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rhHMGB1 drives osteoblast migration in a TLR2/TLR4- and NF-κB-dependent manner.

Li MJ, Li F, Xu J, Liu YD, Hu T, Chen JT - Biosci. Rep. (2016)

Bottom Line: Osteoblast migration is significant in skeletal development.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 in vitro, and TLR2/TLR4-dependent NF-κB pathways are involved in HMGB1-induced osteoblast migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pediatric Orthopedic, Wuhan PuAi Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China.

No MeSH data available.


Related in: MedlinePlus

TLR2- or TLR4-siRNA attenuated the mobility of osteoblasts in vitro(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, TLR2-siRNA or TLR4-siRNA, and allowed to invade materiel for 5 h. Invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet dye, which were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in four randomly selected microscopic fields. One-way ANOVA was performed to determine statistical significance. The increased number of migrated cells was markedly reduced when the cells were pretreated by TLR2- or TLR4-siRNA (**P<0.01). (C) Osteoblasts were incubated with TLR2- or TLR4-siRNA or rhHMGB1 for 24–72 h, and the MTT assay showed that knockdown of TLR2 or TLR4 had no significant effect on cell viability.
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Figure 4: TLR2- or TLR4-siRNA attenuated the mobility of osteoblasts in vitro(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, TLR2-siRNA or TLR4-siRNA, and allowed to invade materiel for 5 h. Invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet dye, which were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in four randomly selected microscopic fields. One-way ANOVA was performed to determine statistical significance. The increased number of migrated cells was markedly reduced when the cells were pretreated by TLR2- or TLR4-siRNA (**P<0.01). (C) Osteoblasts were incubated with TLR2- or TLR4-siRNA or rhHMGB1 for 24–72 h, and the MTT assay showed that knockdown of TLR2 or TLR4 had no significant effect on cell viability.

Mentions: 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 rhHMGB1 and subsequently used in a transwell assay to test for effects on migration. The migration of osteoblasts stimulated with rhHMGB1 (150 μg/l) was enhanced by 2-fold compared with those unstimulated (P<0.05). Additionally, after pretreatment with TLR2- or TLR4-siRNA, the increased number of migrated cells was markedly reduced (P<0.01; Figures 4A and 4B). Second, pretreated osteoblasts were subjected to the MTT assay to examine proliferation. We found that knockdown of TLR2 or TLR4 did not inhibit osteoblast proliferation compared with those cells stimulated only with HMGB1 (Figure 4C). In accord with our previous findings, these data suggest that TLR2- or TLR4-dependent NF-κB signalling pathways are involved in HMGB1-induced osteoblast migration.


rhHMGB1 drives osteoblast migration in a TLR2/TLR4- and NF-κB-dependent manner.

Li MJ, Li F, Xu J, Liu YD, Hu T, Chen JT - Biosci. Rep. (2016)

TLR2- or TLR4-siRNA attenuated the mobility of osteoblasts in vitro(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, TLR2-siRNA or TLR4-siRNA, and allowed to invade materiel for 5 h. Invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet dye, which were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in four randomly selected microscopic fields. One-way ANOVA was performed to determine statistical significance. The increased number of migrated cells was markedly reduced when the cells were pretreated by TLR2- or TLR4-siRNA (**P<0.01). (C) Osteoblasts were incubated with TLR2- or TLR4-siRNA or rhHMGB1 for 24–72 h, and the MTT assay showed that knockdown of TLR2 or TLR4 had no significant effect on cell viability.
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Related In: Results  -  Collection

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Figure 4: TLR2- or TLR4-siRNA attenuated the mobility of osteoblasts in vitro(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, TLR2-siRNA or TLR4-siRNA, and allowed to invade materiel for 5 h. Invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet dye, which were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in four randomly selected microscopic fields. One-way ANOVA was performed to determine statistical significance. The increased number of migrated cells was markedly reduced when the cells were pretreated by TLR2- or TLR4-siRNA (**P<0.01). (C) Osteoblasts were incubated with TLR2- or TLR4-siRNA or rhHMGB1 for 24–72 h, and the MTT assay showed that knockdown of TLR2 or TLR4 had no significant effect on cell viability.
Mentions: 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 rhHMGB1 and subsequently used in a transwell assay to test for effects on migration. The migration of osteoblasts stimulated with rhHMGB1 (150 μg/l) was enhanced by 2-fold compared with those unstimulated (P<0.05). Additionally, after pretreatment with TLR2- or TLR4-siRNA, the increased number of migrated cells was markedly reduced (P<0.01; Figures 4A and 4B). Second, pretreated osteoblasts were subjected to the MTT assay to examine proliferation. We found that knockdown of TLR2 or TLR4 did not inhibit osteoblast proliferation compared with those cells stimulated only with HMGB1 (Figure 4C). In accord with our previous findings, these data suggest that TLR2- or TLR4-dependent NF-κB signalling pathways are involved in HMGB1-induced osteoblast migration.

Bottom Line: Osteoblast migration is significant in skeletal development.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 in vitro, and TLR2/TLR4-dependent NF-κB pathways are involved in HMGB1-induced osteoblast migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic and Spinal Surgery, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Department of Pediatric Orthopedic, Wuhan PuAi Hospital, Tongji Medical College, Huazhong Science and Technology University, Wuhan 430030, China.

No MeSH data available.


Related in: MedlinePlus