Runx2 promotes both osteoblastogenesis and novel osteoclastogenic signals in ST2 mesenchymal progenitor cells

Abstract

We profiled the global gene expression of a bone marrow-derived mesenchymal pluripotent cell line in response to Runx2 expression. Besides osteoblast differentiation, Runx2 promoted the osteoclastogenesis of co-cultured splenocytes. This was attributable to the upregulation of many novel osteoclastogenic genes and the downregulation of anti-osteoclastogenic genes.

Introduction: In addition to being a master regulator for osteoblast differentiation, Runx2 controls osteoblast-driven osteoclastogenesis. Previous studies profiling gene expression during osteoblast differentiation had limited focus on Runx2 or paid little attention to its role in mediating osteoblast-driven osteoclastogenesis.

Methods: ST2/Rx2(dox), a bone marrow-derived mesenchymal pluripotent cell line that expresses Runx2 in response to Doxycycline (Dox), was used to profile Runx2-induced gene expression changes. Runx2-induced osteoblast differentiation was assessed based on alkaline phosphatase staining and expression of classical marker genes. Osteoclastogenic potential was evaluated by TRAP staining of osteoclasts that differentiated from primary murine splenocytes co-cultured with the ST2/Rx2(dox) cells. The BeadChip™ platform (Illumina) was used to interrogate genome-wide expression changes in ST2/Rx2(dox) cultures after treatment with Dox or vehicle for 24 or 48 h. Expression of selected genes was also measured by RT-qPCR.

Results: Dox-mediated Runx2 induction in ST2 cells stimulated their own differentiation along the osteoblast lineage and the differentiation of co-cultured splenocytes into osteoclasts. The latter was attributable to the stimulation of osteoclastogenic genes such as Sema7a, Ltc4s, Efnb1, Apcdd1, and Tnc as well as the inhibition of anti-osteoclastogenic genes such as Tnfrsf11b (OPG), Sema3a, Slco2b1, Ogn, Clec2d (Ocil), Il1rn, and Rspo2.

Conclusion: Direct control of osteoblast differentiation and concomitant indirect control of osteoclast differentiation, both through the activity of Runx2 in pre-osteoblasts, constitute a novel mechanism of coordination with a potential crucial role in coupling bone formation and resorption.

Fig. 1

Establishment of the ST2/Rx2^dox sub-line with conditional Runx2 expression. a ST2/Rx2^dox cells were treated with the indicated concentration of Dox for 48 h, and whole cell extracts were subjected to Western blot analysis with either anti-FLAG antibodies to detect FLAG-Runx2 or with anti-tubulin antibodies as control. b ST2/Rx2^dox cells were treated with 250 ng/mL Dox for 48 h and subjected to RT-qPCR analysis to of Runx2 mRNA. c ALP staining of ST2/Rx2^dox cells following Dox treatment for 48 h. d ST2/Rx2^dox cells were treated with Dox for 48 h and the mRNA level of the indicated genes assessed using RT-qPCR analysis. Bars represent the mean±SD (n=3) from a representative experiment, which was repeated at least three times with similar results. Transcript changes detected by RT-qPCR were significant with p < 0.05. GAPDH was used for normalization, and its expression did not significantly change by Dox treatment

Fig. 2

Unsupervised hierarchical clustering of Runx2-regulated genes. a Hierarchical clustering was carried out using the Minkowski distance function and the Ward clustering method for 216 genes that Runx2 stimulated or inhibited by ≥2.5 fold with p≤0.008 on either day 1 or day 2. The heat map represents z-scores across all 16 samples. b Heat map showing the z-scores for 20 upregulated and 20 downregulated genes with the most significant changes

Fig. 3

Runx2 decreases ST2 cell proliferation. a RT-qPCR analysis of IGF1 mRNA in ST2/Rx2^dox cells treated with Dox or vehicle. b MTT-based cell proliferation assay of ST2/Rx2^dox cultures treated with Dox or vehicle for the indicated time periods. c Representative phase contrast micrographs showing ST2/Rx2^dox cells after 24 and 48 h of treatment with vehicle or Dox

Fig. 4

Runx2 promotes osteoclastogenic potential of ST2 cells. a Primary mouse splenocytes were co-cultured with either the parental ST2 or ST2/Rx2^dox cells in the presence of Dox or vehicle. Osteoclasts are demonstrated by TRAP staining of day 13 cultures after collagenase digestion and washing of the ST2 cells. b Quantitation of TRAP-positive area in the same co-cultures (mean±SD, n=4). c–e RT-qPCR analysis of Runx2-regulated genes related to osteoclastogenesis. RT-qPCR data are presented as the mean±SD. All p values were <0.05, except for Hey1 where p=0.16

Fig. 5

Genes involved in Runx2-mediatd osteoblast-driven osteoclastogenesis. The model shows Runx2-stimulated pro-osteoclastogenic genes on the left and Runx2-inhibited anti-osteoclastogenic genes on the right. A hypothetical Runx2-driven self-limiting negative feedback loop is described at the top based on the microarray data and the reported inhibitory effects of the Notch pathway on Runx2 and on osteoclast differentiation