Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma  Ya-Wei Qiang, Bart Barlogie, Stuart Rudikoff, John D. Shaughnessy  Bone  Volume 42, Issue 4, Pages 669-680 (April 2008) DOI: 10.1016/j.bone.2007.12.006 Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 1 Frizzled (Fz) and LRP5/6 mRNAs are expressed in osteoblast cells. Total RNA was extracted from the indicated cell lines. RT-PCR was performed with mouse (A) and human (B) primers specific for Fz1 through 10 and LRP5/6 (mouse, C; human, D), respectively. GAPDH was included as control. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 2 Canonical Wnt signaling is functional in osteoblast (OB) cells. OB cells were treated with Wnt3a CM or control CM for the indicated time and cell lysate harvested. Lysate protein was subjected to GST–E-cadherin assay and immunoblotting analysis using anti-β-catenin antibody (A) and anti-non-phosphorylated form (B) as in Materials and methods. RT-PCR was performed using specific primers for the indicated mouse (C) and human (D) genes in the indicated cell lines. C2C12 cells transfected using wild type (TOPflash) or mutant (FOPflash) LEF/TCF reporter luciferase constructs and pSV-b-galactosidase vector (transfection efficiency internal control) (E) were treated with Wnt3a CM or control CM prior to determination of luciferase activity. Results are shown as mean±SD (n=3) and are representative of three independent experiments. ⁎⁎p<0.01 versus control. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 3 Dkk-1 and MM patient sera inhibit Wnt3a induced accumulation of β-catenin in OB cells. (A) The indicated cells were treated with recombinant Dkk-1 at the indicated concentrations and then stimulated with Wnt3a or control CM. Proteins in cell lysate were subjected to GST–E-cadherin assay and immunoblotting analysis using anti-non-phosphorylated β-catenin as described in Materials and methods. (B) Dkk1 protein in cell lysate (upper panel) and CM (middle panel) from OPM-2 clones expressing PEF6/V5-His-TOPO-Dkk-1 (Dkk1) or vector (EV) was confirmed by immunoblotting blotting with anti-V5 antibody as described in Materials and methods. (C) C2C12 cells were incubated with Dkk1 or EV CM from OPM-2 transfected cells then treated with Wnt3a or control CM. Proteins in lysates were analyzed as in Fig. 2 using the indicated antibodies. (D) C2C12 cells were incubated with sera from MM patients containing low (L1) or high (H1–4) concentrations of Dkk1 protein. Cells were then treated with Wnt3a or control CM and lysates prepared and analyzed as in C. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 4 Dkk1 inhibits BMP-2 induced alkaline phosphatase activity. C2C12 (A), Saos-2 (B), and hFOB1.19 (C) cells were cultured for 72 h in DMEM with 2% horse serum containing Wnt3a, BMP-2, or Dkk1 either alone or in the indicated combinations. Cells were lysed and ALP activity measured and normalized to protein concentration. Data represent the mean±SD (n=3). ⁎p<0.05, ⁎⁎p<0.01, ⁎⁎⁎p<0.001, and ⁎⁎⁎⁎p<0.00001 versus control or BMP-2 versus BMP-2 plus DKK1. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 5 Blocking the canonical β-catenin pathway inhibits TCF/LEF mediated transcription and BMP-2 induced alkaline phosphatase activity. (A) C2C12 cells were transfected with dominant negative β-catenin (DN-β-Cat) or control (pcDNA4his) constructs and the expressions were confirmed by the indicated antibodies by immunoblotting analysis. (B) The positive clones were co-transfected with wild type (TOPflash) or mutant (FOPflash) LEF/TCF reporter luciferase constructs. After transfection, the cells were treated and subjected to luciferase assay as in Fig. 2 E. C2C12 pcDNAhis or DN-β-Cat clone #4 (C) and #5 (D) cells were cultured in DMEM containing 2% horse serum or 100 ng/ml of BMP-2 for 72 h after which ALP activity was measured. (E) C2C12 cells were transfected with empty vector or Dkk1- and Dkk2-expressing vectors and the expression of the proteins were determined as in Fig. 3B. The positive clones and control vector were treated with BMP-2 and subjected to ALP activity assay as in D. Results are shown as mean±SD (n=3) and are representative of three independent experiments (F). ⁎⁎p<0.01 and ⁎⁎⁎p<0.001 versus control. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 6 Silencing LRP5/6 mRNA blocks BMP-2 induced alkaline phosphatase activity. C2C12 cells were transiently transfected with 1.0 μg/ml (A) or serial concentration of siRNA specific for LRP5 (B) or LRP6 (C). Forty eight hour after transfection RNA was isolated and subjected to RT-PCR (A) or qPCR (B and C) as described in Materials and methods. (D) The cells transfected with 0.25 or 0.5 μg/ml of siRNA specific for LRP5 or LRP6 were cultured in medium containing 100 ng/ml of BMP-2 in DMEM. Cells were lysed after 72 h and alkaline phosphatase activity determined. Data represent the mean±SD (n=3) of representative experiments. ⁎p<0.05, ⁎⁎p<0.01, and ⁎⁎⁎p<0.001 versus control. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Fig. 7 Wnt3a mediated increase in β-catenin is independent of BMP-2. (A) C2C12, hFOB1.19, MG63, and Saos-2 cells were treated with Wnt3a CM, control CM or 100 ng/ml of BMP2. Lysate protein was subjected to GST–E-cadherin assay and immunoblotting analysis by anti-β-catenin antibody as described in Materials and methods. (B) 50 μg aliquots of protein from cell lysates were resolved on 8% SDS-PAGE and analyzed with the indicated antibodies. Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions

Bone 2008 42, 669-680DOI: (10.1016/j.bone.2007.12.006) Copyright © 2007 Elsevier Inc. Terms and Conditions