| 1. BackgroundThe proliferation and migration of osteoblasts, osteoclasts and MSCs is important for both skeletal development and bone fracture healing. Bone function including mechanical support to the soft tissues, protection of the brain and spinal cord, supporting hematopoiesis rely on the continuously updated of bone tissues. The bone renewal consists of bone formation by osteoblast and bone resorption by osteoclast. Imbalance of osteogenesis and bone resorption could bring a series of diseases such as osteogenesis imperfecta, bone nonunion, rheumatoid arthritis, osteoporosis. Osteogenesis imperfecta is a kind of hereditary disease that seriously affects children’s bone development. Bone nonunion, rheumatoid arthritis, osteoporosis are wildspread, costly diseases with poor prognosis that need long time to treat. In addition to the traditional treatment, cell therapy and gene therapy provide new opportunities for treatment of these diseases. In the past years, the major part of treatment is to inhibite the ability of osteoclast, but now how to promote bone formation become more important. Understanding the function of these cells and the regulatory mechanisms will provide an effective way to treat these diseases.Previous studies have shown that many extracellular cytokines, such as BMPs, IGFs, and FGFs are involved in the proliferation and migration of bone cells. Recently, some reports showed that BMP is effective in the treatment of bone defect, bone nonunion and femoral head necrosis. IGFs is significant in the regulation of long bone development. It can induce the differentiation of MSCs to chondrocyte and improve the survive rate of MSCs through the receptors on the surface of MSCs. In the meantime, it can prompt the expression of extracellular matrix. FGFs is the most effective blood vessel factor which also can prompt the proliferation and differentiation of osteoblast and osteocyte.High mobility group box 1 protein (HMGB1) is a nonhistone nuclear protein that is expressed in all eukaryotic cells. It participates many physical and pathologic process such as DNA repair, transcriptional regulation, cell replication and mature through cross talk with transcriptional factors and receptors. HMGB1 can be passively released by necrotic or damaged cells and actively secreted by activated monocytes or macrophages. In the necrosis cells, HMGB1 could be detached from the chromosomes and released into the extracellular as the cell membrane was destroyed. In the activated monocyte and macrophage, HMGB1 can be transferred to the cytoplasm and then be actively secreted once they were methylated. Since it was initially reported as a lethal mediator of sepsis by Wang and colleagues in 1999, extracellular HMGB1 has been reported to act as a novel cytokine that contributes to inflammation, reperfusion after ischemia of skeletal muscle tissue, liver fibrosis, and tumor growth and metastases. That study showed that macrophage can secrete HMGB1 after 8 hours stimulated by the endotoxin. The application of HMGB1 antibody can reduce the mortality of sepsis while HMGB1 may lead to the death of rat. Recombinant human HMGB1 (rhHMGB1) can promote the proliferation and migration of myofibroblasts, skeletal myoblasts, and mesoangioblasts.Recent reports have shown that rhHMGB1 is a factor that can exert bone bioactivity to induce osteoclast formation and differentiation of bone marrow stem cells to osteoblasts. A previous study reported that hypertrophic chondrocytes within growth plates could release HMGB1 to regulate endochondral ossification. Analyses of Hmgb1-/-embryos indicated that long bone development is significantly compromised by HMGB1 deficiency. These findings suggest that extracellular HMGB1 may contribute to embryo skeletal development. HMGB1 can induce the migration and differentiation of MSCs to osteoblast with the inhibition of the proliferation. HMGB1 and LPS can transduce the phenotype of synovial fibroblasts of the osteoarthritis to the fibroblasts of the rheumatoid arthritis and accelerate bone destruction.Extracellular HMGB1 is a ligand for Toll-like receptor 2 (TLR2), TLR4, and the receptor for advanced glycation end products (RAGE); osteoblasts express all of these known receptors for HMGB1. BMSCs could increase the expression of RANKL/OPG and release TNF-a and IL-6 after the treatment of HMGB1. HMGB1 can be released from bone cells, including osteoblasts and osteoclasts, as well as MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells which were stimulated by TNF-a and CHX. The bone of RAGE-/-rat increase and the amount of osteoclast decreased. The bone between the wild type and RAGE-/-did not show any significant difference. The wild expressed Toll like receptors not only participate the immune reaction and tumor immunization, but also the repair of the skeletal muscle after ischemia. A previous study showed that extracellular HMGB1 could promote the repair of femoral head necrosis. LPS can induce the expression of RANKL in osteoblast through Toll like receptor.Together, HMGB1 is significant in the bone tissue and osteoblast express all the known receptors of HMGB1. But the effect of HMGB1 on the migration and proliferation of osteoblast and the molecular mechanism is need to be elucidated. Based on the aforementioned earlier reports, our present study aimed to investigate the potential role and regulatory mechanisms of rhHMGB1 in the proliferation and migration of rat osteoblasts using RNA interfere technique. This study will provide useful information for better treatment of severe bone-related diseases.2. Objective(1)Study on effects of high mobility group protein B1 (HMGB1) on osteoblast migration and proliferation:effects of recombined HMGB1 on the rat osteoblast migration and proliferation in vitro.(2)Study on the molecular mechanisms of HMGB1 on osteoblast migration: The expression of TLR2 and TLR4 in osteoblast were inhibited by siRNA. And then we study the effects of TLR and NF-κB on the migration of osteoblast.3. Materials and Methods(1)Materials:Osteoblasts from SD rats were purchased from Weikai Bioeng (Tianjin, China). Recombinant human HMGB1 was obtained from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), DMEM, a-MEM, and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Antibodies against HMGB1, TLR2, TLR4, NF-κB p65, and β-actin.NE-PER nuclear and cytoplasmic extraction kit (Pierce,Rockford,IL).(2) Cell culture:Osteoblasts were cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS),100 U/ml penicillin, and 100 μg/ml streptomycin and incubated at 37℃ with 5% CO2; medium was replaced every 2 days.(3) TLR2 or TLR4 knockdown by siRNA:Following generally accepted optimization principles for siRNA design, we generated three sequence-specific siRNAs to target either TLR2 or TLR4. Additionally, a negative control siRNA (NC-siRNA) that showed no homology with the human genome was designed as a negative control. The siRNA sequences are listed in text. Osteoblasts were cultured at 37℃ in a 5% CO2 incubator until cells were 70-80% confluent. Subsequently, on-target siRNA or negative control siRNA were transfected into cells using Turbofect siRNA transfection reagent according to the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). After culture for 4-6 h at 37℃, serum-free DMEM was replaced with complete growth medium (DMEM with 10% FBS) and cells were cultured for an additional 48 h.(4) RT-PCR:The mRNA transcript levels of TLR2, TLR4 were measured by quantitative real-time PCR, as described previously. Total RNA was extracted from each group of cells using TRIzol (Invitrogen) according to the manufacturer’s protocol. A total of 1 ug RNA was reverse-transcribed into cDNA using a RevertedAid First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA, USA). Primer sequences are listed in Table 2. Real-time PCR was performed using ABI StepOne Plus (Columbia, MD, USA) and specific primers for target genes and Actin (for endogenous control) in triplicate. PCR products were subjected to melting curve analysis and a standard curve that was generated to confirm amplification.(5) Cell viability assay:The MTT method was used to assess cell viability. Cells were seeded at a concentration of 5×103 cells per well in 96-well plates. After growth for 24 h, cells were treated with the indicated doses of rhHMGBl for 24,48, and 72 h. Cell viability was measured using the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay kit (Sigma) and absorbance was read using a microplate reader (Bio-Rad, Hercules, CA, USA) at 492 nm. All experiments were carried out in triplicate.(6) Cell invasion assay:Cell invasion was studied using a Boyden Transwell chamber assay (polycarbonate membrane inserts with 24 pores, pore size 8.0 μm, membrane insert diameter 6.5 mm). Cells in 100 μL serum-free DMEM media at a density of 1×105 cells/mL were plated in upper chambers that were precoated with Matrigel Basement Membrane Matrix (Sigma). In the lower chamber,500μL DMEM medium with 15% FBS was added as a chemoattractant. After 4-5 h, Matrix gels and cells on the top membrane surface were removed using a cotton swab. Transwell membranes were stained with crystal violet, and cells were counted under a light microscope in four or five randomly selected microscopic fields (Olympus Microscope System, Olympus, Tokyo, Japan).(7)Western blotting:Proteins from whole cell lysates were extracted using a radio-immunoprecipitation assay. Osteoblasts stimulated with rhHMGB1 were lysed in lysis buffer. Supernatants were prepared by centrifugation, electrophoresed on a 10% SDS-polyacrylamide gel, and blotted onto a polyvinylidene difluoride membrane. Immunoblotting was performed using antibodies specific to TLR2, TLR4 and GAPDH, followed by an HRP-conjugated secondary antibody and developed using an ECL detection kit (Abcam). The relative amounts of protein bands on the blots were determined using IPP 6.0 software(Media Cybernetics Inc,Maryland,USA).(8) Cell fraction and immunoblotting:Nuclear and cytosolic factions were prepared using Nuclear and Cytoplasmic Extraction Reagents kit. Lamin B1 and GAPDH were used as markers for nuclear and cytosol proteins, respectively. Lysate proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with TBS containing 0.1% Tween 20 and 5% skimmed milk, and then exposed to the desired primary antibodies. After treatment with anti-rabbit antibodies conjugated with horseradish peroxidase, the immunoreactive bands were visualized by standard ECL method.(9) Statistical analyses:All experiments were performed in triplicate. Data were presented as means ± standard error of the mean (SEM). One-way ANOVA tests were used for statistical analyses that were conducted with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). To identify statistically significant differences, a threshold of P<0.05 was used.4.Results(1).rhHMGB1 promotes osteoblast migration without inhibiting cell viabilityTo 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 4h, where they could be stained dark purple with crystal violet. 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 hours of HMGB1 treatment at 150ug/l (p<0.05). The MTT assay revealed that the viability of osteoblasts was not reduced significantly after treated by rhHMGB1. Therefore,150 μg/l was chosen as an optimal concentration for use in subsequent experiments.(2).Pre-designed siRNAs knockdown expression of TLR2 and TLR4To study the roles of TLR2 and TLR4 in osteoblasts, we examined the expression levels of TLR2 and TLR4 in osteoblasts by RT-PCR and western blotting. The mRNA transcript levels and the protein expression levels of TLR4 were higher than those of TLR2. Next, we measured the mRNA transcript levels and the protein levels of these receptors after transfection with siRNA constructs. TLR2 and TLR4 mRNA transcript levels and protein levels were significantly reduced by specific TLR2-and TLR4-siRNA constructs. Moreover, the TLR2-siRNA392 and TLR4-siRNA703 constructs could reduce the TLR2 and TLR4 mRNA levels by up to 94% and 84%(P<0.05).(3).rhHMGB1 induced NF-κB activation through TLR2 or TLR4 signaling in osteoblasts.To investigate the potential mechanisms whereby HMGB1 can regulate osteoblast migration, we incubated rat osteoblasts with rhHMGB1 at a concentration of 150 μg/L for 24h and detected the levels of NF-κB p65 in the cytosolic and nuclear fractions by immunoblotting. We found that the nuclear translocation of NF-κB p65 subunit increased in response to rhHMGB1 stimulation. Then, we measured the expression of NF-κB p65 when osteoblasts were pretreated with TLR2-or TLR4-siRNA for 24 h, followed by the addition of rhHMGB1 (150 μg/L) to the culture medium for 24 h. Pretreatment with TLR2-or TLR4-siRNA decreased nuclear NF-κB p65 subunit levels (p<0.05), which indicated that rhHMGB1 could induce NF-κB activation via TLR2 or TLR4 in osteoblasts.(4).Involvement of TLR2 and TLR4 in HMGB1-mediated osteoblast migrationFirst, to investigate whether TLR2 or TLR4 signaling 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). 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. In accord with our previous findings, these data suggest that TLR2-or TLR4-dependent NF-κB signaling pathways are involved in HMGB1-induced osteoblast migration.5.ConclusionsIn this study, we found that HMGB1 can promote osteoblast migration. Transwell assay indicated that the migration rate of osteoblasts increases 2.3 fold after HMGB1 treatment. The MTT assay revealed that the viability of osteoblasts was not reduced significantly after treated by rhHMGB1.In our cellular system, the expression level of TLR2 was higher than that of TLR4. Introduction of specific siRNA constructs could effectively inhibit the mRNA and protein expression of the receptor. The results identified that siRNA technique is effective in silencing the expression of targeted genes. Our findings suggest that knockdown of TLR2 or TLR4 could significantly inhibit osteoblast migration and NF-κB p65 subunit nuclear translocation when stimulated by HMGB1.Meanwhile, osteoblast proliferation was not significantly inhibited by HMGB1. These data showed that HMGB1 could promote the migration of osteoblasts through TLR2/4 and NF-κB and did not cause any cytotoxic effects. Recent reports showed that HMGB1 could prompt the migration of MSCs and the differentiation of osteoclast. Therefore, we assumed HMGB1 could participate the process of bone remolding and endochondral ossification by connection of osteoblast and osteoclast. For the first time, we provided evidence that HMGB1 promotes osteoblast migration through the activation of NF-κB. And the results connect osteoblast and immune system through toll like receptors which are the major receptors of immune cells. That how to prevent the pathological bone loss and promote bone repair may represent a therapeutic strategy for treating diseases in which regeneration is impaired. Together, our results demonstrate that HMGB1 promotes the migration of osteoblasts by TLR2/4-dependent signaling pathways that drive the activation of NF-κB, which indicates a significant functional role for HMGB1 in skeletal development and bone restoration. |
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