Peroxiredoxin II negatively regulates BMP2-induced osteoblast differentiation and bone formation via PP2A Cα-mediated Smad1/5/9 dephosphorylation

Kim et al. Experimental & Molecular Medicine (2019) 51:62 https://doi.org/10.1038/s12276-019-0263-x ARTICLE Experimental & Molecular Medicine Open Access Peroxiredoxin II negatively regulates BMP2induced osteoblast differentiation and bone formation via PP2A Cα-mediated Smad1/5/9 dephosphorylation 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Kyeong-Min Kim1,2, Do-Young Kim1,2, Dong-Seok Lee3, Jung-Woo Kim4, Jeong-Tae Koh4, Eun-Jung Kim2,5 and Won-Gu Jang1,2 Abstract Peroxiredoxin II (Prx II), an antioxidant enzyme in the Prx family, reduces oxidative stress by decreasing the intracellular ROS levels. Osteoblast differentiation is promoted by bone morphogenetic protein 2 (BMP2), which upregulates the expression of osteoblast differentiation marker genes, through Smad1/5/9 phosphorylation. We found that Prx II expression was increased by a high dose of lipopolysaccharide (LPS) but was not increased by a low dose of LPS. Prx II itself caused a decrease in the osteogenic gene expression, alkaline phosphatase (ALP) activity, and Smad1/5/9 phosphorylation induced by BMP2. In addition, BMP2-induced osteogenic gene expression and ALP activity were higher in Prx II knockout (KO) cells than they were in wild-type (WT) cells. These inhibitory effects were mediated by protein phosphatase 2A Cα (PP2A Cα), which was increased and is known to induce the dephosphorylation of Smad1/ 5/9. The overexpression of Prx II increased the expression of PP2A Cα, and PP2A Cα was not expressed in Prx II KO cells. Moreover, PP2A Cα reduced the level of BMP2-induced osteogenic gene expression and Smad1/5/9 phosphorylation. LPS inhibited BMP2-induced Smad1/5/9 phosphorylation and the suppressed phosphorylation was restored by the PP2A inhibitor okadaic acid (OA). Bone phenotype analyses using microcomputed tomography (μCT) revealed that the Prx II KO mice had higher levels of bone mass than the levels of the WT mice. We hypothesize that Prx II has a negative role in osteoblast differentiation through the PP2A-dependent dephosphorylation of Smad1/5/9. Introduction Peroxiredoxins (Prxs) are antioxidant enzymes with six subtypes (Prx I–VI) and they mainly reduce the reactive oxygen species (ROS) levels1. These subtypes have different locations within the cell; Prx I, II, and VI are present in the cytoplasm, Prx III plays a role in the mitochondria, Prx IV plays a role in the Golgi apparatus, and Prx V performs an antioxidant function in Correspondence: Eun-Jung Kim () Won-Gu Jang () 1 Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea 2 Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea Full list of author information is available at the end of the article. peroxisomes2. Prx II protects cells from oxidative stress by reducing the levels of ROS3,4. The levels of ROS, which are produced during cellular respiration, are regulated to reduce oxidative damage5. Intracellular ROS is an important regulator of cell proliferation and differentiation in various cells, including stem cells and cancer cells6. In addition, ROS plays a role in the regulation of gene expression as a secondary signal transducer in a variety of cell and biological processes, such as the cytokine response, growth factor and hormone treatment response, ion transport, transcription, neuromodulation, and apoptosis7–9. Oxidative stress occurs by the excessive accumulation of ROS and has been implicated © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Kim et al. Experimental & Molecular Medicine (2019) 51:62 in various metabolic diseases, such as diabetes and osteoporosis10–14. The bone is a dynamic tissue; osteoblast-induced bone formation and osteoclast-induced bone loss occur throughout the lifetime15. Osteoblasts differentiate from mesenchymal stem cells, and they are regulated by hormones and cytokines16,17. BMP2 is one of the most important cytokines in bone repair, bone formation, and osteoblast differentiation18,19. BMP2-induced osteoblast differentiation regulates the transcription of osteogenic genes, such as the DNA-binding protein inhibitor (Id1), distal-less homeobox 5 (Dlx5), and runt-related transcription factor 2 (Runx2) through the phosphorylation of Smad1/5/920,21. These genes also upregulate the expression of next-stage markers, including ALP and OC20,22,23. Protein phosphatases modulate various important cellular processes, such as protein synthesis, the cell cycle, and glycogen metabolism24. PP2A is a large family of heterotrimeric phosphatases in eukaryotic cells that regulate numerous signaling pathways, such as those involved in metabolism, the kinase cascade, cell growth, and cell death25,26. Prx I and Prx V in osteoblasts have been studied27,28, but the role of Prx II in osteoblast differentiation has not yet been elucidated. In this study, we demonstrate that Prx II causes a decrease in BMP2-induced osteoblast differentiation through the upregulation of PP2A Cα expression, which inhibits the phosphorylation of Smad1/5/9. Materials and methods Reagents and antibodies Lipopolysaccharide (LPS) was purchased from SigmaAldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline, penicillin–streptomycin, and 0.25% trypsin-EDTA were obtained from GIBCO-BRL (Grand Island, NY). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Seoul, Korea). Emerald Amp GRPCR Master Mix was purchased from TaKaRa (Shiga, Japan), and AmpiGeneTM qPCR Green Mix Hi-ROX was purchased from Enzo (Farmingdale, NY). Recombinant human BMP2 was purchased from Cowellmedi Co. (Busan, Korea). Antibodies against Prx II and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX). Antibodies against PP2A Cα, Smad, and phospho-Smad (p-Smad) were purchased from Cell Signaling Technology (Cambridge, MA). Cell culture The mouse embryonic mesenchymal stem cell line C3H10T1/2 (ATCC, Manassas, VA) was maintained in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/ml streptomycin in humidified air wi (...truncated)