Sadahiko Masuko

Peripheral and central distribution of TRPV1, substance P and CGRP of rat corneal neurons.

The rat corneal neurons expressing vanilloid receptor TRPV1, substance P (SP) and calcitonin-gene-related peptide (CGRP) were examined. In the cornea, some TRPV1-immunoreactive nerve fibers displayed either SP- or CGRP immunoreactivity also. For observing corneal neuronal elements in the trigeminal ganglion (TG) and in the medulla oblongata, retrograde and anterograde cholera toxin subunit B (CTB) tracing methods combining with triple immunofluorescence technique were performed. The corneal neuronal somata were located in the ophthalmic division of the TG; 37% of them were immunoreactive for TRPV1. One third and three quarters of the corneal TRPV1-immunoreactive neurons co-expressed SP and CGRP, respectively. All of SP-immunoreactive corneal neurons exhibited TRPV1 immunoreactivity. They were predominantly medium-sized (mean +/- SE = 638.2 +/- 49.5 microm(2)) and significantly larger than SP-immunoreactive and TRPV1-immunonegative neurons in the ophthalmic division of the TG. The central projection fibers of corneal neurons co-expressing TRPV1 with SP and CGRP were observed at the subnucleus interpolaris/caudalis transition within trigeminal nucleus. The present study suggests that TRPV1 of the corneal neurons works in close relation to SP and CGRP both in the cornea and CNS for healing and nociceptive transduction.

T helper type 2 differentiation and intracellular trafficking of the interleukin 4 receptor-|[alpha]| subunit controlled by the Rac activator Dock2

The lineage commitment of CD4+ T cells is coordinately regulated by signals through the T cell receptor and cytokine receptors, yet how these signals are integrated remains elusive. Here we find that mice lacking Dock2, a Rac activator in lymphocytes, developed allergic disease through a mechanism dependent on CD4+ T cells and the interleukin 4 receptor (IL-4R). Dock2-deficient CD4+ T cells showed impaired antigen-driven downregulation of IL-4Rα surface expression, resulting in sustained IL-4R signaling and excessive T helper type 2 responses. Dock2 was required for T cell receptor–mediated phosphorylation of the microtubule-destabilizing protein stathmin and for lysosomal trafficking and the degradation of IL-4Rα. Thus, Dock2 links T cell receptor signals to downregulation of IL-4Rα to control the lineage commitment of CD4+ T cells.

DOCK180 Is a Rac Activator That Regulates Cardiovascular Development by Acting Downstream of CXCR4

Rationale: During embryogenesis, the CXC chemokine ligand (CXCL)12 acts on endothelial cells to control cardiac development and angiogenesis. Although biological functions of CXCL12 are exerted in part through activation of the small GTPase Rac, the pathway leading from its receptor CXC chemokine receptor (CXCR)4 to Rac activation remains to be determined. Objective: DOCK180 (dedicator of cytokinesis), an atypical Rac activator, has been implicated in various cellular functions. Here, we examined the role of DOCK180 in cardiovascular development. Methods and Results: DOCK180 associates with ELMO (engulfment and cell motility) through the N-terminal region containing a Src homology 3 domain. We found that targeted deletion of the Src homology 3 domain of DOCK180 in mice leads to embryonic lethality with marked reduction of DOCK180 expression at the protein level. These mutant mice, as well as DOCK180-deficient mice, exhibited multiple cardiovascular abnormalities resembling those seen in CXCR4-deficient mice. In DOCK180 knocked down endothelial cells, CXCL12-induced Rac activation was impaired, resulting in a marked reduction of cell motility. Conclusions: These results suggest that DOCK180 links CXCR4 signaling to Rac activation to control endothelial cell migration during cardiovascular development.

The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca2+‐permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT‐PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild‐type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up‐regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat‐induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.—Aijima, R., Wang, B., Takao, T., Mihara, H., Kashio, M., Ohsaki, Y., Zhang, J.‐Q., Mizuno, A., Suzuki, M., Yamashita, Y., Masuko, S., Goto, M., Tominaga, M., Kido, M. A., The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia. FASEB J. 29, 182–192 (2015). www.fasebj.org

Organ-specific autoimmunity in mice whose T cell repertoire is shaped by a single antigenic peptide

Organ-specific autoimmune diseases have been postulated to be the result of T cell response against organ-specific self-peptides bound to MHC molecules. Contrary to this paradigm, we report here that transgenic mice lacking MHC class I expression and expressing an MHC class II I-A(b) molecule that presents only a single peptide (E alpha 52-68) spontaneously develops peripheral nervous system-specific autoimmune disease with many of the histopathological features found in experimental allergic neuritis. Reciprocal bone marrow chimeras produced using susceptible and resistant lines revealed that bone marrow-derived cells determined disease susceptibility. While the expression of the I-A(b)-E alpha 52-68 complex in the periphery was readily detectable in both lines, its expression on thymic dendritic cells responsible for tolerance induction was markedly lower in the susceptible line than in the resistant line. Consistent with this, CD4(+) T cells that can be activated by the I-A(b)-E alpha 52-68 complex were found in the susceptible line, but not in the resistant line. Such CD4(+) T cells conferred the disease to the resistant line by adoptive transfer, and administration of Ab specific for the I-A(b)-E alpha 52-68 complex inhibited disease manifestation in the susceptible line. These results indicate that disease development involves systemic T cell reactivity to I-A(b)-E alpha 52-68 complex, probably caused by incomplete negative thymocyte selection.

The localization of oxytocin receptors in the islets of Langerhans in the rat pancreas.

In this study, oxytocin receptors (OTRs) in the islets of Langerhans were detected using real-time RT-PCR and immunohistochemical technique. Indeed, OTR mRNA was expressed in the rat pancreas. Double immunohistochemical staining for OTR and either glucagon or insulin demonstrated their co-localization in A-cells or B-cells, respectively. OTR-immunoreactivity in A-cells was stronger than that of B-cells. All A-cells and 94.8% of B-cells were OTR-immunoreactive. We reveal the statistically significant relations of OTR with A-cells and B-cells in the islets of Langerhans. This is the first demonstration of the OTR localization in the islets of Langerhans immunohistochemically. It suggests that oxytocin (OT) is involved in the release of insulin and glucagon.

TRPV2 expression in rat oral mucosa

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Collateral projections from the subfornical organ to the median preoptic nucleus and paraventricular hypothalamic nucleus in the rat.

It is morphologically demonstrated that the subfornical organ (SFO) projects to the paraventricular hypothalamic nucleus (PVN) and also projects to the nucleus preopticus medianus (POMe), a relay nucleus of indirect projections from the SFO to PVN. However, it remains unknown, whether or not SFO neurons project collaterally to the POMe and PVN. To confirm this, a double retrograde labeling method was performed on rats using two fluorescent tracers. One tracer (red-colored FluoSpheres: FSR) was injected into the POMe and the other (Fast Blue: FB) was injected into the unilateral PVN at the same time. As a result, many retrogradely labeled neurons were found in the entire SFO. Of these, some neurons showed both FSR and FB fluorescence. Double-labeled neurons were found in about 8.7% of FSR-labeled neurons and 15.5% of FB-labeled neurons. The existence of double-labeled neurons indicates that single neurons in the SFO project simultaneously to the POMe and PVN via collateral axon branches. The data suggest that there are complicated neuronal pathways originating from the SFO in regulating cardiovascular and body fluid homeostasis.

Developmental expression of oxytocin receptors in the neonatal medulla oblongata and pons

The distribution of oxytocin receptors (OTRs) in the postnatal brain stem of rats was examined by immunohistochemistry. Fibrous or rounded shaped OTR-immunoreactive structures were distributed densely in sensory nuclei, such as the gracile nucleus, the solitary nucleus, and the spinal trigeminal nucleus, evenly in the reticular formation of the medulla oblongata and pons, and moderately in the locus coeruleus during the neonatal period, but disappeared by postnatal day 10. Few OTR-immunoreactive structures were distributed in motor nuclei. Many rounded OTR-immunoreactive structures were discovered layered and partially overlapping with GM-130-immunoreacivity in the neuronal Golgi apparatus, which was confirmed by electron microscopy. The present study suggests that a transient type of OTR may be functioning in neuronal development during the neonatal period.

Populations of lamina II interneurons possessing C-fiber inputs in the spinal dorsal horn of adult rats which received neonatal capsaicin treatments

The development of pain after peripheral nerve and tissue injury involves not only neuronal pathways but also glia. However, uncertainty still remains as to the relative contribution of different types of glial cells in the development of the pain-related enhanced response states. We examined the contribution of glial cells to the central sensitization in the rat spinal dorsal horn which is induced in neuropathic pain and inflammatory pain. In rats subjected to neuropathic pain, the immunoreactivity (IR) of microglial marker OX 42 was largely increased. In rats subjected to inflammatory pain, IR of astosytes marker GFAP was slightly increased. The optically-recorded neuronal excitation induced by single-pulse stimulation to the dorsal root was augmented in rats subjected to neuropathic pain and rats subjected to inflammatory pain by comparison to control rats. The bath application of a microglial inhibitor minocycline and a p38 mitogen-activated protein kinase inhibitor SB203580 inhibited the neuronal excitation in rats subjected to neuropathic pain, but not in control and rats subjected to inflammatory pain. PPADS slightly inhibited the neuronal excitation in all group. The additional perfusion of TNP-ATP in PPADS largely inhibited the neuronal excitation in rats subjected to neuropathic pain. In contrast, an astroglial toxin L-alpha-aminoadipate and a gap junction blocker carbenoxolone inhibited the neuronal excitation in rats subjected to inflammatory pain, but not in control and rats subjected to neuropathic pain. The larger number of cells in the spinal cord slice taken from rats subjected to neuropathic pain showed the Ca2+ signal by puff application of ATP to comparison with control and rats subjected to inflammatory pain. The Ca2+ signal was inhibited by minocycline and TNP-ATP. Research fund: KAKENHI22600005.