Tyrosine Kinase Inhibitors Regulate OPG through Inhibition of PDGFRβ

Abstract

Nilotinib and imatinib are tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). In vitro, imatinib and nilotinib inhibit osteoclastogenesis, and in patients they reduce levels of bone resorption. One of the mechanisms that might underlie these effects is an increase in the production of osteoprotegerin (OPG). In the current work we report that platelet-derived growth factor receptor beta (PDGFRβ) signaling regulates OPG production in vitro. In addition, we have shown that TKIs have effects on RANKL signaling through inhibition of the PDGFRβ and other target receptors. These findings have implications for our understanding of the mechanisms by which TKIs affect osteoclastogenesis, and the role of PDGFRβ signaling in regulating osteoclastogenesis. Further studies are indicated to confirm the clinical effects of PDGFRβ-inhibitors and to elaborate the intracellular pathways that underpin these effects.

Fig 1. Effect of TKIs on OPG in ST2 Cells.

Effect of nilotinib on OPG (A) gene expression and (D) protein production. Figs 1A and D have previously been published [29] (S2 File) and are reproduced for illustrative purposes with permission (S1 File). Effect of imatinib on OPG (B) gene expression and (E) protein production. (C) Effect of bosutinib on OPG gene expression. Gene expression is quantitated relative to the baseline control value. In the case of ST2 cells treated with bosutinib, individual time-points were not significantly different from control, thus the p-value for the overall difference between the treatment group and the control group is shown on Fig 1E. Data are mean ± SEM. ^nsnot significant, *p<0.05, **p<0.01, ***p<0.001 vs untreated control.

Fig 2. Effect of TKIs on Expression of OPG in Primary Cells.

Effect of (A) nilotinib, (B) imatinib, and (C) bosutinib on expression of OPG mRNA in primary rat osteoblasts. (D) Effect of nilotinib and imatinib on expression of OPG mRNA in murine bone marrow. Gene expression is quantitated relative to the baseline control value. Data are mean ± SEM. In the case of primary rat osteoblasts treated with nilotinib, imatinib and bosutinib, individual time-points were not significantly different from control, thus the p-value for the overall difference between the treatment group and the control group is shown on Figs 2A-C. ^nsnot significant, *p<0.05, ***p<0.001 vs untreated control at each time point.

Fig 3. Effect of PDGF-BB (PDGF) on OPG in ST2 Cells.

Effect of PDGF-BB (PDGF) on (A) expression of OPG mRNA and (B) production of OPG protein by ST2 cells. Gene expression is quantitated relative to the baseline control value. Data are mean ± SEM. *p<0.05, ***p<0.001 vs untreated control at each time point.

Fig 4. Effect of PDGF-BB (PDGF), Nilotinib and Imatinib on OPG in ST2 Cells.

Partial reversal of the effect of PDGF 10ng/ml by nilotinib 0.1μM on (A) expression of OPG mRNA in ST2 cells and (E) production of OPG protein by ST2 cells. Reversal of the effect of PDGF 10ng/ml by nilotinib 1.0μM on (B) expression of OPG mRNA in ST2 cells and (F) production of OPG protein by ST2 cells. Partial reversal of the effect of PDGF 10ng/ml by imatinib 0.1μM on (C) expression of OPG mRNA in ST2 cells and (G) production of OPG protein by ST2 cells. Reversal of the effect of PDGF 10ng/ml by imatinib 1.0μM on (D) expression of OPG mRNA in ST2 cells and (H) production of OPG protein by ST2 cells. Gene expression is quantitated relative to the appropriate baseline value. Data are mean ± SEM. ^nsnot significant, *p<0.05, **p<0.01, ***p<0.001 vs PDGF/nilotinib-treated or PDGF/imatinib-treated group. NIL, nilotinib. IM, imatinib.

Fig 5. Effects of PDGFRB Gene Silencing on OPG in ST2 and MC3T3-E1 Cells.

Effects of siRNA targeting PDGFRB on (A) expression of OPG mRNA in ST2 cells and (B) production of OPG protein by ST2 cells. The level of gene silencing achieved is indicated in the second row of the x-axis of each graph. Gene expression is quantitated relative to the baseline control oligo value. Data are mean ± SEM. ***p<0.001 vs control oligo. OPG protein levels were not significantly different at individual time-points between ST2 cells with PDGFRB gene silencing and those with control oligo, thus the p-value for the overall difference between the two groups is shown on Fig 5B. (C) Effects of siRNA targeting PDGFRB on PDGFRβ protein levels. The immunoblot presented is representative of at least three separate experiments. OPG gene expression (D) or protein secretion (E) in PDGFRB shRNA transduced MC3T3-E1 cells. The level of PDGFRB gene expression or protein is shown on the x-axis. Gene expression is quantitated relative to the levels in SHRNA-A control cells at baseline. Data are mean. (F) PDGFRβ protein levels in PDGFRB shRNA transduced MC3T3-E1 cells. Effects of siRNA targeting (G) PDGFRA or (H) ABL on expression of OPG mRNA in ST2 cells. Gene expression is quantitated relative to the baseline control oligo value. Data are mean ± SEM. * p<0.05 vs control oligo. OPG gene expression was not significantly different at individual time-points between ST2 cells with PDGFRA gene silencing and those with control oligo, thus the p-value for the overall difference between the two groups is shown on Fig 5G.

Fig 6. Effect of TKIs and PDGF-BB (PDGF) on RANKL.

Effect of (A) nilotinib, (B) imatinib, (C) bosutinib and (D) PDGF-BB (PDGF) on expression of RANKL mRNA in primary rat osteoblasts. Gene expression is quantitated relative to the baseline control value. Data are mean ± SEM. In the case of primary rat osteoblasts treated with imatinib, individual time-points were not significantly different from control, thus the p-value for the overall difference between the treatment group and the control group is shown on Fig 6B. ^nsnot significant, *p<0.05, **p<0.01, ***p<0.001 vs vs untreated control. Effect of nilotinib and imatinib 1 μM on expression of RANKL mRNA in murine bone marrow (Fig 6E). Data are mean ± SEM. Individual time-points were not significantly different from control, thus the p-value for the overall difference between the treatment group and the control group is shown on 6E. ^nsnot significant vs untreated control. Effect of PDGF 10ng/ml and nilotinib 1.0μM on expression of RANKL mRNA in ST2 cells (Fig 6F). Gene expression is quantitated relative to the appropriate baseline value. Data are mean ± SEM. ^nsnot significant, **p<0.01, ***p<0.001 vs PDGF/nilotinib-treated group. NIL, nilotinib.

Fig 7. Mechanisms by Which Inhibition of PDGFRβ by TKIs or Gene Silencing Inhibits Osteoclastogenesis.

TKIs or PDGFRB gene silencing (SiRNA) inhibit PDGFRβ signaling (1) with a resultant increase in OPG production (2). This reduces RANK-RANKL interaction (3) leading to inhibition of osteoclast differentiation (4). Additionally, TKIs reduce RANKL secretion but not through PDGFRβ-mediated mechanisms.