Notch Signaling Participates in TGF-ß-Induced SOST Expression Under Intermittent Compressive Stress.

Notch signaling is regulated by mechanical stimuli in various cell types. It has previously been reported that intermittent compressive stimuli enhanced sclerostin (SOST) expression in human periodontal ligament cells (hPDLs) by regulating transforming growth factor-ß (TGF-ß) expression. The aim of the present study was to determine the involvement of Notch signaling in the TGF-ß-induced SOST expression in hPDLs. Cells were treated with intermittent compressive stress in a computer-controlled apparatus for 24 h. The mRNA and protein expression of the cells were determined by real-time polymerase chain reaction and Western blot analysis, respectively. In some experiments, the target signaling pathway was impeded by the addition of a TGF-ß receptor kinase inhibitor (SB431542) or a γ-secretase inhibitor (DAPT). The results demonstrated that hPDLs under intermittent compressive stress exhibited significantly higher NOTCH2, NOTCH3, HES1, and HEY1 mRNA expression compared with control, indicating that mechanical stress induced Notch signaling. DAPT pretreatment markedly reduced the intermittent stress-induced SOST expression. The expression of NOTCH2, NOTCH3, HES1, and HEY1 mRNA under compressive stress was significantly reduced after pretreatment with SB431542, coinciding with a reduction in SOST expression. Recombinant human TGF-ß1 enhanced SOST, Notch receptor, and target gene expression in hPDLs. Further, DAPT treatment attenuated rhTGF-ß1-induced SOST expression. In summary, intermittent compressive stress regulates Notch receptor and target gene expression via the TGF-ß signaling pathway. In addition, Notch signaling participates in TGF-ß-induced SOST expression in hPDLs. J. Cell. Physiol. 232: 2221-2230, 2017. © 2016 Wiley Periodicals, Inc.

Novel Epigenetic Modulation Chitosan-Based Scaffold as a Promising Bone Regenerative Material.

Bone tissue engineering is a complicated field requiring concerted participation of cells, scaffolds, and osteoactive molecules to replace damaged bone. This study synthesized a chitosan-based (CS) scaffold incorporated with trichostatin A (TSA), an epigenetic modifier molecule, to achieve promising bone regeneration potential. The scaffolds with various biphasic calcium phosphate (BCP) proportions: 0%, 10%, 20%, and 40% were fabricated. The addition of BCP improved the scaffolds' mechanical properties and delayed the degradation rate, whereas 20% BCP scaffold matched the appropriate scaffold requirements. The proper concentration of TSA was also validated. Our developed scaffold released TSA and sustained them for up to three days. The scaffold with 800 nM of TSA showed excellent biocompatibility and induced robust osteoblast-related gene expression in the primary human periodontal ligament cells (hPDLCs). To evaluate in vivo bone regeneration potential, the scaffolds were implanted in the mice calvarial defect model. The excellent bone regeneration ability was further demonstrated in the micro-CT and histology sections compared to both negative control and commercial bone graft product. New bone formed in the CS/BCP/TSA group revealed a trabeculae-liked characteristic of the mature bone as early as six weeks. The CS/BCP/TSA scaffold is an up-and-coming candidate for the bone tissue engineering scaffold.

NOTCH2 participates in Jagged1-induced osteogenic differentiation in human periodontal ligament cells.

Jagged1 activates Notch signaling and subsequently promotes osteogenic differentiation in human periodontal ligament cells (hPDLs). The present study investigated the participation of the Notch receptor, NOTCH2, in the Jagged1-induced osteogenic differentiation in hPDLs. NOTCH2 and NOTCH4 mRNA expression levels increased during hPDL osteogenic differentiation. However, the endogenous NOTCH2 expression levels were markedly higher compared with NOTCH4. NOTCH2 expression knockdown using shRNA in hPDLs did not dramatically alter their proliferation or osteogenic differentiation compared with the shRNA control. After seeding on Jagged1-immobilized surfaces and maintaining the hPDLs in osteogenic medium, HES1 and HEY1 mRNA levels were markedly reduced in the shNOTCH2-transduced cells compared with the shControl group. Further, shNOTCH2-transduced cells exhibited less alkaline phosphatase enzymatic activity and in vitro mineralization than the shControl cells when exposed to Jagged1. MSX2 and COL1A1 mRNA expression after Jagged1 activation were reduced in shNOTCH2-transduced cells. Endogenous Notch signaling inhibition using a γ-secretase inhibitor (DAPT) attenuated mineralization in hPDLs. DAPT treatment significantly promoted TWIST1, but decreased ALP, mRNA expression, compared with the control. In conclusion, Notch signaling is involved in hPDL osteogenic differentiation. Moreover, NOTCH2 participates in the mechanism by which Jagged1 induced osteogenic differentiation in hPDLs.

Asiaticoside induces osteogenic differentiation of human periodontal ligament cells through the Wnt pathway.

BACKGROUND: Asiaticoside is a compound isolated from Herb Centella asiatica, which has been shown to promote osteogenic differentiation of human periodontal ligament (hPDL) cells. This study investigated the molecular mechanism underlying the asiaticoside-induced osteogenic differentiation of hPDL cells. METHODS: hPDL cells were incubated with various concentrations of asiaticoside to test cell viability by MTT assay. The mRNA expression levels were analyzed by using quantitative real-time polymerase chain reaction (PCR). Osteogenic differentiation was determined by alkaline phosphatase activity assay and alizarin red staining. The subcellular localization of ß-catenin was analyzed by both immunofluorescence and western blot. RESULTS: The results showed that asiaticoside had no effect on the cell viability at any of the tested concentrations. Real-time PCR revealed that osterix (OSX) and dentin matrix protein1 (DMP1) mRNA were significantly enhanced by asiaticoside treatment. Alkaline phosphatase activity and in vitro mineralization were also significantly induced. Interestingly, asiaticoside dose-dependently increased WNT3A mRNA expression, but not WNT5A and WNT10B. The activation of Wnt signaling was shown to result in nuclear accumulation of ß-catenin as evaluated by immunofluorescence staining and western blot analysis. Pre-treatment with recombinant human Dickkopf1 (rhDKK1) inhibited asiaticoside-induced ß-catenin nuclear translocation and osteoblast marker gene expression. Moreover, rhDKK1 attenuated asiaticoside-induced DMP1 protein expression. CONCLUSION: The data demonstrate that asiaticoside induces osteogenic differentiation of hPDL cells by activating the Wnt/ß-catenin signaling pathway. The findings suggest that asiaticoside could be used as a novel therapeutic drug for periodontal tissue regeneration.

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Role of cyclin D1 cytoplasmic sequestration in the survival of postmitotic neurons.

Cyclin D-dependent kinases phosphorylate the retinoblastoma (Rb) protein and play a critical role in neuronal cell cycle control and apoptosis. Here we show that cyclin D1 became predominantly cytoplasmic as primary cortical progenitor cells underwent cell cycle withdrawal and terminal differentiation. Furthermore, ectopically expressed cyclin D1 sequestered in the cytoplasm of postmitotic neurons, whereas it efficiently entered the nucleus of proliferating progenitor cells. Cytoplasmic cyclin D1 were complexed with cyclin-dependent kinase 4 (CDK4), and also with CDK inhibitors, p27(Kip)(I) or p21(Cip)(I), which positively regulate assembly and nuclear accumulation of the cyclin D1-CDK4 complex. Although overexpression of p21(Cip)(I) promoted cyclin D1 nuclear localization, inhibition of either glycogen synthase kinase 3beta- or CRM1-mediated cyclin D1 nuclear export did not, suggesting that the inhibition of its nuclear import, rather than the acceleration of nuclear export, contributes to cytoplasmic sequestration of cyclin D1 in postmitotic neurons. In differentiated progenitor cells, nuclear localization of ectopic cyclin D1 induced apoptosis, and the DNA-damaging compound camptothecin caused nuclear accumulation of endogenous cyclin D1, accompanied by Rb phosphorylation. These results indicate that nuclear accumulation of cyclin D1 is inhibited in postmitotic neurons and suggest a role of its subcellular localization in neuronal death and survival.

[Prevention of cyclin D1 nuclear localization in terminally differentiated neurons].

Terminally differentiated neurons irreversibly withdraw from the cell cycle. The mechanisms governing the activity of cyclin D 1, a key regulator of the cell cycle, during neuronal cell cycle withdrawal are not fully understood. This study shows that cyclin D 1 became predominantly cytoplasmic in differentiated cortical neurons. Cytoplasmic cyclin D 1 assembled with cyclin dependent kinase 4 (CDK 4), and the CDK inhibitors p21Cip1 and p27Kip1. Although forced expression of p 21 caused cyclin D 1 nuclear accumulation, the inhibition of its nuclear export by inhibiting GSK-3 beta activity had no effect. Furthermore, ectopically expressed cyclin D 1 entered the nucleus of proliferating nervous, but not that of differentiated neurons, whereas ectopic cyclin D 1 in quiescent fibroblasts accumulated in the nucleus and induced cell cycle progression. These results indicate that cyclin D 1 nuclear localization is tightly inhibited in terminally differentiated neurons, and suggest that the regulation of its nuclear import plays a role in neuronal cell cycle withdrawal.

Pressure induces interleukin-6 expression via the P2Y6 receptor in human dental pulp cells.

BACKGROUND AND OBJECTIVE: An increase in intrapulpal pressure occurs during inflammation and restorative procedures; however, the role of the pressure on human dental pulp cell (HDPC) is not yet clarified. In this study, the effect of pressure on interleukin-6 (IL-6) expression of HDPCs was examined. DESIGN: HDPCs were applied with pressure (0.7-1.4 g/cm(2)). The level of IL-6 mRNA and protein release was determined by RT-PCR and ELISA, respectively. The signalling pathways were investigated using inhibitors, antagonists, and small interfering RNA. RESULTS: The results showed that pressure up-regulated IL-6 mRNA expression and protein release in a time- and dose-dependent manner. The implication of P2Y receptor was exhibited by a significant inhibition of pressure-induced IL-6 expression by suramin, an antagonist for the non-specific purinergic receptor family. Using loss of function experiments, we showed MRS2578 (a specific P2Y6 antagonist), as well as P2Y6 small interfering RNA, abolished pressure-induced IL-6, whilst MRS2179 (a specific P2Y1 antagonist) and NF449 (a P2X1, P2X3, P2Y1, and P2Y2 antagonist) had no effect. Finally, we demonstrated that either the conditioned medium collected from pressurised dental pulp cells or addition of UDP, a selective agonist of P2Y6, up-regulated IL-6 expression in HDPCs. CONCLUSIONS: These results indicate that pressure could induce IL-6 expression through the P2Y6 receptor in HDPCs, leading to a new insight of the role of pressure on cytokine release during pulpal inflammatory process.

Fibronectin supports TNF-alpha-induced osteopontin expression through beta1 integrin and ERK in HN-22 cells.

The extracellular matrix (ECM), in collaboration with intracellular signal, plays a critical role in the modulation of cellular behavior and function. Herein, we investigated the influence of fibronectin (FN) and tumor necrosis factor-alpha (TNF-alpha) on OPN expression in HN-22, a human head and neck squamous cell carcinoma (HNSCC) cell line. The data showed that TNF-alpha significantly increased OPN expression only in the FN-coated condition. Application of function-blocking antibody directed against beta1 integrin abolished this OPN induction. Moreover, TNF-alpha when added together with activating beta1 integrin antibody is sufficient to induced OPN expression. The combination effect of FN and TNF-alpha was significantly deteriorated by a MEK inhibitor, but not NF-kappaB inhibitor. We further demonstrated that the phosphorylation of ERK1/2 was strongly enhanced by TNF-alpha and FN compared to the application of either one alone. Synergistic effect on ERK1/2 phosphorylation was also detected by TNF-alpha and activating beta1 integrin antibody, whereas inhibitory antibody to beta1 integrin attenuated FN and TNF-alpha-induced phosphorylation of ERK1/2. Our results indicate that FN coordinates TNF-alpha-mediated OPN induction via beta1 integrin-dependent signaling mechanism that activates ERK. The results suggest the critical role of tumor micro-environment signaling networks on the regulation of cytokine expression profiles during tumor progression.

Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells.

The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16(INK4a), a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

Insulin-like growth factor-I attenuates the inhibitory effect of type I collagen through beta1 integrin receptor.

Extracellular matrix and growth factors are the crucial factors that regulate healing and regenerating processes in human periodontal ligament cells. The purpose of this study was to examine the effects of type I collagen and insulin-like growth factor-I (IGF-I) on osteopontin (OPN) expression. The data showed that OPN expression was significantly decreased when cells were cultured on collagen-coated plates. Addition of IGF-I obviously induced OPN expression only in a collagen-coated condition, suggesting an attenuating effect of IGF-I on the decrease of OPN expression. Cells treated with a combination of inhibitory antibody to beta1 integrin and IGF-I showed the same level of OPN expression as those treated with either inhibitory antibody to beta1 integrin or IGF-I alone. These results indicate that IGF-I counteracts with the inhibitory signal from type I collagen through beta1 integrin receptor.

Loss of cyclin E requirement in cell growth of an oral squamous cell carcinoma cell line implies deregulation of its downstream pathway.

Cyclin E and Cdk2 have been shown to play an important role in G1/S transition of the cell cycle. Two E-type cyclins (E1 and E2) have been identified to date and share functionally similarities. Upregulation of these cyclins has been observed frequently in human cancers. We examined the expression profile of cyclin E1 and E2 in cell lines derived from human oral squamous cell carcinoma (SCC), and found that the expression of cyclin E1 protein was hardly detected in HSC-2 cells. Although cyclin E2 was abundantly expressed, histone H1 kinase activities of both E-type cyclins were virtually undetectable in this cell line. Inhibition of cyclin E1, but not that of E2, by using vectors expressing antisense-oriented their cDNAs induced drastic growth suppression on HOC313 cells that express both E-type cyclins. Inhibition of neither cyclin E1 nor E2 suppressed the growth of HSC-2 cells, and compensatory elevation of cyclin E1 was not evident in cyclin E2-inhibited HSC-2 cells. In contrast, HSC-2 cells expressed cyclin D1 and hyperphosphorylated forms of Rb family proteins, and were arrested in G1 by overexpression of p16(INK4), a specific inhibitor against D-type cyclin activity. These results indicate that HSC-2 cells lost proper growth control specifically mediated by cyclin E and suggest that deregulation of its downstream pathway may contribute to tumorigenesis of oral SCC.