Src family kinase/abl inhibitor dasatinib suppresses proliferation and enhances differentiation of osteoblasts

Abstract

Dasatinib, a dual Src family kinase and Abl inhibitor, is being tested clinically for the treatment of prostate cancer bone metastasis. Bidirectional interactions between osteoblasts and prostate cancer cells are critical in the progression of prostate cancer in bone, but the effect of dasatinib on osteoblasts is unknown. We found that dasatinib inhibited proliferation of primary mouse osteoblasts isolated from mouse calvaria and the immortalized MC3T3-E1 cell line. In calvarial osteoblasts from Col-luc transgenic mice carrying osteoblast-specific Col1alpha1 promoter reporter, luciferase activity was inhibited. Dasatinib also inhibited fibroblast growth factor-2-induced osteoblast proliferation, but strongly promoted osteoblast differentiation, as reflected by stimulation of alkaline phosphatase activity, osteocalcin secretion and osteoblast mineralization. To determine how dasatinib blocks proliferative signaling in osteoblasts, we analyzed the expression of a panel of tyrosine kinases, including Src, Lyn, Fyn, Yes and Abl, in osteoblasts. In the Src family kinases, only Src was activated at a high level. Abl was expressed at a low level in osteoblasts. Phosphorylation of Src-Y419 or Abl-Y245 was inhibited by dasatinib treatment. Knockdown of either Src or Abl by lenti-shRNA in osteoblasts enhances osteoblast differentiation, suggesting that dasatinib enhances osteoblast differentiation through inhibition of both Src and Abl.

Figure 1

Dasatinib inhibits calvarial osteoblast (PMOs) proliferation. A, cell density and morphologic characteristics after treatment of PMOs with various concentrations of dasatinib for 72 h. PMOs were cultured in 10% FBS or in 0.1% BSA as indicated. B, quantification of cell numbers. C. Time course of effect of dasatinib on PMOs proliferation. The PMOs were cultured in 10% FBS and treated without or with 50 nM dasatinib. Data are means ± SD. *P < 0.05 vs. control (DMSO). The experiment was repeated three times with similar results.

Figure 2

Dasatinib treatment inhibits the proliferation of PMOs isolated from calvaria of Col-luc transgenic mice. A, DNA construct used for the generation of Col-luc mouse. The proximal 2.3-kb col1α1 promoter was used to drive the luciferase gene expression. B, measurement of luciferase activity in various organs and bioluminescence detection of luciferase expression in 4-day-old Col-luc mice. C, the cell density and luciferase activity of Col-luc PMOs after treatment with various concentrations of dasatinib for 72 h. The Col-luc PMOs were cultured in 10% FBS or in 0.1% BSA as indicated. Data are means ± SD. *P < 0.05 vs. control. The experiment was repeated at least three times with similar results; the data shown are representative of all the findings.

Figure 3

Dasatinib inhibits FGF-2–induced osteoblast proliferation. A, cell density after the MC3T3-E1 cells were treated with or without 50 nM dasatinib in either 0.1% BSA (left panel) or 10% FBS (middle panel). Right panel, time course of effect of dasatinib on MC3T3-E1 cell proliferation. B, PMOs isolated from Col-luc transgenic mice or MC3T3-E1 cells were untreated or treated with 10 ng/mL of FGF-2 in the presence or absence of 50 nM dasatinib. DMSO was used as the vehicle control. Data are means ± SD. *P < 0.05 vs. control. The experiment was repeated twice with similar results; the data shown are representative of all the findings.

Figure 4

Effects of dasatinib on the differentiation of osteoblasts. PMOs and MC3T3-E1 cells were cultured in osteoblast differentiation medium for the times indicated. A, alkaline phosphatase activity in cell lysates. The alkaline phosphatase activities of the control samples are also shown in the insert in Fig. 4A. Data are means ± SD. *P < 0.05 vs. control. B, osteocalcin concentration in the conditioned medium, as measured by using ELISA. The osteocalcin levels of the control samples are also shown in the insert in Fig. 4B. Data are means ± SD. *P < 0.05 vs. control. C, mineralized nodules detected on von Kossa staining. Right panel, higher magnification of mineralized nodules.

Figure 5

Expression of SFKs in osteoblasts. A, Western blot of osteoblast cell lysates with anti–p-SFK antibody. B, immunoprecipitation of Src, Lyn, Fyn, and Yes from MC3T3-E1 cell lysates followed by Western blotting with anti–p-SFK antibody. All the positive cell controls for immunoprecipitation were run on the same gel with MC3T3-E1 immunoprecipitation samples, however, the positive control lanes were separated for clarity purpose.

Figure 6

Effect of Src knock down on osteoblast differentiation. A, Experimental plan for knocking down Src in PMOs and examining its effect on osteoblast differentiation. B, The levels of Src in PMOs treated with lentiviral control shRNA (shC) or lentiviral Src shRNA (shSrc). C, alkaline phosphatase activity in cell lysates. Data are means ± SD. *P < 0.05 vs. control. D, osteocalcin concentration in the conditioned medium, as measured by using ELISA. Data are means ± SD. *P < 0.05 vs. control. E, mineralized nodules detected on von Kossa staining. The experiment was repeated twice with similar results.

Figure 7

Expression of Abl in osteoblasts and effect of Abl knockdown on osteoblast differentiation. A, Immunoblot of PMO or MC3T3-E1 cell lysate with anti–Abl antibody (upper panel). Immunoprecipitation of PMO or MC3T3-E1 cell lysates with anti–Abl and immunoblot with anti–pY245–Abl antibody (lower panel). B, The levels of Abl in PMOs treated with lentiviral control shRNA (shCont) or Abl shRNA (shAbl). C, alkaline phosphatase activity, osteocalcin concentration in the conditioned medium, and von kossa staining of PMOs treated with control shRNA (shCont) or Abl shRNA (shAbl) lentivirus. D, PMOs were treated with lentiviral Src shRNA (shSrc), or Abl shRNA (shAbl), or both. Data are means ± SD. *P < 0.05 vs. control.

Figure 8

Effects of dasatinib on osteonectin expression. Proteins in the conditioned medium from PMOs treated with dasatinib, Src shRNA, Abl shRNA, or both Src shRNA and Abl shRNA were separated on SDS-PAGE and immunoblotted with anti-osteonectin antibody.