scholarly journals Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

1990 ◽  
Vol 111 (5) ◽  
pp. 2077-2088 ◽  
Author(s):  
L S Musil ◽  
B A Cunningham ◽  
G M Edelman ◽  
D A Goodenough
Keyword(s):  
Cell Adhesion ◽  
Gap Junctions ◽  
Gap Junction ◽  
Cell Lines ◽  
Gap Junctional Communication ◽  
Competent Cells ◽  
Deficient Cell ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

scholarly journals Gap Junctions Regulate Extracellular Signal-regulated Kinase Signaling to Affect Gene Transcription

2005 ◽  
Vol 16 (1) ◽  
pp. 64-72 ◽  
Author(s):  
Joseph P. Stains ◽  
Roberto Civitelli
Keyword(s):  
Protein Kinase ◽  
Gap Junctions ◽  
Gap Junction ◽  
Gene Transcription ◽  
Electrical Coupling ◽  
Mitogen Activated Protein Kinase ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Extracellular Signal ◽  
Gap Junctional

Osteoblasts are highly coupled by gap junctions formed by connexin43. Overexpression of connexin45 in osteoblasts results in decreased chemical and electrical coupling and reduces gene transcription from connexin response elements (CxREs) in the osteocalcin and collagen Iα1 promoters. Here, we demonstrate that transcription from the gap junction-dependent osteocalcin CxRE is regulated by extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) cascades. Overexpression of a constitutively active mitogen-activated protein kinase kinase (MEK), Raf, or Ras can increase transcription more than twofold of the CxRE, whereas inhibition of MEK or PI3K can decrease transcription threefold from the osteocalcin CxRE. Importantly, disruption of gap junctional communication by overexpression of connexin45 or treatment with pharmacological inhibitors of gap junctions results in reduced Raf, ERK, and Akt activation. The consequence of attenuated gap junction-dependent signal cascade activation is a decrease in Sp1 phosphorylation by ERK, resulting in decreased Sp1 recruitment to the CxRE and inhibited gene transcription. These data establish that ERK/PI3K signaling is required for the optimal elaboration of transcription from the osteocalcin CxRE, and that disruption of gap junctional communication attenuates the ability of cells to respond to an extracellular cue, presumably by limiting the propagation of second messengers among adjacent cells by connexin43-gap junctions.

scholarly journals Blockade of Brain Stem Gap Junctions Increases Phrenic Burst Frequency and Reduces Phrenic Burst Synchronization in Adult Rat

2003 ◽  
Vol 89 (1) ◽  
pp. 135-149 ◽  
Author(s):  
Irene C. Solomon ◽  
Ki H. Chon ◽  
Melissa N. Rodriguez
Keyword(s):  
Gap Junctions ◽  
Brain Stem ◽  
Gap Junction ◽  
Phrenic Nerve ◽  
Respiratory Rhythm ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Nerve Discharge

Recent investigations have examined the influence of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in vitro using transverse medullary slice and en bloc brain stem-spinal cord preparations obtained from neonatal (1–5 days postnatal) mice. Gap junction proteins, however, have been identified in both neurons and glia in brain stem regions implicated in respiratory control in both neonatal and adult rodents. Here, we used an in vitro arterially perfused rat preparation to examine the role of gap junctional communication on generation and modulation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents. We recorded rhythmic inspiratory motor activity from one or both phrenic nerves before and during pharmacological blockade (i.e., uncoupling) of brain stem gap junctions using carbenoxolone (100 μM), 18α-glycyrrhetinic acid (25–100 μM), 18β-glycyrrhetinic acid (25–100 μM), octanol (200–300 μM), or heptanol (200 μM). During perfusion with a gap junction uncoupling agent, we observed an increase in the frequency of phrenic bursts (∼95% above baseline frequency; P < 0.001) and a decrease in peak amplitude of integrated phrenic nerve discharge ( P < 0.001). The increase in frequency of phrenic bursts resulted from a decrease in both T I ( P < 0.01) and T E ( P < 0.01). In addition, the pattern of phrenic nerve discharge shifted from an augmenting discharge pattern to a “bell-shaped” or square-wave discharge pattern in most experiments. Spectral analyses using a fast Fourier transform (FFT) algorithm revealed a reduction in the peak power of both the 40- to 50-Hz peak (corresponding to the MFO) and 90- to 110-Hz peak (corresponding to the HFO) although spurious higher frequency activity (≥130 Hz) was observed, suggesting an overall loss or reduction in inspiratory-phase synchronization. Although additional experiments are required to identify the specific brain stem regions and cell types (i.e., neurons, glia) mediating the observed modulations in phrenic motor output, these findings suggest that gap junction communication modulates generation of respiratory rhythm and inspiratory motoneuron synchronization in adult rodents in vitro.

Gap junctional communication coordinates vasopressin-induced glycogenolysis in rat hepatocytes

1998 ◽  
Vol 274 (6) ◽  
pp. G1109-G1116 ◽  
Author(s):  
Eliseo A. Eugenín ◽  
Hernan González ◽  
Claudia G. Sáez ◽  
Juan C. Sáez
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Glycogen Content ◽  
Rat Hepatocytes ◽  
Extracellular Loop ◽  
Short Term ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Junctional Protein ◽  
Gap Junctional

Because hepatocytes communicate via gap junctions, it has been proposed that Ca2+waves propagate through this pathway and in the process activate Ca2+-dependent cellular responses. We tested this hypothesis by measuring vasopressin-induced glycogenolysis in short-term cultures of rat hepatocytes. A 15-min vasopressin (10−8 M) stimulation induced a reduction of glycogen content that reached a maximum 1–3 h later. Gap junction blockers, octanol or 18α-glycyrrhetinic acid, reduced the effect by 70%. The glycogenolytic response induced by Ca2+ ionophore 8-bromo-A-21387, which acts on each hepatocyte, was not affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a synthetic peptide homologous to a domain of the extracellular loop 1 of the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced. Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP as a paracrine mediator. We propose that gap junctions allow for a coordinated vasopressin-induced glycogenolytic response despite the heterogeneity among hepatocytes.

scholarly journals Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763 ◽  
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

An inhibition of gap-junctional communication by cadherins

1997 ◽  
Vol 110 (3) ◽  
pp. 301-309 ◽  
Author(s):  
Y. Wang ◽  
B. Rose
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Hepatoma Cells ◽  
Cell Type ◽  
Gap Junctional Communication ◽  
L Cells ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

The action of Ca(2+)-dependent cell-cell adhesion molecules (cadherins) on cell-to-cell channel-mediated intercellular communication was investigated in mouse L and rat Morris hepatoma cells. These cells fail to adhere to one another in aggregation assays and thus seem to lack cell adhesion molecules. Expression of exogenous cadherin induced strong cell-cell adhesion in both cell types, but had opposite effects of communication, causing inhibition in L cells and improvement in hepatoma cells. Both cells express the connexin43 cell-to-cell channel protein. By western blot we found no cadherin-specific changes in connexin43 protein in either cell type, but connexin43 gap junctional plaque staining, i.e. connexin43 localization to cell-cell junctions, was inhibited in L cells and facilitated in hepatoma cells. In addition we found that the inhibitory effect is largely abolished by blockers of glycosylation. Cadherin-cadherin interactions are known to trigger cell type-specific intracellular signal cascades resulting in diverse end effects, and gap junctional communication/plaque formation seems a further example of such cell type-specificity.

scholarly journals Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells.

1995 ◽  
Vol 130 (2) ◽  
pp. 419-429 ◽  
Author(s):  
R Sullivan ◽  
C W Lo
Keyword(s):  
Fusion Protein ◽  
Gap Junctions ◽  
Connexin 43 ◽  
Dominant Negative Effect ◽  
Cell Contact ◽  
Nih3t3 Cells ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Cell Cell

Gap junctions contain membrane channels that mediate the cell-to-cell movement of ions, metabolites and cell signaling molecules. As gap junctions are comprised of a hexameric array of connexin polypeptides, the expression of a mutant connexin polypeptide may exert a dominant negative effect on gap junctional communication. To examine this possibility, we constructed a connexin 43 (Cx43)/beta-galactosidase (beta-gal) expression vector in which the bacterial beta-gal protein is fused in frame to the carboxy terminus of Cx43. This vector was transfected into NIH3T3 cells, a cell line which is well coupled via gap junctions and expresses high levels of Cx43. Transfectant clones were shown to express the fusion protein by northern and western analysis. X-Gal staining further revealed that all of the fusion protein containing cells also expressed beta-gal enzymatic activity. Double immunostaining with a beta-gal and Cx43 antibody demonstrated that the fusion protein is immunolocalized to the perinuclear region of the cytoplasm and also as punctate spots at regions of cell-cell contact. This pattern is similar to that of Cx43 in the parental 3T3 cells, except that in the fusion protein expressing cells, Cx43 expression was reduced at regions of cell-cell contact. Examination of gap junctional communication (GJC) with dye injection studies further showed that dye coupling was inhibited in the fusion protein expressing cells, with the largest reduction in coupling found in a clone exhibiting little Cx43 localization at regions of cell-cell contact. When the fusion protein expression vector was transfected into the communication poor C6 cell line, abundant fusion protein expression was observed, but unlike the transfected NIH3T3 cells, no fusion protein was detected at the cell surface. Nevertheless, dye coupling was inhibited in these C6 cells. Based on these observations, we propose that the fusion protein may inhibit GJC by sequestering the Cx43 protein intracellularly. Overall, these results demonstrate that the Cx43/beta-gal fusion protein can exert a dominant negative effect on GJC in two different cell types, and suggests that it may serve as a useful approach for probing the biological function of gap junctions.

The relationship of gap junctions and compaction in the preimplantation mouse embryo

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 113-118
Author(s):  
David L. Becker ◽  
Catherine Leclerc-David ◽  
Anne Warner
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Mouse Embryo ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Gap Junctional Communication ◽  
Stage Of Development ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional

In the mouse embryo, gap junctions first appear at the 8-cell stage as compaction is about to take place. Compaction of the embryo is important for the differentiation of the first two cell types; the inner cell mass and the trophectoderm. Our studies examine the contribution of gap junctional communication at this stage of development We have characterised the normal sequence of appearance of gap junction protein and its distribution. The extent of communication as shown by the passage of dye between cells has been recorded in both normal embryos and embryos treated with drugs that influence gap junctional communication. Comparisons have been made with embryos that express a lethal gap junction defect and attempts were made to rescue such embryos by increasing their gap junction communication.

Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4703-4714 ◽  
Author(s):  
M. Levin ◽  
M. Mercola
Keyword(s):  
Gene Expression ◽  
Gap Junction ◽  
Large Scale ◽  
Chick Blastoderm ◽  
Gap Junction Channels ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Left And Right ◽  
The Right ◽  
Gap Junctional

Invariant patterning of left-right asymmetry during embryogenesis depends upon a cascade of inductive and repressive interactions between asymmetrically expressed genes. Different cascades of asymmetric genes distinguish the left and right sides of the embryo and are maintained by a midline barrier. As such, the left and right sides of an embryo can be viewed as distinct and autonomous fields. Here we describe a series of experiments that indicate that the initiation of these programs requires communication between the two sides of the blastoderm. When deprived of either the left or the right lateral halves of the blastoderm, embryos are incapable of patterning normal left-right gene expression at Hensen's node. Not only are both flanks required, suggesting that there is no single signaling source for LR pattern, but the blastoderm must be intact. These results are consistent with our previously proposed model in which the orientation of LR asymmetry in the frog, Xenopus laevis, depends on large-scale partitioning of LR determinants through intercellular gap junction channels (M. Levin and M. Mercola (1998) Developmental Biology 203, 90–105). Here we evaluate whether gap junctional communication is required for the LR asymmetry in the chick, where it is possible to order early events relative to the well-characterized left and right hierarchies of gene expression. Treatment of cultured chick embryos with lindane, which diminishes gap junctional communication, frequently unbiased normal LR asymmetry of Shh and Nodal gene expression, causing the normally left-sided program to be recapitulated symmetrically on the right side of the embryo. A survey of early expression of connexin mRNAs revealed that Cx43 is present throughout the blastoderm at Hamburger-Hamilton stage 2–3, prior to known asymmetric gene expression. Application of antisense oligodeoxynucleotides or blocking antibody to cultured embryos also resulted in bilateral expression of Shh and Nodal transcripts. Importantly, the node and primitive streak at these stages lack Cx43 mRNA. This result, together with the requirement for an intact blastoderm, suggests that the path of communication through gap junction channels circumvents the node and streak. We propose that left-right information is transferred unidirectionally throughout the epiblast by gap junction channels in order to pattern left-sided Shh expression at Hensen's node.

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