Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation

Abstract

Osteoblasts express Toll like receptor (TLR) 4 and produce osteoclast-activating cytokines in response to the stimulation by lipopolysaccharide (LPS). It has recently been reported that LPS exerts an inhibitory effect on osteoblast differentiation into osteocytes. However, the molecular mechanisms of this inhibitory effect remain ambiguous. The downstream signals of TLR4 are mediated by adaptor molecules including myeloid differentiation factor 88 (MyD88), leading to the activation of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERKs), whose activation by LPS requires the upstream serine/threonine kinase, Cot/Tpl2. To determine the signal molecules responsible for the inhibitory effects of LPS on osteoblast differentiation, we examined the in vitro differentiation of the primary osteoblasts from myd88^−/− and cot/tpl2^−/− mice. The matrix mineralization by the wild-type and cot/tpl2^−/− osteoblasts was significantly inhibited by LPS, whereas that of myd88^−/− was not affected. During differentiation, LPS suppressed the mRNA expression of runt related transcription factor 2 (Runx2), osterix (Sp7), and activating transcription factor 4 (ATF4) in the wild-type, but not in the myd88^−/− osteoblasts. The inhibitory effect of LPS on the mRNA expression of these transcription factors was absent in the early phase but partially impaired in the late phase of differentiation in the cot/tpl2^−/− osteoblasts. Thus, the inhibitory effect of LPS on osteoblast differentiation is Myd88-dependent, whereas the degree of its requirement for Cot/Tpl2 varies depending on the 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.