doi:10.1016/j.yexcr.2005.09.016 
Copyright © 2005 Elsevier Inc. All rights reserved.
Research Article
Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NFκB
Roman A. Eliseev
, 
, Edward M. Schwarz, Michael J. Zuscik, Regis J. O'Keefe, Hicham Drissi and Randy N. Rosier
Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY 14642, USA
Received 10 August 2005;
revised 23 September 2005;
accepted 26 September 2005.
Available online 2 November 2005.
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Abstract
The transcription factor NFκB is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NFκB and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NFκB regulates the activity of the BMP/Smad signaling. Inhibition of NFκB by overexpression of mIκB leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mIκB (Saos2-mIκB) exhibit higher expression of osteoblast phenotypic genes such as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mIκB cells in comparison to Saos2-EV cells. Inhibition of NFκB signaling in Saos2-mIκB cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mIκB cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NFκB represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.
Keywords: Saos2; Osteosarcoma; Osteoblasts; NFκB; BMP; Smad7
Fig. 1. NFκB activity in Saos2-wt, -EV and -mIκB cells. (A) NFκB activity was measured using the heterologous promoter reporter luciferase assay and pNFκB-luc vector as described in Materials and methods and expressed as relative luminescence units (RLU). Data are Means ± SD (n = 4). *Indicates P < 0.05 when compared to Saos2-EV cells. (B) Immunostaining for p65 NFκB subunit was done using rhodamine-labeled fluorescent secondary antibody. Frames 1, 4, and 7 are representative fluorescent images immunolabeled for p65 in Saos2-wt, -EV, and -mIκB cells respectively. Frames 2, 5, and 8 show these images superimposed with DAPI staining. Frames 3, 6, and 9 are white light images of the same frames. Arrowheads indicate nuclear regions of cells positive for p65 in Saos2-wt and Saos2-EV cells, and negative for p65 in Saos2-mIκB cells.
Fig. 2. Saos2-mIκB cells produce more matrix and collagen I when compared to Saos2-wt or Saos2-EV cells. (A) Representative electron micrographs of Saos2-wt (wt), Saos2-EV (EV), and Saos2-mIκB (mIκB) cells. Arrowheads indicate matrix granules. (B) Representative fluorescent and corresponding white light images of cells immunolabeled for type I collagen. Immunostaining for type I collagen was performed using rhodamine-labeled fluorescent secondary antibody. (C) Type I collagen fluorescence in immunolabeled cells was measured in each frame as described in Materials and methods, normalized to “black level” and to a number of cells in the frame, and plotted as relative fluorescence units (RFU). Data are Means ± SD (n = 20 frames). *Indicates P < 0.05 when compared to Saos2-EV cells. (D) Representative (n = 3) Western blots for type I collagen (Col I) and β-actin in cell lysates. Numbers indicate relative band intensities.
Fig. 3. Real-time RT-PCR assay of BMP-induced expression of osteoblastic marker genes. Cells were treated with 50 ng/ml BMP2 for the indicated times or left untreated (Ct). (A) Total RNA was isolated, reverse-transcribed into cDNA and subjected to real-time RT-PCR analysis as described in detail in Materials and methods. Expression levels of alkaline phosphatase (ALP, upper panel), Runx2 (middle panel) and osteocalcin (OC, bottom panel) were normalized to the expression level of GAPDH and plotted as fold change over control. Data are Means ± SD (n = 4). (B) Alkaline phosphatase activity was measured in cell lysates as described in detail in Materials and methods. Data are means ± SD (n = 3). *Indicates P < 0.05 when compared to Saos2-EV cells.
Fig. 4. Activity of BMP/Smad pathway in Saos2-EV and Saos2-mIκB cells. Cells were treated with 50 ng/ml BMP2 for the indicated times or left untreated (Ct) and following assays were performed: (A) BMP/Smad pathway activity was measured using the promoter reporter luciferase assay and 12 × SBE-luc vector as described in detail in Materials and methods. Data are means ± SD (n = 4). *Indicates P < 0.05 when compared to Saos2-EV cells. (B) Representative (n = 3) Western blots for phospho-Smad1,5,8 (pSmad), total Smad1,5,8, and β-actin in cell lysates.
Fig. 5. Expression of inhibitors of BMP/Smad pathway in Saos2-EV and Saos2-mIκB cells. (A) Expression levels of Smurf1 (Sf1), Smad6 (Sm6), and Smad7 (Sm7) were analyzed by real-time RT-PCR as described in detail in Materials and methods. Data are means ± SD (n = 3). *Indicates P < 0.05 when compared to Saos2-EV cells. (B) Representative (n = 3) Western blots for Smurf1, Smad6, Smad7, and β-actin in lysates from Saos2-EV and Saos2-mIκB cells. Numbers indicate relative band intensities.
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Fig. 6. Molecular manipulation of Smad7 changes BMP/Smad signaling in Saos2 cells. Expression of Smad7 was altered in Saos2-EV and Saos2-mIκB cells by molecular means, and cells were then treated with 50 ng/ml BMP2 or left untreated (Ct). (A) Transient transfection of Saos2-mIκB cells with pCMV-Smad7 construct resulted in overexpression of Smad7 when compared to cells transfected with the empty pCMV vector (CMV) as confirmed by Western blot (n = 3). Numbers here and in panel (C) indicate relative band intensities of Smad7. The blots were reprobed for β-actin to verify equal loading. (B) Overexpression of Smad7 in Saos2-mIκB cells leads to inhibition of BMP/Smad signaling as confirmed with promoter reporter luciferase assay and 12 × SBE-luc vector. (C) Downregulation of Smad7 in Saos2-EV cells achieved with anti-sense oligonucleotides was confirmed by Western blot (n = 3). The blots were reprobed for β-actin to verify equal loading. (D) Anti-sense mediated downregulation of Smad7 in Saos2-EV cells leads to activation of BMP/Smad signaling as confirmed with promoter reporter luciferase assay and 12 × SBE-luc vector. Data in panels (B) and (D) are means ± SD (n = 3). *Indicates P < 0.05 when compared to control.
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