Naoto Ohkura

Gene Expression Analysis of Membrane Transport Proteins in Normal and Lipopolysaccharide-inflamed Rat Dental Pulp

INTRODUCTION Membrane transport proteins (transporters) play a crucial role in the transmembrane uptake and/or efflux of various compounds such as inorganic ions, endogenous bioactive substances such as prostaglandins (PGs), and drugs such as nonsteroidal anti-inflammatory drugs. This study aimed to analyze mRNA expression of selected transporters related to drug disposition and PG transport in normal and lipopolysaccharide (LPS)-inflamed rat incisor pulp. METHODS Pulp tissues were subjected to reverse transcription-polymerase chain reaction (PCR) detection for transporter isoforms belonging to organic anion transporting polypeptide (Oatp), organic anion transporter (Oat), organic cation transporter (Oct), multidrug resistance-associated protein (Mrp), and multidrug resistance protein (Mdr) families. The levels of mRNA expression for PG transporters (Oatp1a5, Oatp1b2, Oatp2a1, Oatp2b1, and Oatp3a1) were compared in normal and LPS-inflamed pulps by using real-time PCR. RESULTS The pulp tissue expressed mRNAs for various transporters belonging to the Oatp, Oat, Oct, Mrp, and Mdr families. LPS inflammation caused significant up-regulation of Oatp2a1 (P < .01) and significant down-regulation of Oatp1a5, Oatp2b1 (P < .01), and Oatp3a1 (P < .05). CONCLUSIONS Rat incisor dental pulp expressed mRNAs for various transporter isoforms. The levels of mRNA expression for PG transporters were significantly up-regulated or down-regulated in LPS-inflamed dental pulp.

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-β1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-β signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-β1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.

Prostaglandin Transporting Protein-mediated Prostaglandin E2 Transport in Lipopolysaccharide-inflamed Rat Dental Pulp

INTRODUCTION The prostaglandin transporter (Pgt) and multidrug resistance-associated protein (Mrp) 4 are membrane transport proteins that play crucial roles in the transmembrane uptake and/or efflux of prostaglandins (PGs). This study attempted to analyze the protein expression of Pgt and Mrp4 and their involvement in PGE2 efflux transport in lipopolysaccharide (LPS)-inflamed rat incisor pulp tissue. METHODS Pulpitis was induced in the upper incisors of Wistar rats by treating them with LPS for 24 hours. The protein expression levels of Pgt, Mrp4, and microsomal PGE synthase (mPGES) were analyzed with immunofluorescent staining. The amount of PGE2 released from the inflamed pulp tissue in the presence or absence of dipyridamole (an Mrp4 inhibitor) was assessed by using an enzyme-linked immunosorbent assay. RESULTS Double immunofluorescence staining revealed that the Pgt, Mrp4, and mPGES immunoreactivity co-localized in CD31-expressing endothelial cells. Moreover, the Mrp4 inhibitor caused a significant decrease in the amount of PGE2 released from the LPS-inflamed pulp (P < .01 at 24 hours). CONCLUSION Pgt, Mrp4, and mPGES expression was detected in the endothelial cells of normal and LPS-inflamed rat incisor pulp tissue, suggesting that these cells are associated with the biosynthesis and transmembrane transport of PGE2. The significant decrease in PGE2 release induced by the Mrp4 inhibitor suggests that Mrp4 contributes to the transport of PGE2 in the transmembrane efflux pathway.

Effects of pulpotomy using mineral trioxide aggregate on prostaglandin transporter and receptors in rat molars

Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

Characterization of Dental Pulp Myofibroblasts in Rat Molars after Pulpotomy

Introduction: Myofibroblasts express alpha smooth muscle actin (&agr;‐SMA) and play a critical role in wound healing. Myofibroblast differentiation is controlled by the joint actions of transforming growth factor beta 1 (TGF‐&bgr;1) and the extradomain A fibronectin splice variant (EDA‐FN). Currently, the contribution of myofibroblasts to dental pulp healing is unknown. Therefore, we analyzed expressional characteristics of &agr;‐SMA–positive cells and investigated TGF‐&bgr;1, EDA‐FN, and &agr;‐SMA expression levels after pulpotomy to better understand dental pulp healing. Methods: The maxillary first molars of 8‐week‐old Wistar rats were pulpotomized with mineral trioxide aggregate. After 1 to 14 days, localization and colocalization of &agr;‐SMA, rat endothelial cell antigen‐1 (as a marker of endothelial cells), neuron‐glial antigen 2 (as a marker of perivascular cells), prolyl‐4‐hydroxylase (P4H, as an additional marker of myofibroblasts), and EDA‐FN were analyzed using immunohistochemistry and double immunofluorescence. Time‐course changes in the messenger RNA expression levels of TGF‐&bgr;1, EDA‐FN, and &agr;‐SMA were evaluated using quantitative real‐time polymerase chain reaction analysis. Results: Spindle‐shaped &agr;‐SMA–positive cells transiently appeared after pulpotomy. These cells initially emerged in the pulp core on day 3 and then accumulated at the wound site by day 5. These cells were isolated from rat endothelial cell antigen‐1 positive cells and did not express neuron‐glial antigen 2 but did express P4H. The messenger RNA levels of TGF‐&bgr;1, EDA‐FN, and &agr;‐SMA were significantly up‐regulated after pulpotomy. EDA‐FN and &agr;‐SMA were colocalized at the wound sites on day 5. Conclusions: In association with up‐regulation of TGF‐&bgr;1 and EDA‐FN expression, &agr;‐SMA and P4H double‐positive cells accumulated at the wound sites after pulpotomy. This suggests that myofibroblasts participate in dental pulp healing. HighlightsDistributional alterations and expressional characteristics of alpha smooth muscle actin (&agr;‐SMA)‐positive cells were analyzed after pulpotomy.Spindle‐shaped &agr;‐SMA–positive cells emerged in the pulp core at 3 days and accumulated at the wound sites at 5 days.These cells were isolated from blood vessels and coexpressed collagen maturation enzymes.The spindle‐shaped &agr;‐SMA–positive cells had myofibroblastic features and may be involved in the healing of injured dental pulp.