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Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ('entry site') to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance. central tolerance | locus decontraction | tissue-restricted antigens doi/ 10.1073/pnas.1009265107

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial gila-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells. adult neurogenesis | coupling | gap junction | astrocyte | patch clamp

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of [sup.35]S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKll and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. in analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses. brain | electrical synapse | gap junction | protein-protein interaction | synaptic plasticity

Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed [beta]-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fuorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the propagation of JGA vascular signals and in the regulation of renin release and blood pressure. gap junction; Cre/loxP technique; lacZ

Author(s): Wilhelm Roell [1, 2, 9]; Thorsten Lewalter [3, 9]; Philipp Sasse [1, 9]; Yvonne N. Tallini [6]; Bum-Rak Choi [7]; Martin Breitbach [1]; Robert Doran [6]; Ulrich M. Becher [...]

Ventricular tachyarrhythmias are the main cause of sudden death in patients after myocardial infarction. Here we show that transplantation of embryonic cardiomyocytes (eCMs) in myocardial infarcts protects against the induction [...]

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a [Cx30.sup.-/-] background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf [Cx30.sup.-/-] mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations. gap junction | hearing rescue | hereditary deafness

Four connexins (Cxs), mouse (m)Cx30.2, Cx40, Cx43, and Cx45, determine cell-cell electrical signaling in mouse heart, and Cx43 and Cx45 are known to form unapposed hemichannels. Here we show that mCx30.2, which is most abundantly expressed in sinoatrial and atrioventricular nodal regions of the heart, and its putative human ortholog, human (h)Cx31.9, also form functional hemichannels, which, like mCx30.2 cell-cell channels, are permeable to cationic dyes up to [approximately equal to]400 Da in size. DAPI uptake by HeLa cells expressing mCx30.2 was >10-fold faster than that by HeLa parental cells. In [Ca.sup.2+]-free medium, uptake of DAPI by HeLaCx30.2-EGFP cells was increased [approximately equal to]2-fold, but uptake by parental cells was not affected. Conversely, acidification by application of C[O.sub.2] reduced DAPI uptake by HeLaCx30.2-EGFP cells but had little effect on uptake by parental cells. Cells expressing mCx30.2 exhibited higher rates of DAPI uptake than did cells expressing any of the other cardiac Cxs. Cardiomyocytes of 2-day-old rats transfected with hCx31.9-EGFP took up DAPI and ethidium bromide 5-10 times faster than wild-type cardiomyocytes. Mefloquine, a close derivative of quinine and quinidine that exhibits antimalarial and antiarrhythmic properties, reduced conductance of cell-cell junctions and dye uptake through mCx30.2 hemichannels with approximately the same affinity (I[C.sub.50] = [approximately equal to]10 [micro]M) and increased dependence of junctional conductance on transjunctional voltage. Unitary conductance of mCx30.2 hemichannels was [approximately equal to]20 pS, about twice the cell-cell channel conductance. Hemichannels formed of mCx30.2 and hCx31.9 may slow propagation of excitation in the sinoatrial and atrioventricular nodes by shortening the space constant and depolarizing the excitable membrane. gap junction | Wenckebach period

In the mammalian heart, gap junction channels between electrically coupled cardiomyocytes are necessary for impulse propagation and coordinated contraction of atria and ventricles. Recently, mouse connexin30.2 (Cx30.2) was shown to be expressed in the cardiac conduction system, predominantly in sinoatrial and atrioventricular (AV) nodes. The corresponding gap junctional channels expressed in HeLa cells exhibit the lowest unitary conductance (9 pS) of all connexin channels. Here we report that Cx30.2 slows down the propagation of excitation through the AV node. Mice expressing a LacZ reporter gene instead of the Cx30.2 coding region (Cx30.[2.sup.Lacz/Lacz]) exhibit a PQ interval that is [approximately equal to] 25% shorter than in WT littermates. By recording atrial, His, and ventricular signals with intracardiac electrodes, we show that this decrease is attributed to significantly accelerated conduction above the His bundle (atrial-His interval: 27.9 [+ or -] 5.1 ms in Cx30.[2.sup.Lacz/Lacz] versus 37.1 [+ or -] 4.1 ms in Cx30.[2.sup.+/+] mice), whereas HV conduction is unaltered. Atrial stimulation revealed an elevated AV-nodal conduction capacity and faster ventricular response rates during induced episodes of atrial fibrillation in Cx30.[2.sup.Lacz/Lacz] mice. Our results show that Cx30.2 contributes to the slowdown of impulse propagation in the AV node and additionally limits the maximum number of beats conducted from atria to ventricles. Thus, it is likely to be involved in coordination of atrial and ventricular contraction and to fulfill a protective role toward pathophysiological states such as atrial tachyarrhythmias (e.g., atrial fibrillation) by preventing rapid conduction to the ventricles potentially associated with hemodynamic deterioration. atrial fibrillation | cardiac connexins | conductive myocardium | coordinated cardiac contraction

Recent research results indicate that glial gap-junction communication is much more complex and widespread than originally thought, and has diverse roles in brain homeostasis and the response of the brain to injury. The situation is far from clear, however. Pharmacological agents that block gap junctions can abolish neuron--glia long-range signaling and can alleviate neuronal damage whereas, intriguingly, opposite effects are observed in mice lacking connexin43, a major gap-junction subunit protein in astrocytes. How can the apparently contradictory results be explained, and how is specificity achieved within the glial gap-junction system? Another key issue in understanding glial connexin function is that oligodendrocytes and astrocytes, each of which express distinct connexin isotypes, are thought to participate in brain homeostasis by forming a panglial syncytium. Molecular analysis has revealed a surprising diversity of connexin expression and function, and this has led to new hypotheses regarding their roles in the brain, which could be tested using new approaches.

Gap junctions are intercellular channels that are formed by the protein family of connexins (Cxs). In mammary tissue, Cx26 and Cx32 are present in the secretory epithelium and Cx43 is localized in the myoepithelium. The expression of Cx26 and Cx32 is induced during pregnancy and lactation, respectively, thus suggesting unique roles for them in the functional development of the gland. The requirement for these connexins was explored using several strains of genetically altered mice: mice with an inactivated Cx32 gene, mice in which the Cx43 gene had been replaced with the Cx32 gene (Cx43KI32 mice) and mice in which the Cx26 gene was specifically ablated in mammary epithelium at different stages of development using Cre-loxP-based recombination. Normal mammary development was obtained in Cx32-null mice and in Cx43KI32 mammary tissue. In contrast, loss of Cx26 in mammary epithelium before puberty resulted in abrogated lobuloalveolar development and increased cell death during pregnancy, which was accompanied by impaired lactation. Loss of Cx26 in mammary epithelium during the later part of pregnancy did not adversely interfere with functional mammary development. These results demonstrate that the presence of Cx26 is critical during early stages but not during the end of pregnancy when the tissue has completed functional differentiation. Cx26 is considered a tumor suppressor gene and Cx26-null mammary tissue was evaluated after five pregnancies. No hyperproliferation or hyperplasia was observed, suggesting that Cx26 does not function as a tumor suppressor. Keywords: Connexins; Gap junction; Mammary gland; Apoptosis; Alveolar development; Differentiation

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands. connexin 26; connexin 32; mouse; tears

Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to 'chemical ischemia' by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18[alpha]-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular [La.sup.3+] also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death. astroglia | dye uptake | dye coupling | Cx43 | phosphorylation

A study was conducted to examine the effect of chronic alterations in cellular communication to the secretion of amylase by pancreatic acinar cells in vivo and the mechanism behind cell uncoupling. The cellular gap junctions and cell-to-cell communication in connexin32-deficient (Cx32) pancreatic acinar cells in mice were characterized. A biochemical assay and a reverse hemolytic plaque assay were used to determine amylase secretion. Findings indicated that absence of Cx32 reduces the amylase secretory activity of the exocrine pancreas.

Gap junctions mediate intercellular communication through the exchange of small diffusible molecules. They are formed after docking of two connexons, which are composed of connexins (Cx). Mice harboring a targeted homozygous defect in gene coding for connexin26, died in utero, although no gross morphological changes were observed between homozygous connexin26-defective embryos and wild-type littermates. It is concluded that the uptake of glucose into the connexin26-deficient mouse embryos was impaired and could not support rapid organogenesis.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average [approximately equal to]17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was [approximately equal to]1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

The bystander effect refers to the process wherein cells that generate herpes simplex virus-thymidine kinase (HSV-tk) kill neighboring cells that do not produce HSV-tk. The potency of such tumor cell killing is enhanced through the transfer of specific cytotoxic agents by gap junctional intercellular communication (GJIC). Bystander killing in gene therapy is attributed to connexin-mediated GJIC.

The difference in compatibility and permeability between connexin channels is useful for understanding the functional and structural differences between murine connexin genes of mammalian cells. Different connexin channels have different passing affinity for cellular molecules while lucifer yellow cells can pass through all connexin channels. It is difficult for ethidium bromide and propidium iodide to pass through these channels. Different connexins form functionally different Gap junctions channels.

Cytoplasts of mouse L929 and Ehrlich ascites tumor cells that contain DNA sequences that cause unrestricted proliferation or immortalization of human lymphocytes after transfection in vitro were investigated. Immortalization may be stimulated by insertional inactivation or operational suppression of genes that have to be expressed during cellular senescence or programmed cell death.

A mouse connexin with a molecular mass of 40.418 kiloDaltons was identified and labeled as Cx40. Its protein and nucleotide sequences were closely related to mouse Cx43 but was found to be more homologous to chick Cx42. Cx40 was observed to be concentrated more densely in the lung areas of the mouse than in other tissues examined. Intercellular channels having distinct characteristics were formed by Cx40 upon expression in Xenopus oocytes. These channels can interact with those of Cx37 forming heterotypic channels with modified voltage-gating properties.

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-[Gamma] (INF-[Gamma]) or tumor necrosis factor-[Alpha] (TNF-[Alpha]) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-[Gamma] plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-[Alpha]] antibody, suggesting the release and autocrine action of TNF-[Alpha]. Treatment with INF-[Gamma] plus TNF-[Alpha] also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18[Alpha]-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-[Gamma] plus LPS or INF-[Gamma] plus TNF-[Alpha]. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response. inflammation | macrophage | connexin | cytokines | dye coupling

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice ([Gjb3.sup.-/-]) were found among the offspring of heterozygous Cx31-deficient parents ([Gjb3.sup.+/-]), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of [Gjb3.sup.-/-] embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving [Gjb3.sup.-/-] embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult [Gjb3.sup.-/-] mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse. [C] 2001 Academic Press Key Words: gap junctions; gene targeting; Connexin31; Gjb3; placenta; inner ear.