Flow cytometry analysis of gap junction-mediated cell–cell communication: Advantages and pitfalls

The gap junction protein, connexin43 (Cx43), plays an important role in skeletal biology. Previously, we have shown that Cx43 can enhance the signaling and transcriptional response to fibroblast growth factor 2 (FGF2) in osteoblasts by increasing protein kinase C-δ (PKCδ) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKCδ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKCδ-independent activation of ERK by FGF2 was confirmed by Western blotting, as was the Cx43-dependent enhancement of ERK activation. Consistent with our prior observations for PKCδ, flow cytometry analyses show that Cx43 overexpression enhances the percentage of phospho-ERK-positive cells in response to FGF2, supporting the notion that shared signals among gap junction-coupled cells result in the enhanced response to FGF2. Western blots and luciferase reporter assays performed on osteoblasts cultured under low-density and high-density conditions revealed that cell-cell contacts are required for Cx43 to amplify ERK activation and gene transcription. Similarly, inhibition of gap junctional communication with the channel blocker 18β-glycyrrhetinic acid attenuates the Cx43-dependent enhancement of Runx2-transcriptional activity. In total, these data underscore the importance of cell-cell communication and activation of the ERK and PKCδ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts.

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Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell–Cell Communication?

International Journal of Molecular Sciences ◽

10.3390/ijms22179169 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9169

Author(s):

Camillo Peracchia

Keyword(s):

Gap Junction ◽

Genetic Diseases ◽

Channel Gating ◽

Cell Communication ◽

Potential Effect ◽

Gap Junction Channel ◽

Direct Cell ◽

Connexin Genes ◽

Cell Cell

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

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Abstract 13799: Electrical Pacing Inhibits Gap Junction-Mediated Cardiac Cell -Cell Communication by Promoting Cx43-Acetylation

Circulation ◽

10.1161/circ.130.suppl_2.13799 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Valerio Azzimato ◽

Viviana Meraviglia ◽

Claudia Colussi ◽

Maria Cristina Florio ◽

Alice Panariti ◽

...

Keyword(s):

Electrical Stimulation ◽

Gap Junction ◽

Cell Communication ◽

Neonatal Rat ◽

Cell Protein ◽

Cardiac Cell ◽

Cx43 Expression ◽

Rat Cardiomyocytes ◽

Electrical Pacing ◽

Cell Cell

Background: Communication among cardiomyocytes depends upon Gap Junction (GJ) protein expression and conductance. Previous studies demonstrated that electrical stimulation can induce GJ remodeling and evidences from neurons also indicate that electrical pacing modifies Lysine acetylase (KAT) and deacetylases (KDAC) activities. Objectives: Aim of the present work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation dependent mechanisms. Methods and Results: Neonatal rat cardiomyocytes (NRCM; n=3) and in HL-1 atrial cells (n=20) were exposed to electrical field stimulation for 24 hours (Ionoptix C-Pace®; 0.5 Hz, 20 V, 0.5 msec pulses). Connexin 43 (Cx43) expression decreased almost 50% in NRCM and 40 % in HL-1; in contrast Cx40 and Cx45 expression was unchanged. Further, confocal microscopy revealed that electrical stimulation induced Cx43 accumulation in the cytoplasm of HL-1 cells. Electrical stimulation significatly down-regulated KDAC activity up to the 30% (n=3), whereas KAT activity was not modified; the net effect was a general increase of cell protein acetylation, confirmed by western blot analysis. Specifically, the pacing-dependent acetylation of Cx43 was proven by immunoprecipitation assay (n=5). Interestingly, our model mimicked the action of the KDAC pan-inhibitors TSA and SAHA on Cx43 expression and intracellular distribution, although we did not observe Cx43 mRNA significant reduction in electrically stimulated cells. In agreement, MG132 proteasome inhibitor (10 μM) restored Cx43 expression level. Finally, also the treatment of paced cells with the KAT inhibitor Anacardic Acid (0.5 μM) was able to rescue Cx43 level (n=4). Intriguingly, preliminary results also indicate lateralization and increased acetylation of Cx43 in the left ventricles of dogs with pacing-induced dilated cardiomyopathy (n=2). Conclusions: In vitro electrical stimulation of cardiac cells promotes Cx43 acetylation, which results in Cx43 down-modulation and intracellular relocalization. These findings suggest that electrical activity-dependent increase in acetylation may represent a novel mechanism for the regulation of cardiomyocyte communication.

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Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src

The Journal of Cell Biology ◽

10.1083/jcb.140.5.1199 ◽

1998 ◽

Vol 140 (5) ◽

pp. 1199-1209 ◽

Cited By ~ 84

Author(s):

Friso R. Postma ◽

Trudi Hengeveld ◽

Jacqueline Alblas ◽

Ben N.G. Giepmans ◽

Gerben C.M. Zondag ◽

...

Keyword(s):

Protein Kinase ◽

Gap Junction ◽

G Protein ◽

Critical Role ◽

Cell Communication ◽

G Protein Coupled Receptors ◽

G Protein Coupled Receptor ◽

Junction Protein ◽

G Protein Coupled ◽

Cell Cell

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca2+, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.

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Substrate Mechanics Dictates Cell-Cell Communication via Gap Junction in Stem Cells from Human Apical Papilla

SSRN Electronic Journal ◽

10.2139/ssrn.3490422 ◽

2019 ◽

Author(s):

Chenchen Zhou ◽

Demao Zhang ◽

Wei Du ◽

Jing Zou ◽

Xiaobing Li ◽

...

Keyword(s):

Stem Cells ◽

Gap Junction ◽

Cell Communication ◽

Apical Papilla ◽

Cell Cell

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Osteogenic Gene Transcription Is Regulated via Gap Junction-Mediated Cell–Cell Communication

Stem Cells and Development ◽

10.1089/scd.2014.0060 ◽

2015 ◽

Vol 24 (2) ◽

pp. 214-227 ◽

Cited By ~ 7

Author(s):

Yoshikazu Mikami ◽

Kiyofumi Yamamoto ◽

Yuko Akiyama ◽

Masayuki Kobayashi ◽

Eri Watanabe ◽

...

Keyword(s):

Gap Junction ◽

Gene Transcription ◽

Cell Communication ◽

Osteogenic Gene ◽

Cell Cell

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Regulation of osteogenic gene transcription via gap junction-mediated cell-cell communication

Journal of Oral and Maxillofacial Surgery ◽

10.1016/j.joms.2014.06.381 ◽

2014 ◽

Vol 72 (9) ◽

pp. e212-e213

Author(s):

Y. Mikami ◽

T. Kitano ◽

M. Asano ◽

K. Komiyama

Keyword(s):

Gap Junction ◽

Gene Transcription ◽

Cell Communication ◽

Osteogenic Gene ◽

Cell Cell

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Arachidonic acid amide inhibitors of gap junction cell-cell communication

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/s0960-894x(99)00148-1 ◽

1999 ◽

Vol 9 (8) ◽

pp. 1151-1154 ◽

Cited By ~ 24

Author(s):

Dale L. Boger ◽

Haruhiko Sato ◽

Aaron E. Lerner ◽

Xiaojun Guan ◽

Norton B. Gilula

Keyword(s):

Arachidonic Acid ◽

Gap Junction ◽

Acid Amide ◽

Cell Communication ◽

Cell Cell

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Modulation of gap junction expression during transient hyperplasia of rat epidermis

Journal of Cell Science ◽

10.1242/jcs.111.10.1395 ◽

1998 ◽

Vol 111 (10) ◽

pp. 1395-1404

Author(s):

B. Risek ◽

A. Pozzi ◽

N.B. Gilula

Keyword(s):

Gap Junction ◽

Growth Response ◽

Phorbol Esters ◽

Expression Patterns ◽

Cell Communication ◽

Nuclear Antigen ◽

Epidermal Keratinocytes ◽

Connexin Expression ◽

Proliferation And Differentiation ◽

Cell Cell

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of alpha1 (Cx43) and beta2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, alpha1 is expressed in the lower, less differentiated portion, while beta2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in alpha1 and beta2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with beta2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in alpha1 and beta2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.

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Gap junction internalization and processing in vivo: a 3D immuno-electron microscopy study

10.1101/2020.06.29.178475 ◽

2020 ◽

Author(s):

Rachael P. Norris ◽

Mark Terasaki

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Connexin 43 ◽

Cell Communication ◽

Membrane Vesicles ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Annular Gap ◽

Cell Cell

AbstractGap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell, called connexosomes or annular gap junctions. Here, we systematically studied the fate of connexosomes in intact ovarian follicles. High pressure frozen, serial sectioned tissue was immunogold labeled for Connexin 43. Within a volume of electron micrographs, every labeled structure was categorized and counted. Surface area measurements indicate that large connexosomes undergo fission. Subsequent modifications are separation of inner and outer membranes, loss of Cx43 from the outer membrane, and outward budding of the modified membranes. We also documented several clear examples of organelle transfer from one cell to another by gap junction internalization. We discuss how connexosome formation and processing may be a novel means for gap junctions to mediate cell-cell communication.

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