Naoko Yamada

The promotional effect of IL-22 on mineralization activity of periodontal ligament cells

OBJECTIVESnInterleukin (IL)-22 acts on non-immune cells to induce anti-microbial responses, protection from tissue damage, and enhance cell regeneration. However, little is known about the involvement of IL-22 in periodontal biology. This study investigated the biological effects of IL-22 on periodontal ligament (PDL) cells as part of studies to assess the involvement of IL-22 in periodontal disease.nnnMATERIALS AND METHODSnGene expression levels of IL-22 and its receptors in PDL cells and gingival tissue samples were evaluated by real-time PCR. Proliferative responses and mineralized-matrix forming activities of PDL cells were examined in the presence and absence of IL-22.nnnRESULTSnIn contrast to the expression of IL-22 receptors detected in PDL tissues and their cell lines, gingival tissues showed modest or no gene expressions of IL-22. The production of several cytokines including IL-11, IL-8 and CCL2 was upregulated by IL-22 treatment of PDL cells in a dose-dependent manner. IL-22 treatment had no effect on the proliferative response in PDL cells. Meanwhile, IL-22 precipitated mineralized nodule formation and induced gene expressions of RUNX2, MSX2 and osteocalcin in PDL cells, suggesting that IL-22 enhances the mineralized matrix-forming activities of PDL cells.nnnCONCLUSIONnIL-22 has the potential to promote mineralizing activity in PDL cells and to develop appropriate regenerative therapy.

Downregulation of matrix metalloproteinase-9 mRNA by valproic acid plays a role in inhibiting the shedding of MHC class I-related molecules A and B on the surface of human osteosarcoma cells

Valproic acid, a histone deacetylase inhibitor, increases the expression of cell surface MHC class I-related chain molecules (MICs) A and B (MICA and B) in osteosarcoma cells and decreases their secretion of soluble MICA and MICB, which are produced by the proteolytic cleavage of cell surface MICs. Osteosarcoma cells have been reported to produce high levels of matrix metalloproteinase (MMP)-2 and -9. In this study, we investigated the involvement of MMP-2 and -9 in the inhibitory action of valproic acid (VPA) on the proteolytic cleavage of cell surface MICs using the U-2 OS and SaOS-2 osteosarcoma cell lines. VPA caused a marked decrease in the expression of MMP-9 mRNA in the U-2 OS and SaOS-2 cells and in the expression of MMP-2 mRNA in the U-2 OS cells, but only a slight decrease in the expression of MMP-2 mRNA in the SaOS-2 cells. The transfection of small interfering RNA (siRNA) for MMP-9 decreased the secretion of soluble MICA and MICB by both U-2 OS and SaOS-2 cells, but that of siRNA for MMP-2 did not. The present study therefore demonstrates that the downregulation of MMP-9 mRNA by VPA plays a role in the inhibitory action of VPA on the secretion of soluble MICA and MICB in osteosarcoma cells.

Valproic acid cooperates with hydralazine to augment the susceptibility of human osteosarcoma cells to Fas- and NK cell-mediated cell death

We investigated the effects of valproic acid (VPA), a histone deacetylase inhibitor, in combination with hydralazine, a DNA methylation inhibitor, on the expression of cell-surface Fas and MHC-class I-related chain molecules A and B (MICA and B), the ligands of NKG2D which is an activating receptor of NK cells, and on production of their soluble forms in HOS, U-2 OS and SaOS-2 human osteosarcoma cell lines. We also examined the susceptibility of these cells to Fas- and NK cell-mediated cell death. VPA did not increase the expression of Fas on the surface of osteosarcoma cells, while hydralazine did, and the combination of VPA with hydralazine increased the expression of cell-surface Fas. In contrast, the combination of VPA with hydralazine did not increase the production of soluble Fas by osteosarcoma cells. Both VPA and hydralazine increased the expression of cell-surface MICA and B in osteosarcoma cells, and their combination induced a greater increase in their expression. VPA inhibited the production of both soluble MICA and MICB by osteosarcoma cells while hydralazine produced no effect. Both VPA and hydralazine enhanced the susceptibility of osteosarcoma cells to Fas- and NK cell-mediated cell death and the combination of VPA with hydralazine further enhanced the effects. The present results suggest that combined administration of VPA and hydrazine is valuable for enhancing the therapeutic effects of immunotherapy for osteosarcomas.

Hypoxia downregulates the expression of cell surface MICA without increasing soluble MICA in osteosarcoma cells in a HIF-1α-dependent manner

Tumor cells express NKG2D ligands on their cell surface, which are the ligands of the activating receptor, NKG2D, that is expressed on the surface of NK cells. The binding of NK cells to tumor cells through the interaction of NKG2D and its ligands induces the cytolysis of the tumor cells. In the present study, we investigated the effects of hypoxia on the expression of NKG2D ligands on the surface of human osteosarcoma cells using three cell lines. To produce hypoxic and normoxic conditions, the osteosarcoma cell lines were cultured under 1 and 20% O2 conditions, respectively. The osteosarcoma cells expressed NKG2D ligands such as MHC class I-related chain molecules A and B (MICA and MICB) and the UL16-binding proteins 1, 2 and 3 (ULBP 1, 2 and 3). MICA was the most frequently expressed NKG2D ligand in the osteosarcoma cells. Hypoxia decreased the expression of cell surface MICA only without increasing the secretion of soluble MICA, which is produced by proteolytic cleavage of cell surface MICA. Hypoxia consistently decreased the susceptibility of the osteosarcoma cells to the cytotoxicity of the NK cells. Hypoxia induced the expression of hypoxia-inducible factor-1α (HIF-1α), and knockdown of the expression of HIF-1α using small interfering RNA increased the expression of cell surface MICA and concomitantly increased the level of soluble MICA. Hypoxia decreased the production of nitric oxide (NO) metabolites (nitrite and nitrate), thus, indicating a decreasing effect on NO production. However, a NO donor, NOC18, decreased the expression of cell surface MICA without any apparent effects on the expression of HIF-1α under both hypoxic and normoxic conditions. The present results indicate that hypoxia downregulates the expression of cell surface MICA without increasing the level of soluble MICA in a HIF-1α-dependent manner and suggest that the effects of hypoxia are not linked to the hypoxia-induced reduction of NO production.

Sodium valproate, a histone deacetylase inhibitor, modulates the vascular endothelial growth inhibitor-mediated cell death in human osteosarcoma and vascular endothelial cells

The level of vascular endothelial growth inhibitor (VEGI) has been reported to be negatively associated with neovascularization in malignant tumors. The soluble form of VEGI is a potent anti-angiogenic factor due to its effects in inhibiting endothelial cell proliferation. This inhibition is mediated by death receptor 3 (DR3), which contains a death domain in its cytoplasmic tail capable of inducing apoptosis that can be subsequently blocked by decoy receptor 3 (DcR3). We investigated the effects of sodium valproate (VPA) and trichostatin A (TSA), histone deacetylase inhibitors, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Consequently, treatment with VPA and TSA increased the VEGI and DR3 expression levels without inducing DcR3 production in the OS cell lines. In contrast, the effect on the HMVE cells was limited, with no evidence of growth inhibition or an increase in the DR3 and DcR3 expression. However, VPA-induced soluble VEGI in the OS cell culture medium markedly inhibited the vascular tube formation of HMVE cells, while VEGI overexpression resulted in enhanced OS cell death. Taken together, the HDAC inhibitor has anti-angiogenesis and antitumor activities that mediate soluble VEGI/DR3-induced apoptosis via both autocrine and paracrine pathways. This study indicates that the HDAC inhibitor may be exploited as a therapeutic strategy modulating the soluble VEGI/DR3 pathway in osteosarcoma patients.

Expression of both hepatocellular carcinoma and cholangiocarcinoma phenotypes in hepatocellular carcinoma and cholangiocarcinoma components in combined hepatocellular and cholangiocarcinoma

Expression of phenotype markers of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) in HCC and CC components of 20 combined hepatocellular and cholangiocarcinomas (CHCs) of the liver was investigated immunohistochemically. Both HCC and CC components of all CHCs expressed at least one of the CC phenotype markers [cytokeratin (CK)-7, CK-19, and carbohydrate (CA) 19-9]. HCC components in 90% of CHCs and CC components in 95% of CHCs expressed at least one of these CC phenotype markers in more than 40% of cancer cells. HCC components in all CHCs expressed at least one of the HCC phenotype markers [hepatocyte antigen (HA), α-fetoprotein (AFP), and canalicular carcinoembryonic antigen]. HCC components in 90% of CHCs and CC components in 75% of CHCs expressed HA, AFP, or both. HCC components in 75% of CHCs and CC components in 60% of CHCs expressed HA, AFP, or both in more than 10% of cancer cells. The present results show that both HCC and CC components of most of the CHCs expressed both HCC and CC phenotypes, supporting the hypothesis that CHC originates from a hepatic progenitor cell capable of differentiating into hepatocytes and cholangiocytes.

A case of mixed ductal-endocrine carcinoma of the pancreas

A 66-year-old male patient underwent a stomach-preserving pancreatoduodenectomy procedure because of a tumor located around the lower bile duct under the diagnosis of carcinoma of the lower bile duct. The tumor (3.5 × 2.5 cm) was found at the head of the pancreas and had invaded the papillae of Vater at the duodenum. Histology findings indicated both ductal adenocarcinoma and endocrine tumor. The ductal adenocarcinoma component expressed carcinoembryonic antigen, cytokeratin (CK)-19, CK-20, carbohydrate 19-9, and amylase, whereas the endocrine component, which occupied about one-third of the tumor, expressed glucagon, neuron-specific enolase, and chromogranin A. The Ki-67 labeling indices of the two components were 49.7% and 5.3%, respectively. Herein, we present this case of mixed ductal-endocrine carcinoma of the pancreas. Our findings indicate that its aggressive mass may be ascribable to the adenocarcinoma component with a high proliferative potential.

Expression of interleukin-34 and colony stimulating factor-1 in the stimulated periodontal ligament cells with tumor necrosis factor-α

Tumor necrosis factor-α (TNF-α) directly and indirectly plays a crucial role in osteoclastogenesis. However, the indirect effects of TNF-α on colony-stimulating factor-1 receptor (CSF-1R)-mediated osteoclastogenesis achieved via periodontal ligament (PDL) cells are not fully understood. We herein examined the potency of osteoclast differentiation and maturation induced by fivefold supernatants in the stimulated human PDL cells with a physiologically high concentration (10xa0ng/mL) of recombinant TNF-α to human peripheral blood monocytes/macrophages in the simultaneous presence of the receptor activator of nuclear factor kappa-B ligand. The number of tartrate-resistant acid phosphatase-positive cells with multiple nuclei, but not those with a single nucleus, was decreased by approximately 50xa0% by neutralization with rabbit IgG against either interleukin-34 (IL-34) or CSF-1. Small and large amounts of IL34 and CSF1 transcripts were measured in the stimulated PDL cells using real-time polymerase chain reaction. The corresponding amounts of proteins to IL34 and CSF1 transcripts were observed in the stimulated PDL cells on immunohistochemical staining or Western blotting. Moreover, 0.13xa0ng/mL of IL-34 and 5.0xa0ng/mL of CSF-1 were measured in the supernatants of the stimulated PDL cells using an enzyme-linked immunosorbent assay. IL-34 derived from the stimulated PDL cells with TNF-α appeared to synergistically function with CSF-1 in the CSF-1R-mediated maturation of osteoclastogenesis.

Phenotypic characteristics and proliferative activity of hyperplastic ductule cells in cholangiofibrosis induced by thioacetamide in rats.

The oral administration of thioacetamide to rats induces cholangiofibrosis characterized by hyperplasia of ductules surrounded by fibrous tissue. In the present study, we examined the expression of markers of cholangiocyte and hepatocyte phenotypes in these hyperplastic ductule cells and their proliferative activity immunohistochemically. The oral administration of thioacetamide to 21-day-old male Fisher 344 rats for 12 weeks induced multiple areas of various sizes with hyperplastic ductules. The ductules consisted of two types of ductules; ductules composed of cholangiocyte-like cuboidal cells with transparent nuclei and cytoplasm, and of intestinal epithelium-like (IE-like) cells of basophilic nuclei and cytoplasm, and the transition of these two types of cells in the same ductule was sometimes observed. The cholangiocyte-like cells expressed cytokeratin (CK)-7, CK-19 and OV-6 (cholangiocyte phenotype markers) but not Hep Par-1 antigen or HNF4α (hepatocyte phenotype markers). In contrast, the IE-like cells expressed Hep Par-1 antigen and HNF4α but not CK-7, CK-19 or OV-6. The examination of Ki-67 expression showed a much higher proliferative activity for the IE-like cells compared to the cholangiocyte-like cells. The present results show that the hyperplastic ductules induced by thioacetamide are composed of IE-like cells with a high proliferative activity expressing the hepatocyte phenotype markers and of cholangiocyte-like cells with a low proliferative activity expressing the cholangiocyte phenotype markers.

Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps.

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.