Abstract
Osteoblast migration is significant in skeletal development. Recently, high mobility group box 1 protein (HMGB1) has been shown to highly expressed in cartilage to regulate endochondral ossification. Nevertheless, whether HMGB1 can modulate osteoblast proliferation and migration is poorly understood, as well as the intracellular signalling pathways that are involved in this process. Herein, we examined the effects of recombinant human HMGB1 (rhHMGB1) on the proliferation and migration of rat osteoblasts and investigated whether Toll-like receptor 2 (TLR2)- and TLR4-dependent signalling pathways are involved in the regulation of intracellular signalling. A transwell chamber assay was used to evaluate the migration of osteoblasts and the MTT assay was used to assess osteoblast proliferation. rhHMGB1 could significantly promote the migration of osteoblasts without inhibiting their proliferation. Meanwhile, rhHMGB1 can increase the nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Specific siRNA constructs that target TLR2 or TLR4 could markedly inhibit HMGB1-induced migration of osteoblasts and HMGB1-enhanced activation of NF-κB. Collectively, HMGB1 could significantly enhance the migration of osteoblasts invitro, and TLR2/TLR4-dependent NF-κB pathways are involved in HMGB1-induced osteoblast migration.
Highlights
The proliferation and migration of osteoblasts is important for both skeletal development and bone fracture healing [1]
Involvement of Toll-like receptor 2 (TLR2) and TLR4 in High mobility group box 1 protein (HMGB1)-mediated osteoblast migration First, to investigate whether TLR2 or TLR4 signalling is involved in HMGB1-induced osteoblast migration, osteoblasts pretreated with TLR2- or TLR4-siRNA were stimulated with Recombinant human HMGB1 (rhHMGB1) and subsequently used in a transwell assay to test for effects
One-way ANOVA was performed to determine statistical significance. rhHMGB1 promotes osteoblast migration in a dose-dependent manner (**P < 0.01), which reached a peak at 150 μg/l. (C) Proliferation assay after incubation with rhHMGB1 for 24–72 h
Summary
The proliferation and migration of osteoblasts is important for both skeletal development and bone fracture healing [1]. Previous studies have shown that many extracellular cytokines, such as bone morphogenetic proteins (BMPs), insulin-like growth factors (IGFs) and wingless and int (Wnt) ligands are involved in the proliferation and migration of bone cells [2,3,4]. HMGB1 can be passively released by necrotic or damaged cells [6] and actively secreted by activated monocytes or macrophages [7] Since it was initially reported as a lethal mediator of sepsis by Wang et al [8], extracellular HMGB1 has been reported to act as a novel cytokine that contributes to inflammation [9,10,11], reperfusion after ischemia of skeletal muscle tissue [12], liver fibrosis [13] and tumour growth and metastases [14,15].
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