CpG-oligodeoxynucleotide inhibits Smad-dependent bone morphogenetic protein signaling: effects on myeloma cell apoptosis and in vitro osteoblastogenesis.
The TLR9 agonist CpG-oligodeoxynucleotide (CpG-ODN) with a phosphorothioate backbone (PTO-CpG-ODN) is evaluated in clinical trials as a vaccine adjuvant or as treatment of cancers. Bone morphogenetic proteins (BMPs) regulate growth and differentiation of several cell types, and also induce apoptosis of cancer cells. Cross-talk between BMP- and TLR-signaling has been reported, and we aimed to investigate whether CpG-ODN influenced BMP-induced osteoblast differentiation or BMP-induced apoptosis of malignant plasma cells. We found that PTO-CpG-ODN inhibited BMP-2-induced osteoblast differentiation from human mesenchymal stem cells. Further, PTO-CpG-ODN counteracted BMP-2- and BMP-6-induced apoptosis of the human myeloma cell lines IH-1 and INA-6, respectively. In contrast, PTO-CpG-ODN did not antagonize the antiproliferative effect of BMP-2 on hMSCs or IH-1 cells. Inhibition of Smad-signaling and p38 MAPK-signaling indicated that apoptosis of IH-1 cells is dependent on Smad-signaling downstream of BMP, whereas the antiproliferative effect of BMP-2 on IH-1 cells also involves p38 MAPK-signaling. Together, the data suggested a specific inhibition by PTO-CpG-ODN on BMP-Smad-signaling. Supporting this we found that PTO-CpG-ODN inhibited BMP-induced phosphorylation of receptor-Smads in human mesenchymal stem cells and myeloma cell lines. This effect appeared to be independent of TLR9 because GpC-ODN and other ODNs with the ability to form multimeric structures inhibited Smad-signaling as efficiently as PTO-CpG-ODNs, and because knockdown of TLR9 by small interfering RNA in INA-6 cells did not blunt the effect of PTO-CpG-ODN. In conclusion, our results demonstrate that PTO-CpG-ODN inhibits BMP-signaling, and thus might provoke unwanted TLR9-independent side effects in patients.
Nørgaard
Nikolai N
NN
Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
Holien
Toril
T
Jönsson
Sofia
S
Hella
Hanne
H
Espevik
Terje
T
Sundan
Anders
A
Standal
Therese
T
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D006131:Growth Inhibitors / Q000494:pharmacology; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D009101:Multiple Myeloma / Q000276:immunology / Q000473:pathology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D054735:Phosphorothioate Oligonucleotides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D051785:Smad Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology; D051217:Toll-Like Receptor 9 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology
D019485:Bone Morphogenetic Proteins; C408982:CPG-oligonucleotide; D006131:Growth Inhibitors; D009838:Oligodeoxyribonucleotides; D054735:Phosphorothioate Oligonucleotides; D034741:RNA, Small Interfering; D051785:Smad Proteins; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9