Bone-Specific Master Transcription Factor Runx2 Regulates Signaling and Metabolism Related Programs in Osteoprogenitors
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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 genes 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. Gene 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 gene ontology 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 collagen 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 Osteocalcin, Osteopontin, some collagens 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