. Forrester W.C. Hinds P.W. Mol. Cell. 2001; 8: 303-316Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 36Jensen E.D. Schroeder T.M. Bailey J. Gopalakrishnan R. Westendorf J.J. J. Bone Miner. Res. 2008; 23: 361-372Crossref PubMed Scopus (115) Google Scholar). For example, Runx2 interacts with GROUCHO/TLE proteins that require the conserved C-terminal VWRPY pentapeptide of Runx2 for gene repression (37Javed A. Guo B. Hiebert S. Choi J.-Y. Green J. Zhao S.-C. Osborne M.A. Stifani S. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell Sci. 2000; 113: 2221-2231Crossref PubMed Google Scholar). Runx2 transduces TGFβ/BMP2 signaling through direct interactions with SMAD proteins that bind to a composite protein domain ("NMTS/SMID") that mediates transcriptional activation and subnuclear targeting (31Javed A. Bae J.S. Afzal F. Gutierrez S. Pratap J. Zaidi S.K. Lou Y. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. Chem. 2008; 283: 8412-8422Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 38Zaidi S.K. Young D.W. Javed A. Pratap J. Montecino M. van W.A. Lian J.B. Stein J.L. Stein G.S. Nat. Rev. Cancer. 2007; 7: 454-463Crossref PubMed Scopus (133) Google Scholar). Furthermore, Runx2 interacts with Yes-associated protein (9Zaidi S.K. Sullivan A.J. Medina R. Ito Y. van Wijnen A.J. Stein J.L. Lian J.B. Stein G.S. EMBO J. 2004; 23: 790-799Crossref PubMed Scopus (321) Google Scholar), a WW domain-containing protein that transduces signals from the SRC/YES family of tyrosine kinases, as well as a growing number of other WW domain-containing proteins (e.g. TAZ, SMURF, and SCHNURRI) that all recognize a PPYP motif linked to the NMTS (39Zhao M. Qiao M. Oyajobi B.O. Mundy G.R. Chen D. J. Biol. Chem. 2003; 278: 27939-27944Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 40Cui C.B. Cooper L.F. Yang X. Karsenty G. Aukhil I. Mol. Cell. Biol. 2003; 23: 1004-1013Crossref PubMed Scopus (165) Google Scholar, 41Jones D.C. Wein M.N. Oukka M. Hofstaetter J.G. Glimcher M.J. Glimcher L.H. Science. 2006; 312: 1223-1227Crossref PubMed Scopus (186) Google Scholar). Despite the growing number of Runx2 partner proteins that modify or modulate its transcriptional activity, there is limited understanding of the signaling pathways and Runx2 cofactors that control the growth regulatory potential of Runx2 and the downstream expression programs that mediate its cellular functions. In this study, we have examined how Runx2 regulates growth of osteoblasts using complementation assays with Runx2 null calvarial osteoprogenitors in which we introduced wild type or mutant Runx2 proteins. Our studies revealed that DNA binding and C-terminal transcriptional functions of Runx2 are essential for cell growth control. Furthermore, our data suggest that Runx2 may regulate G protein-signaling pathways to modify how osteoblasts respond to mitogenic stimuli. Cell Culture—Immortalized Runx2 null cells from mouse calvaria were generated in our laboratory from the fetal calvarial region of Runx2 knock-out mice by stable integration of mTERT as described previously (30Bae J.S. Gutierrez S. Narla R. Pratap J. Devados R. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. Javed A. J. Cell. Biochem. 2007; 100: 434-449Crossref PubMed Scopus (70) Google Scholar). Runx2 null cells were maintained in αMEM supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 30 mm penicillin/streptomycin, and 100 mm l-glutamine at 37 °C and 5% CO2-humidified atmosphere. Mouse MC3T3 osteoblasts (6Galindo M. Pratap J. Young D.W. Hovhannisyan H. Im H.J. Choi J.Y. Lian J.B. Stein J.L. Stein G.S. van Wijnen A.J. J. Biol. Chem. 2005; 280: 20274-20285Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) and primary cultures of calvarial cells from wild type and Runx2 null mice were maintained as described previously (5Pratap J. Galindo M. Zaidi S.K. Vradii D. Bhat B.M. Robinson J.A. Choi J.-Y. Komori T. Stein J.L. Lian J.B. Stein G.S. van Wijnen A.J. Cancer Res. 2003; 63: 5357-5362PubMed Google Scholar, 10Zaidi S.K. Pande S. Pratap J. Gaur T. Grigoriu S. Ali S.A. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19861-19866Crossref PubMed Scopus (65) Google Scholar). Primary cells were expanded in subconfluent culture for three passages before collection and RNA extraction. Adenovirus Constructs, Infections, and Transfections—Adenoviral vectors containing cDNAs of full-length Runx2, key deletion mutants 1–361 (ΔC) and 1–432 (Δ432), as well as several point mutants R182Q (DNA binding domain mutant) (7Young D.W. Hassan M.Q. Pratap J. Galindo M. Zaidi S.K. Lee S.H. Yang X. Xie R. Javed A. Underwood J.M. Furcinitti P. Imbalzano A.N. Penman S. Nickerson J.A. Montecino M.A. Lian J.B. Stein J.L. van Wijnen A.J. Stein G.S. Nature. 2007; 445: 442-446Crossref PubMed Scopus (195) Google Scholar), Y433A (YAP-binding mutant) (42Zaidi S.K. Javed A. Choi J.-Y. van Wijnen A.J. Stein J.L. Lian J.B. Stein G.S. J. Cell Sci. 2001; 114: 3093-3102Crossref PubMed Google Scholar), and H426A/T427A/Y428A (SMAD interaction mutant) (31Javed A. Bae J.S. Afzal F. Gutierrez S. Pratap J. Zaidi S.K. Lou Y. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. Chem. 2008; 283: 8412-8422Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) were each transferred into the AdenoVator™ expression construct (Qbiogene, Irvine, CA) from the corresponding pcDNA expression vectors described in the indicated references from our research group. Viral packaging of the vectors was performed according to the manufacturer's protocol. Virus preparations were purified using a commercial adenovirus purification kit (Promega, Madison, WI). Cells were plated for infections in 6-well plates (12.5 × 104 cells/well). After 24 h, cells were infected with 100 multiplicities of infection of each virus in 600 μl of αMEM complemented with 1% FBS for 4 h. Upon addition of 400 μl of media containing 1% FBS, cells were incubated for an additional 10 h. Cell numbers were determined at daily intervals during the next 4 days after infection to determine growth curves and to perform expression analyses. Infection efficiencies were assessed by expression of a green fluorescent protein (GFP) under the control of an IRES signal, and images of GFP-expressing cells were taken using a Carl Zeiss fluorescence microscope with a SPOT camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Quantitative Real Time Reverse Transcriptase-PCR (qRT-PCR) Analysis—Total RNA for qRT-PCR assays was isolated using TRIzol reagent (Invitrogen), subjected to DNase I digestion, and purified using an RNA purification kit (Zymogen, Orange, CA). Aliquots of RNA (1 μg) were used for reverse transcription (first strand cDNA synthesis kit, Invitrogen) with random hexamer primers. Quantitative PCR was performed with commercially available reagents (Power SYBR Green PCR Master Mix, Applied Biosystems, Foster City, CA) using an automated system (Applied Biosystems 7300 Real Time PCR System) with 0.5 pmol/μl of each of the following primers: Runx2 forward, CGA CAG TCC CAA CTT CCT GT, and reverse, CGG TAA CCA CAG TCC CAT CT; Rgs2 forward, GAG GAG AAG CGG GAG AAA AT, and reverse, GCT TTT CTT GCC AGT TTT GG; osteocalcin forward, CTG ACA AAG CCT TCA TGT CCA A, and reverse, GCG CCG GAG TCT GTT CAC TA; Rgs4 forward, TGC CTT TCT CTC CTC GCT AA, and reverse, GCC GAT GTT TCA TGT CCT TT; Gpr30 forward, CCA AGC CTC AAC ACT CAC AC, and reverse, GAA AAC CAG AAG GGT GGA CA; Rgs5 forward, GCC AGC CAA AAT GTG TAA GG, and reverse, AGC AGA GTC TGG CTT CTG GA; Gpr23 forward, CTG GTG CCA GAG TTT GGT TT, and reverse, TTT TCC CAG AGA GCC TGC TA; Rgs16 forward, GCT CCG ATA CTG GGG GTA TT, and reverse, TTC AGC AGC AAA TCG AAA GA; Gpr54 forward, GGT GCT GGG AGA CTT CAT GT, and reverse, ACA TAC CAG CGG TCC ACA CT; Gna13 forward, CCA CCA TCT ACA GCA ACG TG, and reverse, CCA TGG AGC TGG TTT TTG TT; Gpr64 forward, CTG TGG TTG TGT CCA TCG TC, and reverse, CCA CAT TGC TGT TGA TCC AG; osteopontin forward, ACT CCA ATC GTC CCT ACA GTC G, and reverse, TGA GGT CCT CAT CTG TGG CAT; Wnt7b forward, ATG CCC GTG AGA TCA AAA AG, and reverse, CCT GAC ACA CCG TGA CAC TT; Serpin b2 (PAI2) forward, CAA GAT GGT GCT GGT GAA TG, and reverse, GCT CTC ATG CGA GTT CAC AC; Alk Phos forward, TTG TGC GAG AGA AAG AGA GAG A, and reverse, GTT TCA GGG CAT TTT TCA AGG T; p21 forward, TTG CAC TCT GGT GTC TGA GC, and reverse, TCT GCG CTT GGA GTG ATA GA; Mcox forward, ACG AAA TCA ACA ACC CCG TA, and reverse, GGC AGA ACG ACT CGG TTA TC; Hprt forward, CAG GCC AGA CTT TGT TGG AT, and reverse, TTG CGC TCA TCT TAG GCT TT; histone H4 forward, TGA GCT TCC TTC CTA GTT TGC, and reverse, GCT TAG CAC CAC CCT TAC CA; p27 forward, GTG GAC CAA ATG CCT GAC TC, and reverse, TCT GTT GGC CCT TTT GTT TT; cyclin B2 forward, GCC TCT TGC CTG TCT CAG AA, and reverse, GCT GCA TGA CTT CCA GGA CT. Rodent GAPDH and ribosomal 18 S RNA internal control primers were purchased from Applied Biosystems. The initial denaturation occurred at 95 °C for 10 min followed by 40 cycles of two-step PCR (95 °C for 15 s denaturation and 60 °C for 1 min synthesis). Affymetrix Analysis—Total RNA for Affymetrix analysis (and subsequent qRT-PCR validation) was isolated with TRIzol reagent and purified using the RNeasy mini kit (Qiagen). Approximately 1–5 μg of total RNA was used for two strand cDNA synthesis using the Affymetrix cDNA synthesis kit using oligo(dT) primers (Affymetrix, Santa Clara, CA). Following cRNA synthesis, samples were labeled with biotin using the BioArray HighYield RNA transcript labeling kit (Enzo Lifesciences, Farmingdale, NY) and purified with Affymetrix Cleanup Module for cRNA (Affymetrix). Aliquots of the full-length cRNA products (∼20 μg) were fragmented using metal-induced hydrolysis at a concentration of 1 μg/μl. The quality of cRNA fragments (35–200 bases) was evaluated in 1% formaldehyde/MOPS gel. Aliquots of fragmented cRNA (12 μg) were used for hybridization with Affymetrix cDNA microarrays ("chips") (Mouse Genome 430 2.0 Array) in hybridization mixture for 16 h at 42 °C in a hybridization oven. After hybridization, chips were washed on an Affymetrix Fluidics Station and stained with solutions containing streptavidin/phycoerythrin solution and goat IgG antibody to amplify the signals of the transcripts. The resulting signals were scanned using an Affymetrix scanner, and numerical files (CEL) were generated from the resulting images. Data processing and sample comparisons were performed using an open source library for statistical analysis (Bio-Conductor library for R environment). In brief, robust multiarray average expression measure as part of the Affy software package was applied to calculate the average value of signals on the arrays (43Gautier L. Cope L. Bolstad B.M. Irizarry R.A. Bioinformatics (Oxf.). 2004; 20: 307-315Crossref PubMed Scopus (3876) Google Scholar). Following robust multiarray average background correction, array values were subjected to quantile normalization assuming identical signal distributions in each of the arrays. Statistically significant differences between probe sets were evaluated using Student's t test (p < 0.05). Functional annotation of Affymetrix probe sets and gene ontology relationships between groups of co-regulated genes were assessed using the data base for Annotation, Visualization and Integrated Discovery (DAVID 2.0) (44Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. Genome Biol. 2003; 4: 3Crossref PubMed Google Scholar). Western Blot Analysis—Cell lysates were prepared from cell pellets that were boiled in 100 μl of Direct Lysis buffer (50 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 12% urea, 25 μm MG132, 100 mm dithiothreitol, and 1× Complete protease inhibitors) (Roche Applied Science). Aliquots of each lysate (5 μl) were separated in 10% SDS-PAGE. Proteins were transferred using semi-wet blotting to nitrocellulose membranes (Millipore, Billerica, MA). Phosphate-buffered saline (PBS: 20 mm phosphate, 150 m NaCl, pH 7.4) with 5% nonfat milk was used for 1 h at room temperature to block nonspecific protein binding. Primary and secondary antibodies were used at 1:2,000 dilutions for 1 h room temperature in PBS, 0.1% Tween (PBST) with 1% milk. Signal was detected with ECL (Western Lighting Chemiluminescence Reagent Plus, PerkinElmer Life Sciences). Runx2-specific mouse monoclonal antibodies were a generous gift of Dr. Yoshiaki Ito (Institute for Molecular and Cellular Biology, Singapore). CDK2 rabbit polyclonal antibody (SC-163) was purchased from a commercial vendor (Santa Cruz Biotechnology, Santa Cruz, CA). In Situ Immunofluorescence—Immunofluorescence microscopy was performed according to standard procedures (9Zaidi S.K. Sullivan A.J. Medina R. Ito Y. van Wijnen A.J. Stein J.L. Lian J.B. Stein G.S. EMBO J. 2004; 23: 790-799Crossref PubMed Scopus (321) Google Scholar, 37Javed A. Guo B. Hiebert S. Choi J.-Y. Green J. Zhao S.-C. Osborne M.A. Stifani S. Stein J.L. Lian J.B. van Wijnen A.J. Stein G.S. J. Cell Sci. 2000; 113: 2221-2231Crossref PubMed Google Scholar). For detection of Gpr30 and Rgs2 protein in adenovirus-infected Runx2 null cells, we applied the following procedure. Runx2 null cells were plated on gelatin-coated coverslips at 0.6 × 106 cells per 6-well plate. Cells were infected after 24 h with adenovirus vectors expressing wild type Runx2, the Δ361 mutant, or GFP alone. Cells were fixed with 3.8% formaldehyde in PBS for 10 min and permeabilized with 0.25% Triton X-100 in PBS for 20 min. Nonspecific binding was blocked for 20 min with PBSA solution (PBS containing 0.5% bovine serum albumin). Cells were incubated with the following primary antibodies at a 1:250 dilution in PBSA at 4 °C overnight: rabbit polyclonal specific for Gpr30 (Abcam, Ab-12564) and goat polyclonal specific for Rgs2 (Santa Cruz Biotechnology, C18/sc7678). For Rgs2 and Ki-67 co-staining, MC3T3 cells were plated at ∼0.3 × 106 cells per 6-well plate, fixed 36 h after plating, permeabilized, and blocked as described above. Cells were incubated with a mixture of primary antibodies containing a 1:200 dilution of goat polyclonal anti-Rgs2 antibody (SC-7678, Santa Cruz Biotechnology) plus a 1:100 dilution of rabbit polyclonal anti-Ki67 (Abcam, Ab-15580). Dilutions were made in PBSA, and cells were incubated with antibodies for 1 h at 37 °C. Following 4′,6-diamidino-2-phenylindole staining to define the loc

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