Abstract
Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.
Highlights
Our studies showed that treatment with a Sphingosine-1-phosphate receptor 2 (S1PR2) shRNA, or the specific S1PR2 antagonist, JTE013, suppressed phosphoinositide3 kinase (PI3K), MAPKs, and NF-κB protein kinases induced by an oral bacterial pathogen Aggregatibacter actinomycetemcomitans, subsequently reduced inflammatory cytokines (IL-1β, IL-6, and TNF-α) levels in murine bone marrow-derived monocytes and macrophages (BMMs) compared with controls [20,21]
We observed a reduction of expressions of F-actin, clathrin, EEA1, and syntaxin 6 in murine bone marrow stromal cells (BMSCs) treated with the S1PR2 shRNA compared with murine BMSCs treated with the control shRNA (Figure 5H), which suggests that the S1PR2 shRNA inhibited vesicle trafficking in murine BMSCs cultured in osteogenic media compared with the control shRNA treatment
BMP7, BMPR1A, and BMPRII, but not BMP2 protein expressions in BMSCs compared with murine BMSCs treated with DMSO (Figure 7P), which suggests that the protein synthesis and transport of BMP7, BMPR1A, and BMPRII might be involved in the endoplasmic reticulum (ER)–Golgi signaling pathway. In this in vitro study, we demonstrated that treatment with a S1PR2 specific antagonist (JTE013) or a S1PR2 shRNA increased alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, and enhanced osteogenic gene (ALPL, RUNX2, OCN, OSX) expressions in murine BMSCs cultured in osteogenic media
Summary
The S1P level in the blood is very high (150–1000 nM) because platelets lack S1P lyase and erythrocytes lack both S1P lyase and
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