Thymosin ß4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells.

Thymosin ß4 (Tß4) regulates the expression of molecules associated with dentinogenesis, including bone sialoprotein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between Tß4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate Tß4 mRNA expression in odontoblasts during dentinogenesis and the association between the Tß4 signaling pathway and BSP expression in MDPC­23 odontoblastic cells. Expression and localization of Tß4 mRNA was determined by in situ hybridization during mouse tooth development. The effect of Tß4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC­23 cells. The expression of Tß4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. Tß4 increased BSP mRNA and protein levels in MDPC­23 cells, but this was inhibited by PD98059 or SIS3 treatment. Tß4 increased levels of phosphorylated (p) extracellular signal­regulated kinase (ERK)1/2, pSmad3, pß­catenin, and runt­related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. Tß4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of ß­catenin was decreased. Tß4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. Tß4 induced BSP expression in MDPC­23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.

Thymosin ß4 Reduces H2O2 Induced Oxidative Stress in MC3T3-E1 Cells on Titanium Surface.

Thymosin ß4 (Tß4) is known to inhibit an inflammatory response and to increase the survival of osteoblasts on titanium (Ti) surfaces. Ti is the most widely used graft material in dentistry; however, an inflammatory response induced following implant placement results in the generation of reactive oxygen species (ROS). The oxidative stress from the production of ROS such as nitric oxide (NO) and hydrogen peroxide (H2O2) can damage surrounding cells, resulting in implant failure by decreasing cell viability. Thus, the aim of this study was to determine the biological effects of Tß4 on the oxidative stress induced to MC3T3-E1 preosteoblasts on the Ti surface. Based on an MTT assay and bromodeoxyuridine immunofluorescence staining, Tß4 was found to increase the proliferation of the H2O2-exposed MC3T3-E1 cells on Ti discs. Reverse transcription-polymerase chain reaction and western blot analyses showed that Tß4 decreased the mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in H2O2-exposed MC3T3-E1 cells on the Ti discs. Tß4 inhibited the synthesis of intracellular ROS and the secretion of NO and prostaglandin E2 (PGE2) from H2O2-exposed MC3T3-E1 cells on the Ti discs. In conclusion, Tß4 inhibits H2O2-induced iNOS and COX-2 expression with a decrease in ROS, NO, and PGE2 synthesis, which leads to improved cell survival with low cytotoxicity under an oxidative stress condition in MC3T3-E1 cells on the Ti surface. This suggests that Tß4 may be a crucial molecule to reduce oxidative stress-induced cell damage or hypoxia, leading to promoted osseointegration on the Ti surface during implant placement.

Secretory leukocyte protease inhibitor promotes differentiation and mineralization of MC3T3-E1 preosteoblasts on a titanium surface.

Mineralized bone matrix constituted with collagenous and non-collagenous proteins was synthesized by osteoblasts differentiated from mesenchymal stem cells. Secretory leukocyte protease inhibitor (SLPI), a serine protease inhibitor, promotes cell migration and proliferation, and suppresses the inflammatory response. Recent studies reported that SLPI regulates the formation of dentin and mineralization by odontoblasts and increases the adhesion and viability of preosteoblasts on a titanium (Ti) surface. Ti and its alloys are widely used implant materials in artificial joints and dental implants owing to their biocompatibility with bone. Therefore, this study aimed to examine whether SLPI can be an effective molecule in promoting differentiation and mineralization of osteoblasts on a Ti surface. In order to investigate the effects of SLPI on osteoblasts, an MTT assay, PCR, western blotting and Alizarin Red S staining were performed. The results demonstrated that SLPI increased the viability of osteoblasts during differentiation on Ti discs compared with that of the control. The expression levels of SLPI mRNA and protein were higher than that of the control after treatment of osteoblasts with SLPI on Ti discs during differentiation. SLPI increased the formation of mineralized nodules and mRNA expression of alkaline phosphatase, dentin sialophosphoprotein, dentin matrix protein 1, bone sialoprotein, and collagen I in osteoblasts on Ti discs compared with that of the control. In conclusion, SLPI increases the viability and promotes the differentiation and mineralization of osteoblasts on Ti surfaces, suggesting that SLPI is an effective molecule for achieving successful osseointegration between osteoblasts and a Ti surface.

Function of Secretory Leukocyte Protease Inhibitor (SLPI) in Odontoblast During Mouse Tooth Development.

Secretory leuckocyte protease inhibitor (SLPI) is thought as a regulating protein on the synthesis and degradation of matrix proteins. But there was no report of expression and function of SLPI on the tooth development, especially on the odontoblasts. As observed by in-situ hybridization and immunohistochemical analysis, SLPI was expressed in odontoblasts and predentin on post-natal day 4 (PN4). On PN10, SLPI was observed under the dentin and apical region including odontoblasts processes. Further, on PN15, expression of SLPI was the same pattern compared to PN10. SLPI was expressed under layer of the odontoblasts and in odontoblasts on PN20. Matrix metalloproteinase-2 (MMP-2) and -9 levels in SLPI/MDPC-23 cells were higher than that of the MDPC-23 cells. The gene expression of SLPI, bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I (Col I) was higher in SLPI/MDPC-23 than that of MDPC-23 cells and the expression of dentin sialophosphoprotein (DSPP) was lower in SLPI/MDPC-23. Taken together, our results suggest that SLPI may be a MMP-2 and -9 regulating molecule in odontoblasts during dentin matrix formation and acts as a signaling molecule for dentin matrix related proteins during odontoblasts differentiation and mineralization.

Secretory Leukocyte Protease Inhibitor (SLPI) Increases Focal Adhesion in MC3T3 Osteoblast on Titanium Surface.

An appropriate interaction between implanted materials and the surrounding tissue is essential for successful implantation. Titanium (Ti) and some of its alloys have been used in dentistry and orthopedics as a substitutive material for hard tissue, such as teeth or natural bone. Nevertheless, metal ions released from titanium and alloy implants have adverse biological effects on biological tissues or cells. Secretory leukocyte protease inhibitor (SLPI) promotes cell migration, proliferation and wound healing. FAK and ERK1/2 signaling regulate cell adhesion and proliferation for cell survival. This study evaluated the potential of SLPI as a molecule to increase the cell adhesion on the Ti surface. Compared with the untreated cells, SLPI increased the adhesion of MC3T3-E1 cells to Ti discs, formation of actin stress fibers, paxillin expression and the phosphorylation of FAK. Moreover, SLPI enhanced the level of Grb2 and Ras expression and ERK1/2 phosphorylation in the MC3T3-E1 cells on Ti discs. These results suggest that SLPI can increase the interaction between the implanted Ti material and surrounding bone in orthodontic and dental surgery, making an effective nanomolecule for successful implantation.

660 nm Red LED Induces Secretory Leukocyte Protease Inhibitor (SLPI) in Lipopolysaccharide-Stimulated RAW264.7 Cell.

SLPI acts as a modulator of the innate immune responses of macrophages, neutrophils and odontoblasts, and LPS-inducible anti-inflammatory cytokine to suppress the production of pro-inflammatory products by macrophages. Many studies have revealed the effects of light emitting diodes (LEDs) on the tissue repair and inflammatory responses. Although the anti-inflammatory mechanisms of irradiation with LEDs in gingival fibroblasts are known, the effects of 660 nm red LEDs on the inflammation remain unclear. Moreover, there is no report regarding the molecular mechanism for the relationship between SLPI and biological effects of LEDs. The effects of 660 nm red LEDs on inflammation with SLPI were investigated by examining the effects of 660 nm LED on the SLPI expression of RAW264.7 cells after LPS stimulation. This paper reports that the 660 nm red LED induced SLPI expression or reduced the LPS response, and inhibited NF-κB activation directly, leading to the suppression of pro-inflammatory cytokines, such as TNF-α and IL-1ß, suggesting that it might be a useful wavelength LED for inflammation therapy.

The Effect of Thymosin ß4 for Osteoblast Adhesion on Titanium Surface.

Titanium (Ti) is the most widely used implant material in dentistry and orthopedics but the release of metal ions from Ti implants results in increased bone resorption by enhancing the production of inflammatory cytokines from the macrophages and facilitating osteoclast differentiation. Thymosin ß4 (Tß4) has several biological activities, such as promoting wound healing, angiogenesis, cell proliferation and migration in mammalian cells. This study examined the role of Tß4 in osteoblasts via focal adhesions (FAs) and ERK1/2 signaling related to cell adhesion and proliferation for cell survival on the Ti surface. As a result, cell adhesion and proliferation increased in the Tß4 treated cells (Tß4/MC3T3-E1) but was significantly lower in the Tß4 knock-down cells by Tß4-siRNA (si-Tß4/MC3T3-E1) than that of the untreated cells. The levels of FAK phosphorylation, paxillin expression, and paxillin localization were higher in the Tß4/IMC3T3-E1 cells than that of the untreated cells but lower in the si-Tß4/MC3T3-E1 cells. In addition, the levels of cell proliferation, Grb2 and Ras protein expression and phosphorylation of ERK1/2 were higher in the Tß4/MC3T3-E1 cells than in the untreated cells but lower in the si-Tß4/IMC3T3-E1 cells. These results suggest that Tß4 might be a good nanomolecule that promotes osteoblast survival by facilitating adhesion and proliferation on the Ti surface.

Dynamin II involves in cell migration and actin formation of NIH3T3 cells.

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.

Expression of thymosin ß4 in odontoblasts during mouse tooth development.

Thymosin ß4 (Tß4) is expressed in developing tissue, where it stimulates cell differentiation and migration. Further, Tß4 is expressed during molar development in mice, but the expression and function of Tß4 in odontoblasts during mammalian tooth development have not yet been reported. Therefore, this study examined the expression and function of Tß4 in differentiating odontoblasts during tooth development. As observed by immunohistochemistry, Tß4 was expressed in the oral epithelium and inside cells of the tooth bud on embryonic day 15 (E15). Further, on E17, Tß4 was expressed strongly in the dental lamina and oral epithelium, but only expressed in part of the cells in the outer and inner dental epithelium. Tß4 was strongly expressed in the entire cytoplasm of odontoblasts on postnatal day 1 (PN1) and expressed intensively in the apical area of odontoblasts on PN4. Further, expression of Tß4 was increased gradually in odontoblasts from PN1 to PN21. In an odontoblast cell line, MDPC-23, expression of Tß4 mRNA and protein was increased strongly on day 4 and gradually decreased from day 14. The gene expression of dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I, related with mineralization, was significantly decreased in si-Tß4/MDPC-23 during differentiation compared to that in MDPC-23 cells. Taken together, our results suggest that Tß4 may be involved in oral epithelial cell proliferation at the initial stage of tooth development and regulates the expression and secretion of proteins during odontoblast differentiation.

Delayed healing and induction of secretory leukocyte protease inhibitor in polycystic ovary syndrome rat skin wounds.

Secretory leukocyte protease inhibitor (SLPI) and estrogen promote wound healing through a decrease in the excessive inflammatory response, accelerating re-epithelialization and increasing the amount of collagen deposition. The excessive administration of estradiol valerate (EV) using hormonal therapy decreases the concentration of estrogen abruptly and induces the polycystic ovary syndrome (PCOS). In this study, the PCOS rat skin wound area was wider than that of the normal groups and the rate of keratinocyte migration in PCOS was lower than the normal group. The numbers of inflammatory cells and macrophages recruited in the PCOS group were larger than that of the normal group. More collagen was deposited in the healing area of the normal group than in the PCOS group. The level of SLPI expression was higher in the PCOS group than the normal group after wounding, with the exception of the epithelium. On the other hand, mRNA and protein expression levels of transforming growth factor-ß1 (TGF-ß1) were lower in the PCOS group than in the normal group. Matrix metalloproteinase-2 (MMP-2) and MMP-9 levels in the PCOS group were significantly lower than that of the normal group. Therefore, increased SLPI in PCOS skin wounds may help prevent an excessive inflammatory response and aberrant collagen deposition but not are sufficient to accelerate PCOS skin wound healing, suggesting that SLPI may act as a local rather than a systemic modulating molecule in PCOS rat skin wounds.

Expression of dynamin II in odontoblast during mouse tooth development.

Odontoblasts secrete a collagen-based matrix and release numerous membrane-bound matrix vesicles, which are involved in dentin formation during tooth development. Dynamin II is a GTPase protein that contributes a variety of vesicular budding events, such as endocytotic membrane fission, caveolae internalization and protein trafficking in the Golgi apparatus. However, the expression and function of dynamin II in odontoblasts has not been reported. Therefore, this study examined the expression and possible role of dynamin II in odontoblasts during tooth development and mineralization. The levels of mRNA and protein expression in MDPC23 cells were significantly high at the early stages of differentiation and then decreased gradually thereafter. Immunohistochemistry showed that dynamin II was not expressed near the region of the odontoblasts at embryonic day 17 (E17) and E21. However, dynamin II was expressed strongly in the odontoblast layer at postnatal day 1 (PN1) and decreased gradually at PN3 and PN5. In addition, at PN15 in the functional stage, the dynamin II protein was also expressed in the odontoblast process as well as adjacent to the nuclear region. In conclusion, dynamin II may be involved in the transport of vesicles containing collageneous and non-collageneous proteins for dentin formation in odontoblast, suggesting that it is a good nanomolecule as a candidate to regulate the secretion of collagen on the bone and other nano material.

Induction of secretory leukocyte protease inhibitor (SLPI) in estradiol valerate (EV) induced polycystic ovary.

The excessive administration of estradiol valerate induces polycystic ovary syndrome by formation of follicular cysts. Secretory leukocyte protease inhibitor (SLPI) promotes wound healing by decreasing the excessive inflammatory response, stimulating keratinocyte proliferation and increasing collagen deposition through the inhibition of protease activity. In this study, SLPI expression was high in the ovarian stroma, corpus luteum, unilaminar primary follicle, multilaminar primary follicle and granulose layer of the antral follicle in polycystic ovary (PCO) compared to the normal ovary. SLPI was expressed strongly in the theca around the cyst in PCO compared to the mature follicle in the normal ovary. The levels of SLPI mRNA and protein expression were higher in PCO than in the normal ovary, and the level of MMP-2 expression was lower in PCO. These results showed that the formation of a cyst was initiated from a multilaminar primary follicle and SLPI expression was increased depending on the morphological changes in the follicle and ovarian stroma. Therefore, an increase in SLPI may be related to the suppression of tissue disruption, and act as a protease inhibitor in PCO, suggesting that SLPI increases independently of the estrogen concentration in pathological tissues.

Secretory leukocyte protease inhibitor is associated with MMP-2 and MMP-9 to promote migration and invasion in SNU638 gastric cancer cells.

Secretory leukocyte protease inhibitor (SLPI) protects tissue from proteases, and promotes cell proliferation and healing during inflammatory response. SLPI is also overexpressed in gastric, lung and ovarian cancers, which accelerates the metastasis of cancer cells. Matrix metalloproteinases-2, -9 (MMP-2 and MMP-9) are overexpressed in high metastatic cancers, and promote the migration of cancer cells through collagen degradation. SLPI and MMP-2, -9 are critical factors in stimulating the metastatic processes but there are no reports of a direct correlation between these molecules. Therefore, this study examined the role of SLPI related to MMP-2 and MMP-9 using two gastric cancer cell lines, such as characterized non-metastatic SNU484 and highly metastatic SNU638 cells. SLPI, MMP-2 and MMP-9 mRNA and protein expression were higher in SNU638 cells than in SNU484 cells. In addition, the rate of cell migration and invasion was higher in the SNU638 cells than in SNU484 cells. Interestingly, after treatment with SLPI, the rate of migration and invasion was higher in the SNU484 cells than in the positive control (PC) SNU484 cells. The rate of migration was also higher in the SNU638 cells after SLPI treatment than in the SNU638 cells (PC) but the invasion rate was not changed. The expression and secretion of MMP-2 and MMP-9 as well the rate of cell migration and invasion were significantly lower in SLPI-siRNA transfected SNU638 cells (si-SLPI/SNU638) but higher in SLPI-treated SNU484 cells (SNU484 + SLPI). Strong Elk-1 phosphorylation was detected in SNU484 + SLPI and SNU638 cells but was barely detectable in SNU484 and si-SLPI/SNU638 cells. These results show that SLPI promotes the metastasis of SNU638 gastric cancer cells by increasing MMP-2 and MMP-9 expression through Elk-1 signaling, indicating its role as a signaling molecule not a protease inhibitor.

Temporal induction of secretory leukocyte protease inhibitor (SLPI) in odontoblasts by lipopolysaccharide and wound infection.

INTRODUCTION: The secretory leukocyte protease inhibitor (SLPI) is a bacterial lipopolysaccharide (LPS)-induced product of macrophages that antagonizes the LPS-induced activation of a number of proinflammatory signaling factors. From our previous experiments, it was found that SLPI was expressed slightly in odontoblast-like cells (MDPC-23). Therefore, these experiments were designed to determine the function of SLPI in MDPC-23 and odontoblasts during the inflammatory response caused by infections and wounds. METHODS: MDPC-23 cells were exposed to 100 ng/mL Escherichia coli LPS, and artificial wounds were induced in the right first molar of the maxillary of rats. In addition, a morphological change in the MDPC-23 cells was observed after LPS treatment. MDPC-23 cells were transfected transiently with the nuclear factor kappa-B (NF-kappaB) promoter binding vector. RESULTS: The level of SLPI expression increased strongly 30 minutes after the LPS treatment. Scanning electron microscopy revealed many extensions of the cytoplasmic processes after LPS stimulation. SLPI was expressed along the dentinal tubules and odontoblasts layer in rat teeth after an artificial wound. SLPI also inhibited the LPS-induced activation of NF-kappaB in MDPC-23. CONCLUSIONS: We report for the first time that SLPI is expressed temporally in infected odontoblasts and may participate in the anti-inflammatory response through NF-kappaB signaling in odontoblast-like cells.

Increased association of dynamin II with myosin II in ras transformed NIH3T3 cells.

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin II has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin II and myosin II. The dynamin II expression level was higher when co-expressed with myosin II in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin II interacted with myosin II. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin II and myosin II gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.