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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

Dose-dependent promotion of osteoblast migration by rhHMGB1 without inhibiting cell viability(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, and allowed to invade the matrigel for 4 h. The invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet, and were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in five randomly selected microscopic fields. 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. Results of the MTT assay showed that rhHMGB1 had no significant effect on the cellular proliferation of osteoblasts.
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Figure 1: Dose-dependent promotion of osteoblast migration by rhHMGB1 without inhibiting cell viability(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, and allowed to invade the matrigel for 4 h. The invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet, and were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in five randomly selected microscopic fields. 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. Results of the MTT assay showed that rhHMGB1 had no significant effect on the cellular proliferation of osteoblasts.

Mentions: To examine the effects of rhHMGB1 on the migration and viability of osteoblasts, we used a modified Boyden Chamber system and the MTT assay. We added 0, 50, 100, 150 or 200 μg/l rhHMGB1 to investigate the effects of rhHMGB1 on osteoblast activation. In a transwell chamber, many of the cells had migrated through the pores to the lower side of the membrane by 4 h, where they could be stained dark purple with crystal violet (Figure 1A). As shown in Figure 1B, our findings indicated that rhHMGB1 could promote the migration of osteoblasts in a dose-dependent manner (**P<0.01), which peaked at 150 μg/l. These data showed that the count of cells that migrated across the membrane increased 2.3-fold after 4 h of HMGB1 treatment at 150 μg/l (P<0.05). The MTT assay revealed that the viability of osteoblasts was not reduced significantly after treated by rhHMGB1 (Figure 1C). Therefore, 150 μg/l was chosen as an optimal concentration for use in subsequent experiments.


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)

Dose-dependent promotion of osteoblast migration by rhHMGB1 without inhibiting cell viability(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, and allowed to invade the matrigel for 4 h. The invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet, and were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in five randomly selected microscopic fields. 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. Results of the MTT assay showed that rhHMGB1 had no significant effect on the cellular proliferation of osteoblasts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759610&req=5

Figure 1: Dose-dependent promotion of osteoblast migration by rhHMGB1 without inhibiting cell viability(A) Osteoblasts were seeded into the upper chamber of the transwell, treated with rhHMGB1, and allowed to invade the matrigel for 4 h. The invasive cells that migrated through the basal membrane to the lower surface were stained with crystal violet, and were photographed at 10× magnification. (B) Migrated cells were counted under a light microscope in five randomly selected microscopic fields. 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. Results of the MTT assay showed that rhHMGB1 had no significant effect on the cellular proliferation of osteoblasts.
Mentions: To examine the effects of rhHMGB1 on the migration and viability of osteoblasts, we used a modified Boyden Chamber system and the MTT assay. We added 0, 50, 100, 150 or 200 μg/l rhHMGB1 to investigate the effects of rhHMGB1 on osteoblast activation. In a transwell chamber, many of the cells had migrated through the pores to the lower side of the membrane by 4 h, where they could be stained dark purple with crystal violet (Figure 1A). As shown in Figure 1B, our findings indicated that rhHMGB1 could promote the migration of osteoblasts in a dose-dependent manner (**P<0.01), which peaked at 150 μg/l. These data showed that the count of cells that migrated across the membrane increased 2.3-fold after 4 h of HMGB1 treatment at 150 μg/l (P<0.05). The MTT assay revealed that the viability of osteoblasts was not reduced significantly after treated by rhHMGB1 (Figure 1C). Therefore, 150 μg/l was chosen as an optimal concentration for use in subsequent experiments.

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