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Cell Growth Regulatory Role of Runx2 During Proliferative Expansion of Preosteoblasts

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Cell Growth Regulatory Role of Runx2 During Proliferative Expansion of Preosteoblasts University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 2003-09-23 Cell growth regulatory role of Runx2 during proliferative expansion of preosteoblasts Jitesh Pratap University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Life Sciences Commons, and the Medicine and Health Sciences Commons Repository Citation Pratap J, Galindo M, Zaidi SK, Vradii D, Bhat BM, Robinson JA, Choi J, Komori T, Stein JL, Lian JB, Stein GS, Van Wijnen AJ. (2003). Cell growth regulatory role of Runx2 during proliferative expansion of preosteoblasts. Open Access Articles. Retrieved from https://escholarship.umassmed.edu/oapubs/357 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. [CANCER RESEARCH 63, 5357–5362, September 1, 2003] Cell Growth Regulatory Role of Runx2 during Proliferative Expansion of Preosteoblasts1 Jitesh Pratap,2 Mario Galindo,2 S. Kaleem Zaidi, Diana Vradii, Bheem M. Bhat, John A. Robinson, Je-Yong Choi, Toshisha Komori, Janet L. Stein, Jane B. Lian, Gary S. Stein, and Andre J. van Wijnen3 Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655 [J. P., M. G., S. K. Z., D. V., J. L. S., J. B. L., G. S. S., A. J. v. W.]; Bone Metabolism/Osteoporosis, Women’s Health Research Institute, Wyeth Research, Collegeville, Pennsylvania 19426 [B. M. B., J. A. R.]; Department of Biochemistry, Kyungpook National University, Daegu, Republic of Korea 700-422 [J-Y. C.]; and Department of Molecular Medicine, Osaka University Medical School, Osaka, Japan 565-0871 [T. K.] ABSTRACT is required for normal osteogenesis. Runx2 deficiency or mutations affecting the function of Runx2 protein cause severe bone abnormal- ␣ The Runx2 (CBFA1/AML3/PEBP2 A) transcription factor promotes ities in mouse and human (2–5). Deletion of the COOH terminus of lineage commitment and differentiation by activating bone phenotypic Runx2, which interacts with a series of cell signaling responsive genes in postproliferative osteoblasts. However, the presence of Runx2 in actively dividing osteoprogenitor cells suggests that the protein may also cofactors, generates bone defects that are comparable with the Runx2 participate in control of osteoblast growth. Here, we show that Runx2 is null mouse (5). Runx2 is up-regulated during osteoblast differentia- stringently regulated with respect to cell cycle entry and exit in osteo- tion to support the activation of bone-specific genes. However, Runx2 blasts. We addressed directly the contribution of Runx2 to bone cell is already expressed at early stages of chondrogenesis (6–9), in proliferation using calvarial osteoblasts from wild-type and Runx2-defi- actively proliferating immature osteoblasts (10, 11), and in C2C12 cient mice (i.e., Runx2؊/؊ and Runx2⌬C/⌬C). Runx2⌬C/⌬C mice express a myoblast cells before BMP-2-dependent osteogenic differentiation protein lacking the Runx2 COOH terminus, which integrates several cell (10–12). The expression of Runx2 in distinct proliferating mesenchy- proliferation-related signaling pathways (e.g., Smad, Yes/Src, mitogen- mal cell types does not necessarily result in activation of mature bone activated protein kinase, and retinoblastoma protein). Calvarial cells but phenotypic markers. These observations raise the question of whether not embryonic fibroblasts from Runx2؊/؊ or Runx2⌬C/⌬C mutant mice Runx2 has a regulatory function in proliferating osteoblasts before exhibit increased cell growth rates as reflected by elevations of DNA osteoblast maturation. synthesis and G1-S phase markers (e.g., cyclin E). Reintroduction of Runx2 into Runx2؊/؊ calvarial cells by adenoviral delivery restores strin- In this study, we provide evidence that Runx2 is tightly regulated gent cell growth control. Thus, Runx2 regulates normal osteoblast prolif- during entry into and exit from the cell cycle, and that Runx2 supports eration, and the COOH-terminal region is required for this biological stringent control of osteoblast cell growth. Hence, our results indicate that function. We propose that Runx2 promotes osteoblast maturation at a key Runx2 has a dual biological role in the osteogenic lineage by attenuating developmental transition by supporting exit from the cell cycle and acti- osteoblast growth and promoting bone phenotype maturation. vating genes that facilitate bone cell phenotype development. INTRODUCTION MATERIALS AND METHODS Stringent positive and negative control of the proliferative expan- Cell Growth Analysis. The osteoblastic cell line MC3T3-E1 was main- sion of mesenchymal cells, osteoprogenitor cells, and immature os- tained in ␣-MEM supplemented with 10% FBS.4 Cells were seeded in either teoblasts is critical for normal skeletal development and bone forma- six-well or 100-mm plates at 0.08 ϫ 106 cells/well or 0.4 ϫ 106 cells/plate, tion. Osteoprogenitors represent mesenchymal cells that are respectively. The growth medium was changed every 2 days and cultured until committed to the bone lineage and can differentiate into osteoblasts, confluent (at 8 days). For serum deprivation experiments, cells were grown for ␣ which are the principal cells that contribute to skeletogenesis by 3 days, then washed three times in PBS, and refed with MEM plus 10, 5, 2.5, 1, or 0% FBS. Cells were maintained in culture for 2 days before harvesting. mediating extracellular matrix mineralization. Osteoprogenitors pro- Growth rates were assessed by cell counting and FACS analysis. liferate in response to mitotic growth factors and must expand into the MC3T3 cells were synchronized in the G0/G1 phase of the cell cycle by appropriate number of osteoblasts to support normal formation of serum starvation. Briefly, exponentially growing cells in ␣-MEM plus 10% distinct skeletal elements. Osteoprogenitor expansion reflects the bal- FBS were washed three times in PBS on day 3 and cultured in serum-free ance of cell growth and survival. This balance is controlled by both medium for 48 h. Then, the cells were stimulated to progress through the cell circulating factors (e.g., growth factors, cytokines, and steroid hormones) cycle by removing medium and adding ␣-MEM plus 10% FBS. After serum and tissue architecture-related signals (e.g., cell-cell contact and cell stimulation, cells were harvested at selected time points for Western blot adhesion) that have either growth-stimulatory or inhibitory effects. analysis and FACS analysis. Cell growth control is mediated in part at the transcriptional level, The distribution of cells at specific cell cycle stages was evaluated by flow and there are cell cycle stage-specific demands for de novo synthesis cytometry. Cells were trypsinized, washed with PBS, and fixed in 70% ethanol at Ϫ20°C overnight. Cells were stained with propidium iodide and subjected of proteins (e.g., histones and cyclins; Ref. 1). Yet, there is a paucity to FACS analysis based on DNA content (13). The samples (1 ϫ 106 cells) of data on transcription factors known to control cell growth of were analyzed for cell cycle distribution using the FACStar cell sorter and osteoblasts. The Runt-related transcription factor Runx2 has a well- Consort 30 software (Becton Dickinson, Mountain View, CA). defined role in mediating the final stages of osteoblast maturation and Calvarial osteoblasts were isolated from wild-type and homozygous mouse embryos at 17.5 dpc. Runx2-deficient mice were identified by soft X-ray Received 3/21/03; revised 5/29/03; accepted 6/9/03. analysis and genotyped by using PCR analysis as described previously (5). The costs of publication of this article were defrayed in part by the payment of page Normal diploid osteoblasts are obtained from the central bone area (i.e. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grants AR39588 and AR48818. 4 The abbreviations used are: FBS, fetal bovine serum; FACS, fluorescence-activated 2 These authors contributed equally to this study. cell sorter; TCA, trichloroacetic acid; RT-PCR, reverse transcription-PCR; dpc, days 3 To whom requests for reprints should be addressed, at Department of Cell Biology postcoitum; GFP, green fluorescent protein; FGF, fibroblast growth factor; FGFR, fibro- and Cancer Center, University of Massachusetts Medical School, 55 Lake Avenue North, blast growth factor receptor; TGF, transforming growth factor; CDK, cyclin-dependent Worcester, MA 01655. Phone: (508) 856-5625; Fax: (508) 856-6800; E-mail: andre. kinase; CBFA, core binding factor alpha; PEBP, polyoma enhancer binding protein; [email protected]. AML, acute myelogenous leukemia; BMP, bone morphogenetic protein. 5357 RUNX2 CONTROL OF OSTEOBLAST PROLIFERATION removing suture tissue) of calvaria from 17.5 dpc embryos. Cells were isolated and maintained as described previously (14). Briefly, calvaria were minced and subjected to three sequential digestions (8, 10, and 26 min) with collagenase P (Roche Molecular Biochemicals, Indiana, IN) at 37°C. Osteoblasts in the third digest were collected and resuspended in ␣-MEM supplemented with 10% FBS. Cells were plated at a density of 1 ϫ 106 cells/six-well plate. Measurement of DNA synthesis in calvarial cells was performed by [3H]thymidine incorporation (15). Briefly, calvarial cells isolated from wild- type or homozygotes were plated in 12-well plates at 5 ϫ 104 cell/well. After 24 h, [3H]thymidine was added to culture medium to a final concentration of 5 ␮Ci/ml and incubated at 37°C for 30 min. Medium was removed by aspiration, and cells were washed twice with ice-cold serum-free ␣-MEM. Cells were extracted twice with 10% TCA on ice for 5 min. TCA precipitates were solubilized by adding 10% SDS for 2 min at room temperature. Cells were harvested, and the amount of radioactivity was measured by liquid scintillation counting (Beckman Instruments, Inc., Fullerton, CA).
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