Dasatinib promotes osteogenic differentiation of MSCs from MM patients and healthy donors and of the hMSC-TERT cell line.

<p>(A) Dasatinib upregulates the expression of bone-formation markers in the osteogenic differentiation process. Primary MSCs from MM patients and healthy donors were cultured in osteogenic medium in the presence (2–5 nM) or absence of dasatinib, and total RNA was isolated on days 7 and 14. Real-time qRT-PCR was used to determine the expression of several OB related markers: ALP was determined at day 7, whereas the transcription factors Runx2/Cbfa1 and Osterix (Osx), and collagen I type A 1 (COLIA1) were measured at day 14. Expression levels for each gene were normalized to GAPDH expression and referred to those in the absence of dasatinib. Graphs illustrate mean values from samples from 5 healthy donors and 5 MM patients ± SEM (<i>bars</i>) *<i>P</i><0.05. (B) Dasatinib increases ALP and Runx2/Cbfa1 activities in osteoprogenitor cells. In the hMSC-TERT cell line and in primary hMSCs derived from three myeloma patients and three healthy donors, ALP activity was measured at day 7 (upper graph) and Runx2/Cbfa1 activity was measured at day 14 (lower graph) after the addition of dasatinib to the osteogenic differentiation medium. Data are represented as the mean ± SD from three experiments. (C) Dasatinib (2–5 nM) augments bone matrix mineralization in OBs derived from the hTERT-MSC cell line (left) or MSCs from healthy donors and myeloma patients (right), as assessed by alizarin red staining quantification. Data are represented as the mean ± SD from three experiments with the hMSC-TERT cell line, and as the mean (5 MM patients and 5 healthy donors) ± SEM in experiments with primary MSCs. Statistically significant differences from controls are indicated as *, where <i>P</i> < 0.05. Micrographs show matrix mineralization after alizarin red staining of correspondent differentiated OBs. (D) Both dephospho- and phospho-β-catenin levels were determined by immunoblotting in cytosolic or nuclear lysates of pre-OBs differentiated from the hMSC-TERT cell line in the absence or presence of dasatinib. Histone H1 and α-tubulin were used as loading controls for nuclear or cytosolic protein fractions.