Yukiko Kitase

Mechanical Induction of PGE2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid‐flow shear stress blocks dexamethasone‐induced apoptosis of osteocyte‐like cells (MLO‐Y4). Prostaglandin E2 (PGE2), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone‐induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3‐kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β‐catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid‐induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase‐2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β‐catenin protein expression. β‐catenin siRNA knockdown experiments abrogated the protective effects of PGE2, confirming the central role of β‐catenin in mediating the protection against dexamethasone‐induced cell death. Our data support a central role for PGE2 acting through the cAMP/PKA and β‐catenin signaling pathways in the protection of osteocyte apoptosis by fluid‐flow shear stress.

Smad2 is involved in the apoptosis of murine gingival junctional epithelium associated with inhibition of Bcl-2

OBJECTIVE Gingival junctional epithelium (JE) actively contributes to the homeostasis of the periodontium. Altered activation of TGF-β signalling is implicated in the epithelium from chronic periodontitis. However, little is known about the effects of TGF-β signalling on the JE. In this study, we investigated the relationship between Smad2, which plays an important role in mediating TGF-β signal, and induction of apoptosis in the JE. METHODS K14-Smad2 transgenic mice were used to observe the effect of over-expression of Smad2 driven by CK14 promoter in the JE. We performed TUNEL technique to evaluate the epithelial apoptosis. Expression of apoptosis related genes was examined using real-time PCR and immunofluorescence. RESULTS K14-Smad2 mice showed an increased number of phospho-Smad2 positive JE cells associated with an increase in TGF-β1 expression. K14-Smad2 mice have a significantly higher percentage of TUNEL positive cells in the JE. Immunofluorescence double labelling revealed that TUNEL positive cells showed immunoreactivity to phospho-Smad2. Real-time PCR analysis of apoptosis related gene expression provided evidence of lower expression of Bcl-2 in the gingival tissue from K14-Smad2 mice. There was a strong positive reaction for Bcl-2 protein in the junctional epithelium of wild type mice, while the gingival tissue of K14-Smad2 transgenic mice had only a faint signal for Bcl-2. CONCLUSIONS The present study provided evidence that Smad2 plays a crucial role in the induction of apoptosis in gingival JE through inhibition of Bcl-2.

Microtubule disassembly prevents palatal fusion and alters regulation of the E-cadherin/catenin complex.

During palatal fusion, the midline epithelial seam (MES) degrades to achieve mesenchymal confluence. Epithelial mesenchymal transition (EMT) is one mechanism which is active in MES degradation. TGF-β induces EMT in medial edge epithelium (MEE) by down-regulation of an epithelial marker, E-cadherin. Microtubule disassembly impaired palatal fusion leading to a multi-layered MES in the mid-region. In this study, we investigated the effect of microtubule disruption on the regulation of the E-cadherin/catenin adhesion complex. Nocodazole (NDZ) enhanced the accumulation of the adhesion complex at cell-cell contacts in MEE, while loss of the adhesion complex was observed in the control. NDZ caused aberrant regulation of the E-cadherin transcriptional repressors (Snail and Zeb) and the activator (c-MYC) through inhibition of the TGF-β/SMAD2 signaling pathway, which led to a failure in EMT. These results suggest that the microtubule cytoskeleton plays an important role in mediating TGF-β/SMAD2 signals to control E-cadherin gene expression in MEE during palatal fusion.

Spatiotemporal Localization of Periostin and Its Potential Role in Epithelial-Mesenchymal Transition during Palatal Fusion

The medial epithelial seam (MES) between the palatal shelves degrades during palatal fusion to achieve the confluence of palatal mesenchyme. Cellular mechanisms underlying the degradation of MES have been proposed, such as apoptosis, epithelial-mesenchymal transition (EMT) and migration of medial edge epithelia (MEE). Extracellular matrix components have been shown to play an important role in EMT in many model systems. Periostin (also known as osteoblast-specific factor-2) is a secreted mesenchymal extracellular matrix component that affects the ability of cells to migrate and/or facilitates EMT during both embryonic development and pathologic conditions. In this study, we evaluated the spatiotemporal expression patterns of periostin during mouse palatal fusion by in situ hybridization and immunofluorescence. Periostin mRNA and protein were present in the palatal mesenchyme, the protein being distributed in a fine fibrillar network and in the basement membrane, but absent from the epithelium. During MES degradation, the protein was strongly expressed in the basement membrane underlying the MES and in some select MEE. Confocal microscopic analysis using an EMT marker, twist1, and an epithelial marker, cytokeratin 14, provided evidence that select MEE were undergoing EMT in association with periostin. Moreover, the major extracellular matrix molecules in basement membrane, laminin and collagen type IV were degraded earlier than periostin. The result is that select MEE establish interactions with periostin in the mesenchymal extracellular matrix, and these new cell-matrix interactions may regulate MEE transdifferentiation during palatal fusion.

Multi‐Layered hypertrophied MEE formation by microtubule disruption via GEF‐H1/RhoA/ROCK signaling pathway

Background: Formation of the secondary palate is complex and disturbance during palatal fusion may result in cleft palate. The processes of adhesion, intercalation, and disappearance of medial edge epithelia (MEE) are characterized by morphological changes requiring dynamic cytoskeletal rearrangement. Microtubules are one of the cytoskeletal elements involved in maintenance of cell morphology. Microtubule‐disrupting drugs have been reported to cause craniofacial malformations including cleft palate. The mechanisms underlying the failure of palatal fusion remain poorly understood. We evaluated the effect of nocodazole (NDZ), a drug that disrupts microtubules, on palatal fusion in organ culture. Results: NDZ caused failure of palatal fusion due to the induction of a multi‐layered hypertrophied MEE in the mid‐region of the secondary palatal shelves. Microtubule disruption increased RhoA activity and stress fiber formation. Pharmacological inhibition of the RhoA/ROCK pathway blocked multi‐layered MEE formation. Partial prevention of hypertrophied MEE was observed with Y27632 and cytochalasin, but not with blebbistatin. NDZ induced re‐localization of GEF‐H1 into cytoplasm from cell–cell junctions. Conclusions: The present study provided evidence that the GEF‐H1/RhoA/ROCK pathway plays a pivotal role in linking microtubule disassembly to the remodeling of the actin cytoskeleton, which resulted in a multi‐layered hypertrophied MEE and failure of palatal fusion. Developmental Dynamics 241:1169–1182, 2012.

Smad2 Overexpression Reduces the Proliferation of the Junctional Epithelium

The overexpression of the intracellular signaling molecule of the transforming growth factor–beta family (TGF-β) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE). Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild-type (WT) mice selected as the control group. Samples were stained with hematoxylin and eosin stains and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-Myc as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) μm2 for the WT and 24,937.25 (± 1,965) μm2 for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-Myc expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice. Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-Myc and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest.

Smad2 overexpression induces alveolar bone loss and up regulates TNF-α, and RANKL

OBJECTIVE The aim of the current study was to investigate whether Smad2 overexpression in JE cells induced alveolar bone loss, and to understand the mechanisms regulating the bone loss. METHODS A mouse line was created that used a cytokeratin 14 (K14) promoter to overexpress Smad2 in the epithelium of the transgenic mice (K14-Smad2). Micro CT radiographs (μCT) were used to assess bone loss, bone volume, and bone density. The expression of Tnfα, Il1-β, Ifγ, Rankl, and Opg were assessed by RT-PCR. Western blots were used to detect the protein levels of TNF-α and IL1-β. Tartrate-resistant acid phosphatase (TRAP) was used as a marker for osteoclasts. Wild type (WT) mice were used as controls in all steps of the current study. RESULTS K14-Smad2 mice had 52.5% (±4.2) root exposed compared to 32.4%(±3.2) in the WT mice. There was a significant difference in alveolar bone volume in the K14-Smad2 mice when compared to WT mice 2.65mm3 (±0.3) and 4.3mm3 (±0.35) respectively. K14-Smad2 mice also had reduced bone density 696.8mg/cc (±70) at 12 months when compared to WT mice 845.9mg/cc(±10). The mRNA levels of Tnfα and Rankl increased by 3.26- and 2.5-fold respectively in the K14-Smad2 mice when compared to controls. The protein level of TNF-α was also significantly increased to 2.8-fold in K14-Smad2 mice when compared to WT mice. Smad2 overexpression increased the total numbers of osteoclasts in K14-Smad2 mice (3.4±0.2)-fold when compared to WT mice. CONCLUSION Smad2 overexpression induces alveolar bone loss and increases the numbers of osteoclasts. Also, Smad2 overexpression up-regulates TNF-α and RANKL.

Growth of ovarian cancer xenografts causes loss of muscle and bone mass: a new model for the study of cancer cachexia: ES-2 cells cause cachexia in vivo

Cachexia frequently occurs in women with advanced ovarian cancer (OC), along with enhanced inflammation. Despite being responsible for one third of all cancer deaths, cachexia is generally under‐studied in OC due to a limited number of pre‐clinical animal models. We aimed to address this gap by characterizing the cachectic phenotype in a mouse model of OC.

ACVR2B/Fc counteracts chemotherapy-induced loss of muscle and bone mass

Chemotherapy promotes the development of cachexia, a debilitating condition characterized by muscle and fat loss. ACVR2B/Fc, an inhibitor of the Activin Receptor 2B signaling, has been shown to preserve muscle mass and prolong survival in tumor hosts, and to increase bone mass in models of osteogenesis imperfecta and muscular dystrophy. We compared the effects of ACVR2B/Fc on muscle and bone mass in mice exposed to Folfiri. In addition to impairing muscle mass and function, Folfiri had severe negative effects on bone, as shown by reduced trabecular bone volume fraction (BV/TV), thickness (Tb.Th), number (Tb.N), connectivity density (Conn.Dn), and by increased separation (Tb.Sp) in trabecular bone of the femur and vertebra. ACVR2B/Fc prevented the loss of muscle mass and strength, and the loss of trabecular bone in femurs and vertebrae following Folfiri administration. Neither Folfiri nor ACVR2B/Fc had effects on femoral cortical bone, as shown by unchanged cortical bone volume fraction (Ct.BV/TV), thickness (Ct.Th) and porosity. Our results suggest that Folfiri is responsible for concomitant muscle and bone degeneration, and that ACVR2B/Fc prevents these derangements. Future studies are required to determine if the same protective effects are observed in combination with other anticancer regimens or in the presence of cancer.

Palatal adhesion is dependent on Src family kinases and p38MAPK

During secondary palate development, palatal shelves adhere to each other in the midline to form a midline epithelial seam leading to palatal closure. Cell-cell and cell-extracellular matrix adhesions, which are mediated by cell adhesion receptors, E-cadherin and integrins, are implicated in the process of adhesion of the palatal shelves. Src family kinases (SFK) function downstream of both receptors. In this study, we focused on the role of SFK in the process of palatal adhesion. During palatal adhesion, the expression of SFK mRNA, as well as localization and quantitation of the protein in the activated form, were examined by real-time qPCR and immunofluorescence. Palatal organ cultures were performed to identify the effect of pharmacological inhibition of SFK on palatal adhesion. Activated SFKs were found to be co-localized with adhesion receptors, E-cadherin and integrins in the palatal medial edge epithelium. Src, Fyn and Yes subfamily members were expressed in the palatal tissue. The expression of SFK mRNA and the quantity of the activated form of the protein were upregulated during palatal adhesion. An SFK inhibitor, PP2, blocked palatal adhesion, but another SFK inhibitor, SU6656 was not inhibitory. However, the combination of SU6656 and either of the p38MAPK inhibitors, SB203580 or BIRB0796, showed similar inhibitory effects on palatal adhesion compared to PP2 alone. The p38MAPK inhibitors alone did not alter palatal adhesion. Real-time qPCR revealed that p38MAPK alpha and delta were elevated during palatal adhesion. This study indicates that palatal cell adhesion is dependent on signaling from integrin receptors and E-cadherin through SFK and p38MAPK.