The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Abstract

Background: The proto-oncogene Src is an important non-receptor protein tyrosine kinase involved in signaling pathways that control cell adhesion, growth, migration and differentiation. It negatively regulates osteoblast activity, and, as such, its inhibition is a potential means to prevent bone loss. Dasatinib is a new dual Src/Bcr-Abl tyrosine kinase inhibitor initially developed for the treatment of chronic myeloid leukemia. It has also shown promising results in preclinical studies in various solid tumors. However, its effects on the differentiation of human osteoblasts have never been examined.

Methods: We evaluated the effects of dasatinib on bone marrow-derived mesenchymal stromal cells (MSC) differentiation into osteoblasts, in the presence or absence of a mixture of dexamethasone, ascorbic acid and beta-glycerophosphate (DAG) for up to 21 days. The differentiation kinetics was assessed by evaluating mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers (receptor activator of nuclear factor kappa B ligand [RANKL], bone sialoprotein [BSP], osteopontin [OPN]).

Results: Dasatinib significantly increased the activity of ALP and the level of calcium deposition in MSC cultured with DAG after, respectively, 7 and 14 days; it upregulated the expression of BSP and OPN genes independently of DAG; and it markedly downregulated the expression of RANKL gene and protein (decrease in RANKL/OPG ratio), the key factor that stimulates osteoclast differentiation and activity.

Conclusions: Our results suggest a dual role for dasatinib in both (i) stimulating osteoblast differentiation leading to a direct increase in bone formation, and (ii) downregulating RANKL synthesis by osteoblasts leading to an indirect inhibition of osteoclastogenesis. Thus, dasatinib is a potentially interesting candidate drug for the treatment of osteolysis through its dual effect on bone metabolism.

Figure 1

Quantitative analysis of matrix calcification and alkaline phosphatase activity during osteogenic differentiation of MSC. A: Calcium content of the extracellular matrix of control cell cultures (open bars) and DAG-treated cell cultures (black bars) was determined after 7, 14 and 21 days. B: ALP activity in MSC cultured in control (open bars) or osteogenic (black bars) medium was measured after 7, 14 and 21 days. ALP activity (U, μmol p-nitrophenol released per min) was normalized for 10⁵ cells. A, B: Results are presented as mean ± SEM from 5 independent experiments performed in duplicate; *** p < 0.001 versus the corresponding control.

Figure 2

Semi-quantitative evaluation of expression of osteoblast-related genes in MSC treated or not with DAG for 7, 14 and 21 days. Gene expressions were divided into 3 groups. A: a gene whose expression was downregulated by DAG exposure, B: 4 genes whose expressions did not change under treatment, and C: 6 genes whose expressions were upregulated by treatment. A, B, C: Total RNA was isolated from MSC and subjected to semi-quantitative RT-PCR analysis using specific primers for osteoblast gene markers and β-actin (see Table 1). Amplified products were separated by electrophoresis, quantified by densitometry and results were corrected by comparison with the housekeeping gene. Data are presented as mean ± SEM from 5 independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the corresponding gene expression at time 0.

Figure 3

Scheme indicating that a combination of 5 osteoblast-related parameter levels/activity (Ca⁺⁺ deposition, ALP activity, and RANKL, BSP, OPN gene expression) can be used as an indicator of the stage of MSC differentiation into osteoblasts. Using this setting, 4 stages were defined: (i) undifferentiated MSC (=native stage) expressed high RANKL levels, but no BSP, no OPN, no ALP and no mineralization activity, (ii) osteoprogenitors (=early stage observed after 7 days of DAG treatment) expressed high BSP, moderate RANKL, moderate OPN levels, but had low ALP and no mineralization activity, (iii) mature osteoblasts (=intermediate stage reached after 14 days of DAG treatment) had a high ALP activity, expressed high BSP, high OPN, moderate RANKL levels and had low mineralization activity, and (iv) late osteoblasts (=late stage obtained after 21 days of DAG treatment) had a high mineralization activity, expressed high BSP, high OPN and moderate RANKL levels and had moderate ALP activity.

Figure 4

Proliferation rate of MSC exposed to dasatinib alone or in combination with DAG. A: Cells were treated for 3, 7 and 10 days with increasing concentrations of the drug (10^-9 - 10^-4 M) or vehicle (Control) in complete culture medium. B: Cells were exposed to increasing concentrations of the dasatinib (10^-9 - 10^-4 M) or vehicle (Control) in complete culture medium supplemented or not with DAG for 3, 7 and 10 days. A, B: Cell proliferation was determined by a colorimetric assay using the Cell Proliferation Reagent WST-1. The results are presented as mean ± SEM from 4 independent experiments performed in duplicate; * p < 0.05 versus the corresponding control.

Figure 5

Effect of dasatinib or E804 on Src phosphorylation. A: Cells treated for 30 min or 24 hours with 10^-8, 10^-7 M dasatinib or vehicle (Control) in complete culture medium. B: Cells exposed to 10^-7 M dasatinib and/or DAG or vehicle for 24 hours. C: Cells stimulated for 24 hours with 10^-7 M E804 or vehicle. A, B, C: Proteins (30 μg) were resolved by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Immunodetections were performed using p-Src (Tyr 419) or Src antibodies, secondary antibodies and chemiluminescent substrate. Representative results from 2 independent experiments.

Figure 6

Quantitative determination of matrix calcification and alkaline phosphatase activity in MSC exposed to dasatinib or E804. A: Matrix calcium content of untreated MSC (Control), and cells cultured with 10^-8 M dasatinib, DAG or a combination of both treatments, was assessed after 7, 14 and 21 days. B: ALP activity in MSC cultured in control medium supplemented or not with 10^-8 M dasatinib and/or DAG was measured after 7, 14 and 21 days. ALP activity (U, μmol p-nitrophenol released per min) was normalized. C: ALP activity in MSC exposed to 10^-7 M E804 and/or DAG for 7, 14 and 21 days. ALP activity (U, μmol p-nitrophenol released per min) was normalized normalized for 10⁵ cells. A, B, C: Results are presented as mean ± SEM from 4 independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the corresponding control, and comparing DAG versus Dasatinib+DAG or versus E804+DAG.

Figure 7

Quantitative evaluation of expression of osteoblast-related genes in dasatinib-treated MSC. Cells were treated or not with 10^-8 M dasatinib and DAG, alone or in combination. RNA was isolated from MSC and subjected to real-time quantitative PCR using specific primers for osteoblast gene markers (RANKL, OPG, BSP, OPN) and β-actin (see Table 2). Data are presented as mean ± SEM from 4 independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the corresponding control, and comparing DAG versus Dasatinib+DAG.

Figure 8

Evaluation of RANKL/OPG ratio by ELISA in conditioned medium of MSC exposed to dasatinib. Cells were treated or not with 10^-8 and 10^-7 M dasatinib in the absence of DAG for 3 and 7 days. 24-hours conditioned medium were collected at the end of the treatment time. RANKL and OPG protein levels were measured by ELISA in conditioned medium (1 ml/10⁵ cells) and normalized to total protein content. Data are presented as RANKL/OPG ratio (mean ± SEM from 4 independent experiments). *** p < 0.001 versus the corresponding control.