Transgelin mediates transforming growth factor-ß1-induced proliferation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Human periodontal ligament cells (HPDLCs) express transforming growth factor-ß1 (TGF-ß1) that regulates differentiation and proliferation, and plays key roles in homeostasis of PDL tissue. Transgelin is a cytoskeleton-associated protein with an Smad-binding element in its gene promoter region. In this study, we examined the localization and potential function of transgelin in PDL tissue and cells. MATERIAL AND METHODS: Microarray analysis of HPDLC lines (2-14, 2-23 and 2-52) was performed. Expression of transgelin in HPDLCs was examined by quantitative reverse transcription-polymerase chain reaction, immunofluorescence staining and western blot analysis. Effects of TGF-ß1 and its signaling inhibitor, SB431542, on transgelin expression in HPDLCs were examined by western blot analysis. The effects of transgelin knockdown by small interfering RNA (siRNA) on HPDLC proliferation stimulated by TGF-ß1 were assessed by WST-1 assay. RESULTS: In microarray and quantitative reverse transcription-polymerase chain reaction analyses, the expression levels of transgelin (TAGLN) in 2-14 and 2-23 cells, which highly expressed PDL markers such as periostin (POSTN), tissue non-specific alkaline phosphatase (ALPL), α-smooth muscle actin (ACTA2) and type I collagen A1 (COL1A1), was significantly higher than those in 2-52 cells that expressed PDL markers weakly. Immunohistochemical and immunofluorescence staining revealed expression of transgelin in rat PDL tissue and HPDLCs. In HPDLCs, TGF-ß1 treatment upregulated transgelin expression, whereas inhibition of the type 1 TGF-ß1 receptor by SB431542 suppressed this upregulation. Furthermore, TAGLN siRNA transfection did not promote the proliferation of HPDLCs treated with TGF-ß1. The expression levels of CCNA2 and CCNE1, which regulate DNA synthesis and mitosis through the cell cycle, were also not upregulated in HPDLCs transfected with TAGLN siRNA. CONCLUSION: Transgelin is expressed in PDL tissue and might have a role in HPDLC proliferation induced by TGF-ß1 stimulation.

Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells.

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased ß-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/ß-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

Benzo[a]pyrene/aryl hydrocarbon receptor signaling inhibits osteoblastic differentiation and collagen synthesis of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Cigarette smoking has detrimental effects on periodontal tissue, and is known to be a risk factor for periodontal disease, including the loss of alveolar bone and ligament tissue. However, the direct effects of cigarette smoking on periodontal tissue remain unclear. Recently, we demonstrated that benzo[a]pyrene (BaP), which is a prototypic member of polycyclic aryl hydrocarbons and forms part of the content of cigarettes, attenuated the expression of extracellular matrix remodeling-related genes in human periodontal ligament (PDL) cells (HPDLCs). Thus, we aimed to examine the effects of BaP on the osteoblastic differentiation and collagen synthesis of HPDLCs. MATERIAL AND METHODS: HPDLCs were obtained from healthy molars of three patients, and quantitative reverse transcription-polymerase chain reaction were performed for gene expression analyses of cytochrome P450 1A1 and 1B1, alkaline phosphatase, bone sialoprotein and aryl hydrocarbon receptor (AhR), a receptor for polycyclic aryl hydrocarbons. We have also analyzed the role of the AhR, using 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), which is an AhR antagonist. RESULTS: The treatment of HPDLCs with BaP reduced mRNA expression of osteogenic genes, alkaline phosphatase activity, mineralization and collagen synthesis. The treatment with CH-223191 subsequently restored the observed suppressive effects of BaP on HPDLCs. CONCLUSIONS: The present results suggest that BaP exerts inhibitory effects on the maintenance of homeostasis in HPDL tissue, such as osteoblastic differentiation and collagen synthesis of HPDLCs, and that this signaling pathway could be suppressed by preventing the transactivity of AhR. Future studies may unveil a role for the inhibition of AhR as a promising therapeutic agent for periodontal disease caused by cigarette smoking.

Mechanical induction of interleukin-11 regulates osteoblastic/cementoblastic differentiation of human periodontal ligament stem/progenitor cells.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is continually exposed to mechanical loading caused by mastication or occlusion. Physiological loading is thus considered a key regulator of PDL tissue homeostasis; however, it remains unclear how this occurs. We recently reported that an appropriate magnitude of mechanical stretch can maintain PDL tissue homeostasis via the renin-angiotensin system. In the present study, we investigated the expression of interleukin-11 (IL-11) in human primary PDL cells (HPDLCs) exposed to stretch loading, the contribution of angiotensin II (Ang II) to this event and the effects of IL-11 on osteoblastic/cementoblastic differentiation of human PDL progenitor cells (cell line 1-17). MATERIAL AND METHODS: Human primary PDL cells, derived from human tissues, with or without antagonists against the Ang II receptors AT1 or AT2, were subjected to cyclical stretch loading with 8% elongation for 1 h. Expression of IL-11 was measured by ELISA in these cultures and by immunohistochemistry in the sectioned maxillae of rats. The osteoblastic/cementoblastic potential of cell line 1-17 was determined using cell proliferation, gene expression and Alizarin Red staining. RESULTS: Positive staining for IL-11 was observed in the PDL of rat maxillae and in cultures of HPDLCs. In HPDLCs exposed to stretch, expression of the IL11 gene and the IL-11 protein were up-regulated, concomitant with an increase in Ang II and via AT2. Recombinant human IL-11 (rhIL-11) stimulated an increase in expression of mRNA for the cementoblast-specific marker, CP-23, and for the osteoblastic markers, osteopontin and bone sialoprotein, and promoted proliferation in cell line 1-17. In addition, rhIL-11 also increased the degree of mineralized nodule formation in cell line 1-17 cultures treated with CaCl2 . CONCLUSION: Mechanical loading appears to control proliferation and osteoblastic/cementoblastic differentiation of human PDL stem/progenitor cells through the regulation of Ang II and AT2 by IL-11.

The roles of angiotensin II in stretched periodontal ligament cells.

The loading caused by occlusion and mastication plays an important role in maintaining periodontal ligament (PDL) tissues. We hypothesized that a loading magnitude would be involved in the production of biological factors that function in the maintenance of PDL tissues. Here, we identified up-regulated gene expressions of transforming growth factor-ß1 (TGF-ß1), alkaline phosphatase (ALP), and angiotensinogen in human PDL fibroblastic cells (HPLFs) that were exposed to 8% stretch loading. Immunolocalization of angiotensin I/II (Ang I/II), which was converted from angiotensinogen, was detected in rat PDL tissues. HPLFs that were stimulated by Ang II also increased their gene expressions of TGF-ß1 and ALP. Furthermore, the antagonist for Ang II type 2 receptor, rather than for type 1, significantly inhibited gene expressions induced by the stretch loading. Analysis of these data suggests that Ang II mediates the loading signal in stretched HPLFs to induce expressions of TGF-ß1 and ALP.

An in vitro evaluation of two resin-based sealers on proliferation and differentiation of human periodontal ligament cells.

AIM: To evaluate the effects of a polymethyl methacrylate resin-based sealer [Superbond sealer (SB)] on the proliferation and osteogenic differentiation of human periodontal ligament cells (HPDLCs) in vitro, compared with a methacrylate resin-based sealer [Epiphany SE sealer (EP)]. METHODOLOGY: Human periodontal ligament cells were obtained from of healthy third molar teeth of two participants with informed consent. To determine the effects of the eluent from set resin sealers on HPDLCs, the 7-day-washed (washed) or non-washed freshly prepared (fresh) set SB or EP discs were prepared. Cells cultured on these discs were evaluated by the WST-1 proliferation assay and scanning electron microscopy (SEM). The osteogenic differentiation of HPDLCs on washed SB discs was then evaluated by gene expression analysis of osteopontin (OPN) and osteocalcin (OCN) by using quantitative RT-PCR. RESULTS: Human periodontal ligament cells exhibited growth on washed SB discs, whereas fresh SB and EP discs and washed EP discs inhibited proliferation of HPDLCs. SEM observation revealed that HPDLCs tightly attached and spread on the surface of washed SB discs, whilst no HPDLCs were observed on the surface of fresh and washed EP discs. Furthermore, HPDLCs significantly upregulated gene expressions of OPN and OCN when cultured on washed SB discs in osteogenic differentiation medium for 2 weeks. CONCLUSIONS: Although Superbond sealer initially exerted cytotoxic effects on HPDLCs, these effects were reduced during washing for 7 days compared to EP, which continued to be cytotoxic even though the specimens were washed for the same period of time. Washed Superbond allowed HPDLCs to differentiate into osteogenic cells.

[Immunocytochemical localization of carcinoembryonic antigen (CEA) and transport of CEA into the blood vessels in gastric cancer].

CEA was localized in the luminal border, cytoplasm, but not in mucus, in Signet ring cell carcinoma (sig) and mucinous carcinoma (muc). Electronmicroscopically, CEA was localized in the glycocalyx of the microvilli and microvesicles of the cytoplasm. The histologically different cancer types showed no difference in the localization of T-CEA. We also studied P-CEA elevating factors in 38 CEA-positive (++) patients manifesting subserosal (ss) or deeper invasion. No remarkable findings were obtained. When P-CEA elevating factors were studied in 20 patients with (+) CEA reaction and ss or deeper invasion, we found that the incidence of por was high in P-CEA negative cases. In particular, the por incidence was significantly low (p less than 0.01) in patients with scirrhous type. High P-CEA levels were detected in blood adjacent to the cancer. Among 6 cases with negative P-CEA in the vessels adjacent to the cancer whose T-CEA reactions were (++) or (+), and who manifested ss or deeper invasion, there was a high incidence of por (5 cases) and scirrhous type (4 cases), histologically.

Hirschsprung's disease in an adult. Special reference to histochemical determination of the acetylcholinesterase activity.

A 24-year-old Japanese woman with Hirschsprung's disease was subjected to the combination of barium enema, anorectal manometry, and a histochemical study of acetylcholinesterase (AChE) activity in rectal suction biopsy. The histochemistry revealed a marked increase of AChE positive nerve fibers. The importance of such a study is stressed in order to diagnose Hirschsprung's disease correctly, even in adults. A Z-shaped anastomosis as a modification of Duhamel's method was carried out. The postoperative course was uneventful and she now has normal bowel evacuation.