Attenuation of WNT signaling by DKK-1 and -2 regulates BMP2-induced osteoblast differentiation and expression of OPG, RANKL and M-CSF-5

<p><b>Copyright information:</b></p><p>Taken from "Attenuation of WNT signaling by DKK-1 and -2 regulates BMP2-induced osteoblast differentiation and expression of OPG, RANKL and M-CSF"</p><p>http://www.molecular-cancer.com/content/6/1/71</p><p>Molecular Cancer 2007;6():71-71.</p><p>Published online 30 Oct 2007</p><p>PMCID:PMC2173906.</p><p></p>ml Noggin, or plain M-CM (two columns at the top). Cells were grown for 3 days in Wnt3a-CM or L-CM that did not contain Wnt3a (control). Total RNA was extracted and mRNA levels of M-CSF were determined using real-time RT-PCR. Data are shown as means ± SD based on 5 independent experiments. Number signs and asterisks indicate significant changes (< 0.005) compared to untreated cells and Wnt3a-treated cells, respectively. , Dkk1 restores Wnt3a-dependent decrease in M-CSF protein secreted into the extracellular milieu. Supernatant was harvested and M-CSF was determined using ELISA. Cells were treated and results are presented as described above. , Wnt3a-dependent loss of secreted M-CSF is abrogated in BMP2-treated cells by Dkk1. Cells were pre-incubated as described in panel A. Cells were grown for 3 days in L-CM () or Wnt3a-CM (), both containing 100 ng/ml BMP2 where indicated. Cells cultured in L-CM (no Wnt3a) served as control. Supernatant was harvested and M-CSF was determined using ELISA. Results are shown as means ± SD based on 5 independent experiments. Number signs, asterisks and paragraph symbols denote significant differences (< 0.005) compared to untreated cells, BMP2-treated cells and Wnt3a/BMP2-treated cells, respectively. Note that the control here is identical to the control in panel B and that the top-most bar in the bottom panel is identical to the "Wnt3a" bar in panel B. , Wnt3a-dependent loss of membrane-associated M-CSF is abrogated in BMP2-stimulated cells by treatment with Dkk1. Cells were pre-incubated and cultured as in panel C followed by FACS analysis using antibody to M-CSF (open histograms) or isotype-matched unrelated antibody (filled histograms). BMP2-stimulated cells or cells left untreated demonstrated very small, if any, changes in surface M-CSF expression upon treatment with Dkk1. Cells co-stimulated with BMP2 and Wnt3a, however, showed a marked increase in M-CSF reactivity in presence of Dkk1. Similar results were obtained in three independent experiments