RhoE regulates actin cytoskeleton organization in human periodontal ligament cells under mechanical stress.

OBJECTIVES: RhoE and regulator of G-proteins signalling (RGS) 2 were identified as the up-regulated genes in human periodontal ligament (PDL) cells under compression. RhoE belongs to the Rho GTPase family, and RGS2, a novel family of GTPase-activating proteins, turns off the G-protein signalling. Rho family proteins have recently been known to regulate actin cytoskeleton dynamics in various cell types. In this study, we investigated the involvement of RhoE and RGS2 in the regulation of actin filament organization in the PDL cells under mechanical stress. METHODS: Human PDL cells were cultured and subjected to a static compressive force (3.0g/cm(2)) for 48h. To observe changes in the actin cytoskeleton and the expression of RhoE and RGS2 in response to mechanical stress, immunofluorescence analysis was performed. To examine the role of RhoE and RGS2 in actin filament organization, cells were transfected with antisense S-oligonucleotides (ODNs) to RhoE and RGS2. RESULTS: Compressive force caused a loss and disassembly of actin stress fibres leading to cell spreading. Immunocytochemical study revealed that RhoE and RGS2 expressions were induced by mechanical stress and localized in the perinuclear and in the cell membrane, respectively. The impaired formation of stress fibres caused by compressive forces was recovered by treatment with antisense S-ODN to RhoE to the control levels. However, addition of antisense S-ODN to RGS2 did not affect the stress fibre formation. CONCLUSIONS: These results indicate that the loss and disassembly of stress fibres due to mechanical stress are mediating RhoE signalling, without the exertion of RGS2.

Blood flow distribution of repaired lip in cleft lip patients.

OBJECTIVE: To investigate the blood flow distribution in the repaired lip of cleft patients using a laser Doppler imager and to evaluate the difference in blood flow of the scar tissue between unilateral cleft lip patients with and without cleft palate. MATERIALS AND METHODS: Twenty patients with either unilateral cleft lip only (CL group, n = 8) or cleft lip and palate (CLP group, n = 12) were used as subjects. The blood flow of the upper lip was two-dimensionally visualized by a color scale alongside the corresponding photo image of the tissue surface with laser Doppler imaging. The upper lip photo image was divided into five regions: scar, white lip on the cleft side, white lip on the noncleft side, red lip on the cleft side, and red lip on the noncleft side. The average flux score (AFS), which is proportional to blood flow, was analyzed in each region. RESULTS: The AFS for the scar region was significantly (P < .05) lower than in the other four regions. The AFS for the red lip on the cleft side was not significantly different from that for the noncleft side. The white lip revealed a significantly (P < .05) higher score on the cleft side than on the noncleft side. The AFS ratio (AFS in the scar region/AFS in the white lip region) was significantly (P < .01) lower in the CLP group than in the CL group. CONCLUSIONS: These results suggest that blood flow distribution in the repaired lip might be affected by the anatomic features of the cleft.

Role of regulator of G-protein signaling 2 (RGS2) in periodontal ligament cells under mechanical stress.

Mechanical stress is thought to regulate the expression of genes in the periodontal ligament (PDL) cells. Using a microarray approach, we recently identified a regulator of G-protein signaling 2 (RGS2) as an up-regulated gene in the PDL cells under compressive force. The RGS protein family is known to turn off G-protein signaling. G-protein signaling involves the production of cAMP, which is thought to be one of the biological mediators in response to mechanical stress. Here, we investigated the role of RGS2 in the PDL cells under mechanical stress. PDL cells derived from the ligament tissues of human premolar teeth were cultured in collagen gels and subjected to static compressive force. Compressive force application time-dependently enhanced RGS2 expression and intracellular cAMP levels. To examine the interrelationship between RGS2 and cAMP, the PDL cells were treated with 2',5'-dideoxyadenosine (DDA), an inhibitor of adenyl cyclase, or antisense S-oligonucleotide (S-ODN) to RGS2 under compressive force. DDA dose-dependently inhibited RGS2 stimulated by compressive force. Blockage of RGS2 by antisense S-ODN elevated the cAMP levels compared with controls. These results indicate that cAMP stimulates RGS2 expression, which in turn leads to a decrease in the cAMP production by inactivating the G-protein signaling in the mechanically stressed PDL cells.

Function and regulation of osteopontin in response to mechanical stress.

UNLABELLED: Extensive histological study revealed the impairment of bone remodeling caused by mechanical stress in OPN knockout mice in a tooth movement system. Analysis of OPN promoter transgenic mice showed the mechanical stress response element(s) in the 5.5-kb upstream region. These results were also obtained with the primary cultured cells. INTRODUCTION: Mechanical loading system changes the bone architecture through the stimulation of bone remodeling by the action of a numbers of molecules. Among them, we showed that osteopontin (OPN) plays an important role in response to mechanical loading in rats with an experimental system for tooth movement. The results indicate the important role of OPN in bone remodeling. However, the molecular mechanism of OPN expression in response to mechanical stress is unknown. MATERIALS AND METHODS: OPN knockout mice and transgenic mice carrying green fluorescent protein (GFP) in the control of the OPN promoter were used for analysis. Orthodontic closed coil springs were bonded to the maxillary first molars and incisors for the experimental tooth movement. Spatial expression of GFP and OPN was detected by in situ hybridization. RESULTS: In contrast to wildtype mice, a smaller number of TRACP+ cells was detected in OPN knockout mice after treatment. In GFP-OPN5.5 mice, OPN and GFP mRNA-expressing cells were detected in bone cells after treatment, and the localization of GFP was consistent with that of endogenous OPN. An increase in the co-expression of GFP and OPN was detected when primary cultured osteoblastic cells derived from the transgenic mice were exposed to strain or pressure force. Significant increase in the number of OPN+ osteocyte was detected in the pressure side at 48 h after treatment. At 72 h, increase in the number of TRACP+ cells was detected predominantly in the pressure side. CONCLUSIONS: Bone remodeling in response to mechanical stress was suppressed in OPN knockout mice. These results indicate the critical role of OPN in the process of bone remodeling. The analysis of GFP expression in the promoter transgenic mice indicated the presence of an in vivo mechanical stress response element in the 5.5-kb upstream region of the OPN gene.

Messenger RNA expression of periostin and Twist transiently decrease by occlusal hypofunction in mouse periodontal ligament.

Periostin, which is a secreted protein that supports cell adhesion, is highly expressed in the periodontal ligament (PDL). Twist, a basic helix-loop-helix (bHLH) transcription factor and a negative regulator of osteoblast differentiation, has been found to regulate the periostin gene transcription. Since occlusal force is thought to be important in the homeostasis of the PDL, in this study we investigated the expression of periostin and Twist mRNA in the mouse periodontal tissue following removal of antagonizing teeth. Unilateral maxillary tooth extraction was performed in 3-week-old male mice to produce occlusal hypofunction of the right mandibular molars. The expressions of periostin and Twist mRNA were examined by real time-PCR and in situ hybridization at 12, 24, 72 and 168 h after the tooth extraction. The real-time PCR analysis showed that periostin and Twist mRNA significantly decreased at 24 h to 14.5 and 49.9% of those in control group, respectively. But the recovery began at 72 and 168 h, no significant difference was observed. As determined by in situ hybridization analysis, the number of periostin and Twist mRNA-expressing PDL cells showed a marked decrease at 24 h, although an increase was observed from 72 h until the distribution was almost similar to that of the control group at 168 h. These results suggested that occlusal force might have putative roles in periostin and Twist gene expression in the PDL and the changes in their expression level during hypofunction may be considered a form of adaptation to environmental changes.

MIP-1alpha utilizes both CCR1 and CCR5 to induce osteoclast formation and increase adhesion of myeloma cells to marrow stromal cells.

OBJECTIVES: Macrophage inflammatory protein-1alpha (MIP-1alpha), an osteoclast (OCL) stimulatory factor produced by primary multiple myeloma (MM) cells, increases bone destruction and tumor burden in murine models of MM. Several chemokine receptors (CCR1, CCR5, and CCR9) mediate the effects of MIP-1alpha. In this study, we determined which of these mediates the effects of MIP-1alpha on human OCL formation and myeloma cells. METHODS: We employed RT-PCR analysis, neutralizing antibodies to CCR1 and CCR5 as well as a CCR1-specific antagonist and OCL formation assays to identify the MIP-1alpha receptors involved in MIP-1alpha's effects on myeloma cells and OCL formation. RESULTS: RT-PCR analysis demonstrated that both CCR1 and CCR5 were expressed by highly purified human OCL precursors, myeloma cell lines, and purified marrow plasma cells from MM patients. Neutralizing antibodies to CCR1 or CCR5 inhibited MIP-1alpha-induced OCL formation. Furthermore, monocyte chemotactic protein-3 (MCP-3), which binds CCR1 but not CCR5 and the CCR1-specific antagonist, BX471, markedly inhibited OCL formation stimulated with MIP-1alpha. Anti-CCR1, anti-CCR5, or BX471 also inhibited the upregulation of beta1 integrin mRNA in myeloma cells induced by MIP-1alpha, as well as the adherence of myeloma cells to stromal cells and IL-6 production by stromal cells in response to myeloma cells. CONCLUSION: These data demonstrate that MIP-1alpha utilizes either CCR1 or CCR5 for its effects on OCL formation and myeloma cells, and that blocking either CCR1 or CCR5 inhibits OCL formation and myeloma cell adhesion to stromal cells.

An interdisciplinary approach for improved functional and esthetic results in a periodontally compromised adult patient.

In contemporary dental care, an increasing number of adult patients are seeking orthodontic treatment. In such adult patients, a combined orthodontic and other specialized therapy often offers the best option for achieving a predictable outcome to solve complex clinical problems. This case report demonstrates a combined therapy with orthodontic, periodontic, and implant-prosthodontic treatments in a 56-year six-month-old female patient with mild diastemata in the maxillary anterior region and a missing left maxillary second premolar caused by a periodontal disease with medium bone loss. The patient had improved her oral hygiene condition through periodontal treatment before orthodontic treatment. The patient was orthodontically treated with a maxillary lingual arch and a maxillary edgewise orthodontic appliance. Active orthodontic treatment was completed in 18 months, and an implant-supported prosthesis was placed with a single crown in the region of the left maxillary second premolar. The treatment outcomes, including the periodontal condition and the dental implant treatment, were stable at two years after the active orthodontic treatment. We demonstrate that combined orthodontic-periodontic-implant-prosthodontic treatment can achieve an improved masticatory function, esthetics, occlusion, and periodontal condition.

Eosinophil chemotactic factor-L (ECF-L): a novel osteoclast stimulating factor.

UNLABELLED: Screening a cDNA library enriched for genes expressed in OCLs identified ECF-L. ECF-L enhanced OCL formation without increasing RANKL levels. Anti-ECF-L inhibited RANKL-induced OCL formation. These results support a potent role of ECF-L in osteoclastogenesis. INTRODUCTION: To investigate the molecular mechanisms that control osteoclastogenesis, we developed an immortalized osteoclast (OCL) precursor cell line that forms mature OCLs in the absence of stromal cells and used it to form pure populations of OCLs. MATERIALS AND METHODS: Polymerase chain reaction (PCR) selective cDNA subtraction was used to identify genes that are highly expressed in mature OCLs compared with OCL precursors employing OCL and OCL precursors derived from this cell line. RESULTS: Eosinophil chemotactic factor-L (ECF-L), a previously described chemotactic factor for eosinophils, was one of the genes identified. Conditioned media from 293 cells transfected with mECF-L cDNA, or purified ECF-L Fc protein, increased OCL formation in a dose-dependent manner in mouse bone marrow cultures treated with 10(-10) M 1,25(OH)2D3. OCLs derived from marrow cultures treated with ECF-L conditioned media formed increased pit numbers and resorption area per dentin slice compared with OCLs induced by 1,25(OH)2D3 (p < 0.01). Addition of an antisense S-oligonucleotide to mECF-L inhibited OCL formation in murine bone marrow cultures treated only with 10(-9) M 1,25(OH)2D3 compared with the sense S-oligonucleotide control. Time course studies demonstrated that ECF-L acted at the later stages of OCL formation, and chemotactic assays showed that mECF-L increased migration of OCL precursors. mECF-L mRNA was detectable in mononuclear and multinucleated cells by in situ hybridization. Interestingly, a neutralizing antibody to ECF-L blocked RANKL or 10(-9) M 1,25(OH)2D3-induced OCL formation in mouse bone marrow cultures, although ECF-L did not induce RANKL expression. CONCLUSIONS: These data show ECF-L is a previously unknown factor that is a potent mediator of OCL formation, which acts at the later stages of OCL formation and enhances the effects of RANKL.