• Journal of dental hygiene science
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• v.23 no.4
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• pp.282-295
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• 2023

Background: Secretory leukocyte protease inhibitor (SLPI) protects tissues from proteases and promotes cell proliferation and healing. SLPI also reduces periodontal inflammation and alveolar bone resorption by inhibiting proinflammatory cytokine expression in rat periodontal tissues and osteoblasts. However, little is known of the role of SLPI in the expression of osteoclast regulatory factors from osteoblasts, which are crucial for the interaction between osteoblasts and osteoclasts. Therefore, we aimed to determine the effects of SLPI on the regulation of osteoclasts and osteoblasts in LPS-treated alveolar bone and osteoblasts. Methods: Periodontitis was induced in rats using LPS. After each LPS injection, SLPI was injected into the same area. Immunohistochemical analysis was performed with antibodies against SLPI, RANKL, OPG, and Runx2 in the periodontal tissue. RT-PCR and western blotting were performed to determine the expression levels of SLPI, RANKL, OPG, and Runx2 in LPS- and SLPI/LPS-treated MC3T3-E1 cells. SLPI/LPS-treated MC3T3-E1 cells were also stained with Alizarin Red S. Results: Immunohistochemical analysis showed that the expression levels of SLPI, OPG, and Runx2 were higher while that of RANKL was lower in the LPS/SLPI group relative to those in the LPS group. The mRNA and protein expression of SLPI, OPG, and Runx2 was higher in SLPI/LPS/MC3T3-E1 cells than in LPS/MC3T3-E1 cells, and RANKL expression was lower. During differentiation, OPG and Runx2 protein levels were higher whereas RANKL levels were lower in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 cells on days 0, 4, 7, and 10. In addition, mineralization and matrix deposition were higher in SLPI/LPS/MC3T3-E1 than in LPS/MC3T3-E1 on days 7 and 10. SLPI decreased RANKL expression in LPS-treated alveolar bone and osteoblasts but increased the expression of OPG and Runx2. Conclusion: SLPI can be considered as a regulatory molecule that indirectly regulates osteoclast activation via osteoblasts and promotes osteoblast differentiation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.33 no.4
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• pp.381-385
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• 2007

Bone is a dynamic organ that bone remodeling occurs throughout life and involves the process in which the bone matrix is broken down through resorption by osteoclasts and then built back again through bone formation by osteoblasts. Usually these two processes balance each other and a stable level of bone mass is maintained. We here discuss transcription factors involved in regulating the osteoblast differentiation pathway. Runx2 is a transcription factor which is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Its companion subunit, Cbf${\beta}$ is needed for an early step in osteoblast differentiation pathway. Whereas Osterix(Osx) is a new identified osteoblast-specific transcription factor which is required for the differentiation of preosteoblasts into more mature and functional osteoblasts. We also discuss other transcription factors, Msx1 and 2, Dlx5 and 6, Twist, and Sp3 that affect skeletal patterning and development. Understanding the characteristics of mice in which these transcription factors are inactivated should help define their role in bone physiology and pathology of bone defects.

• Journal of Korean Medicine Rehabilitation
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• v.30 no.4
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• pp.1-16
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• 2020

Objectives The main purpose of this study was to evaluate the bone healing effect of Pahyeolsandong-tang (PHT)(Poxiesanteng-tang) extract in tibia fracture-induced mice. Methods PHT was extracted using a solution of 35% ethanol in 60℃ for 8 hours. Mice were randomly divided into 4 groups (normal, control, PHT 50 and PHT 100). Mice of experimental groups were medicated with PHT 50 or 100 mg/kg for 7 to 21 days. To clarify the effect of bone fracture healing, relative messenger RNA (mRNA) expressions of osteocalcin (OCN), runt-related transcription factor 2 (Runx2), osterix (OSX), Sox9, collagen type II alpha 1 chain (Col2a1), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) were examined. Results In in vitro experiment, relative mRNA expression of OCN, Runx2, Col2a1 was significantly increased in PHT treated group to compare with control differentiation group. In in vivo experiment, relative mRNA expression of OCN, Runx2, OSX, Sox9, Col2a1, RANKL, OPG was significantly increased in PHT treated group. Conclusions This study showed that PHT accelerates bone fracture healing through the activation of osteoclasts and osteoblasts. It was showed that PHT significantly promotes osteoblasts differentiation by osteoblast differentiation markers such as OCN, Runx2, Col1a2. Also it was investigated that PHT had stimulatory effect on osteoblasts function through enhancing OCN, Runx2, OSX, Sox9, Col2a1 and, osteoclasts function through enhancing RANKL and OPG markers. PHT effectively promotes bone fracture healing process through activation of osteoblasts and osteoclasts.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.2
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• pp.290-303
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• 2006

Tooth eruption is a complex and tightly regulated process that involves cells of the tooth organ and the surrounding alveolus. Osteoclast precursors must be recruited into the dental follicle prior to the onset of eruption. This function of dental follicle may be regarded as the ability of bone remodeling characterized by the interaction of osteoclasts and osteoblasts. This is because tooth eruption is a localized event in which many of the genes required for eruption are expressed in the dental follicle. RANKL is a membrane-bound protein that is a member of the TNF ligand family. which is present on bone marrow stromal cells and osteoblasts, and induces osteoclast formation and activation from precursor cell. The biologic effect of RANKL is inhibited by OPG and, in bone, the relative ratio of RANKL and OPG modulates osteoclastogenesis. To evaluate the roles of RANKL and OPG in tooth eruption and the relations with the expression pattern of Runx2, in situ hybridization was performed with mandibles of mice at postnatal stage 1, 3, 5, 7, 9 and 11. mRNA of RANKL, OPG, and Runx2 are expressed in dental follicle and surrounding tissue from P1 to 11. To determine the sites of osteoclastic activity during tooth eruption, mandibles were dissected. Peak osteoclastic activity in alveolar bone along the occlusal and basal regions was observed from P5 to 9, with osteoclasts in these regions being large and strongly TRAP-positive The specific spatio-temporal expression patterns of RANKL, OPG, and Runx2 in our study suggest that tooth eruption could be progressed through the interactions of molecular signaling among dental follicle, dental organ and alveolar bone, furthermore it means that dental follicle is quite important in tooth eruption In addition, it indicates that these genes (RANKL, OPG, and Runx2) play critical roles in tooth eruption.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.23
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• pp.10187-10192
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• 2015

Cytochrome P450 (CYP) enzymes are a large family of constitutive and inducible mono-oxygenase enzymes that play a central role in the oxidative metabolism of both xenobiotic and endogenous compounds. Several CYPs are involved in metabolism of oxysterols, which are cholesterol oxidation products whose expression may be dysregulated in inflammation-related diseases including cancer. This study focused on CYP39A1, which can metabolize 24-hydroxycholesterol (24-OH) that plays important roles in the inflammatory response and oxidative stress. We aimed to investigate the expression status of CYP39A1 and its transcription factor (RUNX2) in relation to clinical significance in cholangiocarcinoma (CCAs) and to determine whether 24-OH could induce oxidative stress in CCA cell lines. Immunohistochemistry showed that 70% and 30% of CCA patients had low and high expression of CYP39A1, respectively. Low expression of CYP39A1 demonstrated a significant correlation with metastasis. Our results also revealed that the expression of RUNX2 had a positive correlation with CYP39A1. Low expression of both CYP39A1 (70%) and RUNX2 (37%) was significantly related with poor prognosis of CCA patients. Interestingly, oxidized alpha-1 antitrypsin (ox-A1AT), an oxidative stress marker, was significantly increased in CCA tissues in which CYP39A1 and RUNX2 were down regulated. Additionally, immunocytochemistry showed that 24-OH could induce ox-A1AT in CCA cell lines. In conclusion, our study revealed putative roles of the CYP39A1 enzyme in prognostic determination of CCAs.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.4
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• pp.643-653
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• 2003

Fibroblast growth factor (FGF) / FGF receptor (FGFR) mediated signaling is required for skeletogenesis in cluding intramembranous and endochondral ossifications Runx2 ($Cbfa1/Pebp2{\alpha}A/AML3$) is an essential transcription factor for osteoblast differentiation and bone formation. Murine calvaria and mandible are concurrently undergoing both intramembranous bone and cartilage formations in the early developmental stage. However the mechanism by which these cartilage formations are regulated remains unclear. To elucidate the effect of FGF signaling on development of cranial sutural cartilage and Meckel's cartilage and to understand the role of Runx2 in these process, we have done both in vivo and in vitro experiments. Alcian blue staining showed that cartilage formation in sagittal suture begins from embryonic stage 16 (E16), Meckel's cartilage formation in mandible from E12. We analyzed by in situ hybridization the characteristics of cartilage cells that type II collagen, not type X collagen, was expressed in sagittal sutural cartilage and Meckel's cartilage. In addition, Runx2 was not expressed in Meckel's cartilage as well as sagittal sutural cartilage, except specific expression pattern only surrounding both cartilages. FGF signaling pathway was further examined in vitro. Beads soaked in FGF2 placed on the sagittal suture and mandible inhibited both sutural and Meckel's cartilage formations. We next examined whether Runx2 gene lies in FGF siganling pathway during regulation of cartilage formation. Beads soaked in FGF2 on sagittal suture induced Runx2 gene expression. These results suggest that FGF signaling inhibits formations of sagittal sutural and Meckel's cartilages, also propose that FGF siganling is involved in the proliferation and differentiation of chondroblasts through regulating the transcription factor Runx2.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.3
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• pp.169-176
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• 2010

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects $1{\alpha}$, 25-dihydroxyvitamin $D_3$ ($1{\alpha},25(OH)_2D_3$)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of $1{\alpha},25(OH)_2D_3$-induced tartrate-resistant acid phosphatase-positive multinucleated cells and $1{\alpha},25(OH)_2D_3$-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) and Runx2 expressions were down-regulated in response to $1{\alpha},25(OH)_2D_3$. Knockdown of Runx2 inhibited $1{\alpha},25(OH)_2D_3$-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of $1{\alpha},25(OH)_2D_3$-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.

• Journal of Korean Medicine Rehabilitation
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• v.31 no.1
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• pp.1-16
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• 2021

Objectives This study was designed to evaluate the effects of Danggwisu-san (Dangguixu-san, DG) on bone repair from femur fracture in mice. Methods Mice were randomly divided into 4 groups (normal, control, positive control and DG 300 mg/kg-treated group). In order to investigate the effects of DG on gene expressions in experimental animals with fracture, we measured the levels of bone morphogenetic protein-2 (BMP2), cyclooxygenase-2 (COX2), Sox9, collagen type II alpha 1 chain (Col2a1), runt-related transcription factor 2 (Runx2), osterix genes. After the cytotoxicity test, we analyzed the levels of expression of osteocalcin and Runx2, and tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine. The process of fusion in the fracture was also investigated by gross examination. Results Through in vivo BMP2, COX2 gene expression significantly decreased. Sox9 significantly increased. Col2a1, Runx2, osterix gene expression also increased as well, but there was no statistical significance. The degree of unilateral fracture fusion investigated by gross examination was significantly faster than those of the other groups. Through in vitro the level of TNF-α in macrophages was increased by DG in a dose-dependent mannerand and 250 and 500 ㎍/mL showed statistical significance. Osteocalcin and Runx2 genes expressions increased when DG was treated in osteoblasts. Conclusions DG promotes the healing of the fracture through the expression of bone repair-related genes and TNF-α production. This study may set the foundation for the clinical application of DG to the patients with bone fractures.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1063-1066
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• 2016

Longan (Dimocarpus longan Lour.) has been used as a traditional oriental medicine and possesses a number of physiological activities. In this study, we used cell-based herbal extract screening to identify longan fruit extract (LFE) as an activator of osteoblast differentiation. LFE up-regulated alkaline phosphatase (ALP) activity, induced mineralization, and activated Runx2 gene expression in MC3T3-E1 cells. Furthermore, treatment of MC3T3-E1 cells with LFE promoted the phosphorylation of extracellular signal-regulated kinase1/2 (Erk1/2); however, abrogation of Erk1/2 activation with PD98059 resulted in down-regulation of the phospho-SMAD1/5/8 and Runx2 levels, which in turn reduced the ALP activity. Our findings suggest that LFE exerts its osteogenic activity through activation of the ERK signaling pathway and may have potential as an herbal therapeutic or a preventive agent for the treatment of osteoporosis.

• Molecules and Cells
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• v.43 no.10
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• pp.889-897
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• 2020

K-RAS is frequently mutated in human lung adenocarcinomas (ADCs), and the p53 pathway plays a central role in cellular defense against oncogenic K-RAS mutation. However, in mouse lung cancer models, oncogenic K-Ras mutation alone can induce ADCs without p53 mutation, and loss of p53 does not have a significant impact on early K-Ras-induced lung tumorigenesis. These results raise the question of how K-Ras-activated cells evade oncogene surveillance mechanisms and develop into lung ADCs. RUNX3 plays a key role at the restriction (R)-point, which governs multiple tumor suppressor pathways including the p14^ARF-p53 pathway. In this study, we found that K-Ras activation in a very limited number of cells, alone or in combination with p53 inactivation, failed to induce any pathologic lesions for up to 1 year. By contrast, when Runx3 was inactivated and K-Ras was activated by the same targeting method, lung ADCs and other tumors were rapidly induced. In a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS-driven human lung tumors, Runx3 was inactivated in both adenomas (ADs) and ADCs, whereas K-Ras was activated only in ADCs. Together, these results demonstrate that the R-point-associated oncogene surveillance mechanism is abrogated by Runx3 inactivation in AD cells and these cells cannot defend against K-Ras activation, resulting in the transition from AD to ADC. Therefore, K-Ras-activated lung epithelial cells do not evade oncogene surveillance mechanisms; instead, they are selected if they occur in AD cells in which Runx3 has been inactivated.