RESUMEN
Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.
Asunto(s)
Neuronas , Protocadherinas , Neuritas , Cuerpo Celular , Comunicación CelularRESUMEN
Sweat plays a critical role in human body, including thermoregulation and the maintenance of the skin environment and health. Hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion, resulting in severe skin conditions (pruritus and erythema). Bioactive peptide and pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and identified to activate adenylate cyclase in pituitary cells. Recently, it was reported that PACAP increases sweat secretion via PAC1R in mice and promotes the translocation of AQP5 to the cell membrane through increasing intracellular [Ca2+] via PAC1R in NCL-SG3 cells. However, intracellular signaling mechanisms by PACAP are poorly clarified. Here, we used PAC1R knockout (KO) mice and wild-type (WT) mice to observe changes in AQP5 localization and gene expression in sweat glands by PACAP treatment. Immunohistochemistry revealed that PACAP promoted the translocation of AQP5 to the lumen side in the eccrine gland via PAC1R. Furthermore, PACAP up-regulated the expression of genes (Ptgs2, Kcnn2, Cacna1s) involved in sweat secretion in WT mice. Moreover, PACAP treatment was found to down-regulate the Chrna1 gene expression in PAC1R KO mice. These genes were found to be involved in multiple pathways related to sweating. Our data provide a solid basis for future research initiatives in order to develop new therapies to treat sweating disorders.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Sudor , Ratones , Humanos , Animales , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Sudor/metabolismo , Sudoración , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Hipófisis/metabolismoRESUMEN
Dry eye disease (DED) is caused by a reduction in the volume or quality of tears. The prevalence of DED is estimated to be 100 million in the developed world. As aging is a risk factor for DED, the prevalence of DED is expected to grow at a rapid pace in aging populations, thus creating an increased need for new therapies. This review summarizes DED medications currently in clinical use. Most current medications for DED focus on stimulating tear secretion, mucin secretion, or suppressing inflammation, rather than simply replenishing the ocular surface with moisture to improve symptoms. We recently reported that the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) induces tear secretion and suppresses corneal injury caused by a reduction in tears. Moreover, it has been reported that a PACAP in water and a 0.9% saline solution at +4 °C showed high stability and achieved 80-90% effectiveness after 2 weeks of treatment. These results reveal PACAP as a candidate DED medication. Further research on the clinical applications of PACAP in DED is necessary.
Asunto(s)
Síndromes de Ojo Seco/tratamiento farmacológico , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/uso terapéutico , Animales , Síndromes de Ojo Seco/patología , Humanos , Modelos Biológicos , Soluciones Oftálmicas/farmacología , Soluciones Oftálmicas/uso terapéutico , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Transducción de Señal/efectos de los fármacos , Lágrimas/efectos de los fármacosRESUMEN
The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line-PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3ß (GSK-3ß) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3ß as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3ß inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10-7 M, 10-8 M, and 10-9 M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10-7 M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3ß-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proyección Neuronal/efectos de los fármacos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas del Tejido Nervioso/genética , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Células PC12 , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1 receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1 receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1 receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.
Asunto(s)
Asma/metabolismo , Bronquios/metabolismo , Músculo Liso/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Tensoactivos/metabolismo , Animales , Hiperreactividad Bronquial/metabolismo , Ratones , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Hipersensibilidad Respiratoria/metabolismo , Péptido Intestinal Vasoactivo/metabolismoRESUMEN
Prostaglandin D2 (PGD2) is one of the key lipid mediators of allergic airway inflammation, including bronchial asthma. However, the role of PGD2 in the pathogenesis of asthma is not fully understood. In the present study, the effect of PGD2 on smooth muscle contractility of the airways was determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In isolated bronchial smooth muscles (BSMs) of naive mice, application of PGD2 (10-9â»10-5 M) had no effect on the baseline tension. However, when the tissues were precontracted partially with 30 mM K⺠(in the presence of 10-6 M atropine), PGD2 markedly augmented the contraction induced by the high K⺠depolarization. The PGD2-induced augmentation of contraction was significantly inhibited both by 10-6 M laropiprant (a selective DP1 antagonist) and 10-7 M Y-27632 (a Rho-kinase inhibitor), indicating that a DP1 receptor-mediated activation of Rho-kinase is involved in the PGD2-induced BSM hyperresponsiveness. Indeed, the GTP-RhoA pull-down assay revealed an increase in active form of RhoA in the PGD2-treated mouse BSMs. On the other hand, in the high Kâº-depolarized cultured human BSM cells, PGD2 caused no further increase in cytosolic Ca2+ concentration. These findings suggest that PGD2 causes RhoA/Rho-kinase-mediated Ca2+ sensitization of BSM contraction to augment its contractility. Increased PGD2 level in the airways might be a cause of the AHR in asthma.
Asunto(s)
Bronquios/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Prostaglandina D2/farmacología , Animales , Atropina/farmacología , Hiperreactividad Bronquial/metabolismo , Humanos , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Potasio/farmacología , Cultivo Primario de Células , Receptores de Prostaglandina/efectos de los fármacosRESUMEN
Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. PACAP shows particularly high homology (~ 68%) to VIP. Because of the high homology of the amino acid sequences of PACAP and VIP, these peptides share three class B-G-protein coupled receptors: the PAC1-Receptor (PAC1-R), the VPAC1-Receptor (VPAC1-R) and VPAC2-Receptor (VPAC2-R). These receptors have high homology to each other, and their high homology is utilized for these discoveries. This review provides mainly an overview of the history of the discovery of PACAP and its three receptors.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Animales , HumanosRESUMEN
BACKGROUND: The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections. RESULTS: We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity. CONCLUSIONS: Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.
Asunto(s)
Cadherinas/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Cadherinas/genética , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Electrofisiología , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vías Nerviosas/fisiología , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , ADN Metiltransferasa 3BRESUMEN
Clustered protocadherins (cPcdhs) comprising cPcdh-α, -ß, and -γ, encode a large family of cadherin-like cell-adhesion molecules specific to neurons. Impairment of cPcdh-α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh-α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh-α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh-α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh-α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.
Asunto(s)
Cadherinas/metabolismo , Cuerpos Geniculados/patología , Terminales Presinápticos/patología , Retina/patología , Trastornos de la Visión/metabolismo , Trastornos de la Visión/patología , Agudeza Visual , Animales , Cadherinas/genética , Calcio/metabolismo , Ratones , Ratones Noqueados , Trastornos de la Visión/fisiopatología , Corteza Visual/patologíaRESUMEN
Diverse protocadherin-alpha genes (Pcdha, also called cadherin-related neuronal receptor or CNR) are expressed in the vertebrate brain. Their genomic organization involves multiple variable exons and a set of constant exons, similar to the immunoglobulin (Ig) and T-cell receptor (TCR) genes. This diversity can be used to distinguish neurons. Using polymorphisms that distinguish the C57BL/6 and MSM mouse strains, we analyzed the allelic expression of the Pcdha gene cluster in individual neurons. Single-cell analysis of Purkinje cells using multiple RT-PCR reactions showed the monoallelic and combinatorial expression of each variable exon in the Pcdha genes. This report is the first description to our knowledge of the allelic expression of a diversified receptor family in the central nervous system. The allelic and combinatorial expression of distinct variable exons of the Pcdha genes is a potential mechanism for specifying neuron identity in the brain.
Asunto(s)
Cadherinas/genética , Variación Genética , Neuronas/metabolismo , Animales , Exones , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Células de Purkinje/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
[This corrects the article DOI: 10.1016/j.isci.2022.105766.].
RESUMEN
Galanin-like peptide (GALP) is a neuropeptide that was first isolated and identified from the porcine hypothalamus. Studies have described an anti-obesity effect of GALP. We previously found that intracerebroventricular administration of GALP in mice resulted in an increase in respiratory exchange rate 12 to 16 h later. GALP may also affect glucose metabolism, but the detailed mechanism has not been elucidated. In this study, we investigated the effects of GALP on glucose and lipid metabolism in the liver. Nine-week-old male C57BL / 6 J mice were administered a single intracerebroventricular dose of saline or GALP and dissected 16 h later. There were no significant between-group differences in body weight and blood glucose levels. With regard to gene and protein expression, G6Pase associated with hepatic gluconeogenesis was significantly reduced in the GALP group. In addition, the hepatokines selenoprotein P and fetuin-A, which induce insulin resistance in the liver, were significantly decreased in the GALP group. These results suggest that intracerebroventricular administration of GALP decreases the expression of key hepatokines, thereby enhancing glucose metabolism.
Asunto(s)
Péptido Similar a Galanina , Masculino , Animales , Ratones , Porcinos , Ratones Endogámicos C57BL , Péptido Similar a Galanina/farmacología , Hígado , Peso Corporal , GlucosaRESUMEN
Clustered protocadherin is a family of cell-surface recognition molecules implicated in neuronal connectivity that has a diverse isoform repertoire and homophilic binding specificity. Mice have 58 isoforms, encoded by Pcdhα, ß, and γ gene clusters, and mutant mice lacking all isoforms died after birth, displaying massive neuronal apoptosis and synapse loss. The current hypothesis is that the three specific γC-type isoforms, especially γC4, are essential for the phenotype, raising the question about the necessity of isoform diversity. We generated TC mutant mice that expressed the three γC-type isoforms but lacked all the other 55 isoforms. The TC mutants died immediately after birth, showing massive neuronal death, and γC3 or γC4 expression did not prevent apoptosis. Restoring the α- and ß-clusters with the three γC alleles rescued the phenotype, suggesting that along with the three γC-type isoforms, other isoforms are also required for the survival of neurons and individual mice.
RESUMEN
Functional neural circuits in the cerebral cortex are established through specific neural connections between excitatory and various inhibitory cell types. However, the molecular mechanisms underlying synaptic partner recognition remain unclear. In this study, we examined the impact of clustered protocadherin-γ (cPcdhγ) gene deletion in parvalbumin-positive (PV+) cells on intralaminar and translaminar neural circuits formed between PV+ and pyramidal (Pyr) cells in the primary visual cortex (V1) of male and female mice. First, we used whole-cell recordings and laser-scan photostimulation with caged glutamate to map excitatory inputs from layer 2/3 to layer 6. We found that cPcdhγ-deficient PV+ cells in layer 2/3 received normal translaminar inputs from Pyr cells through layers 2/3-6. Second, to further elucidate the effect on PV+-Pyr microcircuits within intralaminar layer 2/3, we conducted multiple whole-cell recordings. While the overall connection probability of PV+-Pyr cells remained largely unchanged, the connectivity of PV+-Pyr was significantly different between control and PV+-specific cPcdhγ-conditional knock-out (PV-cKO) mice. In control mice, the number of reciprocally connected PV+ cells was significantly higher than PV+ cells connected one way to Pyr cells, a difference that was not significant in PV-cKO mice. Interestingly, the proportion of highly reciprocally connected PV+ cells to Pyr cells with large unitary IPSC (uIPSC) amplitudes was reduced in PV-cKO mice. Conversely, the proportion of middle reciprocally connected PV+ cells to Pyr cells with large uIPSC amplitudes increased compared with control mice. This study demonstrated that cPcdhγ in PV+ cells modulates their reciprocity with Pyr cells in the cortex.
Asunto(s)
Parvalbúminas , Protocadherinas , Ratones , Femenino , Masculino , Animales , Parvalbúminas/metabolismo , Potenciales Postsinápticos Inhibidores , Células Piramidales/fisiología , Corteza Cerebral/metabolismo , Interneuronas/metabolismoRESUMEN
Many studies have investigated the beneficial effects of n-3 polyunsaturated fatty acids, such as their potential for lowering lipid levels and reducing diabetes risk. However, few studies have specifically examined docosapentaenoic acid (DPA), an n-3 polyunsaturated fatty acid with limited availability in its pure form. We hypothesized that DPA would have lipid-lowering effects and improve insulin resistance in KK/Ta mice. To test our hypothesis, 7-week-old KK/Ta mice were fed a high-fat diet for 12 weeks to induce obesity before being divided into 3 groups and fed an experimental diet for 10 weeks. The experimental diets were: LSO, using lard and safflower oil as fat sources; SO, in which lard in the LSO diet was replaced with safflower oil; and DPA, in which lard in the LSO diet was replaced with DPA oil. After 10 weeks, plasma triglyceride and total cholesterol concentrations were significantly decreased in the DPA group, but not in the SO group. Sterol regulatory element-binding protein-1 and stearoyl-CoA desaturase-1 gene expressions involved in fatty acid synthesis in the liver were significantly lower in the DPA group compared with the LSO group. Plasma glucose concentrations were significantly decreased in both the SO group and the DPA group compared with the LSO group, whereas plasma insulin concentrations were significantly decreased in the DPA group alone. These results indicate that DPA has plasma lipid-lowering and hypoglycemic effects, possibly from suppression of fatty acid synthesis in the liver.
Asunto(s)
Diabetes Mellitus , Ácidos Grasos Omega-3 , Animales , Ratones , Glucemia/metabolismo , Aceite de Cártamo , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Omega-3/farmacología , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Diabetes Mellitus/metabolismo , Hígado/metabolismo , Metabolismo de los LípidosRESUMEN
The clustered protocadherins (Pcdhs), Pcdh-α, -ß, and -γ, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-α regulatory element in vitro, affects especially the expression of specific Pcdh-α isoforms in vivo. We also identified a Pcdh-ß cluster control region (CCR) containing six HS sites (HS16, 17, 17', 18, 19, and 20) downstream of the Pcdh-γ cluster. This CCR comprehensively activates the expression of the Pcdh-ß gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-γ isoforms and does not affect Pcdh-α expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-γ cluster to activate the upstream Pcdh-ß genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-ß expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.
Asunto(s)
Cadherinas/genética , Familia de Multigenes , Neuropéptidos/genética , Animales , Proteínas Relacionadas con las Cadherinas , Línea Celular Tumoral , Regulación de la Expresión Génica , Ratones , Neuronas , Protocadherinas , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Forces associated with blood flow are crucial not only for blood vessel development but also for regulation of vascular pathology. Although there have been many studies characterizing the responses to mechanical stimuli, molecular mechanisms linking biological responses to mechanical forces remain unclear. Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion adaptor protein proposed as a candidate for a mediator of mechanotransduction. In the present study, we generated Hic-5-deficient mice by targeted mutation. Mice lacking Hic-5 are viable and fertile, and show no obvious histological abnormalities including vasculature. However, after wire injury of the femoral artery in Hic-5 deficient mice, histological recovery of arterial media was delayed due to enhanced apoptosis of vascular wall cells, whereas neointima formation was enhanced. Stretch-induced apoptosis was enhanced in cultured vascular smooth muscle cells (vascular SMCs) from Hic-5 deficient mice. Mechanical stress also induced the alteration of intracellular distribution of vinculin from focal adhesions to the whole cytoplasm in SMCs. Immunoelectron microscopic study of vascular SMCs from a wire-injured artery demonstrated that vinculin was dispersed in the nucleus and the cytoplasm in Hic-5-deficient mice whereas vinculin was localized mainly in the sub-plasma membrane region in wild type mice. Our findings indicate that Hic-5 may serve as a key regulator in mechanosensitive vascular remodeling.
Asunto(s)
Apoptosis/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Animales , Apoptosis/genética , Southern Blotting , Western Blotting , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Proteínas con Dominio LIM , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Inmunoelectrónica , Modelos Biológicos , Miocitos del Músculo Liso/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Vinculina/metabolismoRESUMEN
The process of sweating plays an important role in the human body, including thermoregulation and maintenance of the environment and health of the skin. It is known that the conditions of hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion and can result in severe skin conditions such as pruritus and erythema, which significantly reduce the patient's quality of life. However, there are many aspects of the signaling mechanisms in the process of sweating that have not been clarified, and no effective therapies or therapeutic agents have yet been discovered. Previously, it was reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes sweating, but details of the underlying mechanism has not been clarified. We used immortalized human eccrine gland cells (NCL-SG3 cell) to investigate how sweat secretion is induced by PACAP. Intracellular Ca2+ levels were increased in these cells following their exposure to physiological concentrations of PACAP. Intracellular Ca2+ was not elevated when cells were concomitantly treated with PA-8, a specific PAC1-R antagonist, suggesting that PAC1-R is involved in the elevation of intracellular Ca2+ levels in response to PACAP treatment. Furthermore, immunocytochemistry experiments showed that aquaporin-5 was translocated from the cytoplasm to the cell membrane by PACAP. These results suggest that PACAP acts on eccrine sweat glands to promote sweat secretion by translocation of aquaporin-5 to the cell membrane in response to increased levels of intracellular Ca2+. These findings also provide a solid basis for future research initiatives to develop new therapies to treat sweating disorders.
Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Glándulas Sudoríparas/efectos de los fármacos , Acuaporina 5/metabolismo , Calcio/metabolismo , Línea Celular Transformada , Humanos , Transporte de Proteínas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Glándulas Sudoríparas/citología , Glándulas Sudoríparas/metabolismoRESUMEN
The clustered protocadherin-alpha (Pcdha) genes, which are expressed in the vertebrate brain, encode diverse membrane proteins whose functions are involved in axonal projection and in learning and memory. The Pcdha cluster consists of 14 tandemly arranged genes (Pcdha1-Pcdha12, Pcdhac1, and Pcdhac2, from 5' to 3'). Each first exon (the variable exons) is transcribed from its own promoter, and spliced to the constant exons, which are common to all the Pcdha genes. Cerebellar Purkinje cells show dual expression patterns for Pcdha. In individual Purkinje cells, different sets of the 5' genes in the cluster, Pcdha1-12, are randomly expressed, whereas both 3' genes, Pcdhac1 and Pcdhac2, are expressed constitutively. To elucidate the relationship between the genomic structure of the Pcdha cluster and their expression in Purkinje cells, we deleted or duplicated multiple variable exons and analyzed the expression of Pcdha genes in the mouse brain. In all mutant mice, transcript levels of the constant exons and the dual expression patterns were maintained. In the deletion mutants, the missing genes were flexibly compensated by the remaining variable exons. On the other hand, in duplication mutants, the levels of the duplicated genes were trimmed. These results indicate that the Pcdha genes are comprehensively regulated as a cluster unit, and that the regulators that randomly and constitutively drive Pcdha gene expression are intact in the deleted or duplicated mutant alleles. These dual regulatory mechanisms may play important roles in the diversity and fundamental functions of neurons.
Asunto(s)
Cadherinas/fisiología , Eliminación de Gen , Duplicación de Gen , Regulación de la Expresión Génica , Familia de Multigenes , Animales , Southern Blotting , Western Blotting , Proteínas de Unión al ADN , Femenino , Humanos , Hibridación in Situ , Integrasas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/fisiología , Células de Purkinje , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Olfactory sensory neurons (OSNs) that express the same odorant receptor project their axons to specific glomeruli in the main olfactory bulb. Protocadherin-alpha (Pcdha) proteins, diverse cadherin-related molecules that are encoded as a gene cluster, are highly concentrated in OSN axons and olfactory glomeruli. Here, we describe Pcdha mutant mice, in which the constant region of the Pcdha gene cluster has been deleted by gene targeting. The mutant mice show abnormal sorting of OSN axons into glomeruli. There are multiple, small, extraneous glomeruli for the odorant receptors M71 and MOR23. These abnormal patterns of M71 and MOR23 glomeruli persist until adulthood. Many M71 glomeruli, but apparently not MOR23 glomeruli, are heterogeneous in axonal innervation. Thus, Pcdha molecules are involved in coalescence of OSN axons into OR-specific glomeruli of the olfactory bulb.