Current status and factors of periodontal disease among Japanese high school students: a cross-sectional study.

INTRODUCTION: This study aimed to investigate the prevalence of periodontal disease and the factors of the disease among high school students. METHOD: The participants were all students aged 15-18 years (n = 1202) at a high school in Japan. The data on oral health perceptions and behaviours were collected by a questionnaire survey. The prevalence of periodontal disease among them was investigated with the partial community periodontal index (PCPI). A logistic regression analysis was used to identify the factors associated with the PCPI. RESULTS: A total of 1069 students (88.9%) participated in this study. The prevalence of gingival bleeding, calculus, pocket depth of 4-5 mm, and pocket depth of 6 mm or more were 44.2%, 42.2%, 11.4%, and 1.6%, respectively. Approximately one-third of the students had a fear of dental treatment, and only 28.4% used dental floss. The results of logistic regression analysis, adjusted for sex and school year, showed that not visiting dentists regularly, not using dental floss, brushing teeth for less than 5 min, fear of dental treatment, and drinking sports drinks frequently were positively associated with periodontal conditions. CONCLUSION: This study identified a high prevalence of periodontal disease among Japanese high school students aged 15-18 years and its risk factors, such as poor oral health behaviours and fear of dental treatment.

Effects of root-end filling materials on vascular endothelial cell proliferation and tube formation.

Background/purpose: Regarding root-end filling materials in apical surgery, sealing ability and biocompatibility are useful for treatment. Angiogenesis, which occurs in the process of periapical wound healing, is closely related to bone formation. In this study, we investigated the effects of root-end filling materials on vascular endothelial cell proliferation and angiogenesis. Materials and methods: Mineral trioxide aggregate (MTA), 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane (4-META/MMA-TBB) resin, Super EBA, and CS-BG-multi, bioactive glass-related materials, were used. After curing, each material was soaked in a medium for 1 or 7 days, and then cultured for 1-7 days to investigate the effects on human umbilical vein endothelial cell (HUVEC) proliferation, angiogenesis, and vascular endothelial growth factor receptors (VEGFRs) mRNA expression. Results: In the 1-day soaked sample, there was significantly less proliferation in MTA and Super EBA on day 7 of culture. In the 7-day soaked sample, there was significantly less proliferation in Super EBA and CS-BG-multi on day 7 of culture. Tube formation was significantly high in MTA in both the 1-day and 7-day soaked samples, significantly high in SB in the 1-day soaked sample, and significantly low in Super EBA in both the 1-day and 7-day soaked samples. CS-BG-multi was comparable to the control. VEGFR-1 and VEGFR-2 mRNA expressions showed an upward trend in MTA, and a trend similar to the control in SB. Conclusion: MTA and 4-META/MMA-TBB resin had a higher pro-angiogenic effect while Super EBA had a less pro-angiogenic effect. CS-BG-multi had low toxicity on tube formation of HUVEC.

The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate.

Background/purpose: 5' Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methods: MC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. Results: The results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. Conclusion: AMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.

Sphingosine-1-phosphate receptor 2 agonist induces bone formation in rat apicoectomy and alveolar bone defect model.

Background/purpose: Sphingosine-1-phosphate (S1P) is a lipid mediator that exerts its physiological functions in vivo through receptors. In the bone, S1P induces osteoblast differentiation. Here, we investigated the effects of S1P receptor agonists on the expression of osteoblast differentiation markers locally in the bone. Then, a rat apicoectomy and alveolar bone defect model was established to extend S1P applications to endodontics, and the effect of local administration of S1P receptor agonist on postoperative bone formation was examined. Materials and methods: Sphingosine-1-phosphate receptor (S1PR) 1/S1PR3 agonists, S1PR2 agonists, and their signal-related agents were intraperitoneally administered to mice. Using the mRNA collected from the tibial bone, the expression of osteoblast differentiation markers was evaluated by real-time reverse-transcriptase quantitative polymerase chain reaction. An apicoectomy and alveolar bone defect model was established on the mesial root of the rat mandibular first molar. Bone formation parameters were measured by micro-computed tomography analysis 3 weeks after the procedure. Results: Intraperitoneal administration of S1PR2 agonist significantly increased the mRNA expression of osteoblast differentiation markers, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin, in the local tibial bone of mice. The S1PR2/Rho-associated coiled-coil forming kinase (ROCK) signaling was thought to be involved in the upregulated mRNA expression of ALP, OPN, and BSP. In the rat apical defects, bone formation parameters significantly increased following local administration of S1PR2 agonist. Conclusion: In the rat apicoectomy and alveolar bone defect model, therapeutic agents targeting S1PR2 agonist are effective against osteogenesis.

Space-making effect for new bone formation by suppressing scar contraction of mucosal epithelium of rat tooth extraction wound using diode laser and CO2 laser treatment.

Background/purpose: As an extraction wound closes, the mucosal epithelium of the tooth extraction wound impedes the space for new bone formation by invading into the extraction socket. Thus, the height of the alveolar crest decreases, causing significant depression of the alveolar mucosa. In this study, we created a rat tooth extraction model and examined the effects of laser irradiation by CO2 and diode on the dynamics of myofibroblast expression through α-SMA, and TGF-ß1. Materials and methods: After tooth extraction of five-week-old male Wistar rats, they were divided into two laser treatment groups (CO2 laser or diode laser was irradiated into tooth extraction socket) and non-laser treatment group (control group). Surrounding tissues, including the extraction socket, were removed at 3, 5, 7, and 21 days after tooth extraction and the expression of α-SMA and TGF-ß1 was verified using immunohistological techniques (6 animals in each group and each period, 72 animals in total). Results: α-SMA-positive cells and TGF-ß1-positive areas were significantly lower in the two laser treatment groups than in the control group. Particularly, the diode group almost had no TGF-ß1-positive areas on the 21st day when healing after tooth extraction was deemed to be completed. Conclusion: Both CO2 and diode laser irradiation of tooth extraction wounds decreases α-SMA-positive cells and TGF-ß1-positive areas. Further, it causes a decrease in myofibroblast expression and suppresses the invasion of mucosal epithelium into the extraction socket. Therefore, laser irradiation may exert a space-making effect for new bone formation and also contribute to socket preservation.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Dental regenerative therapy targeting sphingosine-1-phosphate (S1P) signaling pathway in endodontics.

The establishment of regenerative therapy in endodontics targeting the dentin-pulp complex, cementum, periodontal ligament tissue, and alveolar bone will provide valuable information to preserve teeth. It is well known that the application of stem cells such as induced pluripotent stem cells, embryonic stem cells, and somatic stem cells is effective in regenerative medicine. There are many somatic stem cells in teeth and periodontal tissues including dental pulp stem cells (DPSCs), stem cells from the apical papilla, and periodontal ligament stem cells. Particularly, several studies have reported the regeneration of clinical pulp tissue and alveolar bone by DPSCs transplantation. However, further scientific issues for practical implementation remain to be addressed. Sphingosine-1-phosphate (S1P) acts as a bioactive signaling molecule that has multiple biological functions including cellular differentiation, and has been shown to be responsible for bone resorption and formation. Here we discuss a strategy for bone regeneration and a possibility for regenerative endodontics targeting S1P signaling pathway as one of approaches for induction of regeneration by improving the regenerative capacity of endogenous cells. SCIENTIFIC FIELD OF DENTAL SCIENCE: Endodontology.

Development of an error-detection examination for conservative dentistry education.

OBJECTIVE: For dental students, textbooks and lectures provide basic knowledge, and simulated and actual clinical training provide learning in technical and communication skills. At our college, conservative dentistry is taught in the third and fourth years of a 6-year undergraduate degree. Clinical training is undertaken subsequently in the fifth year and includes cavity preparation and composite resin filling tasks. However, despite the clinical importance of a full understanding surrounding these procedures, sixth-year students occasionally provide incorrect answers regarding these procedures in assessments. Although they demonstrated a basic understanding of the procedures, they may have forgotten the acquired knowledge during their clinical training. Therefore, we developed an error-detection examination to evaluate and improve fifth-year students' knowledge. METHODS: Written detailed treatment procedures for standardized, typical, cases were presented to students. Some critical steps were intentionally written incorrectly, and students had to identify and correct these. After correcting the steps, students gave a presentation to their peers on their corrections. This was followed by a summary of the correct answers and a short lecture by the teacher. Students then completed a questionnaire investigating their experience of the examination. RESULTS: Students misunderstood some key treatment steps, such as pretreatment of composite resin filling, amalgam removal, and ceramic inlay fitting. The questionnaire revealed that this method of testing applied knowledge was new to students and helped them to identify knowledge gaps. The test also increased their motivation to study conservative dentistry. Students were open to taking similar tests in different areas. CONCLUSION: Although conservative dentistry is a basic field of dental treatment, mistakes in treatment can lead to early treatment failure or reduce the lifetime of a restored tooth. Therefore, students need to have a deep understanding of procedures. Error-detection examinations may help students identify knowledge gaps and provide useful feedback to teachers to identify areas that they should stress in earlier years.

Induction of bone repair in rat calvarial defects using a combination of hydroxyapatite with phosphatidylserine liposomes.

Phosphatidylserine (PS)-normally present on the inner leaflet of the plasma membrane-translocates to the outer leaflet at an early stage of apoptosis. PS-containing liposomes (PSLs) can mimic the effect of apoptotic cells in inducing the secretion of prostaglandin E2 from phagocytes and inhibiting the maturation of dendritic cells and osteoclast precursors. The present study attempted to evaluate the effect of calcium phosphate (in the form of hydroxyapatite [HAP]) in the presence or absence of PSLs for repair of rat calvarial bone defects. The defects, each 5 mm in diameter, were created in the calvaria parietal bone of 8-week-old Wistar rats and subjected to one of the following treatments: no augmentation (Sham), HAP alone, or a mixture of HAP and PSL (HAP+PSL). Micro-computed tomography data showed that the HAP+PSL complexes promoted greater bone regeneration in comparison with either the Sham procedure or HAP alone at 4 and 8 weeks after implantation. The regeneration of calvarial bone defects induced by PSLs was mediated partly through upregulation of the osteogenic marker Alkaline Phosphatase, Type I collagen, osteocalcin, Runx2, and Osterix mRNAs. These data are the first to show that PSLs can influence bone regeneration by regulating osteoblast differentiation.

Angiopoietin-like protein 2 is a positive regulator of osteoblast differentiation.

INTRODUCTION AND AIMS: Several studies have reported that angiopoietin-like protein 2 (Angptl2) is expressed abundantly in adipocytes and is associated with adipose tissue inflammation. In the present study, we found that osteoblasts and mesenchymal stem cells also expressed Angptl2 at high levels. The aim of this study was to understand the role of Angptl2 in osteoblastic cell differentiation. METHODS: Angptl2 expression was examined during osteoblast and adipocyte differentiation. The role of Angptl2 on cell differentiation and associated signaling was analyzed by gene knockdown using Angptl2 small interfering ribonucleic acid (siRNA). RESULTS: Angptl2 was highly expressed in MC3T3-E1 cells, ST2 cells and primary osteoblasts, but not in RAW264 cells. Inhibition of Angptl2 expression using siRNA markedly inhibited alkaline phosphatase (ALP) expression and osteoblastic differentiation in MC3T3-E1, ST2 cells and primary osteoblasts. Angptl2 siRNA also inhibited adipocyte differentiation in ST2 cells. Treatment of MC3T3-E1 cells with Angptl2 siRNA led to the down-regulation of the activities of several cell signaling pathways, including extracellular signal-regulated kinase (ERK), Jun amino-terminal kinase (JNK), Akt, and nuclear factor kappa B (NF-κB) signals. It also down-regulated the expression of Osterix, but not that of runt-related transcription factor 2 (Runx2), suggesting that Angptl2 is a positive activator of Osterix and its down-stream signals. Treatment of MC3T3-E1 cells with anti-Angptl2 antibodies suppressed ALP gene expression. In addition, treatment of Angptl2 siRNA-treated cells with culture supernatants of normal MC3T3-E1 cells restored ALP gene expression, indicating that Angptl2 acts in an autocrine manner. CONCLUSIONS: The results suggest that Angptl2 is an autocrine positive regulator of cell differentiation. Thus, it is suggested that Angptl2 regulates not only adipose tissue metabolism but also bone metabolism.

Celecoxib inhibits osteoblast maturation by suppressing the expression of Wnt target genes.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to impair bone healing. We previously reported that in colon cancer cells, celecoxib, a COX-2-selective NSAID, inhibited the canonical Wnt/ß-catenin signaling pathway. Since this pathway also plays an important role in osteoblast growth and differentiation, we examined the effect of celecoxib on maturation of osteoblast-like cell line MC3T3-E1. Celecoxib induced degradation of transcription factor 7-like 2, a key transcription factor of the canonical Wnt pathway. Subsequently, we analyzed the effect of celecoxib on two osteoblast differentiation markers; runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), both of which are the products of the canonical Wnt pathway target genes. Celecoxib inhibited the expression of both RUNX2 and ALP by suppressing their promoter activity. Consistent with these observations, celecoxib also strongly inhibited osteoblast-mediated mineralization. These results suggest that celecoxib inhibits osteoblast maturation by suppressing Wnt target genes, and this could be the mechanism that NSAIDs inhibit bone formation and fracture healing.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts.

Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that also plays crucial roles in bone regeneration. Recently, we reported that the S1P receptors S1PR1 and S1PR2 were mainly expressed in osteoblast-like cells, and that the S1P/S1PR1 signaling pathway up-regulated osteoprotegerin and osteoblast differentiation. However, the involvement of S1P/S1PR2 signaling in osteoblast differentiation is not well understood. Here we investigate the role of S1P/S1PR2-mediated signaling in osteoblast differentiation and clarify the underlying signaling mechanisms. We found that an S1P/S1PR2/Gi-independent signaling pathway activated RhoA activity, leading to phosphorylation of Smad1/5/8 in mouse osteoblast-like MC3T3-E1 cells and primary osteoblasts. Furthermore, this signaling pathway promoted nuclear translocation of Smad4, and increased the amount of Smad6/7 protein in the nucleus. S1P also up-regulated runt-related transcription factor 2 (Runx2) expression through S1PR2/RhoA/ROCK/Smad1/5/8 signaling. Moreover, we found that S1P partially triggered S1PR2/RhoA/ROCK pathway leading to bone formation in vivo. These findings suggest that S1P induces RhoA activity, leading to the phosphorylation of Smad1/5/8, thereby promoting Runx2 expression and differentiation in osteoblasts. Our findings describe novel molecular mechanisms in S1P/S1PR2-mediated osteoblast differentiation that could aid future studies of bone regeneration.

Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

Sphingosine-1-phosphate inhibits differentiation of C3H10T1/2 cells into adipocyte.

Mesenchymal stem cells (MSCs) can differentiate into a number of cell types, including adipocytes and osteoblasts. MSC differentiation into adipocytes inhibits osteogenic differentiation and vice versa. Therefore, understanding the mechanisms of MSC differentiation at the signaling level can lead to the development of novel therapeutic strategies toward tissue regeneration. Sphingosine-1-phosphate (S1P) is a signaling molecule that regulates many cellular responses, including cellular differentiation. However, the effects of S1P on MSC differentiation are largely unknown. The purpose of study was to investigate whether S1P drives MSCs toward either adipogenic or osteogenic differentiation, and if so, to clarify the underlying signaling mechanisms for such differentiation. We found that S1P inhibited adipogenic differentiation of C3H10T1/2 multipotent stem cells, while promoting their osteogenic differentiation. During adipogenic differentiation, S1P suppressed the cAMP accumulation in a Gi-protein-dependent manner. The Gi-dependent S1P signaling suppressed C/EBPß expression, which is essential for adipogenic differentiation. Furthermore, S1P did not affect cAMP-independent adipogenic differentiation. These findings suggest that S1P suppresses cAMP accumulation, leading to inhibition of C/EBPß expression, thereby resulting in decreased adipogenic differentiation of C3H10T1/2 cells. Thus, our findings provide novel molecular mechanisms as regards how S1P inhibits adipogenic differentiation of C3H10T1/2 cells, indicating a potential beneficial role for regeneration and repair of tissues.

Sphingosine-1-phosphate promotes the nuclear translocation of ß-catenin and thereby induces osteoprotegerin gene expression in osteoblast-like cell lines.

Sphingosine-1-phosphate (S1P) is a well-known signaling sphingolipid and bioactive lipid mediator. Recently, it was reported that S1P inhibits osteoclast differentiation and bone resorption. On the other hand, S1P effects on osteoblasts and bone formation are little known. In this study, we investigated the effects of S1P on osteoblasts, using two osteoblast-like cell lines, SaOS-2 and MC3T3-E1. S1P activated phosphatidylinositol 3-kinase (PI3K)/Akt signaling, leading to the inhibition of glycogen synthase kinase-3ß and the nuclear translocation of ß-catenin, followed by the increase of the transcriptional activity by ß-catenin/T-cell factor complex formation in both SaOS-2 cells and MC3T3-E1 cells. The inhibitors of PI3K and Akt suppressed S1P-induced nuclear localization of ß-catenin. We further investigated the effects of PI3K/Akt signaling on the Wnt/ß-catenin signaling pathway, since ß-catenin takes a central role in this signaling pathway. Both inhibitors for PI3K and Akt suppressed the nuclear localization of ß-catenin and T-cell factor transcriptional activity induced by Wnt-3a. S1P increased the amount of osteoprotegerin at both mRNA and protein levels, and increased the activity of alkaline phosphatase, leading to the mineralization. These findings suggest that S1P activates the PI3K/Akt signaling pathway leading to the promotion of nuclear translocation of ß-catenin in osteoblast-like cells, resulting in the upregulation of osteoptotegerin and osteoblast differentiation markers including alkaline phosphatase, probably relating to the inhibition of osteoclast formation and the mineralization, respectively.

Differentiation-inducing factor-1 suppresses the expression of c-Myc in the human cancer cell lines.

Differentiation-inducing factor-1 (DIF-1), a morphogen for Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by suppressing the Wnt/ß-catenin signaling pathway. In this study, we examined the effect of DIF-1 on c-Myc, a target gene product of the Wnt/ß-catenin signaling pathway, mainly using HCT-116 colon cancer cells. DIF-1 strongly reduced the amount of c-Myc protein in time- and concentration-dependent manners and reduced c-Myc mRNA expression by inhibiting promoter activity through the TCF binding sites. The effect of DIF-1 on c-Myc was also confirmed using the human cervical cell line HeLa. Pretreatment with the proteasome inhibitor MG132 or glycogen synthase kinase-3ß (GSK-3ß) inhibitors (LiCl and SB216763) attenuated the effect of DIF-1, suggesting that DIF-1 induced c-Myc protein degradation through GSK-3ß activation. Furthermore, we examined whether c-Myc was involved in the anti-proliferative effect of DIF-1 using c-Myc-overexpressing cells and found that c-Myc was associated with the anti-proliferative effect of this compound. These results suggest that DIF-1 inhibits c-Myc expression by inhibiting promoter activity and inducing protein degradation via GSK-3ß activation, resulting in the inhibition of cell proliferation. Since c-Myc seems to be profoundly involved in accelerated proliferation of various malignant tumors, DIF-1 may have a potential to develop into a novel anti-cancer agent.

Involvement of GSK-3beta and DYRK1B in differentiation-inducing factor-3-induced phosphorylation of cyclin D1 in HeLa cells.

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.