ISSN 0233-7657. Biopolymers and Cell. 2010. Vol. 26. N 4

Bone-specific master transcription factor Runx2 regulates signaling and metabolism related programs in osteoprogenitors

N. M. Teplyuk1, 2, V. I. Teplyuk2

1University of Massachusetts Medical School 55, Lake Ave North, 01655, Worcester, MA, USA 2Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine 150, Zabolotnogo str., Kiev, Ukraine, 03680 [email protected]

Aim. Runx2 (AML3) transcription factor is the key regulator of osteoblastic lineage progression and is indispensable for the formation of mineral bones. Runx2 expression increases during differentiation of osteoblasts to induce osteoblast-specific necessary for the production and deposition of bone mineral matrix. However, Runx2 is also expressed at a lower level in early osteoprogenitors, where its function is less understood. Here we study how Runx2 determines the early stages of osteoblastic commitment using the model system of Runx2 re-introduction in mouse calvaria cells with Runx2 null background. Method. Affymetrix analysis, Western blot analysis and quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis were employed. Results. expression profiling by Affymetrix microarrays revealed that along with the induction of extracellular matrix and bone mineral deposition related phenotypic markers, Runx2 regulates several cell programs related to signaling and metabolism in the early osteoprogenitors. Particularly, Runx2 regulates transcription of genes involved in G-protein coupled signaling network, FGF and BMP/TGF beta signaling pathways and in biogenesis and metabolism pathways of steroid hormones. Conclusion. The data indicate that the lineage specific program, regulated by the master regulatory transcription factor, includes the regulation of cellular signaling and metabolism which may allow the committed cell to react and behave differently in the same microenvironment. Keywords: osteoblast progenitors, Runx2, signaling.

Introduction. Bone development, repair and remode- transcription or by epigenetic remodeling of gene regu- ling homeostasis require constant differentiation of latory sequences [3]. mesenchymal stem cells through osteoprogenitors to Multiple Runx2 target genes are responsible for bo- mature osteoblasts and osteocytes. Consistent with the ne mineral production at the late stages of osteoblasts recent discoveries that a cell fate can be programmed maturation. However, Runx2 expression starts very by only few master transcriptional regulators, the cell early along osteoblastic lineage, and its role at the early fate along osteoblastic lineage is regulated by Runx2 steps of osteogenic commitment is scarcely understo- and Osterix factors, both indispensable for bone for- od. This research is focused on the fundamental me- mation [1, 2]. chanisms of osteoprogenitors programming by Runx2. Runx2 factor regulates expression of multiple tar- We have compared the transcriptomes of Runx2 null get genes via direct activation and repression of genes osteoprogenitor cells before and after their reconsti- tution with wild type Runx2 protein or its non-func- Ó Institute of Molecular Biology and Genetics NAS of Ukraine, 2010 tional mutant using Affymetrix microarrays. We have

273 TEPLYUK N. M., TEPLYUK V. I.. found that several clusters of functionally related genes Western blot analysis. Cell lysates were prepared respond to Runx2 re-introduction. They include the from cell pellets that were boiled in 100 ml of Direct genes related to the osteoblast-specific signaling net- Lysis buffer (50 mM Tris-HCl, pH 6.8, 2 % SDS, 10 % work, hormones biosynthesis and general metabolism. Glycerol, 12 % Urea, 25 mM MG132, 100 mM DTT These genes define osteoblast cell identity in the bone and 1 ´ Complete protease inhibitors) («Roche», USA). microenvironment. Aliquots of each lysate (5 ml) were separated in 10 % Materials and methods. Cell culture and adeno- sodium dodecyl sulfate (SDS)-polyacrylamide gel virus infections. Runx2 null calvaria osteoprogenitors electrophoresis (PAGE). Proteins were transferred cell line was developed previously from the fetal cal- using semi-wet blotting to nitrocellulose membranes varial region of Runx2 knock-out mice by stable inte- («Millipore», USA). Phosphatase buffered saline gration of Tert [4]. Runx2 null cells were maintained in (PBS) with 5 % non-fat milk was used for 1 h at room aMEM supplemented with 10 % fetal bovine serum temperature to block non-specific protein binding. (FBS) («Atlanta Biologicals», USA), 30 mM penicil- Primary and secondary antibodies were used at 1:2,000 lin-streptîmycin and 100 mM L-glutamine at 37 °C and dilutions for 1 h room temperature in PBS/0.1 %

5 % CO2 humidified atmosphere. Adenoviral vectors Tween (PBST) with 1 % milk. Signal was detected with containing cDNAs of full length Runx2 and its C- ECL (Perkin Elmer Western Lighting Chemilumines- terminal deletion mutants 1–361 (DC) were each cence Reagent Plus, «Perkin Elmer», USA). RUNX2- transferred into the AdenoVatorTM expression construct specific mouse monoclonal antibodies were a generous («Qbiogene», USA) from the corresponding pcDNA gift of Dr. Yoshiaki Ito (Institute for Molecular and expression vectors described previously. Cellular Biology, Singapore). CDK2 rabbit polyclonal Cells were plated for infections in 6-well plates antibodies (SC-163) were purchased from Santa Cruz (12.5×104 cells/well). After 24 h, cells were infected Biotechnology, Santa Cruz, USA. with 100 MOI of each virus in 600 ml of aMEM media Quantitative real-time reverse transcriptase PCR complemented with 1 % FBS for 4 h. Upon addition of (qRT-PCR) analysis. qRT-PCR was performed to vali- 400 ml media containing 1 % FBS, cells were incubated date changes for the sub-set of Runx2 responsive ge- for additional 10 h. nes. Specific qPCR primers were designed using Pri- Affymetrix analysis. Total RNA for Affymetrix mer 3 software. Total RNA for qRT-PCR assays was analysis (and subsequent qRT-PCR validation) was isolated as described, subjected to DNaseI digestion isolated with Trizol reagent and purified using the and purified using an RNA purification kit («Zy- RNeasy Mini Kit («Qiagen», USA). Analysis of gene mogen», USA). Aliquots of RNA (1 mg) were used for expression using Mouse Genome 430 2.0 Array was reverse transcription (First strand cDNA synthesis kit, performed as described earlier [5]. Data processing and «Invitrogen», USA) with random hexamer primers. sample comparisons were performed using an open Quantitative PCR was performed with Power SYBR source library for statistical analysis (BioConductor Green PCR Master Mix («Applied Biosystems», USA) library for R environment; http://www.bioconductor. using an automated system (Applied Biosystems 7300 org). Following Robust Multi-array Average expres- Real Time PCR System) with 0.5 pmoles/ml of the spe- sion measurement (RMA) and background correction, cific gene primers. the array values were subjected to quantile normaliza- Results and discussion. Functional clustering of tion assuming identical signal distributions in each of Runx2 responsive genes in osteoprogenitors. To inves- the arrays. Statistically significant differences between tigate Runx2 involvement in the programming of os- probe sets were evaluated using Student’s T test (p < teoprogenitors, we used the model system of early < 0.05). Functional annotation of Affymetrix probe sets mouse calvaria osteoprogenitors with Runx2 null back- and relationships between groups of co- ground. These cells are blocked at the very early stage regulated genes were assessed using the Database for of osteogenic lineage because of the absence of Runx2 Annotation, Visualization and Integrated Discovery gene; although they deposit into extracel- (DAVID 2.0) (http://david.abcc.ncifcrf.gov) [6]. lular matrix, they do not express other phenotypic mar-

274 BONE-SPECIFIC MASTER TRANSCRIPTION FACTOR RUNX2

A Runx2-WT Runx2-D361 GFP-EV

Fig. 1. Runx2 overex- pression in Runx2 null mouse calvaria cells: A – Runx2 wild type (WT), C-terminal truncated B C Runx2 mutant (aa 1– 361, DC) or GFP control Protein: RNA: protein were expressed via adenoviral infections 1 l 1 l 6 6 a T a T 3 s 3 n n s u -W D ig -W D ig u in Runx2 null cells (åqu- r 2 - s 2 - -s ir i x 2 - x 2 v v n x P n x P al infection efficiency o u n F u n F o u u N N R R G R R G was determined by GFP signal); B, C – åxoge- Runx2 Runx2 nous Runx2 protein and RNA expression levels were detected by Wes- Cdk2 Gapdh tern blot and RT-PCR respectively kers and are not able to differentiate and mineralize in Early osteoblast phenotypic genes are induced by osteogenic media (ascorbic acid and beta-glycero- Runx2 in osteoblasts progenitors. As we expected, the phosphate). After cells reconstitution with exogenous- functional clustering of results has shown that the ly expressed Runx2 protein the block is released and genes of the early/middle osteoblast differentiation cells progress along osteoblatic lineage, differentiate to encoding , , some normal osteoblasts and mineralize in osteogenic media. and Matrix metalloproteinases, were robustly induced To assess the global changes in gene expression du- by Runx2 within 1 day after Runx2 re-introduction ring the osteoblatic lineage the genome-wide Affy- (Fig. 2). The genes characteristic for the late diffe- metrix expression profiling was performed with the rentiation stage encoding Bone sialoprotein, Alkaline investigated cells reconstituted with either wild type phosphatase have not revealed detectable expression (WT) Runx2 protein or with its C-terminally truncated (data not shown). deletion mutant (Runx2-DC), which is non-functional Matrix metalloproteinases Mmp9, Mmp13 and so- because of the absence of transactivation and repres- me other collagen matrix processing enzymes (i. e. sion domains (Fig. 1). Loxl2, Loxl3, Tissue Inhibitors of Matrix Metallopro- Data analysis after Runx2 WT and Runx2-DC teinases Timp1 and Timp3) were robustly induced by introduction revealed total 1828 probe-sets and 1438 Runx2, while others (i. e. Timp2) were inhibited. Con- up- and down-regulated genes (with more than 1.4 fold sistently, a sub-set of bone-related Runx2 responsive changes, p < 0.05) (Table). Functional clustering of genes like secreted phosphoprotein (Spp1, Osteopon- these genes has shown that several clusters of func- tin), Bone gamma-carboxyglutamate (Bglap1, Osteo- tionally related genes are responsive to Runx2 in- calcin) were also up-regulated. Some genes in the list troduction, and include the genes related to cell signa- encode other master fate-determining transcription fac- ling pathways, general metabolism, transport, cell cyc- tor in osteoblasts acting up- and downstream of Runx2 le and osteoblast phenotype. The changes in expression protein (e. g. Core binding factor beta (Cbfb), Sp7 or of selected subset of 180 genes were validated by quan- Osterix). titative PCR (qPCR) analysis and revealed a significant Runx2 programs bone-related signaling network in consistency with the data obtained by microarray. osteoprogenitor cell. Along with the cluster of osteo-

275 TEPLYUK N. M., TEPLYUK V. I.

The number of genes up- and down-regulated by Runx2 in osteoprogenitors

Genes Functional cluster Probe-sets Total Up-regulation Down-regulation

Total change 1828 1438 660 778

Not annotated yet 211 198 75 123

Genes with unknown function 234 191 94 97

Miscellaneous 147 121 47 74

Signaling network 268 192 75 117

Biosynthesis/metabolism 135 102 60 42

Cytokines/Inflammatory response 121 87 38 49

Growth/Cell cycle 93 70 41 29

Cytoskeleton/transport/proteins sorting 90 68 27 41

Proteasome degradation 70 57 21 36

Channels/transporters 77 56 22 34

Bone phenotypic genes 61 45 22 23

RNA binding/splicing/metabolism 51 41 27 14

Myogenic phenotype 46 36 21 15

Adhesion/Migration/communications 42 32 3 29

Steroids biosynthesis/metabolism 37 31 16 15

Heat shock/Chaperones 47 30 25 5

Apoptosis 38 30 16 14

Function in nervous system 32 26 8 18

Translation 28 25 22 3 blast phenotypic gene markers, several other big clus- down-regulated in osteoprogenitors. Some of these ge- ters of interrelated genes were responsive to Runx2. nes are known to play a role in osteoblastic cells (e. g. The biggest of these clusters includes genes coding for non-genomic Gpr30, Gper), while cell signaling pathways, namely transmembrane recep- the function of other G-proteins coupled receptors (e. g. tors, extra- and intracellular modulators of their activi- Gpr54, Gpr23) in bone has to be determined. The four ty, cytoplasmic secondary messengers and the nuclear of the Small Regulators of G-proteins (Rgss) are rep- effectors. Among the most affected signaling pathways ressed by Runx2: Rgs2, 4, 5 and 16 (Fig. 3). Notably, we identified all known critical signaling components Rgs2 and Rgs4 are known as molecular switches in osteogenesis, including FGF, BMP/TGFbeta, Wnt directing the signal along different ways from PTH/ and small G-proteins coupled signaling (e. g. PTH/ PTHrP receptor [7, 8], the critical modulator of PTHrP, non-genomic steroid receptor Gpr30, cAMP osteoblastic differentiation, growth and function [9]. signaling, growth factors FGF and EGF signaling). Further investigation is needed to determine if these We further arranged a sub-cluster of Runx2 res- changes affect signaling pathway from PTH/PTHrP ponsive genes related to G-protein coupled signaling in receptor during differentiation. osteoblasts (Fig. 3). A bunch of G-protein coupled re- The second sub-cluster includes the extracellular ceptors and their interacting G-proteins are up- and matrix/proteoglycan genes known as the regulators of

276 BONE-SPECIFIC MASTER TRANSCRIPTION FACTOR RUNX2

FGF, Wnt and BMP signaling, critical in the bone cells critical role in osteoblasts development and bone for- [10, 11]. Heparane sulfate proteoglycanes (Syndeca- mation, both in vivo and in vitro [13]. nes) physically interact with FGF receptors as well as Moreover, proper hormone signaling involving all FGF ligands and regulate signaling activity depending classes of steroids is critical for bone development and on the types of Heparane sulfate chains present [12]. homeostasis. For example deficiency of Estrogen or The enzymes modifying chemical composition of Androgen receptor leads to severe osteoporosis; defi- Heparane sulfate chains components along with ciency of Vitamin D is well known to cause Rickets, Proteoglycans co-repressors themselves are Runx2 while glucocorticoid treatment is associated with glu- responsive (Fig. 4), indicating that Runx2 plays a role cocorticoid-induced osteoporosis. as a modulator of the whole FGF signaling system. Taken together, these results guided us to a novel Runx2 modulates steroid hormones biosynthesis fundamental concept that the master transcriptional and cholesterol metabolism machinery in osteoproge- regulator (i. e. Runx2) changes representation of the nitors. The second big cluster of genes responsive to total signaling network of the cell, and that such change Runx2 includes the genes responsible for cellular me- may be a principal part of the fate-determining program tabolism (Table), particularly for lipid/fatty acid, su- to define cell identity in the environment. In other gars and energy metabolism, red-ox homeostasis and words, due to reprogramming its signaling network, cholesterol/steroid hormones biogenesis (Fig. 5). The- Runx2 expressing osteoprogenitor reacts and behaves re are genes coding for the enzymes for several con- differently from its mother Mesenchymal stem cell in secutive steps of cholesterol biosynthesis (Fdps, Lss, the same bone microenvironment, retaining the osteo- Cyp51, Dhcr) following by Cholesterol downstream progenitor identity memory. The retention of such conversion to Pregnenolone (Cyp11a1, Fdxr) are indu- memory can be realized by chromatin remodeling (as ced by Runx2. Interestingly, the genes related to bone- Runx2 is a part of chromatin modification complexes) catabolic glucocorticoids signaling, including Gluco- as well as by the retention of Runx2 on mitotic chromo- corticoid receptors (Nr3c1, Nr2c2) and Glucocorti- somes through progenitor cells generations. coid-induced gene (Gig1, Zfp704) are inhibited by At the same time, using bone as a model system, it Runx2 expression (Fig. 5). Taken together these data is a part of broader biological problem of understan- suggest that Runx2 may promote the endogenous bio- ding the complete mechanism of cell specialization and genesis of some anabolic steroid hormones derivatives (re)programming by master regulatory factors. Still far of cholesterol in differentiating osteoblasts, while re- from understanding, this question is of the critical pressing signaling from catabolic glucocorticoids. importance in the new and fast growing field of stem We performed genome-wide screening of the genes cell-based regenerative medicine, which has tremen- that are directly or indirectly regulated by the bone dous potential for improving both healthcare and hu- master regulatory transcription factor Runx2 at the man wellbeing. early stage of osteoblastic commitment. We found that several interrelated functional gene programs are archi- Í. Ì. Òåïëþê , Â. ². Òåïëþê tected by the presence of Runx2 in osteoprogenitors, Òðà íñêðèïö³éíèé ôàê òîð Runx2 ðå ãó ëþº ãå íå òè÷í³ including early steps of bone matrix production, regu- ïðî ãðà ìè, ïî â’ÿ çàí³ ³ç ñèã íàë³íãîì ³ ìå òà áîë³çìîì lation of osteoblastic cell cycle, general metabolism, ïî ïå ðåä íèê³â îñòå îá ëàñò³â steroids biogenesis and the membrane/intracellular sig- Ðå çþ ìå naling network. Ìåòà. Òðà íñêðèïö³éíèé ôàêòîð Runx2 (AML3) º âàæ ëè âèì ðå - Genetic alterations of osteoblast specific signaling ãóëÿ òî ðîì äèôå ðåíö³àö³¿ îñòåîá ëàñò³â, íåîáõ³äí èì äëÿ ôîð - pathways that we have identified – FGF, PTH/PTHrP, ìó âàí íÿ ê³ñòîê. Åêñïðåñ³ÿ ãåíà Runx2 çðîñòຠâ ïðî öåñ³ äè- ôåðåíö³àö³¿ îñòåîá ëàñò³â ³ ïðèçâî äèòü äî àêòè âàö³¿ îñòåî- BMP, TGFb and Wnt signaling, cause hereditary syn- áëàñò-ñïåöèô³÷íèõ ãåí³â, â³äïîâ³äàëüíèõ çà ïðîäó êó âàí íÿ ì³íå- dromes with prominent bone abnormalities. Also, there ðàëü íî ãî ìàò ðèê ñó. Ìåòà ðî áî òè ïî ëÿ ãà ëà ó âèç íà ÷åíí³ ôóíêö³¿ ãåíà Runx2 ó ïî ïå ðåä íèêàõ îñòå îáëàñò³â, äå â³í åêñïðå- are tremendous amount of accumulated evidences that ñóºòüñÿ íà äîñèòü íèçü êî ìó ð³âí³. Ìåòî äè . Ìè äîñë³äèëè each of above-mentioned signaling pathways plays a ôóíêö³þ ãåíà Runx2 íà ðàíí³õ ñòàä³ÿõ ðîçâèò êó îñòå îáëàñò³â

277 TEPLYUK N. M., TEPLYUK V. I.

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Figures to article by N. M. Teplyuk, V. I. Teplyuk b a b a 1 1 1 1 b a 1 1 D D D D _ _ _ _ D D T T _ _ C C W W G G D D Day (numeric) Cat_WT Cat_DC Cat_GFP Dmp1 Dentin matrix protein 1 Dmp1 Dentin matrix protein 1 Dkk2 Dickkopf homolog 2 Cl15a1 Collagen, type XV, alpha 1 Adam 8 A disintegrin and metallopeptidase domain 8 Runx1 Runt related transcription factor 1 Col4a3bp Collagen, type IV, alpha 3 binding protein Runx1 Runt related transcription factor 1 Lepre1 Leprecan 1 Figf C-fos induced growth factor Figf C-fos induced growth factor Figf C-fos induced growth factor Adam 9 A disintegrin and metallopeptidase domain 9 (meltrin gamma) Timp2 Tissue inhibitor of metalloproteinase 2 Adam12 A disintegrin and metallopeptidase domain 12 (meltrin alpha) Timp2 Tissue inhibitor of metalloproteinase 2 Pcolce2 Procollagen C – endopeptidase enchancer 2 Adam19 A disintegrin and metallopeptidase domain 19 (meltrin beta) Cbfb Core binding factor beta Adam19 A disintegrin and metallopeptidase domain 19 (meltrin beta) Col4a5 Collagen, type IV, alpha 5 Timp2 Tissue inhibitor of metalloproteinase 2 Id1 Inhibitor of DNA binding 1 Col4a5 Collagen, type IV, alpha 5 Id3 Inhibitor of DNA binding 3 Col3a1 Collagen, type III, alpha 1 Timp2 Tissue inhibitor of metalloproteinase 2 Mmp9 Matrix metallopeptidase 9 Efhd1 EF-hand domain containing 1 Loxl2 Lysyl oxidase-like 2 Timp3 Tissue inhibitor of metalloproteinase 3 Mmp13 Matrix metallopeptidase 13 Slc6a4 Solute carrier family 6 ( transporter, serotonin) member 4 Enpp1 Ectonucleotide pyrophosphatase/ phosphodiesterase 1 Jam2 Junction adhesion molecule 2 Timp3 Tissue inhibitor of metalloproteinase 3 Gdf15 Growth differentiation factor 15 Loxl3 Lysyl oxidase-like 3 Timp3 Tissue inhibitor of metalloproteinase 3 Timp3 Tissue inhibitor of metalloproteinase 3 Bglap1 Bone gamma carboxyglutamate protein 1 (Osteocalcin) Spp1 Secreted phosphoprotein 1 (Osteopontin) Fig. 2. Osteoblast phe- Trpv2 Transient receptor potential cation channel, subfamily V, member 2 Grem1 Gremlin 1 notypic markers respon- Plod2 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 Plod2 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 sive to Runx2 WT ver- Timp1 Tissue inhibitor of metalloproteinase 1 Sp7 Sp transcription factor 7 (Osterix) sus Runx2 DC non-func- Col2a1 Collagen type II, alpha 1 Adamts4 A disintegrin-like and metallopeptidase (reprolysin-like) with throbmospondin type 1, motif 4 tional mutant as was Adamts4 A disintegrin-like and metallopeptidase (reprolysin-like) with throbmospondin type 1, motif 4 Loxl2 Lysyl oxidase-like 2 detected by functional Col6a2 Collagen type VI, alpha 2 Col6a2 Collagen type VI, alpha 2 clustering of Affymetrix Col6a1 Collagen type VI, alpha 1 P4ha2 Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha II polypeptide microarrays data

Galr2 receptor 2 Kiss1r Kiss 1 receptor (Gpr54) Gtpbp4 Gtp binding protein 4 Sgsm3 Small G-protein signaling modulator 3 Gna13 Guanine nucleotide binding protein, alpha 13 Drg2 Developmentally regulated GTP binding protein 2 Creb1 cAMP responsive element binding protein 1 Gpr137b G-protein coupled receptor 137b Gpr137b G-protein coupled receptor 137b Rgl1 Ral guanine nucleotide dissociation stimulator-like 1 Gper G-protein-coupled estrogen receptor 1 (Gpr30) Creb1 cAMP responsive element binding protein 1 Crem cAMP responsive element modulator Rgs5 Regulator of G-protein signaling 5 Gnaq Guanine nucleotide binding protein (G-protein), alpha q polypeptide Rgs16 Regulator of G-protein signaling 16 Rgs16 Regulator of G-protein signaling 16 Rgs16 Regulator of G-protein signaling 16 Gng2 Guanine nucleotide binding protein (G-protein), gamma 2 Gprc5b G-protein-coupled estrogen receptor, family C, group 5, member B Sox4 SRY-box containing gene 4 Gng2 Guanine nucleotide binding protein (G-protein), gamma 2 Gnaq Guanine nucleotide binding protein (G-protein), alpha q polypeptide Adcy7 Adenylate cyclase 7 Adcy7 Adenylate cyclase 7 Gng2 Guanine nucleotide binding protein (G-protein), gamma 2 Gng2 Guanine nucleotide binding protein (G-protein), gamma 2 Crem cAMP responsive element modulator Gng12 Guanine nucleotide binding protein (G-protein), gamma 12 Adcy7 Adenylate cyclase 7 Rgs2 Regulator of G-protein signaling 2 Rgs2 Regulator of G-protein signaling 2 Mrgprf MAS-related GPR, member F Gpr124 G-protein-coupled estrogen receptor 124 Rgs2 Regulator of G-protein signaling 2 S1pr2 Sphingosine-1-phosphate receptor 2 Mt2 Metallothionein 2 Rgs5 Regulator of G-protein signaling 5 Camp Cathelicidin antimicrobial Rgs5 Regulator of G-protein signaling 5 Rgs5 Regulator of G-protein signaling 5 Sox4 SRY-box containing gene 4 Gpr64 G-protein-coupled estrogen receptor 64 Sox4 SRY-box containing gene 4 Sox4 SRY-box containing gene 4 Rgs4 Regulator of G-protein signaling 4 Fig. 3. Runx2-responsi- S1pr1 Sphingosine-1-phosphate receptor 1 Sox4 SRY-box containing gene 4 ve genes related to G- Rgs4 Regulator of G-protein signaling 4 protein coupled signa-

–3.5 –1.5 0 –1.5 –3.0 ling in osteoprogenitors ISSN 0233-7657. Biopolymers and Cell. 2010. Vol. 26. N 4 b a b a 1 1 1 1 b a 1 1 D D D D _ _ _ _ D D T T _ _ C C W W G G D D Day (numeric) Cat_WT Cat_DC Cat_GFP

Lama3 , alpha 3 Nid1 Nidogen 1 Vcan Versican Vcam1 Vascular cell adhesion molecule 1 Chst1 Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 Lama1 Laminin, alpha 1 Vcam1 Vascular cell adhesion molecule 1 Sulf1 Sulfatase 1 Hs6st1 Heparan sulfate 6-O-sulfotransferase 1 Sulf1 Sulfatase 1 Sulf2 Sulfatase 2 Cd24a Cd24 antigen Cd24a Cd24 antigen Cd24a Cd24 antigen Vcam1 Vascular cell adhesion molecule 1 Lama4 Laminin, alpha 4 Lama4 Laminin, alpha 4 Sulf2 Sulfatase 2 Clec11a C-type lectin domain family 11, member a Fmod Fibromodulin Tspan6 Tetraspanin 6 Tspan6 Tetraspanin 6 Osgepl1 O-sialoglycoprotein endopeptidase-like 1 Mgea5 Meningioma expressed antigen 5 (hyaluronidase) Sdc1 Syndecan1 Sdc1 Syndecan1 Osgepl1 O-sialoglycoprotein endopeptidase-like 1 Osgepl1 O-sialoglycoprotein endopeptidase-like 1 Sdc1 Syndecan1 Sdc1 Syndecan1 Fig. 4. Runx2-respon- Gpc6 Glypican 6 sive genes related to Sdc4 Syndecan4 Sdc2 Syndecan2 FGF/proteoglycan sig- naling in osteoproge- –3.5 –1.5 0 –1.5 –3.0 nitors

Day (numeric) Cat_WT Cat_DC Cat_GFP

Sc5d Sterol-C5-desaturase homolog Lss Lanosterol synthase Tsc22d3 Tsc22 domain family, member 3 Cyp11a1 Cytochrom P450, family 11, subfamily a, polypeptide 1 Aldh3a1 Aldehydedehydrogenase family 3, subfamily A1 Cyp39a1 Cytochrom P450, family 39, subfamily a, polypeptide 1 Scarb1 Scavenger receptor class b, member 1 Fkbp4 Fk506 binding protein 4 Fdxr Ferredoxin reductase Cyp51 Cytochrom P450, family 51 Fdps Farnesyl diphosphate synthetase Dhcr7 7-dehydrocholesterol reductase Lss Lanosterol synthase Gper G-protein coupled estrogen receptor 1 Ebpl Emopamil binding protein-like Hsd3b7 Hydroxy-delta 5-sterol dehydrogenase 3-beta and steroid delta isomerase Osbpl2 Oxysterol binding protein-like 2 Zfp407 Zinc finger protein 407 Nr3c1 Nuclear receptor subfamily 3, group C, member 1 Ch25h Cholesterol 25-hydroxylase Nrip1 Nuclear receptor interacting protein 1 Osbpl3 Oxysterol binding protein-like 3 Cyp1b1 Cytochrom P450, family 1, subfamily b, polypeptide 1 Nr3c1 Nuclear receptor subfamily 3, group C, member 1 Por Cytochrome P450 oxidoreductase Ldlr Low density lipoprotein receptor Cyp20a1 Cytochrom P450, family 20, subfamily a, polypeptide 1 Osbpl6 Oxysterol binding protein-like 6 Cyp1b1 Cytochrom P450, family 1, subfamily b, polypeptide 1 Fig. 5. Runx2-respon- Nr3c1 Nuclear receptor subfamily 3, group C, member 1 sive genes related to Nr3c2 Nuclear receptor subfamily 3, group C, member 2 Nr3c1 Nuclear receptor subfamily 3, group C, member 1 steroid hormones sig- naling and metabolism –3.5 –1.5 0 –1.5 –3.0 in osteoprogenitors