Relative contribution of cell contact pattern, specific PKC isoforms and gap junctional communication in tight junction assembly in the mouse early embryo

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

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103 GAP JUNCTIONAL INTERCELLULAR COMMUNICATION IS DISPENSIBLE DURING REGULATION OF TIGHT JUNCTION MEMBRANE ASSEMBLY BY CELL CONTACT PATTERN AND PKC SIGNALING

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab103 ◽

2005 ◽

Vol 17 (2) ◽

pp. 202

Author(s):

J. Eckert ◽

A. Mears ◽

I. Cameron ◽

T. Fleming

Keyword(s):

Tight Junction ◽

Intercellular Communication ◽

Cell Contact ◽

Contact Pattern ◽

Gap Junctional Intercellular Communication ◽

Membrane Assembly ◽

Junctional Proteins ◽

Pkc Signaling ◽

Gap Junctional

Contact symmetries are involved in regulating cell lineage segregation during blastocyst biogenesis when tight junction (TJ) membrane assembly is restricted to the epithelial trophectoderm (TE). Manipulation of cell contact patterns by immunosurgical isolation of inner cell masses (ICMs) providing a contact-free cell surface serves as a switch to induce TE differentiation upon in vitro culture. In this model, protein kinase C (PKC)-mediated signaling up-regulates TJ membrane assembly. Whether signaling via gap junctional intercellular communication (GJIC) affects these processes is controversial. The current study investigates the interrelationship between changes in cell contact pattern, PKC signaling, and GJIC on TE differentiation and TJ assembly. Eight-cell embryos flushed from MF1 mice were cultured in T6/BSA to time development to early blastocyst stage (<2 h of cavitation). Laser confocal microscopy (BioRad MRC 600, BioRad Laboratories, Inc., Hertfordshire, UK) after immunostaining with antibodies against PKCδ, θ, λ, or ζ isoforms (Transduction Labs, Oxford, UK or Sigma) or junctional proteins (E-cadherin, ZO-2, Occludin, ZO-1α+, Desmoplakin) combined with ALEXA 488 conjugated secondary antibodies (Cambridge Bioscience, Oxford, UK) was used to determine the distribution of PKCs and junctional proteins in intact blastocysts and fully and partially isolated ICMs after immunosurgery and in vitro culture in DMEM + 10% FCS (Eckert et al. 2004 Reproduction 127, 653). While broad PKC activators (1 μM 12-O-tetradecanoylphorbol-13-acetate or Indolactam; Calbiochem, Nottingham, UK) accelerate membrane assembly of the TJ proteins ZO-2 and ZO-1α+ in fully isolated ICMs this up-regulation was suppressed in intact blastocysts (n = 32–47 per treatment and antibody) and in partially isolated ICMs (remnants of lysed TE remaining surrounding the ICM; n = 17–21 per treatment and antibody) for up to 24 h with no TJ protein detectable within the ICM, even after two consecutive rounds of TE lysis (n = 13–22 per treatment and antibody). When GJIC was inhibited during blastocyst formation in vitro and in cultured fully isolatd ICMs by 18 α-glycyrrhetinic acid (AGA, 65 μM; Sigma), cavitation rate and distribution of PKCs or junction assembly were not affected compared to controls (70–80% cavitated with characteristic distribution of junctional proteins and PKCs; P > 0.05, ANOVA; n = 15–20 per treatment, antibody, and cell contact pattern). When GJIC inhibition by AGA was confirmed by Lucifer yellow (Sigma) injection (no dye transfer in 82–100%, n = 14–17 per contact pattern), GJIC was also absent in 50% of fully isolated ICMs without AGA treatment, suggesting that cell contact modulation may affect GJIC. Taken together, our data suggest that cell contact pattern regulates TJ assembly via PKC signaling pathways and may also affect GJIC. GJIC appeared dispensable during cavitation, TJ assembly, and PKC signaling. A better understanding of the interrelationships between different signaling mechanisms may help to improve embryo culture methods and viability. Funding by the Wellcome Trust and MRC is gratefully acknowledged.

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Cell-cell interactions in regulating lung function

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00172.2004 ◽

2004 ◽

Vol 287 (3) ◽

pp. L455-L459 ◽

Cited By ~ 26

Author(s):

Scott Boitano ◽

Zeenat Safdar ◽

Donald G. Welsh ◽

Jahar Bhattacharya ◽

Michael Koval

Keyword(s):

Barrier Function ◽

Cell Contact ◽

Permeability Barrier ◽

Washington Dc ◽

Gap Junctional Communication ◽

Metabolic Coupling ◽

Junctional Communication ◽

Barrier Formation ◽

Gap Junctional ◽

Cell Cell

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.

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Role of gap junctions during early embryo development

Reproduction ◽

10.1530/rep.1.00277 ◽

2005 ◽

Vol 129 (2) ◽

pp. 129-135 ◽

Cited By ~ 63

Author(s):

Franchesca D Houghton

Keyword(s):

Gap Junctions ◽

Embryo Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Early Embryo ◽

Preimplantation Development ◽

Gap Junctional Communication ◽

Junctional Communication ◽

Gap Junctional

Gap junctional communication plays a central role in the maintenance of cellular homeostasis by allowing the passage of small molecules between adjacent cells. Gap junctions are composed of a family of proteins termed connexins. During preimplantation development several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. Although, many of the connexin genes have been disrupted using homologous recombination in embryonic stem cells to obtain unique phenotypes, none of these studies has demonstrated a specific role for connexins during preimplantation development in the null mutants. This review surveys evidence for the involvement of gap junctional communication during embryo development highlighting discrepancies in the literature. Although some evidence suggests that gap junctions may be dispensable during preimplantation development this is difficult to envisage particularly for the process of cavitation and the maintenance of homeostasis between the differentiated trophectoderm cells and the pluripotent inner cell mass cells of the blastocyst.

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Manipulation of gap junctional communication during compaction of the mouse early embryo

Development ◽

10.1242/dev.91.1.283 ◽

1986 ◽

Vol 91 (1) ◽

pp. 283-296

Author(s):

Harry Goodall

Keyword(s):

Cell Adhesion ◽

Early Embryo ◽

Embryonal Carcinoma Cell ◽

Cell Coupling ◽

Calcium Dependent ◽

Gap Junctional Communication ◽

Embryonal Carcinoma Cell Line ◽

Junctional Communication ◽

Junctional Coupling ◽

Gap Junctional

Three treatments that prevent cell flattening during compaction of the mouse preimplantation embryo were assessed for their effects on the onset of gap junctional communication. Medium low in calcium (LCM) and an antiserum to an embryonal carcinoma cell line (anti-EC; Johnson et al. 1979) both prevented the establishment of coupling between blastomeres of the 8-cell embryo as assessed by transmission of carboxyfluorescein or by ionic coupling. Since neither of these agents prevents the contact-mediated induction of cell polarity that occurs at this stage, it is concluded that the induction of this process is not signalled via gap junctions. A monoclonal antibody (ECCD-1; Yoshida-Noro, Suzuki & Takeichi, 1984), that recognizes more specific components of the calcium-dependent cell adhesion system, failed to prevent the onset of junctional coupling. This suggests that the onset of junctional coupling is not dependent upon extensive cell apposition and that the requirement for extracellular Ca2+ resides at a level other than that of cell adhesion. Moreover, neither LCM nor anti-EC could reverse cell coupling once it had become established despite their complete reversal of cell flattening.

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

The Journal of Cell Biology ◽

10.1083/jcb.130.2.419 ◽

1995 ◽

Vol 130 (2) ◽

pp. 419-429 ◽

Cited By ~ 39

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.

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