Plasminogen/plasmin modulates bone metabolism by regulating the osteoblast and osteoclast function.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Targeted drug delivery system for oral cancer therapy using sonoporation.

Ultrasound-mediated destruction of microbubbles has been proposed as an innovative non-invasive drug delivery system for cancer therapy. We developed a specific drug delivery system for squamous cell carcinoma that uses sonoporation with the anti-epidermal growth factor receptor (EGFR) antibody. Administration of a low dose of bleomycin (BLM) by sonoporation with the anti-EGFR antibody produced a marked growth inhibition of Ca9-22 cells in vitro. In addition, scanning electron microscopic analysis revealed apparent surface deformation of Ca9-22 cells treated with sonoporation in the presence of the antibody. Interestingly, the population of apoptotic cells was remarkably increased when a low dose of BLM was delivered using sonoporation with the Fab fragment of the anti-EGFR antibody. These findings indicate that sonoporation with the Fab fragment makes it possible to administer drugs into cells more efficiently and specifically, suggesting a novel application for chemotherapy and gene therapy treatments for oral squamous cell carcinoma.

CCAAT/Enhancer-binding protein beta regulates expression of human T1R3 taste receptor gene in the bile duct carcinoma cell line, HuCCT1.

The T1R family (T1R1, T1R2 and T1R3 receptors) has a role in the detection of umami and sweet tastes in taste buds. Although T1R3 is also expressed in the intrahepatic bile duct, the expression patterns of T1R1 and T1R2 in this region have not been determined. In addition, the mechanisms of transcriptional regulation of the human T1R3 gene (Tas1r3) have not been elucidated. In this study, we determined the expression patterns of T1R2 and T1R3 in human liver and the function of C/EBPbeta in Tas1r3 promoter activity. Immunohistochemistry showed that T1R2 and T1R3 were expressed in the intrahepatic bile duct. 5'-RACE analysis revealed that the transcriptional start sites of Tas1r3 were located 67 bp and 176 bp upstream of the ATG. Promoter analysis of Tas1r3 was performed using the luciferase reporter assay and EMSA in the Tas1r3-expressing cell line, HuCCT1. The 226-bp region upstream of the ATG had promoter activity, and C/EBPbeta activated the Tas1r3 promoter activity in HuCCT1 cells. These results show that T1R2 and T1R3 receptors, in addition to their role in taste perception, may also have a role in intrahepatic cholangiocytes. C/EBPbeta was identified as the transcription factor regulating Tas1r3 promoter activity in HuCCT1 cells.

Expression of ATP-gated P2X3 receptors in rat gustatory papillae and taste buds.

It has recently become evident that ATP and other extracellular nucleotides could play an important role in signal transductions. ATP mediates excitatory signaling by means of P2X receptors. P2X3, one of its subtypes, a membrane ligand-gated ion channel, is strongly expressed in peripheral sensory neurons. The aim of the present study was to examine the distribution of nerve fibers expressing P2X3 receptors in taste buds in the gustatory papillae and soft palate of rats by immunohistochemistry. We found that the fluorescence ATP marker quinacrine stained subsets of taste bud cells. Numerous nerve fibers innervating taste buds were intensely immunostained with the P2X3 receptor antibody. These nerve fibers ascended among intragemmal cells and terminated just below the taste pores. In order to examine whether P2X3 receptors are involved in signal modulation within taste buds, we used fluorescent double stainings to analyze the distribution of P2X3 receptors and their relationship to alpha-gustducin immunopositive taste receptor cells. Many varicose nerve fibers expressing P2X3 receptor-immunoreactivities were entangled with alpha-gustducin-immunopositive taste receptor cells and ended closely below the taste pores. In fungiform papillae, nerve fibers expressing both P2X3 receptors and PGP 9.5 were observed. In contrast, only PGP 9.5 immunoreactive nerve fibers were recognized in filiform papillae. These results suggest that P2X3 receptors might be involved in taste transmission pathways within taste buds. ATP may act as a neurotransmitter, co-transmitter, or neuromodulator at P2X3 receptors to generate activating gustatory nerve fibers.

In vitro differentiation of dental epithelial progenitor cells through epithelial-mesenchymal interactions.

In developing teeth, dental epithelial progenitor cells differentiate through sequential and reciprocal interactions with neural-crest-derived mesenchyme. However, the molecular mechanisms involved in cell differentiation are not well understood. Continuously growing teeth are useful in the study of differentiation of dental progenitor cells. In rat lower incisors, ameloblasts originate from the dental epithelial adult stem cell compartment referred to as the 'apical bud'. To elucidate the mechanism of ameloblast differentiation, we designed a primary culture system and confirmed the differentiation of dental epithelial cells through interaction with mesenchymal cells. Cytokeratin was used as a marker for epithelial cells, nerve growth factor receptor p75 for inner enamel epithelial (IEE) cells, and ameloblastin for ameloblasts. The apical bud cells could only differentiate into IEE cells and, within 10 days, into ameloblasts expressing ameloblastin in the presence of dental papilla cells. Interestingly, the IEE cells could proliferate transiently and differentiate into ameloblasts in the presence or absence of dental papilla cells. These results suggest that apical bud cells can enter the ameloblast cell lineage through interaction with mesenchymal cells. IEE cells, on the other hand, are already committed to differentiate into ameloblasts. This culture system is useful in future studies of ameloblast differentiation.

Expression of P2Y1 receptors in rat taste buds.

Extracellular nucleotides such as ATP are the signaling molecules which bind to membrane receptors (P2X ligand-gated ion channels and G-protein-coupled P2Y families). In the gustatory system, it is known that P2X receptors are expressed exclusively in nerve fibers innervating the taste buds. Also, P2Y receptors are suggested to play some important roles in taste signal transductions on the basis of the physiological studies. In the present study, we examined the expression patterns of P2Y1 receptor subtype by using reverse transcript polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR analyses showed that P2Y1 receptor mRNAs appeared in circumvallate papillae. P2Y1 receptor mRNA was detected in a subset of taste bud cells by in situ hybridization. By immunohistochemical analyses, P2Y1 receptor was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. We showed that ATP induced a biphasic intracellular Ca2+ increase in taste cells by a Ca2+ imaging method. Furthermore, we showed by double-immunolabeling methods that P2Y1-expressing cells coexpressed both IP3R3 and SNAP-25. These results suggest that ATP may activate P2Y receptors resulting in Ca2+ release from internal stores via IP3R3. Since many SNAP-25-immunoreactive taste bud cells coexpressed P2Y1 immunoreactivity, it is suggested that P2Y1-expressing cells may possess synapses with afferent nerve fibers. The results of the present study suggest that P2Y1 receptor may play some roles in ATP-mediated signal transductions between taste bud cells and afferent taste fibers.

Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.

Epithelial stem cells in teeth.

Many tissues and organs maintain a process known as homeostasis, in which cells are replenished as they die as a result of apoptosis or injury. The continuously growing mouse incisors are an excellent model for studying the molecular mechanisms of cell homeostasis, renewal, and repair. We elucidated these mechanisms in mouse incisors by detecting adult stem cells and analyzing the stem cell lineage by bromodeoxyuridine (BrdU) labeling analysis. The stem cells divide slowly, giving rise to a daughter cell that remains in the cervical loop and a second daughter cell that enters the zone of rapidly dividing inner enamel epithelial cells (transit-amplifying cell population). During subsequent rounds of cell division, the latter cells move toward the incisal end and differentiate into ameloblasts that form the enamel matrix. Recent evidence from gene knockout mice suggests that fibroblast growth factor (Fgf10) plays an important role in the formation and maintenance of stem cells in the development of mouse incisors. The role of dental stem cells in odontogenic tumors is discussed.

Fine structural and histochemical study of lingual taste organs of Rana catesbeiana (Anura: Ranidae) transplanted to liver.

Autologous tongues were transplanted onto the liver of the anuran Rana catesbeiana, specimens of which were sacrificed at intervals from 6 hr to 5 months after transplantation. Light and electron microscopy as well as histochemistry disclose that the grafts start to organize into cysts after 14 days. The taste organs occur in all grafts irrespective of the age of the graft. All surviving taste organs exhibit intense adenosine triphosphatase (ATPase) activity and contain fluorescent basal cells of the usual type. Ultrastructurally, the taste organs are composed of three distinct types of cells that lack nerves. The taste and basal cells retain the characteristic dense-core granules in their cytoplasm through the experimental periods. The present results suggested that the taste organs of Rana can survive ectopically in the liver for up to 5 months.