Masae Kitagawa

Periostin Promotes Invasion and Anchorage-Independent Growth in the Metastatic Process of Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer. Typically, HNSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. However, molecular mechanisms associated with the invasion and metastasis of HNSCC remain poorly understood. Here, we identified periostin as an invasion-promoting factor in HNSCC by comparing the gene expression profiles between parent HNSCC cells and a highly invasive clone. Indeed, periostin overexpression promoted invasion and anchorage-independent growth both in vitro and in vivo in HNSCC cells. Moreover, periostin-overexpressing cells spontaneously metastasized to cervical lymph nodes and to the lung through their aggressive invasiveness in an orthotopic mouse model of HNSCC. Interestingly, periostin was highly expressed in HNSCCs in comparison with normal tissues, and the level of periostin expression was well correlated with the invasiveness of HNSCC cases. In summary, these findings suggest that periostin plays an important role in the invasion and anchorage-independent growth of HNSCC.

Periostin is frequently overexpressed and enhances invasion and angiogenesis in oral cancer

Oral squamous-cell carcinoma (OSCC) is one of the most common types of human cancer. Typically OSCC cells show persistent invasion that frequently leads to local recurrence and distant lymphatic metastasis. We previously identified Periostin as the gene demonstrating the highest fold change expression in the invasive clone by comparing the transcriptional profile of parent OSCC cell line and a highly invasive clone. Here, we demonstrated that Periostin overexpression enhanced invasiveness in oral cancer cell lines. To know the role of Periostin in invasion, angiogenesis and metastasis in OSCC cases, we first examined the expression of Periostin mRNA in 31 OSCC cases by RT–PCR and Periostin protein in 74 OSCC cases by immunohistochemistry. Then, we compared the Periostin expression with invasion pattern, metastasis and blood vessel density. Periostin mRNA and protein overexpression were frequently found in OSCC cases and Periostin expression was well correlated with the invasion pattern and metastasis. Moreover, blood vessel density of Periostin-positive cases was higher than those of Periostin-negative cases. Interestingly, recombinant Periostin enhanced capillary formation in vitro in a concentration-dependant manner. In summary, these findings suggest that Periostin may promote invasion and angiogenesis in OSCC, and that Periostin can be a strong marker for prediction of metastasis in oral cancer patients.

Role of Cks1 overexpression in oral squamous cell carcinomas: cooperation with Skp2 in promoting p27 degradation.

Down-regulation of p27 is frequently observed in various cancers due to an enhancement of its degradation. Skp2 is required for the ubiquitination and consequent degradation of p27 protein. Another protein called Cks1 is also required for p27 ubiquitination in the SCF(Skp2) ubiquitinating machinery. In the present study, we examined Cks1 expression and its correlation with p27 in oral squamous cell carcinoma (OSCC) derived from tongue and gingiva. By immunohistochemical analysis, high expression of Cks1 was present in 62% of OSCCs in comparison with 0% of normal mucosae. In addition, 65% of samples with low p27 expression displayed high Cks1 levels. Finally, Cks1 expression was well correlated with Skp2 expression and poor prognosis. To study the role of Cks1 overexpression in p27 down-regulation, we transfected Cks1 with or without Skp2 into OSCC cells. Cks1 transfection could not induce a p27 down-regulation by itself, but both Cks1 and Skp2 transfection strongly induced. Moreover, we inhibited Cks1 expression by small interference RNA (siRNA) in OSCC. Cks1 siRNA transfection induced p27 accumulation and inhibited the growth of OSCC cells. These findings suggest that Cks1 overexpression may play an important role for OSCC development through Skp2-mediated p27 degradation, and that Cks1 siRNA can be a novel modality of gene therapy.

Characterization of established cementoblast-like cell lines from human cementum-lining cells in vitro and in vivo.

To study cellular characteristics of human cementoblasts using a cellular model is important for understanding the mechanisms of homeostasis and regeneration of periodontal tissues. However, at present no immortalized human cementoblast cell line has been established due to limitation of the life span. In the present study, therefore, we attempted to establish human cementoblast-like cell lines by transfection with telomerase catalytic subunit hTERT gene. Two stable clones (HCEM-1 and -2) with high telomerase activity were obtained and they grew over 200 population doublings without significant growth retardation. The expression of mRNA for differentiation markers, type I collagen, alkaline phosphatase (ALP), runt-related transcription factor 2, osteocalcin, bone sialoprotein and cementum-derived protein was revealed in these clones by RT-PCR. Moreover, these cells showed high ALP activity and calcified nodule formation in vitro. Interestingly, HCEM-2 showed cementum like formation on the surface of hydroxyapatites granules by subcutaneous transplantation into immunodeficient mice with hydroxyapatite granules. Thus, we established human cementoblast-like cell lines. We suggest that HCEM cell lines can be useful cell models for investigating the characteristics of human cementoblasts.

Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell growth of oral cancer cells by inhibiting p27 degradation.

S phase kinase–interacting protein 2 (Skp2), an F box protein, is required for the ubiquitination and consequent degradation of p27. It is well known that reduced expression of p27 is frequently observed in various cancers including oral squamous cell carcinoma and is due to an enhancement of its protein degradation. Our previous study showed that overexpression of Skp2 was frequently found in oral squamous cell carcinoma and inversely correlated with p27 expression. Recently, a technique known as RNA interference has been successfully adapted to mammalian cells. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27 down-regulation in oral squamous cell carcinoma. We used a siRNA plasmid vector, which has an advantage over synthetic siRNAs in determining the effects of decreasing the high constitutive levels of Skp2 protein in oral squamous cell carcinoma. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in oral squamous cell carcinoma cells. Moreover, p27 protein in Skp2 siRNA-transfected cells is more stabilized than that in control siRNA-transfected cells. Interestingly, Skp2 siRNA inhibited the cell proliferation of oral squamous cell carcinoma cells both in vitro and in vivo. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel modality of cancer gene therapy for suppression of p27 down-regulation.

Brain-derived Neurotrophic Factor Stimulates Bone/Cementum-related Protein Gene Expression in Cementoblasts

Brain-derived neurotrophic factor (BDNF), recognized as essential in the developing nervous system, is involved in differentiation and proliferation in non-neuronal cells, such as endothelial cells, osteoblasts, and periodontal ligament cells. We have focused on the application of BDNF to the regeneration of periodontal tissue and indicated that BDNF promotes the regeneration of experimentally created periodontal defects. Cementoblasts form cementum, mineralized tissue, which is key to establishing a functional periodontium. The application of BDNF to the regeneration of periodontal tissue requires elucidation of the mechanism by which BDNF regulates the functions of cementoblasts. In this study, we examined how BDNF regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase (ALP), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2)) in cultures of immortalized human cementoblast-like (HCEM) cells. BDNF elevated the mRNA levels of ALP, OPN, and BMP-2 in HCEM cells. Small interfering RNA (siRNA) for TRKB, a high affinity receptor of BDNF, siRNA for ELK-1, which is a downstream target of ERK1/2, and PD98059, an ERK inhibitor, obviated the increase in the mRNA levels. BDNF increased the levels of phosphorylated ERK1/2 and Elk-1, and the blocking of BDNF signaling by treatment with siRNA for TRKB and PD98059 suppressed the phosphorylation of ERK1/2 and Elk-1. Furthermore, BDNF increased the levels of phosphorylated c-Raf, which activates the ERK signaling pathway. These findings provide the first evidence that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the BDNF-induced mRNA expression of ALP, OPN, and BMP-2 in HCEM cells.

Brain-derived neurotrophic factor protects cementoblasts from serum starvation-induced cell death.

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNFs actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009.

Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice.

Background Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW. Methods To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy. Results Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1β (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion. Conclusions Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

Infection with Porphyromonas gingivalis exacerbates endothelial injury in obese mice.

Background A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. Methods In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. Results Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. Conclusions Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.

Synthetic ameloblastin peptide stimulates differentiation of human periodontal ligament cells.

OBJECTIVE This study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC). DESIGN We used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, we examined quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey-Lowry enzymological method, mineral nodule formation by Dahls method, and expression of mineralization-related genes by RT-PCR. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide. RESULTS At all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by an anti-ameloblastin antibody, which caused ALP levels to revert to their approximate levels in the untreated condition. At concentrations greater than 1ng/ml, the peptide promoted mineralized nodule formation of PDLC. And the peptide induced higher expressions of ALP and bone sialoprotein (BSP) than the control. CONCLUSION Our results show that the ameloblastin peptide upregulate ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide promotes the differentiation activity of PDLC.