Toll-like receptor-4-dependence of the lipopolysaccharide-mediated inhibition of osteoblast differentiation

Abstract

pathogenic factor affecting bone fracture healing. Therefore, the effect of LPS on bone metabolism is relevant for bone healing. In this study, we investigated the effect of LPS on the expression of Toll-like receptor (TLR)-4 (an LPS receptor) by using real-time quantitative PCR and western blotting. We also examined the regulatory role of LPS in osteoblast differentiation by measuring the ALP activity, matrix mineralization, and ALP, OCN, and Runx2 mRNA (essential factors affecting osteoblast differentiation) expression in LPS-treated mouse osteoblast MC3T3-E1 cells. We also evaluated the effect of TLR-4 on LPS-mediated inhibition of osteoblast differentiation using RNA interference. LPS promotes TLR-4 mRNA and protein expression in MC3T3-E1 cells (P < 0.05, P < 0.01 or P < 0.001), and inhibits osteoblast differentiation by downregulating matrix mineralization and ALP activity (P < 0.05, P < 0.01 or P < 0.001), and suppressing the expression ALP, OCN, and Runx2 mRNA in MC3T3-E1 cells (P < 0.05 or P < 0.01). Conversely, RNAi-mediated TLR-4 knockdown abrogates the LPS-mediated inhibition of osteoblast differentiation (P < 0.05 or P < 0.01). In summary, LPS was shown to inhibit osteoblast differentiation by suppressing the expression of ALP, OCN, and Runx2 in a TLR-4-dependent manner. The results of this study may provide insights into the signal pathway of LPS-induced bone loss or delayed bone fracture healing.

Role of Toll-Like Receptor 4 on Osteoblast Metabolism and Function

“…This was also observed in mouse MSCs where TLR4 activation also up-regulated its own expression ( Huang et al, 2014 ). Moreover TLR4 activation in the mouse osteoblastic cell line MC3T3-E1 induced its own expression ( Liu et al, 2016 ). These apparent divergent results regarding TLR4 expression upon its stimulation might be explained according to different incubation times, agonist concentration, as well as cell-specific TLR4 expression rates.…”

“…This was consistent with other reports about TRIF-independent inflammatory responses elicited by TLR4 activation in mouse osteoblasts ( Sato et al, 2004 ). According to the TLR4-mediated inhibition of certain anabolic processes, TLR4 activation in differentiating mouse primary osteoblasts ( Bandow et al, 2010 ), mouse osteoblastic cell line MC3T3-E1 ( Liu et al, 2016 ), or in mouse MSCs ( Chen et al, 2014 ) inhibited the matrix mineralization ( Bandow et al, 2010 ), whereas this activation did not inhibit the matrix mineralization of mouse MyD88 -/- -derived primary osteoblasts ( Bandow et al, 2010 ). Moreover, unlike occurred in MyD88 -/- osteoblasts, TLR4 activation of wild type osteoblasts up-regulated the mRNA of the activating transcription factor 4 (ATF4), as well as down-regulated osteoblastic transcription factors like the runt related transcription factor 2 (Runx2), and osterix (Sp7) ( Bandow et al, 2010 ).…”

S100A9 Increases IL‐6 and RANKL Expressions through MAPKs and STAT3 Signaling Pathways in Osteocyte‐Like Cells

“…Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg), a major pathogenic component, affects bone metabolisms and aggravates periodontitis. Liu et al have recently reported that bacterial LPS inhibits osteoblastic differentiation and alkaline phosphatase (ALP) activity by suppressing the expressions of osteocalcin, Runx2, and ALP, and then downregulates bone matrix mineralization [10]. Moreover, LPS induces inflammatory cytokine levels, such as IL-1β, IL-6, IL-8, TNF-α, and S100A8/S100A9 from various cells in periodontal tissues [11][12][13].…”

TLR4-Induced Inflammation Is a Key Promoter of Tumor Growth, Vascularization, and Metastasis

“…However, all positive cells share the basic traits of this pathway such as responsiveness to LPS reflected by activation of transcription factors of the NF-κB family and production of inflammatory cytokines. During inflammation, TLR4-expressing normal cells significantly increase secretion of inflammatory mediators [25,30] due to co-upregulated TLR4 [31], its intracellular adapters [32], and co-receptors [10] which enhances cooperation among the pathway's components. Likewise, malignant TLR4-positive cells express much higher levels of inflammatory proteins as compared with their normal counterparts [33,34].…”

“…Macrophages are natural responders to TLR4 ligands having the highest expression of this receptor. Under inflammatory conditions, TLR4 is further upregulated by LPS [31] and cytokines [195] through positive feedback loops. Tumor-associated macrophages (TAMs) are well-known promoters of metastasis through secretion of growth-promoting cytokines, proteases [196], and immunosuppressive factors [197].…”