RESUMO
Mucociliary clearance, which is conducted by beating cilia cooperating with the surface mucous layer, is a major host defense mechanism of the airway epithelium. Ezrin, a crosslinker between membrane proteins and the actin cytoskeleton, is located in microvilli and around the basal bodies in airway ciliary cells. It is also likely that ezrin plays an important role in apical localization of ß2 adrenergic receptor (ß2AR) in airway ciliary cells. Here, we studied the physiological roles of ezrin by using trachea and airway epithelial cells prepared from ezrin-knockdown (Vil2kd/kd) mice. The trachea and airway ciliary cells of Vil2kd/kd mice presented a normal morphology and basal body orientation, suggesting that ezrin is not directly involved in development and planar cell polarity of cilia. Procaterol stimulates ciliary beating (frequency and amplitude) via ß2AR in the airway ciliary cells. In the Vil2kd/kd mice, airway ciliary beating stimulated with procaterol was partly inhibited due to the impairment of cell surface expression of ß2AR. These results suggest that ezrin regulates the beating of airway ciliary cells by promoting the apical surface localization of ß2AR. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cílios , Procaterol , Animais , Cílios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Camundongos , Procaterol/metabolismo , Procaterol/farmacologia , Traqueia/metabolismoRESUMO
Ciliary beating in the airway epithelium plays an important role in preventing infection by eliminating small particles and pathogens. Stimulation of ß2 adrenergic receptor (ß2AR) increases [cAMP]i levels and strongly activates this ciliary beating. ß2AR is localized to the apical membrane of the airways by indirectly binding to ezrin, an actin-binding protein. Ezrin takes active phosphorylated and inactive dephosphorylated states at Thr-567. Previously we showed that procaterol-stimulated ciliary beating was impaired in the ezrin-knockdown mice. In this study, we examined the roles of ezrin and its phosphorylation in regulating ciliary beating by using NSC305787, an ezrin inhibitor, in normal human airway epithelial cells (NHBE). We found that NSC305787 inhibits the phosphorylation of ezrin with an IC50 of 50 µM in NHBE. Treatment with NSC305787 for 4 h or more decreased the expression of ß2AR in the cell membrane and induced vesicle- or dot-like expression of ezrin and ß2AR inside the cell. As a result, inhibition of ezrin phosphorylation by NSC305787 attenuated the effect of procaterol-induced activation of ciliary beating in both frequency and distance indices.
Assuntos
Adamantano/análogos & derivados , Cílios , Proteínas do Citoesqueleto , Procaterol , Quinolinas , Camundongos , Humanos , Animais , Cílios/metabolismo , Procaterol/farmacologia , Procaterol/metabolismo , FosforilaçãoRESUMO
Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.
Assuntos
Primatas , Sêmen , Animais , Masculino , Diferenciação Celular , Células Germinativas , Células-Tronco Embrionárias/metabolismoRESUMO
Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED50 value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A2B receptor (A2BR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of A2BR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through A2BR on the cilia, and activation of PKA.
Assuntos
Adenosina , Animais Recém-Nascidos , Encéfalo , Cílios , Proteínas Quinases Dependentes de AMP Cíclico , Receptor A2B de Adenosina , Animais , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/fisiologia , Receptor A2B de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adenosina/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Camundongos , Células Cultivadas , Transdução de Sinais/efeitos dos fármacos , Antagonistas do Receptor A2 de Adenosina/farmacologia , Colforsina/farmacologia , Epêndima/metabolismo , Epêndima/citologiaRESUMO
Single cilia, 100 nm in diameter and 10 µm in length, were isolated from mouse tracheae with Triton X-100 (0.02%) treatment, and the effects of pH on ciliary beating were examined by measuring the ciliary beat frequency (CBF) and the ciliary bend distance (CBD-an index of amplitude) using a high-speed video microscope (250 fps). ATP (2.5 mM) plus 8Br-cAMP (10 µM) reactivated the CBF and CBD in the isolated cilia, similar to the cilia of in vivo tracheae. In the reactivated isolated cilia, an elevation in pH from 7.0 to 8.0 increased the CBF from 3 to 15 Hz and the CBD from 0.6 to 1.5 µm. The pH elevation also increased the velocity of the effective stroke; however, it did not increase the recovery stroke, and, moreover, it decreased the intervals between beats. This indicates that H+ (pHi) directly acts on the axonemal machinery to regulate CBF and CBD. In isolated cilia priorly treated with 1 µM PKI-amide (a PKA inhibitor), 8Br-cAMP did not increase the CBF or CBD in the ATP-stimulated isolated cilia. pH modulates the PKA signal, which enhances the axonemal beating generated by the ATP-activated inner and outer dyneins.
Assuntos
Trifosfato de Adenosina , Cílios , AMP Cíclico , Traqueia , Animais , Cílios/efeitos dos fármacos , Cílios/metabolismo , Trifosfato de Adenosina/metabolismo , Concentração de Íons de Hidrogênio , Traqueia/metabolismo , Traqueia/efeitos dos fármacos , Camundongos , AMP Cíclico/metabolismo , MasculinoRESUMO
An application of CO2/HCO3--free solution (Zero-CO2) did not increase intracellular pH (pHi) in ciliated human nasal epithelial cells (c-hNECs), leading to no increase in frequency (CBF) or amplitude (CBA) of the ciliary beating. This study demonstrated that the pHi of c-hNECs expressing carbonic anhydrase IV (CAIV) is high (7.64), while the pHi of ciliated human bronchial epithelial cells (c-hBECs) expressing no CAIV is low (7.10). An extremely high pHi of c-hNECs caused pHi, CBF and CBA to decrease upon Zero-CO2 application, while a low pHi of c-hBECs caused them to increase. An extremely high pHi was generated by a high rate of HCO3- influx via interactions between CAIV and Na+/HCO3- cotransport (NBC) in c-hNECs. An NBC inhibitor (S0859) decreased pHi, CBF and CBA and increased CBF and CBA in c-hNECs upon Zero-CO2 application. In conclusion, the interactions of CAIV and NBC maximize HCO3- influx to increase pHi in c-hNECs. This novel mechanism causes pHi to decrease, leading to no increase in CBF and CBA in c-hNECs upon Zero-CO2 application, and appears to play a crucial role in maintaining pHi, CBF and CBA in c-hNECs periodically exposed to air (0.04% CO2) with respiration.
Assuntos
Bicarbonatos , Dióxido de Carbono , Anidrase Carbônica IV , Cílios , Células Epiteliais , Mucosa Nasal , Humanos , Concentração de Íons de Hidrogênio , Dióxido de Carbono/metabolismo , Cílios/metabolismo , Bicarbonatos/metabolismo , Células Epiteliais/metabolismo , Mucosa Nasal/metabolismo , Mucosa Nasal/citologia , Anidrase Carbônica IV/metabolismo , Anidrase Carbônica IV/genética , Células Cultivadas , Simportadores de Sódio-Bicarbonato/metabolismo , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Ependymal cilia play pivotal roles in cerebrospinal fluid flow. In the primary culture system, undifferentiated glial cells differentiate well into ependymal multiciliated cells (MCCs) in the absence of fetal bovine serum (FBS). However, the substances included in FBS which inhibit this differentiation process have not been clarified yet. Here, we constructed the polarized primary culture system of ependymal cells using a permeable filter in which they retained ciliary movement. We found that transforming growth factor-ß1 (TGF-ß1) as well as Bone morphogenetic protein (BMP)-2 inhibited the differentiation with ciliary movement. The inhibition on the differentiation by FBS was recovered by the TGF-ß1 and BMP-2 inhibitors in combination.
Assuntos
Proteína Morfogenética Óssea 2 , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Diferenciação Celular , Proteína Morfogenética Óssea 2/farmacologia , Neuroglia/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.
Assuntos
Ambroxol , Animais , Camundongos , Ambroxol/farmacologia , Cálcio/metabolismo , Células Cultivadas , Cílios/fisiologia , Células Epiteliais , Concentração de Íons de Hidrogênio , Pulmão , Camundongos Endogâmicos CBARESUMO
Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca2+ concentration ([Ca2+]i). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca2+ channels (CaVs) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca2+]i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca2+]i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of CaVs by α7-nAChRs. Experiments using a high K+ solution with/without nifedipine (155.5 mM K+) showed that the activation of CaVs enhances CBF and CBD via an [Ca2+]i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by CaV1.2/α7-nAChR/ACh. In conclusion, a novel Ca2+ signalling pathway in airway cilia, CaV1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways.
Assuntos
Acetilcolina , Canais de Cálcio Tipo L , Cílios , Mucosa Respiratória , Receptor Nicotínico de Acetilcolina alfa7 , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Animais , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Colinérgicos/farmacologia , Cílios/efeitos dos fármacos , Cílios/fisiologia , Interleucina-13/metabolismo , Camundongos , Agonistas Nicotínicos/farmacologia , Nifedipino/farmacologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/fisiologia , Receptor Nicotínico de Acetilcolina alfa7/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Ezrin is one of the members of the ezrin/radixin/moesin (ERM) family of proteins. It was originally discovered as an actin-binding protein in the microvilli structure about forty years ago. Since then, it has been revealed as a key protein with functions in a variety of fields including cell migration, survival, and signal transduction, as well as functioning as a structural component. Ezrin acts as a cross-linker of membrane proteins or phospholipids in the plasma membrane and the actin cytoskeleton. It also functions as a platform for signaling molecules at the cell surface. Moreover, ezrin is regarded as an important target protein in cancer diagnosis and therapy because it is a key protein involved in cancer progression and metastasis, and its high expression is linked to poor survival in many cancers. Small molecule inhibitors of ezrin have been developed and investigated as candidate molecules that suppress cancer metastasis. Here, we wish to comprehensively review the roles of ezrin from the pathophysiological points of view.
Assuntos
Actinas , Proteínas dos Microfilamentos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Celular/metabolismo , Proteínas do Citoesqueleto , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismoRESUMO
Small inhaled particles, which are entrapped by the mucous layer that is maintained by mucous secretion via mucin exocytosis and fluid secretion, are removed from the nasal cavity by beating cilia. The functional activities of beating cilia are assessed by their frequency and the amplitude. Nasal ciliary beating is controlled by intracellular ions (Ca2+, H+ and Cl-), and is enhanced by a decreased concentration of intracellular Cl- ([Cl-]i) in ciliated human nasal epithelial cells (cHNECs) in primary culture, which increases the ciliary beat amplitude. A novel method to measure both ciliary beat frequency (CBF) and ciliary beat distance (CBD, an index of ciliary beat amplitude) in cHNECs has been developed using high-speed video microscopy, which revealed that a decrease in [Cl-]i increased CBD, but not CBF, and an increase in [Cl-]i decreased both CBD and CBF. Thus, [Cl-]i inhibits ciliary beating in cHNECs, suggesting that axonemal structures controlling CBD and CBF may have Cl- sensors and be regulated by [Cl-]i. These observations indicate that the activation of Cl- secretion stimulates ciliary beating (increased CBD) mediated via a decrease in [Cl-]i in cHNECs. Thus, [Cl-]i is critical for controlling ciliary beating in cHNECs. This review introduces the concept of Cl- regulation of ciliary beating in cHNECs.
Assuntos
Cloretos/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Mucosa Nasal/metabolismo , Biomarcadores , Cílios/ultraestrutura , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/metabolismo , Fenômenos Mecânicos , Microscopia de Vídeo , Modelos BiológicosRESUMO
In Ts1Rhr, a Down syndrome model mouse, the airway ciliary beatings are impaired; that is, decreases in ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of ciliary beat amplitude)). A resumption to two copies of the Pcp4 gene on the Ts1Rhr trisomic segment (Ts1Rhr:Pcp4+/+/-) rescues the decreases in CBF and CBA that occur in Ts1Rhr. In airway cilia, upon stimulation with procaterol (a ß2-agonist), the CBF increase is slower over the time course than the CBA increase because of cAMP degradation by Ca2+/calmodulin-dependent phosphodiesterase 1 (PDE1) existing in the metabolon regulating CBF. In Ts1Rhr, procaterol-stimulated CBF increase was much slower over the time course than in the wild-type mouse (Wt) or Ts1Rhr:Pcp4+/+/-. However, in the presence of 8MmIBMX (8-methoxymethyl isobutylmethyl xanthine, an inhibitor of PDE1) or calmidazolium (an inhibitor of calmodulin), in both Wt and Ts1Rhr, procaterol stimulates CBF and CBA increases over a similar time course. Measurements of cAMP revealed that the cAMP contents were lower in Ts1Rhr than in Wt or in Ts1Rhr:Pcp4+/+/-, suggesting the activation of PDE1A that is present in Ts1Rhr airway cilia. Measurements of the intracellular Ca2+ concentration ([Ca2+]i) in airway ciliary cells revealed that temperature (increasing from 25 to 37 °C) or 4αPDD (a selective transient receptor potential vanilloid 4 (TRPV4) agonist) stimulates a larger [Ca2+]i increase in Ts1Rhr than in Wt or Ts1Rhr:Pcp4+/+/-. In airway ciliary cells of Ts1Rhr, Pcp4-dose dependent activation of TRPV4 appears to induce an increase in the basal [Ca2+]i. In early embryonic day mice, a basal [Ca2+]i increased by PCP4 expressed may affect axonemal regulatory complexes regulated by the Ca2+-signal in Ts1Rhr, leading to a decrease in the basal CBF and CBA of airway cilia.
Assuntos
Cálcio/metabolismo , Cílios/metabolismo , Síndrome de Down/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Calmodulina/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPV/metabolismo , Traqueia/metabolismoRESUMO
Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl--free and HCO3- -free solutions. The responses were almost completely blocked by TTX (10-6 M) but not by atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT3 and 5-HT4), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors (Vipr) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl-/ HCO3- secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl-/ HCO3- secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25. NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl-/ HCO3- secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.
Assuntos
Ânions/metabolismo , Sistema Nervoso Entérico/metabolismo , Íleo , Vias Neurais/metabolismo , Neurotensina/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Animais , Hormônios Gastrointestinais/metabolismo , Íleo/inervação , Íleo/fisiologia , Mucosa Intestinal/metabolismo , Ratos , Receptores de Neurotensina/metabolismoRESUMO
Moesin is expressed in several types of cells including epithelial and endothelial cells. Several groups reported that moesin plays important roles in the regulation of the cellular motility, and the process of internalization of membrane proteins. However, the physiological roles of moesin in the kidney still remain unclear. Herein, we examined the physiological function of moesin in the kidney using moesin knockout (Msn -/y ) mice. There was no obvious abnormality in the renal morphology of Msn -/y mice. However, we found that Msn -/y mice exhibited mild hyperchloremia, and reduced glomerular filtration rate compared to wild type (WT) mice. Absolute electrolytes excretions of NaCl in Msn -/y mice were not significantly changed compared to WT mice. In the renal medulla, moesin was detected in thick ascending limb of Henle (TALH) as previously reported. To determine the physiological function of moesin in TALH, we examined the expression and subcellular localization of NKCC2 in Msn -/y mice. Interestingly, apical surface expression level, but not total expression of NKCC2 was increased in Msn -/y mice. Subcellular fractionation of renal medulla lysate and internalization assay using tubular suspension showed that the process of NKCC2 endocytosis is impaired. Since the distribution of NKCC2 in lipid raft fractions was decreased in Msn -/y mice, moesin may regulate the NKCC2 distribution to microdomain. These results suggest that moesin regulates the internalization of NKCC2. Furthermore, euhydration by water loading caused hyponatremina in Msn -/y mice, suggesting that dysfunction of moesin is associated with the nephrogenic syndrome of inappropriate antidiuresis (NSIAD).
Assuntos
Extremidades/fisiologia , Alça do Néfron/metabolismo , Proteínas dos Microfilamentos/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Endocitose/fisiologia , Células Endoteliais/metabolismo , Medula Renal/metabolismo , Masculino , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Ezrin/radixin/moesin (ERM) proteins function as general cross-linkers between plasma membrane proteins and the actin cytoskeleton and are involved in the functional expression of membrane proteins on the cell surface. They also integrate Rho guanosine 5'-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rho-related proteins. They act as protein kinase A (PKA)-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation. Therefore, ERM proteins seem to play important roles in the membrane transport of electrolytes by ion channels and transporters. In this review, we focus on the pathophysiological roles of ERM proteins in in vivo studies and introduce the phenotypes of their knockout and knockdown mice.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/patologia , Colestase/genética , Colestase/metabolismo , Colestase/fisiopatologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Osteomalacia/genética , Osteomalacia/metabolismo , Osteomalacia/fisiopatologia , Transporte Proteico/fisiologia , Distribuição Tecidual/fisiologia , Quinases Associadas a rho/metabolismoRESUMO
Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid that possesses many pharmacological effects, including increasing bile flow, changing the hydrophobicity of the bile acid pool, and modulation of the immune response. UDCA has been approved for treating cholestatic liver disease, such as primary biliary cholangitis. However, several unanticipated severe side effects of UDCA are observed in cholestatic patients, and its pharmacological benefits remain controversial. We reported that ezrin-knockdown (Vil2kd/kd) mice exhibited severe hepatic injury because of a functional disorder in bile duct fluidity and alkalinity regulation, resembling human intrahepatic cholestatic disease. Here we used Vil2kd/kd mice as a cholestatic model to investigate the pharmacological effects of UDCA. We investigated the effects of oral and parenteral administration of UDCA on Vil2kd/kd mice. In Vil2kd/kd mice, fed a 0.5% (w/w) UDCA diet for 3 weeks, hepatic injury was exacerbated, although oral administration of a lower dose of UDCA slightly improved hepatic function in Vil2kd/kd mice. On the other hand, intraperitoneal administration of UDCA (50 mg/kg/d) ameliorated hepatic function and markedly reduced periductal fibrosis and cholangiocyte proliferation in Vil2kd/kd mice although biliary pH and HCO3- concentration were not improved. The expression levels of inflammatory and profibrotic genes were also significantly decreased in these mice. Furthermore, UDCA prevented cholangiocytes from hydrophobic bile acid-induced cytotoxicity independent of extracellular pH in in vitro experiments. These results suggest that an appropriate dosage of UDCA can ameliorate the intrahepatic cholestasis in Vil2kd/kd mice without changing the biliary bicarbonate secretion.
Assuntos
Colagogos e Coleréticos/uso terapêutico , Colestase Intra-Hepática/tratamento farmacológico , Ácido Ursodesoxicólico/uso terapêutico , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Bicarbonatos/metabolismo , Bile/metabolismo , Células Cultivadas , Colagogos e Coleréticos/farmacologia , Colestase Intra-Hepática/sangue , Colestase Intra-Hepática/metabolismo , Colestase Intra-Hepática/patologia , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Ácido Ursodesoxicólico/farmacologiaRESUMO
UNLABELLED: Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. CONCLUSION: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.
Assuntos
Ductos Biliares/patologia , Bile , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/patologia , Proteínas do Citoesqueleto/genética , Animais , Epitélio/patologia , Fibrose , Camundongos , Camundongos KnockoutRESUMO
OAT-PG is a kidney-specific prostaglandin transporter and exclusively expressed at the basolateral membrane of proximal tubules in rodent kidneys. We previously reported that OAT-PG was dominantly expressed in the male kidney similar to the other SLC22 family proteins as organic anion transporter (OAT) 1 and OAT3. Recently, Wegner et al. revealed that a transcription factor, B-cell CLL/lymphoma 6 (BCL6), is associated with the male-dominant expressions of OAT1 and OAT3 in the rat kidney. Here, we performed the luciferase assay to investigate whether OAT-PG is also transcriptionally regulated by BCL6. However, the promoter activity of OAT-PG was not directly affected by BCL6 overexpression nor the testosterone treatment, suggesting that different regulatory mechanisms underlie the male-dominant transcriptional regulation of OAT-PG compared to those of OAT1 and OAT3. We newly found that adrenalectomy (Adx) of male rat caused a significant reduction of OAT-PG expression without any significant changes in the OAT1 and OAT3 expressions, and it was recovered by the dexamethasone administration. Furthermore, the renocortical PGE2 concentration was markedly increased in Adx male rat, concomitant with the downregulation of OAT-PG, and it was reduced to the basal level by dexamethasone treatment. In the luciferase assay, dexamethasone stimulated OAT-PG promoter activity but not OAT1. The luciferase activity responsiveness to dexamethasone was significantly reduced by the deletion of glucocorticoid response elements in the OAT-PG promoter region. These results suggest that glucocorticoid plays an important role in the regulation of the renocortical PGE2 concentration by the transcriptional regulation of OAT-PG in the rat kidney.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Rim/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Feminino , Rim/efeitos dos fármacos , Rim/fisiologia , Masculino , Gambás , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Testosterona/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Ezrin cross-links plasma membrane proteins with the actin cytoskeleton. In the kidney, ezrin mainly localizes at the brush border membrane of proximal tubules with the scaffolding protein, Na(+)/H(+) exchanger regulatory factor (NHERF) 1. NHERF1 interacts with the sodium/phosphate cotransporter, Npt2a. Defects in NHERF1 or Npt2a in mice cause hypophosphatemia. Here we studied the physiological role of ezrin in renal phosphate reabsorption using ezrin knockdown mice (Vil2). These mice exhibit hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma phosphate concentrations were ascribed to defects in urinary phosphate reabsorption. Immunofluorescence and immunoblotting indicated a marked reduction in renal Npt2a and NHERF1 expression at the apical membrane of proximal tubules in the knockdown mice. On the other hand, urinary loss of calcium was not found in Vil2 mice. Plasma concentrations of 1,25-dihydroxyvitamin D were elevated following reduced plasma phosphate levels, and mRNA of the vitamin D-dependent TRPV6 calcium channel were significantly increased in the duodenum of knockdown mice. Expression of TRPV6 at the apical membrane, however, was significantly decreased. Furthermore, tibial bone mineral density was significantly lower in both the adult and young Vil2 mice. These results suggest that ezrin is required for the regulation of systemic phosphate and calcium homeostasis in vivo.
Assuntos
Cálcio/metabolismo , Proteínas do Citoesqueleto/fisiologia , Citoesqueleto/fisiologia , Homeostase/fisiologia , Túbulos Renais Proximais/metabolismo , Fosfatos/metabolismo , Animais , Densidade Óssea/fisiologia , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Modelos Animais de Doenças , Duodeno/metabolismo , Feminino , Hipocalcemia/metabolismo , Hipofosfatemia/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismoRESUMO
BACKGROUND: Prostaglandin (PG)-specific organic anion transporter (OAT-PG) is a recently identified renal transporter involved in the local clearance of prostaglandin E2 (PGE2). Since the renal biosynthesis of PGE2 is not increased during pregnancy, this transporter expression would affect the gestational changes in the renal PGE2 content. METHODS: Kidneys from rats at different gestational stages were used to examine gestational changes in the renocortical PGE2 concentration. The renal expression of OAT-PG and the enzymes for PGE2 synthesis was also examined sequentially, together with the gestational changes in renal renin production. RESULTS: The renocortical PGE2 concentration was significantly increased during midterm to late pregnancy, with a maximum increase of 47.6 ± 11.5% from the virgin value. Although the expression of the enzymes, such as cyclooxygenases and PG synthases, was not increased, that of OAT-PG was significantly decreased throughout pregnancy, inversely correlating with changes in the renocortical PGE2 concentration. Renal renin production was significantly increased during pregnancy. CONCLUSION: This study demonstrated for the first time that the tissue PGE2 concentration was increased in pregnant rat kidneys, which may be associated with the gestational rise in glomerular filtration rate. The decreased expression of OAT-PG was thought to be responsible for the increased tissue PGE2 content.