Connexin Mutants Compromise the Lens Circulation and Cause Cataracts through Biomineralization

Gap junction-mediated intercellular communication facilitates the circulation of ions, small molecules, and metabolites in the avascular eye lens. Mutants of the lens fiber cell gap junction proteins, connexin46 (Cx46) and connexin50 (Cx50), cause cataracts in people and in mice. Studies in mouse models have begun to elucidate the mechanisms by which these mutants lead to cataracts. The expression of the dominant mutants causes severe decreases in connexin levels, reducing the gap junctional communication between lens fiber cells and compromising the lens circulation. The impairment of the lens circulation results in several changes, including the accumulation of Ca2+ in central lens regions, leading to the formation of precipitates that stain with Alizarin red. The cataract morphology and the distribution of Alizarin red-stained material are similar, suggesting that the cataracts result from biomineralization within the organ. In this review, we suggest that this may be a general process for the formation of cataracts of different etiologies.

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

Development ◽

10.1242/dev.126.21.4703 ◽

1999 ◽

Vol 126 (21) ◽

pp. 4703-4714 ◽

Cited By ~ 8

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

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1754-1763.1990 ◽

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.

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Distribution, propagation, and coordination of contractile activity in lymphatics

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.4.h1283 ◽

1993 ◽

Vol 264 (4) ◽

pp. H1283-H1291 ◽

Cited By ~ 47

Author(s):

D. C. Zawieja ◽

K. L. Davis ◽

R. Schuster ◽

W. M. Hinds ◽

H. J. Granger

Keyword(s):

Gap Junction ◽

Conduction Velocity ◽

Contractile Activity ◽

Lymph Flow ◽

Rat Small Intestine ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Gap Junctional ◽

Flow Stream

The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent lymphangions in spontaneously contracting lymphatics (n = 51). Diameter index, contraction frequency, and the percentage of the intersegmental contractions that were propagated and coordinated (PP) were determined at both sites. The conduction velocity of the contractile activity and the percentage of the coordinated contractions that were propagated both antegrade to the direction of lymph flow and retrograde to the flow stream were determined. The results indicate that 1) 80-90% of the lymphatic contractions in the vessels we evaluated were propagated, 2) the wave of contractile activity propagated both centrally and peripherally, and 3) the conduction velocity of the contractile activity was approximately 4-8 mm/s. We tested the hypothesis that gap junctional communication is responsible for the coordination of the contractile event. To accomplish this, we used the gap junction blockers n-heptanol and oleic acid. PP was 90 +/- 4% under normal conditions and fell to a minimum value of 55 +/- 7% during the gap junction blockade. These results indicate that gap junctional communication played an important role in the propagation and coordination of contractions that occurred in spontaneously active lymphatics.

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The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

The Journal of Cell Biology ◽

10.1083/jcb.139.7.1785 ◽

1997 ◽

Vol 139 (7) ◽

pp. 1785-1792 ◽

Cited By ~ 174

Author(s):

Xiaojun Guan ◽

Benjamin F. Cravatt ◽

George R. Ehring ◽

James E. Hall ◽

Dale L. Boger ◽

...

Keyword(s):

Gap Junction ◽

Glial Cells ◽

Wave Transmission ◽

Calcium Wave ◽

Mediated Communication ◽

Gap Junctional Communication ◽

Gap Junction Communication ◽

Junctional Communication ◽

Nervous System Function ◽

Gap Junctional

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

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

The Journal of Cell Biology ◽

10.1083/jcb.111.5.2077 ◽

1990 ◽

Vol 111 (5) ◽

pp. 2077-2088 ◽

Cited By ~ 477

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.

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Mechanisms for the coordination of intercellular calcium signaling in insulin-secreting cells

Journal of Cell Science ◽

10.1242/jcs.110.4.497 ◽

1997 ◽

Vol 110 (4) ◽

pp. 497-504 ◽

Cited By ~ 2

Author(s):

D. Cao ◽

G. Lin ◽

E.M. Westphale ◽

E.C. Beyer ◽

T.H. Steinberg

Keyword(s):

Gap Junction ◽

Islet Cells ◽

Cytosolic Calcium ◽

Calcium Waves ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Junction Protein ◽

Insulin Secreting Cells ◽

Gap Junction Protein ◽

Gap Junctional

Insulin-mediated increases in cytosolic calcium are synchronized among the cells in a pancreatic islet, and result in pulsatile secretion of insulin. Pancreatic beta cells express the gap junction protein connexin43 and are functionally coupled, making gap junctional communication a likely mechanism for the synchronization of calcium transients among islet cells. To define the mechanism by which pancreatic islet cells coordinate calcium responses, we studied mechanically-induced intercellular calcium waves in the communication-deficient rat insulinoma cell line RINm5f, and in RINm5f cells transfected with the gap junction protein connexin43. Both RINm5f and RINm5f cells transfected with connexin43 propagated calcium waves that required release of calcium from intracellular stores, did not involve gap junctional communication, and appeared to be mediated by autocrine activity of secreted ATP acting on P2U purinergic receptors. Connexin43 transfectants also propagated calcium waves that required gap junctional communication and influx of extracellular calcium through voltage-gated calcium channels. Gap junction-dependent intercellular calcium waves were inhibited by preventing plasma membrane depolarization. These studies demonstrate two distinct pathways by which insulin-secreting cells can coordinate cytosolic calcium rises, and show that it is by ionic traffic that gap junctions synchronize calcium-dependent events in these cells.

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Transforming Growth Factor-β3 Increases Gap-Junctional Communication among Folliculostellate Cells to Release Basic Fibroblast Growth Factor

Endocrinology ◽

10.1210/en.2005-0122 ◽

2005 ◽

Vol 146 (9) ◽

pp. 4054-4060 ◽

Cited By ~ 12

Author(s):

Nurul Kabir ◽

Kirti Chaturvedi ◽

Lian Sheng Liu ◽

Dipak K. Sarkar

Keyword(s):

Growth Factor ◽

Gap Junction ◽

Fibroblast Growth Factor ◽

Basic Fibroblast Growth Factor ◽

Connexin 43 ◽

Fibroblast Growth ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Gap Junctional ◽

Gap Junction Inhibitor

Abstract Folliculostellate (FS) cells are known to communicate with each other and with endocrine cells via gap junctions in the anterior pituitary. We investigated whether TGFβ3 and estradiol, known to regulate FS cell production and secretion of basic fibroblast growth factor (bFGF), increases gap junctional communication to alter bFGF secretion from FS cells. FS cells in monolayer cultures were treated with TGFβ3 or vehicle alone for 24 h and then microinjected with Lucifer Yellow and high-molecular-weight Texas Red dextran. Ten minutes later the transfer of dye among adjacent cells was recorded with a digital microscope. TGFβ3 increased the transfer of dye. The TGFβ3-neutralizing antibody and the gap junction inhibitor octanol reduced the effect of TGFβ3 on the transfer of dye. The TGFβ3-induced transfer of dye was unaltered by simultaneous treatment with estradiol. The steroid alone also had no effect. TGFβ3 increased total and phosphorylated levels of connexin 43. Estradiol treatment did not produce any significant changes on basal or TGFβ3-induced increases in connexin 43 levels. The gap-junction inhibitor octanol reduced TGFβ3-increased levels of bFGF in FS cells. Taken together, these results suggest that TGFβ3 may act on FS cells to increase gap-junctional communication to maximize its effect on bFGF secretion.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-04-0339 ◽

2005 ◽

Vol 16 (1) ◽

pp. 64-72 ◽

Cited By ~ 89

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.

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Shapes of astrocyte networks in the juvenile brain

Neuron Glia Biology ◽

10.1017/s1740925x06000081 ◽

2006 ◽

Vol 2 (1) ◽

pp. 3-14 ◽

Cited By ~ 65

Author(s):

VANESSA HOUADES ◽

NATHALIE ROUACH ◽

PASCAL EZAN ◽

FRANK KIRCHHOFF ◽

ANNETTE KOULAKOFF ◽

...

Keyword(s):

Gap Junction ◽

Brain Function ◽

Connexin 43 ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Junction Protein ◽

Gap Junction Protein ◽

High Level ◽

Gap Junctional ◽

Neuronal Compartments

The high level of intercellular communication mediated by gap junctions between astrocytes indicates that, besides individual astrocytic domains, a second level of organization might exist for these glial cells as they form communicating networks. Therefore, the contribution of astrocytes to brain function should also be considered to result from coordinated groups of cells. To evaluate the shape and extent of these networks we have studied the expression of connexin 43, a major gap junction protein in astrocytes, and the intercellular diffusion of gap junction tracers in two structures of the developing brain, the hippocampus and the cerebral cortex. We report that the shape of astrocytic networks depends on their location within neuronal compartments in a defined brain structure. Interestingly, not all astrocytes are coupled, which indicates that connections within these networks are restricted. As gap junctional communication in astrocytes is reported to contribute to several glial functions, differences in the shape of astrocytic networks might have consequences on neuronal activity and survival.

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