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 Ca(2+)-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.

In vitro assembly properties of human type I and II hair keratins.

Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two-dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro intermediate filament (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

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.

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.

The mouse Murr1 gene is imprinted in the adult brain, presumably due to transcriptional interference by the antisense-oriented U2af1-rs1 gene.

The mouse Murr1 gene contains an imprinted gene, U2af1-rs1, in its first intron. U2af1-rs1 shows paternal allele-specific expression and is transcribed in the direction opposite to that of the Murr1 gene. In contrast to a previous report of biallelic expression of Murr1 in neonatal mice, we have found that the maternal allele is expressed predominantly in the adult brain and also preferentially in other adult tissues. This maternal-predominant expression is not observed in embryonic and neonatal brains. In situ hybridization experiments that used the adult brain indicated that Murr1 gene was maternally expressed in neuronal cells in all regions of the brain. We analyzed the developmental change in the expression levels of both Murr1 and U2af1-rs1 in the brain and liver, and we propose that the maternal-predominant expression of Murr1 results from transcriptional interference of the gene by U2af1-rs1 through the Murr1 promoter region.

Peptidergic and catecholaminergic synaptic contacts onto nucleus preopticus medianus neurons projecting to the subfornical organ in the rat.

The nucleus preopticus medianus (POMe) is known to be a key site in regulation of cardiovascular and body fluid homeostasis. To clarify the regulation mechanism to the POMe, the innervation pattern of synapses made by axon terminals immunoreactive to beta-endorphin, neuropeptide Y and tyrosine hydroxylase onto POMe neurons projecting to the subfornical organ (SFO) was investigated in the rat. After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the SFO, many neurons were retrogradely labeled in the POMe, more frequently in its dorsal part. Electron microscopy of the POMe revealed that beta-endorphin- and tyrosine hydroxylase-immunoreactive axon terminals formed predominantly axo-somatic synapses, and neuropeptide Y-immunoreactive axon terminals formed more axo-dendritic than axo-somatic synapses with retrogradely labeled neurons. The present localization patterns of POMe neurons retrogradely labeled from the SFO and the type of synapses of axon terminals immunoreactive to three neurochemical markers on these neurons were compared to those of POMe neurons retrogradely labeled from the paraventricular hypothalamic nucleus demonstrated in our previous report.

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.

Changes in Surface Morphology of Myogenic Cells during the Cell Cycle, Fusion and Myotube Formation: (scanning electron microscopy/cell cycle/myogenic cell fusion/myotube/acetylcholine receptor).

The surface morphology of chick myogenic cells during development in cell culture was examined by scanning electron microscopy. Myoblasts at the G1 and S phases of the cell cycle had a relatively smooth surface. In late G2 and mitosis, they had many microvilli and some blebs on their surfaces. Ca2+ -deficient fusion-arrested myoblasts had a relatively smooth surface. When the cells underwent cell fusion, many microvilli, small spherical protrusions, and some blebs appeared on their surface. In newly formed myotubes, the surface over the nucleus was smooth whereas that over perinuclear regions had many flat excrescences and other surface protrusions. This mosaic appearance of the surface was less prominent in striated myotubes. Scanning electron microscopy combined with fluorescence microscopy using rhodamine-labeled erabutoxin b revealed that sites of accumulation of acetylcholine receptor had a smooth surface. These results suggest that changes in surface structure occur in association with the cell cycle, fusion and subsequent development of myotubes.

Developing patterns of nitric oxide synthesizing neurons in the rat striatum: histochemical analysis.

The prenatal and postnatal development of NADPH-diaphorase (NADPH-d)/neuronal nitric oxide synthase (nNOS) positive neurons was studied in the striatum of rats. NADPH-d was demonstrated enzyme histochemically and nNOS immunohistochemically using a polyclonal antibody. NADPH-d neurons appeared in the ventrolateral part of the striatum on embryonic day 18 (E18). Thereafter, the number of NADPH-d neurons increased and began to distribute homogeneously in the striatum. The density of NADPH-d neurons became highest at postnatal day 5 (P5) and then decreased as the volume of the striatum continued to increase. The number of NADPH-d neurons reached its peak around 3-4 weeks after birth. The sizes of NADPH-d neurons were measured. The NADPH-d neurons grew larger until P14 (mean area 260 microm(2)) and became smaller thereafter (mean area 170 microm(2)). Patches of high NADPH-d activity and tyrosine hydroxylase (TH) immunoreactivity were also examined in the developing striatum. The distributions of NADPH-d patches overlapped with those of TH-immunoreactive patches by P10. The spatiotemporal appearance of nNOS and overlapping of nNOS patchy distribution with TH point to an important role of NO and to an interaction between nNOS and DA fibers during development of the striatum.

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.

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.

Cell-type-specific excitatory and inhibitory circuits involving primary afferents in the substantia gelatinosa of the rat spinal dorsal horn in vitro.

The substantia gelatinosa (SG) of the spinal dorsal horn shows significant morphological heterogeneity and receives primary afferent input predominantly from A delta- and C-fibres. Despite numerous anatomical and physiological studies, correlation between morphology and functional connectivity, particularly in terms of inhibitory inputs, remains elusive. To compare excitatory and inhibitory synaptic inputs on individual SG neurones with morphology, we performed whole-cell recordings with Neurobiotin-filled-pipettes in horizontal slices from adult rat spinal cord with attached dorsal roots. Based on dendritic arborization patterns, four major cell types were confirmed: islet, central, radial and vertical cells. Dorsal root stimulation revealed that each class was associated with characteristic synaptic inputs. Islet and central cells had monosynaptic excitatory inputs exclusively from C-afferents. Islet cells received primary-afferent-evoked inhibitory inputs only from A delta-fibres, while those of central cells were mediated by both A delta- and C-fibres. In contrast, radial and vertical cells had monosynaptic excitatory inputs from both A delta- and C-fibres and inhibitory inputs mediated by both fibre types. We further characterized the neurochemical nature of these inhibitory synaptic inputs. The majority of islet, central and vertical cells exhibited GABAergic inhibitory inputs, while almost all radial cells also possessed glycinergic inputs. The present study demonstrates that SG neurones have distinct patterns of excitatory and inhibitory inputs that are related to their morphology. The neurotransmitters responsible for inhibitory inputs to individual SG neurones are also characteristic for different morphological classes. These results make it possible to identify primary afferent circuits associated with particular types of SG neurone.

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-4Ralpha 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-4Ralpha. Thus, Dock2 links T cell receptor signals to downregulation of IL-4Ralpha to control the lineage commitment of CD4+ T cells.

Cystatin C stimulates the differentiation of mouse osteoblastic cells and bone formation.

Cystatin C (CysC) is a natural cysteine proteinase inhibitor that suppresses the differentiation and bone-resorptive function of osteoclasts. By contrast, the effect of CysC on the differentiation and bone-formative function of osteoblasts has not been elucidated thoroughly. We examined the effects of CysC on mouse osteoblastic cells using in vitro cultures from bone marrow and calvaria and ex vivo calvarial cultures. CysC-stimulated cells showed increased alkaline phosphatase (ALP) activity, mineralization of the new bone matrix, and calvarial bone formation. The cells treated with CysC immunodepleted by anti-CysC antibody (iCysC) and a chemical papain-like cysteine proteinase inhibitor, E-64, did not induce mineralization. Elevated mRNA levels of bone morphogenetic protein (BMP)-2, the differentiation marker osteocalcin, and a master osteogenic transcription factor, Runx2, were observed in CysC-treated cells. These results suggest that CysC affects the BMP signaling cascades in osteoblastic cells and then promotes osteoblast differentiation, mineralization, and bone formation.

Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor.

Clearance of apoptotic cells by macrophages is considered important for prevention of inflammatory responses leading to tissue damage. The phosphatidylserine receptor (PSR), which specifically binds to phosphatidylserine (PS) exposed on the surface of apoptotic cells, mediates uptake of apoptotic cells in vitro, yet the physiologic relevance of PSR remains unknown. This issue was addressed by generating PSR-deficient (PSR(-/-)) mice. PSR(-/-) mice exhibited severe anemia and died during the perinatal period. In the PSR(-/-) fetal livers, erythroid differentiation was blocked at an early erythroblast stage. In addition, PSR(-/-) embryos exhibited thymus atrophy owing to a developmental defect of T-lymphoid cells. Clearance of apoptotic cells by macrophages was impaired in both liver and thymus of PSR(-/-) embryos. However, this did not induce up-regulation of inflammatory cytokines. These results indicate that during embryonic development, PSR-mediated apoptotic cell uptake is required for definitive erythropoiesis and T lymphopoiesis, independently of the prevention of inflammatory responses.

Characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse.

Human 11p15.5, as well as its orthologous mouse 7F4/F5, is known as the imprinting domain extending from IPL/Ipl to H19. OBPH1 and Obph1 are located beyond the presumed imprinting boundary on the IPL/Ipl side. We determined full-length cDNAs and complete genomic structures of both orthologues. We also investigated their precise imprinting and methylation status. The orthologues resembled each other in genomic structure and in the position of the 5' CpG island and were expressed ubiquitously. OBPH1 and Obph1 were predominantly expressed from the maternal allele only in placenta, with hypo- and not differentially methylated 5' CpG islands in both species. These results suggested that the imprinting domain would extend beyond the presumed imprinting boundary and that methylation of the 5' CpG island was not associated with the imprinting status in either species. It remains to be elucidated whether the gene is under the control of the KIP2/LIT1 subdomain or is regulated by a specific mechanism. Analysis of the precise genomic sequence around the region should help resolve this question.