DIP/WISH deficiency enhances synaptic function and performance in the Barnes maze.

BACKGROUND: DIP (diaphanous interacting protein)/WISH (WASP interacting SH3 protein) is a protein involved in cytoskeletal signaling which regulates actin cytoskeleton dynamics and/or microtubules mainly through the activity of Rho-related proteins. Although it is well established that: 1) spine-head volumes change dynamically and reflect the strength of the synapse accompanying long-term functional plasticity of glutamatergic synaptic transmission and 2) actin organization is critically involved in spine formation, the involvement of DIP/WISH in these processes is unknown. RESULTS: We found that DIP/WISH-deficient hippocampal CA1 neurons exhibit enhanced long-term potentiation via modulation of both pre- and post-synaptic events. Consistent with these electrophysiological findings, DIP/WISH-deficient mice, particularly at a relatively young age, found the escape hole more rapidly in the Barnes maze test. CONCLUSIONS: We conclude that DIP/WISH deletion improves performance in the Barnes maze test in mice probably through increased hippocampal long-term potentiation.

The TRPV4 channel contributes to intercellular junction formation in keratinocytes.

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca(2+) dynamics. We found that TRPV4 interacts with beta-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca(2+)-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca(2+) increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.

Perception of Cleft Palate Speech by Japanese Listeners-an Assessment of Palatalized Articulations.

To examine how people react to palatalized articulation, we used one cleft palate speech (CPS) sample of palatalized articulation that was purchased in Japan and one recorded sample of speech from a non-cleft palate individual. Study design The two speech samples were rated by 137 native listeners. Each participant rated the set of speech samples for 10 features using a 10-point scale. Alpha factor analysis was performed. Results Two factors were extracted from the entire set of features with alpha factor analysis. Conclusions Although native listeners could not distinguish between CPS and non-CPS using the psychometrical measurements applied in this study, this method of analyzing speech represents a useful technique for planning treatments in cleft disorder patients.

DIP/WISH-deficient mice reveal Dia- and N-WASP-interacting protein as a regulator of cytoskeletal dynamics in embryonic fibroblasts.

DIP/WISH binds to mammalian diaphanous and N-WASP, and functions as a scaffold protein by binding to Nck protein (called SPIN90). In addition, DIP/WISH accelerates actin polymerization through integration with N-WASP and is involved in cytoskeletal dynamics. We previously reported that DIP controls the activities of Rho GTPases in a Src-dependent manner, and accordingly contributes to cell motility (Meng et al. 2004). Here, we made the mice lacking DIP/WISH and demonstrated that DIP/WISH is critical for cell motility and adhesion by using murine embryonic fibroblasts (MEF). Rho activity was higher in DIP/WISH-deficient MEF cells even before platelet-derived growth factor (PDGF) or adhesion stimulation. Cell motility and adhesion were impaired in DIP/WISH-deficient MEF cells, and the MEF cells moved little probably due to the deficiency of tail retractions although they had many small membrane ruffles. Consistent with high Rho activity, DIP/WISH-deficient MEF cells exhibited many stress fibers due to clustering pre-existing actin filament. Thus, DIP/WISH is a negative regulator of Rho and modulates cell adhesion by controlling the integration of adhesion molecules.

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein.

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.

Effects of skin surface temperature on epidermal permeability barrier homeostasis.

Members of the transient receptor potential (TRP) family are temperature sensors, and TRPV1, V3, and V4 are expressed in epidermal keratinocytes. To evaluate the influence of these receptors on epidermal permeability barrier homeostasis, we kept both hairless mouse skin and human skin at various temperatures immediately after tape stripping. At temperatures from 36 to 40 degrees C, barrier recovery was accelerated in both cases compared with the area at 34 degrees C. At 34 or 42 degrees C, barrier recovery was delayed compared with the un-occluded area. 4Alpha-phorbol 12,13-didecanone, an activator of TRPV4, accelerated barrier recovery, whereas ruthenium red, a blocker of TRPV4, delayed barrier recovery. Capsaicin, an activator of TRPV1, delayed barrier recovery, whereas capsazepin, an antagonist of TRPV1, blocked this delay. 2-Aminoethoxydiphenyl borate and camphor, TRPV3 activators, did not affect the barrier recovery rate. As TRPV4 is activated at about 35 degrees C and above, whereas TRPV1 is activated at about 42 degrees C and above, these results suggest that both TRPV1 and TRPV4 play important roles in skin permeability barrier homeostasis. Previous reports suggest the existence of a water flux sensor in the epidermis, and as TRPV4 is known to be activated by osmotic pressure, our results indicate that it might be this sensor.

TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion.

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCepsilon-mediated phosphorylation at S800.

Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.

Structure and function of TRPV1.

Capsaicin, the main ingredient in hot chili peppers, elicits a sensation of burning pain by selectively activating sensory neurons that convey information about noxious stimuli to the central nervous system. The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), is predicted to have six transmembrane (TM) domains and a short, pore-forming hydrophobic stretch between the fifth and sixth TM domains, and is activated not only by capsaicin but also by heat (>43 degrees C), acid and various lipids. Within the TPRV1 protein, many regions and amino acids involved in specific functions (multimerization, capsaicin action, proton action, heat activation, desensitization, permeability, phosphorylation and modulation by lipids) have been identified since the cloning in 1997. Given the fact that TRPV1 is a key molecule in peripheral nociception, these regions and amino acids could prove useful for the development of novel anti-nociceptive or anti-inflammatory agents.

Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins.

Prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2) are major inflammatory mediators that play important roles in pain sensation and hyperalgesia. The role of their receptors (EP and IP, respectively) in inflammation has been well documented, although the EP receptor subtypes involved in this process and the underlying cellular mechanisms remain to be elucidated. The capsaicin receptor TRPV1 is a nonselective cation channel expressed in sensory neurons and activated by various noxious stimuli. TRPV1 has been reported to be critical for inflammatory pain mediated through PKA- and PKC-dependent pathways. PGE2 or PGI2increased or sensitized TRPV1 responses through EP1 or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE2 or PGI2, the temperature threshold for TRPV1 activation was reduced below 35 degrees C, so that temperatures near body temperature are sufficient to activate TRPV1. A PKA-dependent pathway was also involved in the potentiation of TRPV1 through EP4 and IP receptors upon exposure to PGE2 and PGI2, respectively. Both PGE2-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in TRPV1-deficient mice and EP1-deficient mice. IP receptor involvement was also demonstrated using TRPV1-deficient mice and IP-deficient mice. Thus, the potentiation or sensitization of TRPV1 activity through EP1 or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2 or PGI2.

Regulation mechanisms of vanilloid receptors.

The capsaicin receptor TRPV1 (also known as the vanilloid receptor VR1) is a non-selective cation channel and is activated not only by capsaicin but also by noxious heat or protons. Tissue damage associated with infection, inflammation or ischaemia, produces an array of chemical mediators that activate or sensitize nociceptor terminals. An important component of this pro-algeic response is ATP. In cells expressing TRPV1, ATP increased the currents evoked by capsaicin or protons through activation of P2Y metabotropic receptors in a PKC-dependent manner. In the presence of ATP, the temperature threshold for TRPV1 activation was reduced from 42 degrees C to 35 degrees C, such that normal body temperature could activate TRPV1. Functional interaction between P2Y receptors and TRPV1 was confirmed in a behavioural analysis using TRPV1-deficient mice. Direct phosphorylation of TRPV1 by PKC was confirmed biochemically and the two serine residues involved were identified. Extracellular Ca2+ -dependent desensitization of TRPV1 is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. The Ca2+ -binding protein calmodulin binds to the C-terminus of TRPV1. We found that disruption of the calmodulin binding segment prevented TRPV1 desensitization even in the presence of extracellular Ca2+.

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner.

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.

[Molecular mechanisms of nociception].

Capsaicin, the main ingredient in 'hot' chili peppers, elicits burning pain by activating nociceptors. The cloned capsaicin receptor (TRPV1) is a nonselective cation channel with six transmembrane domains, and is activated not only by capsaicin but also by noxious heat (> 43 degrees C) or protons (acidification), both of which cause pain in vivo. Furthermore, analyses of mice lacking VR1 showed that VR1 is essential for selective modalities of pain sensation and for tissue injury-induced thermal hyperalgesia. Tissue damage produces an array of chemical mediators that activate or sensitize nociceptor terminals to elicit pain. Important components of this pro-algesic response are ATP and bradykinin. In cells expressing TRPV1, ATP or bradykinin increased the currents evoked by capsaicin or protons through activation of metabotropic P2Y or B2 bradykinin receptors. In the presence of ATP or bradykinin, the temperature threshold for VR1 activation was reduced from 42 degrees C to 30-35 degrees C, such that normally non-painful normal body temperatures were capable of activating TRPV1, thereby leading to the sensation of pain. Direct phosphorylation of TRPV1 by PKC epsilon was confirmed and the involved two serine residues were determined. This represents a novel mechanism through which ATP or bradykinin in response to tissue trauma might trigger the sensation of pain.

Structural determinant of TRPV1 desensitization interacts with calmodulin.

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca2+-dependent desensitization of TRPV1 observed in patch-clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca2+-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.

The Rho GTPase effector protein, mDia, inhibits the DNA binding ability of the transcription factor Pax6 and changes the pattern of neurite extension in cerebellar granule cells through its binding to Pax6.

mDia, one of the target proteins of the GTPase Rho, is known to be involved in cytoskeletal reorganization and cytokinesis. Here, we report that mDia enters the nucleus and binds to the transcription factor, Pax6. In cultured non-neuronal cells, overexpression of mDia with Pax6 causes redistribution of some Pax6 molecules from the nucleus to the cytosol and decreases Pax6 transcriptional activity. Because Pax6 functions in the early central nervous system morphogenesis, we also examined the effects of mDia on endogenous Pax6 localization and neurite extension in cerebellar granule cells. Here too, Pax6 was partially mislocalized to the cytosol, and its expression level was decreased by mDia overexpression. In addition, mDia overexpression in these cells led to increased neurite branching and length. These results strongly suggest that mDia influences Pax6-induced transcriptional activity and axonal pathfinding in a way opposite from ROCK (Rho kinase) and that it may act via Pax6 to modulate early neuronal development.

Direct phosphorylation of capsaicin receptor VR1 by protein kinase Cepsilon and identification of two target serine residues.

The capsaicin receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that ATP, one of the inflammatory mediators, potentiates the VR1 currents evoked by capsaicin or protons and reduces the temperature threshold for activation of VR1 through metabotropic P2Y(1) receptors in a protein Kinase C (PKC)-dependent pathway, suggesting the phosphorylation of VR1 by PKC. In this study, direct phosphorylation of VR1 upon application of phorbol 12-myristate 13-acetate (PMA) was proven biochemically in cells expressing VR1. An in vitro kinase assay using glutathione S-transferase fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxyl terminus of VR1 were phosphorylated by PKCepsilon. Patch clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites showed that two Ser residues, Ser(502) and Ser(800) were involved in the potentiation of the capsaicin-evoked currents by either PMA or ATP. In the cells expressing S502A/S800A double mutant, the temperature threshold for activation was not reduced upon PMA treatment. The two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.