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1.
Stem Cells ; 35(4): 859-871, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27870307

RESUMO

Gap junctional intercellular communication (GJIC) has been suggested to be involved in early embryonic development but the actual functional role remained elusive. Connexin (Cx) 43 and Cx45 are co-expressed in embryonic stem (ES) cells, form gap junctions and are considered to exhibit adhesive function and/or to contribute to the establishment of defined communication compartments. Here, we describe the generation of Cx43/Cx45-double deficient mouse ES cells to achieve almost complete breakdown of GJIC. Cre-loxP induced deletion of both, Cx43 and Cx45, results in a block of differentiation in embryoid bodies (EBs) without affecting pluripotency marker expression and proliferation in ES cells. We demonstrate that GJIC-incompetent ES cells fail to form primitive endoderm in EB cultures, representing the inductive key step of further differentiation events. Lentiviral overexpression of either Cx43 or Cx45 in Cx43/45 mutants rescued the observed phenotype, confirming the specificity and indicating a partially redundant function of both connexins. Upon differentiation GJIC-incompetent ES cells exhibit a strikingly altered subcellular localization pattern of the transcription factor NFATc3. Control EBs exhibit significantly more activated NFATc3 in cellular nuclei than mutant EBs suggesting that Cx-mediated communication is needed for synchronized NFAT activation to induce orchestrated primitive endoderm formation. Moreover, pharmacological inhibition of NFATc3 activation by Cyclosporin A, a well-described inhibitor of calcineurin, phenocopies the loss of GJIC in control cells. Stem Cells 2017;35:859-871.


Assuntos
Comunicação Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Endoderma/embriologia , Endoderma/metabolismo , Junções Comunicantes/metabolismo , Animais , Apoptose , Biomarcadores/metabolismo , Calcineurina/metabolismo , Diferenciação Celular , Proliferação de Células , Conexina 43/metabolismo , Conexinas/metabolismo , Endoderma/citologia , Gastrulação , Lentivirus/metabolismo , Camundongos , Mutagênese/genética , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais
2.
J Mol Cell Cardiol ; 65: 19-32, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24060583

RESUMO

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and a major cause of stroke. In the mammalian heart the gap junction proteins connexin40 (Cx40) and connexin43 (Cx43) are strongly expressed in the atrial myocardium mediating effective propagation of electrical impulses. Different heterozygous mutations in the coding region for Cx40 were identified in patients with AF. We have generated transgenic Cx40A96S mice harboring one of these mutations, the loss-of-function Cx40A96S mutation, as a model for atrial fibrillation. Cx40A96S mice were characterized by immunochemical and electrophysiological analyses. Significantly reduced atrial conduction velocities and strongly prolonged episodes of atrial fibrillation were found after induction in Cx40A96S mice. Analyses of the gating properties of Cx40A96S channels in cultured HeLa cells also revealed significantly lower junctional conductance and enhanced sensitivity voltage gating of Cx40A96S in comparison to Cx40 wild-type gap junctions. This is caused by reduced open probabilities of Cx40A96S gap junction channels, while single channel conductance remained the same. Similar to the corresponding patient, heterozygous Cx40A96S mice revealed normal expression levels and localization of the Cx40 protein. We conclude that heterozygous Cx40A96S mice exhibit prolonged episodes of induced atrial fibrillation and severely reduced atrial conduction velocities similar to the corresponding human patient.


Assuntos
Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Conexinas/genética , Sistema de Condução Cardíaco/fisiopatologia , Mutação/genética , Animais , Fibrilação Atrial/diagnóstico por imagem , Fibrilação Atrial/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Eletrocardiografia , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/patologia , Fibrose Endomiocárdica/fisiopatologia , Mapeamento Epicárdico , Junções Comunicantes/genética , Células HeLa , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Humanos , Ativação do Canal Iônico , Camundongos , Camundongos Transgênicos , Transporte Proteico , Fatores de Tempo , Transfecção , Ultrassonografia , Proteína alfa-5 de Junções Comunicantes
3.
Hum Mol Genet ; 20(1): 28-39, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20926451

RESUMO

Mutations in the GJB2 gene coding for connexin26 (Cx26) can cause a variety of deafness and hereditary hyperproliferative skin disorders in humans. In this study, we investigated the Cx26S17F mutation in mice, which had been identified to cause the keratitis-ichthyosis-deafness (KID) syndrome in humans. The KID syndrome is characterized by keratitis and chronic progressive corneal neovascularization, skin hyperplasia, sensorineural hearing loss and increased carcinogenic potential. We have generated a conditional mouse mutant, in which the floxed wild-type Cx26-coding DNA can be deleted and the Cx26S17F mutation is expressed under control of the endogenous Cx26 promoter. Homozygous mutants are not viable, whereas the surviving heterozygous mice show hyperplasia of tail and foot epidermis, wounded tails and annular tail restrictions, and are smaller than their wild-type littermates. Analyses of auditory brainstem responses (ABRs) indicate an ∼35 dB increased hearing threshold in these mice, which is likely due to the reduction of the endocochlear potential by 20-40%. Our results indicate that the Cx26S17F protein, which does not form functional gap junction channels or hemichannels, alters epidermal proliferation and differentiation in the heterozygous state. In the inner ear, reduced intercellular coupling by heteromeric channels composed of Cx26S17F and Cx30 could contribute to hearing impairment in heterozygous mice, while remaining wild-type Cx26 may be sufficient to stabilize Cx30 and partially maintain cochlear homeostasis. The phenotype of heterozygous mice resembles many of the symptoms of the human KID syndrome. Thus, these mice represent an appropriate model to further investigate the disease mechanism.


Assuntos
Conexinas/genética , Animais , Conexina 26 , Surdez/genética , Modelos Animais de Doenças , Células-Tronco Embrionárias , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Perfilação da Expressão Gênica , Ictiose/genética , Ceratite/genética , Antígeno Ki-67/análise , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Antígeno Nuclear de Célula em Proliferação/análise , Recombinação Genética/genética
4.
Cell Death Dis ; 9(6): 630, 2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29795380

RESUMO

Correction to: NPG Asia Materials (2018) https://doi.org/10.1038/s41427-018-0014-9 published online on 16 April 2018.

5.
Cell Death Dis ; 8(6): e2845, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569788

RESUMO

Several mutant mice have been generated to model connexin (Cx)-linked skin diseases; however, the role of connexins in skin maintenance and during wound healing remains to be fully elucidated. Here we generated a novel, viable, and fertile mouse (Cx26CK14-S17F/+) with the keratitis-ichthyosis-deafness mutant (Cx26S17F) driven by the cytokeratin 14 promoter. This mutant mouse mirrors several Cx26-linked human skin pathologies suggesting that the etiology of Cx26-linked skin disease indeed stems from epidermal expression of the Cx26 mutant. Cx26CK14-S17F/+ foot pad epidermis formed severe palmoplantar keratoderma, which expressed elevated levels of Cx26 and filaggrin. Primary keratinocytes isolated from Cx26CK14-S17F/+ neonates exhibited reduced gap junctional intercellular communication and migration. Furthermore, Cx26CK14-S17F/+ mouse skin wound closure was normal but repaired epidermis appeared hyperplastic with elevated expression of cytokeratin 6. Taken together, we suggest that the Cx26S17F mutant disturbs keratinocyte differentiation and epidermal remodeling following wound closure. We further posit that Cx26 contributes to epidermal homeostasis by regulating keratinocyte differentiation, and that mice harboring a disease-linked Cx26 mutant display epidermal abnormalities yet retain most wound healing properties.


Assuntos
Conexinas/genética , Surdez/genética , Epiderme/metabolismo , Ictiose/genética , Ceratodermia Palmar e Plantar/genética , Cicatrização/genética , Animais , Diferenciação Celular , Conexina 26 , Conexinas/metabolismo , Surdez/metabolismo , Surdez/patologia , Modelos Animais de Doenças , Epiderme/patologia , Feminino , Proteínas Filagrinas , Efeito Fundador , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Expressão Gênica , Humanos , Ictiose/metabolismo , Ictiose/patologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratina-14/genética , Queratina-14/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Ceratodermia Palmar e Plantar/metabolismo , Ceratodermia Palmar e Plantar/patologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Cultura Primária de Células , Regiões Promotoras Genéticas
6.
J Invest Dermatol ; 136(3): 574-583, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26777423

RESUMO

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.


Assuntos
Canais de Cálcio/genética , Conexinas/genética , Epiderme/metabolismo , Predisposição Genética para Doença , Ceratite/genética , Animais , Permeabilidade da Membrana Celular/genética , Conexina 26 , Junções Comunicantes/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Desequilíbrio Hidroeletrolítico/genética , Desequilíbrio Hidroeletrolítico/fisiopatologia
7.
Circ Cardiovasc Genet ; 8(1): 21-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25504652

RESUMO

BACKGROUND: Sudden infant death syndrome (SIDS) describes the sudden, unexplained death of a baby during its first year of age and is the third leading cause of infant mortality. It is assumed that ≤20% of all SIDS cases are because of cardiac arrhythmias resulting from mutations in ion channel proteins. Besides ion channels also cardiac gap junction channels are important for proper conduction of cardiac electric activation. In the mammalian heart Connexin43 (Cx43) is the major gap junction protein expressed in ventricular cardiomyocytes. Recently, a novel Connexin43 loss-of-function mutation (Cx43E42K) was identified in a 2-month-old SIDS victim. METHODS AND RESULTS: We have generated Cx43E42K-expressing mice as a model for SIDS. Heterozygous cardiac-restricted Cx43E42K-mutated mice die neonatally without major cardiac morphological defects. Electrocardiographic recordings of embryonic Cx43+/E42K mice reveal severely disturbed ventricular activation, whereas immunohistochemical analyses show normal localization and expression patterns of gap junctional Connexin43 protein in the Cx43E42K-mutated newborn mouse heart. CONCLUSIONS: Because we did not find heterogeneous gap junction loss in Cx43E42K mouse hearts, we conclude that the Cx43E42K gap junction channel creates an arrhythmogenic substrate leading to lethal ventricular arrhythmias. The strong cardiac phenotype of Cx43E42K expressing mice supports the association between the human Cx43E42K mutation and SIDS and indicates that Connexin43 mutations should be considered in future studies when SIDS cases are to be molecularly explained.


Assuntos
Arritmias Cardíacas , Conexina 43 , Junções Comunicantes , Mutação de Sentido Incorreto , Morte Súbita do Lactente/genética , Substituição de Aminoácidos , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Conexina 43/biossíntese , Conexina 43/genética , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Humanos , Lactente , Camundongos , Camundongos Transgênicos
8.
FEBS Lett ; 589(15): 1904-10, 2015 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-26070424

RESUMO

The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.


Assuntos
Cálcio/metabolismo , Conexinas/genética , Surdez/metabolismo , Modelos Animais de Doenças , Epiderme/metabolismo , Ictiose/metabolismo , Ceratite/metabolismo , Metabolismo dos Lipídeos , Animais , Conexina 26 , Feminino , Masculino , Camundongos , Microscopia de Fluorescência
9.
FEBS Lett ; 588(9): 1795-801, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24685692

RESUMO

Distinct mutations in the gap junction protein connexin30 (Cx30) can cause the ectodermal dysplasia Clouston syndrome in humans. We have generated a new mouse line expressing the Clouston syndrome mutation Cx30A88V under the control of the endogenous Cx30 promoter. Our results show that the mutated Cx30A88V protein is incorporated in gap junctional plaques of the epidermis. Homozygous Cx30A88V mice reveal hyperproliferative and enlarged sebaceous glands as well as a mild palmoplantar hyperkeratosis. Additionally, homozygous mutant mice show an altered hearing profile compared to control mice. We conclude that the Cx30A88V mutation triggers hyperproliferation in the skin and changes the cochlear homeostasis in mice.


Assuntos
Conexinas/genética , Displasia Ectodérmica/genética , Perda Auditiva/genética , Mutação de Sentido Incorreto , Glândulas Sebáceas/patologia , Animais , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Conexina 30 , Estudos de Associação Genética , Folículo Piloso/metabolismo , Folículo Piloso/patologia , Humanos , Camundongos , Camundongos Transgênicos
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