Masatake Asano

Phosphorylation of Extracellular Signal-Regulated Kinase in medullary and upper cervical cord neurons following noxious tooth pulp stimulation.

The phosphorylated Extracellular Signal-regulated Kinase (pERK) and Fos expression and masticatory muscle activity were analyzed in rats with capsaicin-induced acute inflammation of the tooth pulp in order to clarify the role of the spinal trigeminal nucleus and upper cervical spinal cord in tooth pulp pain. Digastric and masseteric muscle activities were significantly increased following capsaicin injection into the molar tooth pulp but not after vehicle treatment. The pERK-like immunoreactive (LI) neurons were observed in the subnuclei interpolaris-caudalis transition (Vi/Vc) zone, the paratrigeminal nucleus (Pa5) and the superficial laminae of the caudal Vc/C2 zone. The pERK expression was detected as early as 2 min and peaked at 5 min after capsaicin or vehicle injection. The pERK expression in the Vi/Vc zone and Pa5 was bilateral, whereas it was predominantly ipsilateral in the caudal Vc/C2 zone. The capsaicin treatment of the whisker pad produced pERK expression in the rostro-caudal middle portion of the ipsilateral Vc, but small number of pERK-LI cells were observed after vehicle treatment. The pERK expression was similar in the Vi/Vc zone following capsaicin injection into the upper or lower molar tooth pulp, whereas the pERK expression was in the lateral portion of the caudal Vc/C2 zone after upper molar injection and restricted to the medial portion of the Vc/C2 zone after the lower molar capsaicin. These data were confirmed with Western blots. There were differences in the distribution of Fos protein-like immunoreactive (LI) cells and pERK-LI cells following tooth pulp stimulation. After capsaicin application into the upper molar tooth pulp, no pERK-LI cells were observed in the ventral part of the Vi/Vc zone, whereas many Fos protein-LI cells were expressed in this region. The difference in the distribution pattern of pERK- and Fos protein-LI cells in the Vi/Vc zone suggests their differential temporal expression profiles after capsaicin. The present findings suggest that tooth-pulp-driven neurons in the spinal trigeminal nucleus are involved in tooth pulp pain through activation of the intracellular signal transduction pathway that involves earlier ERK phosphorylation and subsequent Fos expression.

Nerve Growth Factor Contribution via Transient Receptor Potential Vanilloid 1 to Ectopic Orofacial Pain

It is well known that oral inflammation causes tenderness in temporomandibular joints or masseter muscles. The exact mechanism of such an orofacial ectopic hyperalgesia remains unclear. Here, we investigated the functional significance of interaction of nerve growth factor (NGF) and transient receptor potential vanilloid 1 (TRPV1) in relation to heat hyperalgesia in the whisker pad skin caused by complete Freunds adjuvant (CFA) injection into the lower lip. CFA injection induced heat hyperalgesia of the ipsilateral whisker pad skin. Moreover, it leads to enhancement of spontaneous activity and heat responses in trigeminal ganglion (TG) neurons that was elicited by heat stimulation of the whisker pad skin. The heat hyperalgesia was dose-dependently reversed by intraperitoneal TRPV1 antagonist administration, also diminished by neutralizing anti-NGF antibody administration into the lower lip and intraganglionic administration of K252a, a tyrosine kinase receptor inhibitor. Nerve fibers in bundle of mandibular nerve and TG neurons that innervates the whisker pad skin and lower lip both expressed labeled NGF, which was administrated into the lower lip. Moreover, the NGF concentrations in ophthalmic-maxillary and mandibular divisions of the TG increased after CFA injection into the lower lip. The number of TRPV1-positive neurons that innervates the whisker pad skin and lower lip was increased after CFA injection into the lower lip, and this increase was annulled by anti-NGF administration. The present findings suggest that inflammation in the lower lip induces release of NGF that regulates TRPV1 expression in TG neurons. This TRPV1 overexpression may underlie ectopic heat hyperalgesia in the whisker pad skin.

Bone morphogenetic protein 2 and dexamethasone synergistically increase alkaline phosphatase levels through JAK/STAT signaling in C3H10T1/2 cells

Alkaline phosphatase (ALP) is generally believed to be a faithful marker of osteoblast differentiation, and its expression is induced by bone morphogenetic protein‐2 (BMP‐2) and dexamethasone (Dex). However, the effects of combined administration of BMP‐2 and Dex on ALP transcription have not been extensively examined. In this study, we found that BMP‐2 and Dex synergistically increase ALP levels in mouse C3H10T1/2 pluripotent stem cells. However, switching from one inducer to the other, by adding BMP‐2 or Dex to cell cultures at different times, was no more effective than continuous treatment with either inducer alone. A significant induction of ALP mRNA expression was observed only in cells continuously treated with both inducers. This result suggests that both BMP‐2 and Dex may act in the same pathway or at the same stage of differentiation. A luciferase assay using ALP promoter deletion constructs showed that a region of the promoter containing a putative signal transducer and activator of transcription 3 (STAT3) response element (SRE) responds to treatment with a combination of BMP‐2 and Dex. Furthermore, a ChIP assay indicated that STAT3 bound to the SRE. In addition, a STAT3 siRNA suppressed the synergistic effect of BMP‐2 and Dex on ALP levels. These results indicate that STAT3 may play an important role in regulating ALP expression. To our knowledge, this is the first time that STAT3 has been implicated in the regulation of ALP expression by BMP‐2 and Dex. These findings raise the possibility of developing new strategies for the enhancement of bone formation using a combination of BMPs and Dex. J. Cell. Physiol. 223: 123–133, 2010.

The polymeric immunoglobulin receptor (secretory component) in a human intestinal epithelial cell line is up‐regulated by interleukin‐1

Secretory component (SC or polymeric immunoglobulin receptor) on mucosal epithelial cells mediates transcytosis of polymeric immunoglobulin into external fluids and functions as a receptor for polymeric immunoglobulin. SC expression in a human colonic adenocarcinoma cell line, HT‐29 has been reported to be up‐regulated by various cytokines, such as interferon‐&ggr;, tumour necrosis factor‐&agr; and interleukin‐4 (IL‐4). However, up‐regulation of SC by IL‐1 is controversial. In this study, we investigated the effect of human recombinant IL‐1 alone on SC expression in HT‐29 cells in detail. Immunocytochemistry and Northern blot analysis revealed that IL‐1&bgr; increased both the number of SC‐positive cells and SC mRNA expression. Enzyme‐linked immunosorbent assay revealed that IL‐1&bgr; enhanced secretion by HT‐29 cells in both time‐ and dose‐dependent manners. IL‐1&agr; had the same effects on HT‐29 cells. Northern blot analysis demonstrated that cycloheximide and actinomycin D abolished the effect of IL‐1. Moreover, we detected IL‐1 receptor (IL‐1R) type I mRNA in HT‐29 cells by polymerase chain reaction (PCR) and sequenced the PCR‐amplified product. We think that it reflects the possibility of the presence of IL‐1R in HT‐29 cells. From these data, we concluded that IL‐1&bgr; and IL‐1&agr; play regulatory roles in SC expression, and their effects depend on de novo protein synthesis and transcription.

Functional role of transforming growth factor‐β type III receptor during palatal fusion

The molecular regulation of palatogenesis continues to be an active area of investigation to provide a foundation for understanding the molecular etiology of cleft palate. Transforming growth factor (TGF) ‐β type III receptor (TβR‐III) has been shown to be specifically expressed in the medial edge epithelium at critical stages of palatal shelf adherence during palatogenesis. The aim of this study was to examine TβR‐III mRNA localization and expression levels in vivo and to determine the requirement for TβR‐III expression during palatal fusion in vitro. TβR‐III gene expression was analyzed by in situ hybridization in tissue specimens and real‐time reverse transcriptase‐polymerase chain reaction using specific cells in the palatal shelf isolated by laser capture microdissection. TβR‐III was knocked down in embryonic day (E) 13 palatal shelves in organ culture. Palatal shelf organ cultures were treated with small interfering RNA (siRNA) at final concentrations of 300, 400, and 500 nM, respectively. The treatment with siRNA specific for TβR‐III decreased the amount of protein by approximately 75%. The reduction in TβR‐III resulted in a delay in the process of palatal fusion compared with control. The protein expression of phospho‐Smad2 was decreased in the TβR‐III siRNA group. In addition, palatal organ cultures treated with TβR‐III siRNA + rhTGF‐β3 completely fused by 72 hr in vitro. These results support our hypothesis that TβR‐III has a critical role in the process of palatal fusion. Developmental Dynamics 236:791–801, 2007.

Retinoic acid enhances the gene expression of human polymeric immunoglobulin receptor (pIgR) by TNF‐α

In this study, the detailed mechanisms for the effects of vitamin A on the expression of polymeric immunoglobulin receptor (pIgR) were examined. Expression of the pIgR by tumour necrosis factor (TNF‐α) was enhanced by the addition of all‐trans retinoic acid (ATRA) or 9‐cis retinoic acid (9CRA). This enhancement was mediated mainly by RARα, and regulated at the transcriptional level. Transcription factor nuclear factor‐κB (NF‐κB) binding and activation were not influenced by addition of ATRA. These data imply that RA, in combination with TNF‐α, could up‐regulate the expression of pIgR. In addition, we hypothesize that up‐regulation of pIgR by RA is controlled through the RAR‐dependent signalling pathway and that it plays a role in enhancement of mucosal immunity.

Poly I:C-induced expression of intercellular adhesion molecule-1 in intestinal epithelial cells

Intercellular adhesion molecul‐1 (ICAM‐1) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily of adhesion molecules and plays perdominant roles in recruitment and trafficking of leucocytes to sites of inflammation. ICAM‐1 expression in intestinal epithelial cells (IECs) is enhanced by several stimuli, such as proinflammatory cytokines, bacterial infections or pathogen‐associated molecular patterns. One of these stimuli, double‐stranded RNA (dsRNA), is a by‐product of viral replication and can be recognized by its cognate receptor Toll‐like receptor 3 (TLR‐3). In spite of expression of both TLR‐3 and ICAM‐1 in IECs, correlation between TLR‐3‐signalling and ICAM‐1 expression has never been examined in IECs. In the present study, we investigated whether poly I:C, an analogue of dsRNA, can stimulate the expression of ICAM‐1 in IEC line, HT‐29. Poly I:C‐stimulation up‐regulated the expression of ICAM‐1 mRNA by real‐time polymerase chain reaction. Enhanced expression of ICAM‐1 was confirmed in protein level by immunofluoresense cell staining and enzyme‐linked immunosorbent assay by measuring the released soluble ICAM‐1 in culture supernatant. As the stimulation effect was reduced by pre‐treatment of the cells with anti‐TLR‐3 antibody, poly I:C‐binding signal was thought to be sensed by TLR‐3 on the surface of HT‐29. The results of luciferase assay and nuclear factor kappa‐b (NF‐kB) inhibitor treatment experiments indicated that the downstream signal was mainly transduced by transcription factor, NF‐kB. All these results demonstrated the connection between TLR‐3 signalling and ICAM‐1 expression in HT‐29 cells and indicated the importance of coordinated function of both innate and adaptive immunity against viral infections.

Immune and Endocrine Function in Patients With Burning Mouth Syndrome

Objectives:Research suggests that varied etiologic factors are responsible for burning mouth syndrome (BMS). We examined the role of immune and endocrine function in the pathology of BMS. Methods:We conducted a case-control study to evaluate immune (lymphocyte subpopulations) and endocrine (hypothalamus-pituitary-adrenal axis and sympathetic-adrenomedullary system) function in 47 female BMS patients and 47 age-matched female controls presenting at an university clinic. Psychological state was assessed with the Zung Self-Rating Depression Scale and Taylor Manifest Anxiety Scale. Results:BMS patients were significantly more anxious than controls (P=0.011). Plasma adrenaline level was significantly lower (P=0.020) in BMS patients than in controls, and linear regression analysis of all patients combined revealed that depression level was significantly positively associated with plasma noradrenaline and cortisol levels (P=0.002 and 0.001, respectively). However, as compared with controls, BMS patients had a significantly lower CD8(+) cell count (P<0.001) and a significantly higher CD4/CD8 ratio (P=0.002). Discriminant analysis revealed that CD8(+) cell count and CD4/CD8 ratio were independent variables that distinguished BMS patients from controls. Discussion:The immunoendocrine system is substantially involved, and may have a key role, in the mechanism of chronic pain in BMS patients. Immune function was significantly and specifically suppressed in BMS, although the hypothalamic-pituitary-adrenal axis and sympathetic nervous system were predominantly activated by psychological stress that was not specific to BMS.

Multiple cleavage sites for polymeric immunoglobulin receptor

Human polymeric immunoglobulin receptor (pIgR) was expressed in baby hamster kidney (BHK) cells using a recombinant vaccinia virus transfection system. Cleavage of pIgR on the cell surface was partially inhibited by the proteinase inhibitor, leupeptin. We addressed the question whether some particular regions of pIgR could affect the efficient cleavage of this molecule, with the following results: (1) a mutant lacking the entire cytoplasmic region resulted in release of secretory component (SC) into the culture supernatant much faster than wild‐type; (2) a pIgR mutant lacking the entire extracellular domain 6, the region containing the susceptible cleavage sites, could be cleaved and released as a mutant SC. The transport kinetics of this mutant between endoplasmic reticulum (ER) and Golgi or Golgi and the cell surface was equivalent to wild‐type pIgR. Our results indicate that although the main cleavage site is in domain 6, at least one other cleavage site may exist.

Tumor Markers in Human Renal Cell Carcinoma

Localization of tumor markers in human renal cell carcinomas (RCC) was studied by an immunohistochemical method using 12 different monoclonal antibodies (MAbs) recognizing carbohydrate antigens, and 2 polyclonal antibodies against S-100 protein and neuron-specific enolase (NSE), respectively. 115D8, DF3 and the MAb to epithelial membrane antigen (EMA) reacted with 9 of 13 (115D8), 6 of 13 (DF3) and 5 of 12 (MAb to EMA) cases of RCC, respectively. S-100 protein was also found in 10 of 13 cases of RCC. Further immunohistochemical studies showed that tumor cells of all 13 RCCs were strongly positive for NSE. Serum NSE levels of patients with RCC were examined by radioimmunoassay. This examination revealed that increased levels of NSE were detected in 11 of 17 sera of patients with RCC. Positive rates for patients in stages II, III and IV were 100% (10/10). On the other hand, increased levels of CA15-3 were detected in only 2 of 17 sera by enzyme immunoassay. Our results indicate that NSE may be a useful marker for human RCC, especially for those tumors that have broken through the renal capsule.