Mechanism of inhibition of junctional communication between animal cells by phorbol ester

Abstract. The tumour promotor 4 β -phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

Mechanism of inhibition of junctional communication between animal cells by phorbol ester

The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.     

The effects of connexin phosphorylation on gap junctional communication

Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34(cdc2)/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60(src) kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.     

Effects of neuroinflammation on glia-glia gap junctional intercellular communication: a perspective

Gap junctions serve as intercellular conduits that allow for the direct transfer of small molecular weight molecules (up to 1 kDa) including ions involved in cellular excitability, metabolic precursors, and second messengers. The observation of extensive intercellular coupling and large numbers of gap junctions in the central nervous system (CNS) suggests a syncytium-like organization of glial compartments. Inflammation is a hallmark of various CNS diseases such as bacterial and viral infections, multiple sclerosis, Alzheimer's disease, and cerebral ischemia. A general consequence of brain inflammation is reactive gliosis typified by astrocyte hypertrophy and proliferation of astrocytes and microglia. Changes in gap junction intercellular communication as reflected by alterations in dye coupling and connexin expression have been associated with numerous CNS inflammatory diseases, which may have dramatic implications on the survival of neuronal and glial populations in the context of neuroinflammation. A review of the effects of inflammatory products on glia-glia gap junctional communication and glial glutamate release is presented. In addition, the hypothesis of a "syncytial switch" based upon differential regulation of gap junction expression in astrocytes and microglia during normal CNS homeostasis and neuroinflammation is proposed.     

Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes

Innumerable toxic substances present in the environment inhibit gap junctions, intercellular membrane channels that play fundamental roles in coordinated function of cells and tissues. Included are persistent organochlorine compounds, which pose health risks to humans and animals owing to their widespread use, bioaccumulation, and ability to inhibit gap junction channel-mediated intercellular communication in liver, lung, skin, heart, and brain cells. In this study, the organochlorine xenobiotics dieldrin and endosulfan, at micromolar concentrations, were found to inhibit gap junction-mediated intercellular communication and induce hypophosphorylation of connexin 43 in cultured rat astrocytes, the predominant cell type in the brain coupled through gap junctions. This inhibition of gap junctional communication was substantially reduced by preincubation with chaetoglobosin K (ChK), a bioactive natural produce previously shown to have ras tumor suppressor activity. Chaetoglobosin K also prevented dieldrin and endosulfan-induced hypophosphorylation of connexin 43 and prevented dieldrin-induced connexin 43 plaque dissolution in both astrocytes and cultured liver epithelial cells. The results suggest that stabilization of the native, phosphorylated form of connexin 43 by ChK may contribute to its ability to prevent organochlorine-induced inhibition of gap junction-mediated communication and dissolution of gap junction plaques within the plasma membrane. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pharmacological evidence for system-dependent involvement of protein kinase C isoenzymes in phorbol ester-suppressed gap junctional communication

Several phorbol esters are potent activators of protein kinase C. They down-regulate gap junctional intercellular communication and induce phosphorylation of connexin43, but the sensitivity and extent of responses vary much between systems. We asked whether the total protein kinase C enzyme activity or the protein kinase C isoenzyme constitution was of importance for such variations. Some fibroblastic culture systems were compared. It was concluded that the total protein kinase C enzyme activity did not determine the sensitivity to phorbol esters. Furthermore, the use of isotype-specific inhibitors of protein kinase C indicated that protein kinase C alpha, delta, and epsilon may be involved to different extents in different fibroblastic systems in the response to phorbol esters.     

Involvement of gap junctional communication in secretion

Glands were the first type of tissues in which the permissive role of gap junctions in the cell-to-cell transfer of membrane-impermeant molecules was shown. During the 40 years that have followed this seminal finding, gap junctions have been documented in all types of multicellular secretory systems, whether of the exocrine, endocrine or pheromonal nature. Also, compelling evidence now indicates that gap junction-mediated coupling, and/or the connexin proteins per se, play significant regulatory roles in various aspects of gland functions, ranging from the biosynthesis, storage and release of a variety of secretory products, to the control of the growth and differentiation of secretory cells, and to the regulation of gland morphogenesis. This review summarizes this evidence in the light of recent reports.     

Dissociation of PDGF receptor tyrosine kinase activity from PDGF-mediated inhibition of gap junctional communication

Gap junction-mediated intercellular communication (GJC) may play an important role in cell proliferation and transformation since GJC is inhibited by growth factors, oncogenes, tumor promoters, and carcinogens. We have studied inhibition of GJC by platelet-derived growth factor-BB (PDGF) in the mouse fibroblast cell line C3H/10T1/2 and have sought to determine whether PDGF-induced inhibition of GJC is mediated by the PDGF receptor tyrosine kinase (RTK). PDGF-mediated inhibition of GJC was rapid and transient, with maximal inhibition occurring 40 min after PDGF addition and GJC returning to control levels after 70 min. The effect of PDGF on GJC was concentration-dependent, with maximal inhibition of 90% or greater occurring at 10 ng/ml PDGF. Stimulation of RTK activity, as determined by antiphosphotyrosine immunoblot analysis of PDGF receptor and the receptor substrates phospholipase C-γl (PLC-γl) and guanosine triphosphatase activating protein (GAP), was also concentration-dependent. Inhibition of GJC required a greater concentration of PDGF than did stimulation of RTK activity. The tyrosine kinase inhibitor genistein blocked PDGF-induced RTK activity, as measured by PDGF receptor, PLC-γl, and GAP tyrosine phosphorylation, in a concentration-dependent manner but did not affect PDGF-mediated inhibition of GJC. Genistein alone had no effect on GJC or PDGF receptor expression. PDGF treatment in the presence or absence of genistein resulted in phosphorylation of the connexin 43 protein on nontyrosine residues. These results suggest that inhibition of GJC by ligand-activated PDGF receptor is dissociable from the RTK activity responsible for PDGF, PLC-γl, and GAP phosphorylation. © 1994 Wiley-Liss, Inc.     

Retinaldehyde, a potent inhibitor of gap junctional intercellular communication

Retinaldehyde and retinoic acid are derivatives of vitamin A, and retinaldehyde is the precursor for the synthesis of retinoic acid, a well-known inhibitor of gap junctional intercellular communication. In this investigation, we asked the question if retinaldehyde has similar effects on gap junctions. Gap junctional intercellular communication was measured by scrape-loading and preloading dye-transfer methods, and studies were carried out mainly on cultured liver epithelial cells. Retinaldehyde was found to be a more potent inhibitor (dye transfer reduced by 50% at 2.8 μM) than retinoic acid (dye transfer reduced by 50% at 30 μM) and glycyrrhetinic acid (dye transfer reduced by 50% at 65 μM). Both the 11-cis and all-trans forms of retinaldehyde were equally effective. Retinaldehyde inhibited dye transfer of both anionic Lucifer yellow and cationic Neurobiotin. Inhibition by retinaldehyde developed in less than two minutes at 50 μM, but unlike the reported case with retinoic acid, recovery was slower, though full. In addition to liver epithelial cells, retinaldehyde inhibited gap junctional communication in lens epithelial cells, retinal pigment epithelial cells and retinal ganglion cells.     

Reversal of the TPA-induced inhibition of gap junctional intercellular communication by Chaga mushroom (Inonotus obliquus) extracts: effects on MAP kinases

Chaga mushroom (Inonotus obliquus) has continued to receive attention as a folk medicine with indications for the treatment of cancers and digestive diseases. The anticarcinogenic effect of Chaga mushroom extract was investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The cells were pre-incubated with Chaga mushroom extracts (5, 10, 20 microg/ml) for 24 h and this was followed by co-treatment with Chaga mushroom extracts and TPA (12-O-tetradecanoylphorbol-13-acetate, 10 ng/ml) for 1 h. The inhibition of GJIC by TPA (12-O-tetradecanoylphorbol-13-acetate), promoter of cancer, was prevented with treatment of Chaga mushroom extracts. Similarly, the increased phosphorylated ERK1/2 and p38 protein kinases were markedly reduced in Chaga mushroom extracts-treated cells. There was no change in the JNK kinase protein level, suggesting that Chaga mushroom extracts could only block the activation of ERK1/2 and p38 MAP kinase. The Chaga mushroom extracts further prevented the inhibition of GJIC through the blocking of Cx43 phosphorylation. Indeed cell-to-cell communication through gap junctional channels is a critical factor in the life and death balance of cells because GJIC has an important function in maintaining tissue homeostasis through the regulation of cell growth, differentiation, apoptosis and adaptive functions of differentiated cells. Thus Chaga mushroom may act as a natural anticancer product by preventing the inhibition of GJIC through the inactivation of ERK1/2 and p38 MAP kinase.     

Dynamic gap junctional communication: a delimiting model for tissue responses.

Gap junctions are aqueous intercellular channels formed by a diverse class of membrane-spanning proteins, known as connexins. These aqueous pores provide partial cytoplasmic continuity between cells in most tissues, and are freely permeable to a host of physiologically relevant second messenger molecules/ionic species (e.g., Ca2+, IP3, cAMP, cGMP). Despite the fact that these second messenger molecules/ionic species have been shown to alter junctional patency, there is no clear basis for understanding how dynamic and transient changes in the intracellular concentration of second messenger molecules might modulate the extent of intercellular communication among coupled cells. Thus, we have modified the tissue monolayer model of Ramanan and Brink (1990) to account for both the up-regulatory and down-regulatory effects on junctions by second messenger molecules that diffuse through gap junctions. We have chosen the vascular wall as our morphological correlate because of its anisotropy and large investment of gap junctions. The model allows us to illustrate the putative behavior of gap junctions under a variety of physiologically relevant conditions. The modeling studies demonstrated that transient alterations in intracellular second messenger concentrations are capable of producing 50-125% changes in the number of cells recruited into a functional syncytial unit, after activation of a single cell. Moreover, the model conditions required to demonstrate such physiologically relevant changes in intercellular diffusion among coupled cells are commonly observed in intact tissues and cultured cells.     

Ilimaquinone inhibits gap junctional communication in a connexin isotype-specific manner

Ilimaquinone (IQ) and brefeldin A (BFA) disrupt the Golgi complex structure and block protein transport to the plasma membrane, and inhibit gap junctional communication. HeLa cells expressing rat connexin26, 32, or 43, or mouse connexin31, 36, 45, or 57, were used to study the response patterns of gap junctional communication (dye transfer) to ilimaquinone, brefeldin, and the potent protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). 12-O-Tetradecanoylphorbol-13-acetate (followed for 2 h) caused dose- and time-dependent decreases in communication for five of seven connexins, the unresponsive being connexin45 and 57. Brefeldin (followed for 6 h) caused dose- and time-dependent decreases in communication for six of seven connexins, the exception being connexin26. These results are consistent with Golgi-mediated transport to the cell membrane for all connexins except connexin26. In contrast, ilimaquinone (followed for 6 h) caused a rapid (15-30 min) and nearly complete inhibition of dye transfer through connexin43 channels. For the other connexins, there was a slow and weak response for connexin26, 31, and 32, reaching 65-70% of control communication level, while connexin36, 45, and 57 were unresponsive. Thus, among the tested connexins, ilimaquinone has a strong specificity for connexin43, and the mechanism appears independent of the Golgi complex and of protein kinase C.     

Role of gap junctional intercellular communication in radiation-induced bystander effects in human fibroblasts

Involvement of gap junctional intercellular communication (GJIC) in bystander responses of confluent human fibroblasts irradiated with a carbon-ion beam was investigated. It was found that the lower the radiation dose, the higher the yield of radiation-induced micronuclei per nuclear traversal, suggesting the existence of bystander effects. This low-dose sensitivity was increased when GJIC was enhanced by treating cells with 8-Br-cAMP, but it was partly reduced by treating cells with DMSO, an effective scavenger of reactive oxygen species (ROS). Moreover, no low-dose sensitivity was observed when cells were treated with 100 micro M lindane, an inhibitor of GJIC. The survival of irradiated cells was increased by DMSO but was not influenced significantly by cAMP or lindane. On the other hand, G(1)-phase arrest was detected in the irradiated cells, and it was enhanced by cAMP. In contrast, this arrest was reduced or almost eliminated by DMSO or lindane, respectively, even when cells were irradiated with such a high dose that each cell received five nuclear traversals on average. Thus the bystander responses occurred after both low-dose and relatively high-dose irradiation. Our results indicated that both GJIC and ROS contributed to the radiation-induced bystander effect, but gap junctional channels might play an essential role by modulating the release of radiation-induced signaling factors.     

Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation

Loss of intercellular communication via gap junctions has been correlated with progression of cells to a malignant phenotype. Here, we show that peroxynitrite, a mediator of toxicity in inflammatory processes, diminishes gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells, assayed by the scrapeloading dye-transfer technique as well as by microinjection of a fluorescent dye into single cells. Exposure of cultured cells to a steady-state concentration of peroxynitrite of 1.6 microM for 4 min or to 3-morpholinosydnonimine (SIN-1) at 0.5 mM strongly diminished GJIC. These concentrations of peroxynitrite or SIN-1 were not cytotoxic. When cells were grown in a medium supplemented with sodium selenite (0.1-1 microM) for 72 h, substantial protection was afforded against the decrease in GJIC by peroxynitrite. Thus, peroxynitrite can disrupt GJIC, and selenium-containing proteins protect.     

Dicumarol is a potent reversible inhibitor of gap junctional intercellular communication

Dicumarol [3,3'-methylene-bis(4-hydroxycoumarin)] is a potent inhibitor of NAD(P)H:quinone oxidoreductase-1. Exposure of rat liver epithelial cells or of human skin fibroblasts to dicumarol resulted in a rapid and complete inhibition of connexin-43-dependent gap junctional intercellular communication (GJC). GJC was restored within 60min following removal of dicumarol. The concentration of dicumarol required for half maximal inhibition of GJC was 3muM, making dicumarol about 10-fold more effective in blocking GJC than 1-octanol and flufenamic acid, known inhibitors of GJC. Warfarin, a related coumarin derivative, also attenuated GJC, yet very high concentrations of 5-10mM were required. Dicumarol-induced downregulation of GJC was found not to be due to an interference with pathways enhancing the phosphorylation of connexin-43, such as epidermal growth factor receptor and extracellular signal-regulated kinase pathways. Rather, inhibition of GJC by dicumarol was paralleled by a reversible loss of a phosphorylated form ("P2") of connexin-43.     

Radiation-induced bystander effects in malignant trophoblast cells are independent from gap junctional communication

It is controversially discussed that irradiation induces bystander effects via gap junction channels and/or diffusible cellular factors such as nitric oxide or cytokines excreted from the cells into the environment. But up to now the molecular mechanism leading to a bystander response is not well understood. To discriminate between both mechanisms of bystander response, (i) mediated by gap junctional communication and/or (ii) mediated by diffusible molecules, we used non-communicating Jeg3 malignant trophoblast cells transfected with inducible gap junction proteins, connexin43 and connexin26, respectively, based on the Tet-On system. We co-cultivated X-ray irradiated and non-irradiated bystander Jeg3 cells for 4 h, separated both cell populations by flow cytometry and evaluated the expression of activated p53 by Western blot analysis. The experimental design was proven with communicating versus non-communicating Jeg3 cells. Interestingly, our results revealed a bystander effect which was independent from gap junctional communication properties and the connexin isoform expressed. Therefore, it seems more likely that the bystander effect is not mediated via gap junction channels but rather by paracrine mechanisms via excreted molecules in Jeg3 cells.     

The effects of phorbol ester on alloantigen presentation

B cells and Ia+ thyroid cells fail to stimulate alloreactive T cells in a primary mixed leukocyte reaction (MLR) and fail to activate some allo-class II (I-A) reactive T cell hybridomas. We now demonstrate that B cells can specifically stimulate a primary MLR in combination with the phorbol ester, PMA, but not with interleukin 1 (IL 1) or calcium ionophore. The primary MLR induced with B cells plus PMA can be blocked by either monoclonal anti-I-A or anti-L3T4 antibodies. In contrast, thyroid cells that can be induced to express Ia antigens after incubation with interferon-gamma fail to stimulate a primary MLR even in the presence of PMA or IL 1. We confirmed these observations by using the alloreactive T cell hybridoma, HTB-9.3, which does not react to stimulator B cells. In the presence of PMA, however, this I-Ab-specific hybridoma line was able to respond to relevant but not to control stimulator B cells. Furthermore, the response of HTB-9.3 to B cells plus PMA was also blocked by anti-I-A or anti-L3T4 antibody. In contrast to B cells, Ia+ thyroid cells could not activate HTB-9.3 even in the presence of PMA or IL 1. The data indicate that for primary class II restricted allo-responses, B cells provide signals that can be complemented with the phorbol ester PMA, whereas Ia+ thyroid cells do not, suggesting the existence of additional requirements for T cell activation.     

The effects of cryptorchidism on the regulation of steroidogenesis and gap junctional communication in equine testes

INTRODUCTION: Evidence collected over the years has demonstrated that cryptorchidism is associated with a defect in spermatogenesis and, as a consequence, with either reduced fertility or infertility. However, the effect of cryptorchidism on Leydig cell function is less clear. The aim of our study therefore was to investigate the regulation of steroid hormone biosynthesis and, additionally, intercellular communication in the cryptorchid equine testes. MATERIAL AND METHODS: Testes of mature bilaterally cryptorchid horse and healthy stallions were used for this study. The expression of luteinising hormone receptor (LHR), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), aromatase and connexin43 (Cx43) was detected by means of immunohistochemistry. Testosterone and oestradiol levels were measured in testicular homogenates using appropriate radioimmunoassays. RESULTS: In the testes of both normal and cryptorchid stallions, immunostaining for LHR, 3beta-HSD and aromatase was confined to the Leydig cells. In the cryptorchid horse, the intensity of the staining for LHR and 3beta-HSD was weaker, whereas the staining for aromatase was clearly stronger than that of the normal stallion. Radioimmunological analysis revealed disturbance of the androgen-oestrogen balance in the cryptorchid testes. Additionally, in both the seminiferous tubules and interstitial tissue of the cryptorchid a clear reduction of the Cx43 signal was observed. CONCLUSIONS: Decreased expression of LHR and 3beta-HSD and increased expression of aromatase in the cryptorchid testes suggest that hormonal imbalance was caused both by reduced testosterone synthesis and by increased androgen aromatisation. Impaired expression of Cx43 in the seminiferous tubules as well as in the interstitial tissue of the cryptorchid horse indicates that cryptorchidism affects intercellular communication in the testes.     

Limitations of the scrape-loading/dye transfer technique to quantify inhibition of gap junctional intercellular communication

Gap junctional intercellular communication (GJIC) is recognized as playing an important role in normal cell proliferation and development. Chemically induced alteration of GJIC has been proposed to be associated with abnormal cellular growth and/or tumor promotion. Several in vitro assays are currently used to determine the effects of chemicals on GJIC between cultured mammalian cells. One of these assays, the scrape-loading dye transfer (SLIDT) technique, is based on monitoring the transfer of the fluorescent dye Lucifer yellow from one cell into adjacent cells via functional gap junctions. The objective of our study was to evaluate and compare various approaches for quantifying results obtained with the SL/DT technique. Confluent cultures of either WB rat liver epithelial cells or LC-540 rat leydig cells were exposed to the animal tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), solvent (0.1% ethanol), or culture medium for one hour at 37° C prior to analysis of GJIC. Inhibition of dye transfer was clearly evident following TPA exposure. Quantification of this dye transfer was assessed via four approaches: manually counting the number of labeled cells; measuring the distance of dye travel from the scrape line; quantifying the amount of cellular dye uptake; and determining the distribution of dye away from the scrape line. Our results suggest that while the SL/DT technique can be effectively used as a tool to determine the qualitative presence or absence of GJIC, its use in quantifying changes in GJIC following chemical exposure is limited. Since concentration-dependent responses are critical in chemical testing, application of the SLIDT method should be restricted to a screening assay for qualitatively assessing the presence or absence of GJIC. Another assay (e.g., electrical coupling, microinjection, metabolic cooperation, radioactive metabolite transfer, or fluorescence redistribution after photobleaching) should be considered to quantify changes in GJIC and construct chemical concentration-response curves.Abbreviations FBS, fetal bovine serum - GJIC, gap junctional intercellular communication - HBSS, Hank's balanced saline solution - SL/DT, scrape-loading/dye transfer - TPA, 12-O-tetradecanoylphorbol-13-acetate.