Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

Abstract

Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)-induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2(dox) cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2-driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT-qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co-treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox-driven alkaline phosphatase (ALP) activity in control ST2/Rx2(dox) cells, but not in cells in which Wif1 was silenced. Unlike its anti-mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S-phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2-driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2(dox) cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 232: 145-153, 2017. © 2016 Wiley Periodicals, Inc.

Fig. 1

GCs synergize with Runx2 in stimulating a select gene set. (A) ST2/Rx2^dox mesenchymal pluripotent cells were treated for 48 h in quadruplicate with vehicle control (C), dox (to induce Runx2; D), the synthetic GC dex (1 μM; G), or dox and dex together (DG). Global gene expression was profiled using the Illumina BeadChip platform and the response of genes to dex in the presence of dox is illustrated as a volcano plot, with red and blue symbols representing genes that were stimulated or repressed, respectively, by dox alone (D/C ≥ 2 or D/C ≤ −2, respectively; FDR P < 0.05). Data representing genes that did not significantly respond to dox alone by these criteria are in gray. NC, no change. Triangles represent genes that were strongly stimulated by dex in the absence of dox (G/C ≥ 5, FDR P < 0.05). Dashed lines highlight a set of 8 genes at the top right that were strongly stimulated (≥ 8 fold; P < 1e–08) by dex in the presence of dox. (B–G) ST2/Rx2^dox cells were treated for 24, 48, or 72 h with dox and/or dex at the depicted concentrations, and expression of the indicated genes was measured by RT-qPCR. Data are means and SDs (n = 3) relative to the control values at 24 h, defined as 1. [Color figure can be viewed at wileyonlinelibrary.com].

Fig. 2

Robust synergistic stimulation of Wif1 by Runx2 and GCs is unique among Wnt antagonists. (A) ST2/Rx2^dox cells were treated for 72 h as indicated and Wifi protein levels were assessed by Western blot analysis of either whole cell extracts (WCE) or conditioned media (CM). Western blot analysis of β-actin in the cell layer was carried out as control. (B–D) Expression of mRNAs for the indicated Wnt antagonists was measured as in Figure 1B–G. Data represent the means and SDs (n = 3) relative to the control values at 48 h (defined as I).

Fig. 3

Synergistic stimulation of Wif1 by Runx2 and GCs in primary osteoblast cultures. Pre-osteoblasts isolated from newborn mouse calvariae were transiently transfected with RUNX2 or a control plasmid and then treated for 72 h with 1.0 μM dex or vehicle. Expression of the indicated genes was assessed by either Western blot analysis of whole cell extracts (A) or (B–E). (F, G) Untransfected cells were treated with differentiation medium on day 2 of culture. Dex (1 μM) or vehicle was added for 72 h before lysis, followed by Western blot analysis of Runx2 (F) and RT-qPCR analysis of Wif1 (G) on days 5 and 7. Bars represent mean ± SD (n = 3).

Fig. 4

Effect of Wif1 silencing on Wnt target gene expression in ST2 cells expressing Runx2 and treated with GCs. A–B. ST2/Rx2^dox cells were transduced with lentiviruses encoding a nonspecific hairpin RNA (shNS) or either of two hairpins (shWif1¹ or shWif1²) targeting distinct regioion in Wif1 mRNA. The derived shRNA-expressing sub-lines were treated for 72 h with dox and/or dex as indicated and Wif1 expression was measured by RT-qPCR (A, Mean ± SD; n = 3) and by Western blotting (B). +/− signs indicate presence or absence of dox and dex in both A and B. The RNA data in A are corrected for 18S RNA and Coomassie blue-stained proteins that remained in the SDS–PAGE gels after transfer are shown as a loading control in B. (C, D) The RNAs from Figure 4A were subjected to RT-qPCR analysis of Axin2 and Ccnd1 (mean ± SD, n = 3).

Fig. 5

Wif1 silencing mitigates GC-mediated suppression of Runx2-driven osteoblast differentiation. (A) ST2/Rx2^dox cells expressing a non-specific shRNA (shNS) or one that targets Wif1 mRNA (shWif1¹) were treated for 6 days by dox and/or dex as indicated, and then subjected to ALP staining. (B) ALP activity was assessed as in A, and the stained area was quantified using the ImageJ software. Bars represent Mean ± SD values from three independent experiments, where the average stained area in the dox-treated cultures is defined as 100. *P < 0.05.

Fig. 6

Wif1 silencing mitigates GC-mediated suppression of Runx2-driven proliferation in mesenchymal pluripotent cells undergoing osteoblast differentiation. Quadruplicate ST2/Rx2^dox cells expressing either shNS or shWif1 as in Figure 4 were treated as indicated commencing one day after plating (defined as Day 0). (A) Percentage of S-phase cells was determined by propidium iodide staining and flow cytometry. (B) Cell growth rates were calculated based on MTT assays as the differences between the day 4 values and the respective day 0 values (just prior to commencement of treatment), or between the day 7 values and the respective day 4 values. *P < 0.05. Complete raw data from the FACS and the MTT assays are presented in Supplemental Tables S3 and S4, respectively.

Fig. 7

GCs hijack Runx2. (A) Working model. Black arrow depicts adaptive circuits that enhance osteoblast-driven bone formation in response to bone loss (by any mechanism). GCs cause bone loss through a myriad of reported mechanisms (1), and at the same time synergize with Runx2 (2) to stimulate Wif1. Stimulation of Runx2 by homeostatic mechanisms is now hijacked to exacerbate GIO. (B) Schematic depiction of the transient expression of Runx2 at the initiation of osteoblast differentiation and the downregulation of Runx2 expression and/or genomic occupancy as the osteogenic program progresses (Komori, 2010; Meyer et al., 2014; Yu et al., 2015). C, Illustrative representation of results (Smith et al., 2000; Gabet et al., 2011; Mikami et al., 2011) suggesting that GCs do not inhibit osteoblast differentiation in vitro when administered at or after a time point that coincides with Runx2 down-regulation.