1. Myeloma-derived Dickkopf-1 disrupts Wnt-regulated osteoprotegerin and RANKL production by osteoblasts: a potential mechanism underlying osteolytic bone lesions in multiple myeloma
by Ya-Wei Qiang, Yu Chen, Owen Stephens, Nathan Brown, Bangzheng Chen, Joshua Epstein, Bart Barlogie, and John D. Shaughnessy
Blood
Volume 112(1):
July 1, 2008
©2008 by American Society of Hematology

2. Wnt3a induced increase in OPG mRNA and protein in osteoblast progenitor cells.
Wnt3a induced increase in OPG mRNA and protein in osteoblast progenitor cells. C2C12 cells (A,C) and Saos-2 cells (B,D) were treated with serial concentrations of recombinant Wnt3a for indicated times. The OPG mRNA (A,B) was amplified by qPCR analysis. The supernatant of treated cells (C,D) was harvested and subjected to ELISA for measurement of OPG protein. Protein in lysate (1 mg) was subjected to the GST-E-cadherin assay. After SDS-PAGE analysis, uncomplexed β-catenin was detected by anti–β-catenin antibody (C,D). The results are means plus or minus SD (n = 4). Results are representative of 3 independent experiments (*P < .05 vs control).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

3. DKK-1 inhibition of Wnt3a induced OPG mRNA and protein in osteoblast cells.
DKK-1 inhibition of Wnt3a induced OPG mRNA and protein in osteoblast cells. C2C12 (A) and Saos-2 (B) cells were stimulated with or without Wnt3a for 8 hours after prior treatment with recombinant DKK-1 for 1 hour at indicated concentrations and then lysed. A total of 0.5 mg of protein from cell lysates was subjected to the GST-E-cadherin assay. After SDS-PAGE, uncomplexed β-catenin was detected with anti–β-catenin antibody. The cells were cultured at 105/well in 6-well plate for 24 hours, and 100 ng/mL of Dkk1 was added for 1 hour followed by addition of 100 ng/mL of rWnt3a for 48 or 72 hours. Total RNA was isolated from treated C2C12 (C) and Saos-2 (D) cells after 48 hours and OPG mRNA was quantified. The supernatant of C2C12 (E) and Saos-2 (F) cells treated, as above for 72 hours, was harvested and subjected to ELISA for measurement of OPG. The results are shown as means plus or minus SD (n = 3). Results are representative of 3 independent experiments (**P < .01, ***P < .001, vs control).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

4. Ectopic expression of DKK1 diminished Wnt3a induced OPG mRNA and protein in osteoblast cells.
Ectopic expression of DKK1 diminished Wnt3a induced OPG mRNA and protein in osteoblast cells. The expression of DKK family members in C2C12 (A) and human osteoblast cell lines (B) as determined by RT-PCR analysis are presented. Concentration of DKK1 protein in culture supernatant of indicated cell lines by ELISA analysis (C). C2C12 cells were stable transfected with an empty vector or DKK1-expressing vector. DKK1 protein expression was detected by the anti-V5 antibody (D). DKK1 protein in the supernatant of the clones was determined by ELISA (E). The cells were treated with recombinant 100 ng/mL of rWnt3a. Relative OPG mRNA (F) and OPG protein concentration (G) was measured by qPCR or ELISA analysis as described in Figure 1. Data represent the means plus or minus SD (n = 3) of representative experiments (*P < .05, ***P < .001, and vs control).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

5. Knockdown DKK1 by shRNA restored Wnt3a-induced OPG in osteoblasts.
Knockdown DKK1 by shRNA restored Wnt3a-induced OPG in osteoblasts. C2C12 cells were transiently infected with supernatant containing control siRNA (shCont) or shRNA specific for DKK1 for indicated times. Total RNA was then isolated and subjected to RT-PCR for detecting DKK1 mRNA (A). cDNA from 24 hours was subject to qPCR to confirm DKK1 mRNA expression (B). Supernatants of the cells were harvested and subjected to ELISA for measuring DKK1 protein (C). The infected cells were treated with rWnt3a for 48 hours and RNA and supernatants were harvested and subjected to qPCR and ELISA analysis for OPG mRNA (D) and protein (E). Data represent the means plus or minus SD (n = 3) of representative experiments (**P < .01, ***P < .001, vs control).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

6. Coculturing osteoblast cells with DKK1- expressing MM cells inhibited Wnt3a-induced OPG. A MM cell line OPM-2 was transfected with pEF6 vector (designated OPM-2/EV) or the vector containing Dkk1 cDNA (OPM-2/DKK1).
Coculturing osteoblast cells with DKK1- expressing MM cells inhibited Wnt3a-induced OPG. A MM cell line OPM-2 was transfected with pEF6 vector (designated OPM-2/EV) or the vector containing Dkk1 cDNA (OPM-2/DKK1). DKK1 protein from OPM-2/EV or OPM-2/DKK1 cell lysates of selected clones was determined by Western blot analysis using anti-V5 antibody as described in “Immunoblotting analysis and GST-E cadherin binding assay” (A). The concentration of DKK1 protein in culture supernatants in OPM-2/EV and OPM-2/DKK1 cells was measured by ELISA analysis (B). C2C12 cells were cocultured with OPM-2/EV or OPM-2/DKK1 cells in the presence of rWnt3a or control for the indicted times. OPG synthesis in these cells, as measured by qPCR, is presented (C). Supernatants of the C2C12 were harvested and subjected to ELISA analysis to measure OPG protein concentration (D). The results are shown as means plus or minus SD (n = 4). Results are representative of 3 independent experiments (**P < .01, ***P < .001, vs control). C2C12 cells were cultured with primary CD138-positive plasma cells from 2 MM patients (P#1 and P#2) for 48 hours in the presence or absence of rWnt3a for 48 hours. The OPG mRNA in C2C12 cells was determined by qPCR (E). OPG protein levels were measured by ELISA analysis (F).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

7. Neutralization of DKK1 rescued OPG expression in osteoblasts grown in the presence of MM sera or primary MM cells.
Neutralization of DKK1 rescued OPG expression in osteoblasts grown in the presence of MM sera or primary MM cells. C2C12 cells were treated with BM sera (50% diluted with serum free DMEM medium) from MM patients (n = 8) containing low (L; 2.7 to 8.5 ng/mL) or high (H; to ng/mL) concentration of DKK1 or recombinant DKK1 (100 ng/mL) as a positive control or normal sera (50% diluted with serum free DMEM medium) for 2 hours. Then rWnt3a or control vehicle was added to the cell culture media, as described above, for 48 hours (A). The cells were treated with rWnt3a or control vehicle for 48 hours after prior treatment with 25% sera from MM patients (n = 21) containing mouse Ig or anti-DKK1 antibody or with control IgG for 2 hours (B). OPG mRNA was determined by qPCR from the RNA (**P < .01, ***P < .001, vs control). Error bars represent SD. C2C12 cells were cocultured with CD138-positive plasma cells (PC) from MM or the lymphoma ST486 cell line (negative control) in the presence or absence of Wnt3a, control IgG or anti-DKK1 antibody for 48 hours. Plasma cells in suspension were removed and harvested. The supernatants were harvested by centrifugation for 10 minutes. C2C12 cells were washed with BPS and homogenized for isolation of RNA. OPG mRNA was determined by qPCR analysis from total RNA isolated from C2C12 cells (C), and OPG protein in cell culture supernatants was measured by ELISA analysis (D).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology

8. DKK1 and sera from MM patients inhibits Wnt3a-induced suppression of RANKL in osteoblast.
DKK1 and sera from MM patients inhibits Wnt3a-induced suppression of RANKL in osteoblast. C2C12 (A), Saos-2 (B), and MG63 (C) cells were treated with rWnt3a or Wnt3a-CM (as indicated) or Cont-CM for 48 hours after prior treatment with 100 ng/mL of DKK1 protein for 2 hours. RANKL mRNA was analyzed by qPCR. C2C12 cells transfected with empty vector (pEF/EV) or the vector carrying DKK1 cDNA (pEF/DKK1) were cultured in presence or absence of rWnt3a protein (100 ng/mL). The RNA and supernatant were harvested and subjected to qPCR analysis to determine RANKL mRNA (D) or ELISA to measure RANKL protein (E). C2C12 cells were treated with rWnt3a protein for 48 hours after prior incubation with sera (50% diluted with fresh serum free DMEM medium) from MM patients (n = 8) containing low (< 10 ng/mL) or high concentration of DKK1 (> 100 ng/mL) or normal sera (50% diluted with serum free DMEM medium) for 2 hours (F). Total RNA was isolated and subjected to cDNA synthesis. RANKL mRNA was amplified by qPCR analysis. The results are shown as means plus or minus SD (n = 3; *P < .01, **P < .001, vs control).
Ya-Wei Qiang et al. Blood 2008;112:
©2008 by American Society of Hematology