Biochemical and Biophysical Research Communications
Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation
Research highlights
► LPS inhibits osteoblast differentiation through down-regulating Runx2, Osterix (Sp7), and ATF4 expression. ► Myd88 is absolutely essential for LPS-induced inhibition of osteoblast differentiation. ► Myd88 is essential for LPS-induced down-regulation of Runx2, Osterix (Sp7), and ATF4 expression. ► Cot/Tpl2 kinase is required for LPS-induced down-regulation of Runx2, Osterix (Sp7), and ATF4 expression in the early phase of osteoblast differentiation. ► Cot/Tpl2 is only partially responsible for LPS-induced down-regulation of Runx2, Osterix (Sp7), and ATF4 expression in the late osteoblast differentiation phase.
Introduction
Lipopolysaccharide (LPS) from Gram-negative bacteria cell wall has been identified as an important pathogenic factor in bone loss diseases, such as inflammatory arthritis [1] and periodontitis [2]. One of the many known functions of LPS is the stimulation of bone resorption by osteoclasts. LPS stimulates osteoblasts to produce or secrete interleukin (IL)-1, IL-6, prostagrandin (PG) E2 and receptor activator of nuclear factor-kappa B ligand (RANKL) [3], [4], [5], [6], all of which induce osteoclast maturation and/or activation. Intriguingly, several recent studies demonstrated that the osteogenic differentiation of osteoblasts is inhibited by LPS, which may be considered as another mechanism of the LPS-induced bone loss [7], [8]. However, little is known about the intracellular signals of LPS mediating the inhibitory effect on osteoblast differentiation.
LPS is sensed by Toll like receptor (TLR) 4 on the cell surface in collaboration with other molecules, such as CD14, MD-2, and LPS-binding protein (LBP) [9], [10], [11]. Downstream signals of TLR4 is mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), Toll receptor–IL-1 receptor domain containing adapter protein (TIRAP)/MyD88-adapter-like (Mal), Toll receptor–IL-1 receptor domain containing adaptor protein inducing interferon-β (TRIF), and TRIF-related adaptor molecule (TRAM) [12], [13], [14], [15]. The further downstream signals of TLR4 include the activation of nuclear transport of nuclear factor-kappa B (NF-κB) and a set of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases [16], [17], [18], [19]. We and others have previously demonstrated that a serine-threonine kinase, Cot/Tpl2, is indispensable for the LPS-induced activation of ERKs in macrophages, dendritic cells, and osteoblasts [20], [21], [22]. Activated Cot/Tpl2 in these types of cells regulates production of various cytokines, such as tumor necrosis factor (TNF) α, IL-12, IL-23, and RANKL, in response to LPS [21], [22], [23].
In our present study, to explore the inhibitory mechanisms of LPS on osteoblast differentiation, we have examined the effects of LPS on the differentiation of primary osteoblasts from myd88−/− and cot/tpl2−/− mice.
Section snippets
Animals
C57BL/6 mice (wild-type) were obtained from Japan Clea (Tokyo, Japan). The cot/tpl2−/− mice in the C57BL/6 background were generated and maintained as described previously [24]. The myd88−/− mice in the C57BL/6 background were kindly provided by Dr. Shizuo Akira (Osaka University, Japan).
Antibodies and reagents
Antibodies and phosphor-specific antibodies against JNKs, ERKs, and p38 kinases as well as anti-IκB antibody were purchased from Cell Signaling Technology (Danvers, MA). The antibody against
The effects of LPS on differentiation in mouse primary osteoblasts
Primary osteoblasts were collected from wild-type, cot/tpl2−/−, and myd88−/−newborn mouse calvalia, and induced to differentiate with ascorbic acid for 2 weeks with or without LPS. The calcific substances of the differentiated cells were visualized by Von Kossa staining (Fig. 1A), and the differentiation-associated ALP activity was also measured (Fig. 1B). Wild-type, cot/tpl2−/−, and myd88−/− osteoblasts showed similar matrix mineralization and ALP activity in the absence of LPS. On the other
Discussion
In present study, we have shown that LPS effectively suppressed osteoblast differentiation, confirming the previous reports [7], [8]. The similar inhibitory effect was also observed for synthetic lipid A, a pure TLR4 agonist, demonstrating that the suppression of osteoblast differentiation was through TLR4. Furthermore, our results have demonstrated that LPS strongly inhibits the gene expression of runx2, sp7 and atf4, all of which are essential transcriptional factors in osteoblast
Acknowledgments
We thank Ms. Mai Nakashima for the secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant 20592176) to K.B.
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