Endothelin-1 induces interleukin-18 expression in human osteoblasts.

OBJECTIVE: Both endothelin-1 (ET-1) and interleukin (IL)-18 induce osteoblast proliferation under normal and pathophysiological conditions. In the present study, we explored the interaction between the two proteins by examining the effect of ET-1 on IL-18 expression in cultured human osteoblasts. METHODS: Human osteoblasts were treated with ET-1 in different concentrations (1, 10, 20, 40, or 50 nM) for different length of time (1, 6, 12, 18, or 24 h) in the presence or absence of ET A receptor (ETAR) blocker BQ123, ET B receptor (ETBR) blocker BQ788, p38 mitogen-activated protein kinase (MAPK) siRNA, or different kinase inhibitors. RESULTS: ET-1 increased the IL-18 mRNA level in a statistically significant dose- and time-dependent manner within 18 h, which was reflected in dose-dependent induction of the human IL-18 gene promoter activity and IL-18 protein/secreted protein expression. BQ123 (1µ M) and p38 MAPK siRNA and inhibitor PD169316 (25 µM) completely abolished the promoting effect of ET-1 on IL-18 expression. [(3)H]thymidine incorporation assays showed that ET-1 lost a major part (57%) of its promoting effect on osteoblast proliferation when the endogenous IL-18 expression in osteoblasts was knocked down by 75%. CONCLUSIONS: ET-1 induces IL-18 expression in human osteoblasts at the gene promoter/transcription level via ETAR by a p38 MAPK-dependent mechanism, and that IL-18 mediates a major part of ET-1 induced osteoblast proliferation. This study provides the first evidence of interaction between ET-1 and IL-18 in osteoblast and adds new insights into bone physiology and pathophysiology.

[Effect of compressive stress on the expression of DNAX-activating protein 12 and tartrate-resistant acid phosphatase in mouse monocyte RAW264.7].

OBJECTIVE: To investigate the expression of DNAX-activating protein 12 (DAP12) and tartrate-resistant acid phosphatase (TRAP) in mouse monocyte RAW264.7 subjected to compressive stress. METHODS: Mouse monocyte RAW264.7 was subjected to four-point bending system. The expression of DAP12 and TRAP mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR) and the expression of DAP12 protein by Western Blotting after 0, 3, 6 and 12h's compressive stress. RESULTS: After RAW264.7 cells were cultured by osteoclast cell culture fluid, the amount of the cells increased, and the volume enlarged, and the number of nuclei per osteoclast increased in vitro. The expression of DAP12 and TRAP mRNA and DAP12 protein of RAW264.7 cells subjected to compressive stress increased along with the time (P < 0.05). CONCLUSION: The mouse monocyte RAW264.7 can differentiate into osteoclast in vitro, and high expression of DAP12 exists in the process of osteoclast differentiation.

[Effect of Erigeron Breviscapus on the expression of OPG/RANKL/RANK in osteoblasts and pre-osteoclasts in vitro].

OBJECTIVE: To study the effect of Erigeron Breviscapus (EB) at different concentrations and different intervention time points on the mRNA and protein expression of OPG/RANKL/RANK in MG63 osteoblast-like cells and RAW264. 7 pre-osteoclast cells cultured in vitro, thus exploring roles EB played in bone rebuilding and its mechanisms. METHODS: MG63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro. The 3rd passage cells were divided into the control group and different experimental groups. Total RNA and protein were respectively isolated from cells treated with different concentrations of EB (0, 0.001, 0.01, 0.1, and 1.0 mg/mL) for 48 h. Meanwhile, the protein was extracted from 0 and 1 mg/mL EB groups at 12, 24, and 48 h respectively. Expression of OPG mRNA and RANKL mRNA in MG63 osteoblast-like cells, and expression of RANK mRNA in RAW264.7 pre-osteoclast cells were detected by semi-quantitative RT-PCR. Expression of OPG protein and RANKL protein in MG63 osteoblast-like cells, and expression of RANK protein in RAW264. 7 pre-osteoclast cells were detected by Western blot. RESULTS: Along with increased EB concentration, expression of OPG mRNA and protein in MG63 osteoblast-like cells was gradually lowered (P < 0.05) after 48-h intervention of EB, the expression of RANKL mRNA and protein in MG63 osteoblast-like gradually increased (P < 0.05); the expression of RANK mRNA in RAW264.7 pre-osteoclast cells increased (P < 0.05). But the expression of RANK mRNA was slightly lower in the 0.1 mg/mL EB group than in the 0.01 mg/mL EB group, and the expression of RANK protein in RAW264.7 pre-osteoclast cells gradually increased (P < 0.05). After treatment with 1 mg/mL EB for 12, 24, 48 h, the expression of OPG protein in MG63 osteoblast-like cells gradually decreased as time went by (P < 0.05), and the expression of RANKL protein in MG63 osteoblast-like and RANK protein in RAW264.7 pre-osteoclast cells gradually increased (P < 0.05). The expression of RANKL protein in RAW264.7 pre-osteoclast cells increased as time went by (P < 0.05). CONCLUSION: EB could inhibit the expression of OPG in osteoblasts in a dose- and time-dependent manner, promote the expression of RANKL in osteoblasts and the secretion of RANK in pre-osteoclast, indicating EB might play roles in promoting bone resorption.

[Effects of DNAX-associated protein 12 signal pathways on differentiation of mouse monocytes RAW264.7 into osteoclasts by tensile strain].

OBJECTIVE: To explore the effect of DNAX-associated protein 12 (DAP12) pathway on the transformation from mouse monocytes RAW264.7 to osteoclasts induced by tensile strain. METHODS: DAP12shRNA plasmid was constructed and introduced to RAW264.7 cells. Then we supplied tensile strain to RAW264.7 cells by four-point bending system. The mRNA or protein expression of DAP12, tartrate-resistant acid phosphatase (TRAP), tyrosine kinases Btk and Tec and nuclear facior of activated T cells 1 (NFATc1) was measured by reverse transcription PCR (RT-PCR) and Western blotting respectively. RESULTS: The expression of DAP12 mRNA (0.112 ± 0.025) and protein (0.193 ± 0.015) both declined sharply after plasmid being introduced into monocytes RAW264.7 (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, tensile strain-induced TRAP mRNA expression of RAW264.7 cells increased at 6 h (0.671 ± 0.031) and 12 h (0.800 ± 0.043) (P < 0.05), but it was weaker than non-RNA-interference-groups at each time point (P < 0.05). After silencing DAP12 expression in RAW264.7 cells by RNA interference, the expressions of Btk, Tec, NFATc1 increased as time passed (6, 12 h) (P < 0.05), but the expressions on corresponding time decreased sharply compared with those in control groups (P < 0.05). CONCLUSIONS: DAP12 pathway play an important role in regulating osteoclast differentiation induced by tensile strain.

[Effect of parathyroid hormone related protein on proliferation of human osteoblast-like cell under tension force].

OBJECTIVE: To investigate the effect of parathyroid hormone related protein (PTHrP) on proliferation of human osteoblasts (MG-63) under the circumstance of tension force in vitro. METHODS: An apparatus was designed and fabricated by which force was loaded onto the cultured cells in vitro. Reverse transcription-polymerase chain reaction (RT-PCR) was used for measuring the expression of PTHrP mRNA and c-fos mRNA. The effect of tension force and different PTHrP dose(0, 0.01, 0.1, 1 nmol/L) on the proliferation of human osteoblasts were examined using flow cytometry. RESULTS: Various forces of the mechanical stretching exerted different influences on the intensities of the mRNA' expression. The strain of 12% induced the most remarkable mRNA' expression. The mitogenesis happened in the group with tension force (12%) combined with PTHrP was more active than that in the group with PTHrP or tension' force only. Tension force combined with PTHrP induced significantly more c-fos mRNA than that of tension force only. CONCLUSION: The mechanical stretching can inevitably influence the expression of PTHrP mRNA. The most active mitogenesis happened in the group with tension force combined with PTHrP. The effect may be related with the signaling pathways of c-fos.

[Effect of continuously compressive pressure on the expression of RANKL mRNA in human periodontal ligament cells in vitro].

OBJECTIVE: To determine the effect of continuously compressive pressure (CCP) on the expression of receptor activator of nuclear factor kappa B ligand (RANKL) in human periodontal ligament cells (HPDLCs) and to investigate the role of RANKL in alveolar bone rebuilding during orthodontic tooth movement. METHODS: The primary HPDLCs were isolated from human periodontal ligament by explanting enzymatic digestion with trypsin and collagenase to establish a pressure model. Top-bottom axial pressures (1, 2, and 3 g/cm(2)) were laid on HPDLCs for 0.5, 1.5, 6, 12, 24, and 48 h, respectively. The RANKL expression was identified by the reverse transcription-polymerase chain reaction (RT-PCR) at the mRNA level. RESULTS: The expression of RANKL mRNA significantly increased in a time-dependent manner (P<0.01), so did the value of pressure, especially in the 2 g/cm(2) group (P<0.05). CONCLUSION: CCP can up-regulate the expression of RANKL mRNA in human periodontal ligament cells.

[Tension-force induced cyclooxygenase-2 expression mediated by microfilament in human periodontal ligament fibroblast].

OBJECTIVE: To study the role of microfilament polymerization in menchanotransduction by human periodontal ligament fibroblast (hPDLFs). METHODS: In tension-force group, hPDLFs were treated by tension-force values of 18% for 8 h, 16 h, 24 h. In tension-force and inhibitor group, the sample was treated with 5 microg/mL cytochalasin B before using tension-forece. Each sample was collected and the expression of cyclooxygenase-2 was measured by using immunohistoche staining. RESULTS: In tension-force group, the expression of cyclooxygenase-2 enhanced with the extension of loading time. In tension-force and inhibitor group, cyclooxygenase-2 expression was depressed and had no relation with loading time. CONCLUSION: Tension-force induced cyclooxygenase-2 expression is mediated by microfilament, disruption of the microfilament polymerization will destroy mechanotransduction in hPDLFs.

[Effect of erigeron breviscapus on the expression of vascular endothelial growth factor in the periodontal tissues of rabbits during orthodontic tooth movement].

OBJECTIVE: To detect the effect of erigeron breviscapus on the expression of vascular endothelial growth factor (VEGF) in the periodontal tissues during orthodontic tooth movement. METHODS: 45 rabbits were divided into 3 groups (groups A, B and C). Groups A and B included experimental group of 1, 3, 7 and 14 days respectively. The mandibular first molar of each experimental rabbit was observed. The rabbits of group A and group B received iontophoresis with erigeron breviscapus into the right (group A-R and group B-R) and with normal sodium into the left as the control (group A-L and group B-L). Additionally, the rabbits of group B were designed orthodontic appliance, by which 0.78 N mesial force was applied to pull the mandibular first molars. Group C, group of 0 day, was no iontophoresis and orthodontic appliance as the control. After killed on schedule, the amount of experimental tooth movement was measured and the expression of VEGF was examined by immunohistochemical method. RESULTS: The amount of experimental tooth movement increased successively from 1 to 14 days. The differences among days 3, 7 and 14 were significant in the comparison between group B-R and group B -L (P < 0.01). The expression of VEGF in groups A-R and B-L enhanced apparently compared with that in groups C and A-L (P < 0.01), but that in group B-R was the most apparent (P < 0.01). The expression of VEGF reached the peak level on day 3 in groups A-R and B-R (P < 0.01), but it reached the peak level on day 7 in group B-L (P < 0.01). CONCLUSION: Erigeron breviscapus by iontophoresis can accelerate orthodontic tooth movement, and can meanwhile up-regulate the expression of VEGF in periodontium in the earlier period of orthodontic tooth movement. Thus it can be presumed that one of its mechanisms for erigeron breviscapus to accelerate orthodontic tooth movement is erigeron breviscapus effects the metabolism and differentiation of osteoblast and osteoclast through up-regulating the expression of VEGF in periodontium.