- Research article
- Open Access
Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells
© Nihei et al; licensee BioMed Central Ltd. 2010
- Received: 24 August 2009
- Accepted: 15 January 2010
- Published: 15 January 2010
We investigated the effects of the signaling molecules, cyclic AMP (cAMP) and protein-kinase C (PKC), on gap junctional intercellular communication (GJIC) between thymic epithelial cells (TEC).
Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC.
Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.
- Vasoactive Intestinal Peptide
- Thymic Epithelial Cell
- Lucifer Yellow
Intercellular communication mediated by gap junctions has been considered ubiquitous during the development, maturation, homeostasis and death of diverse cell types and tissues in metazoa [1–7]. These junctions are membrane specializations located in cell-cell contact regions, where intercellular hydrophilic conduits, assembled as dodecameric protein complexes, directly connect the cytosols of adjacent cells . Each complex is composed by two hexameric hemichannels, the connexons, one in each cell [9, 10]. In vertebrates, members of the connexin protein family form these channels, which in rodents has at least 20 isoforms [11, 12]. Topologically, the connexin protein contains four hydrophobic transmembrane domains (M1 to M4), two conserved extracellular loops (E1-E2), one intracellular loop and intracellular C- and N-terminal domains . With an estimated permeability limited to molecules below approximately 1 kDa, these intercellular channels allow cells to share metabolites such as glucose and nucleotides, buffer ions such as K+ and H+, and convey important intracellular second messengers such as calcium, cyclic 5'-adenosine monophosphate (cAMP) and 1,4,5-inositol-trisphosphate (IP3) [14–18]. Physiologically, gap junctions have been associated with diverse phenomena such as transmission of electrical signals (as electrotonic synapses) and intercellular calcium waves, metabolic and ionic coupling, and cellular synchronization [19–22]. In this respect, loss or dysfunction of gap junctions have been related to distinct diseases [23–28].
Gap junction channels may be modulated at different levels. Gap junction channel gating, i.e., shifting between open and closed states, is regulated by voltage, intracellular pH (pHi) and Ca2+ ([Ca2+]i), and phosphorylation [29–31]. It has been suggested that the connexin C-terminal and the intracellular loop of the protein are associated with gap junction channel sensitivity to pHi and [Ca2+]i, while the M1 domain, the N-terminal and the E1 domain have been associated with the voltage sensor [13, 29]. The C-terminal domain exhibits diverse kinase recognition motifs, which allow channel regulation by threonine/serine and tyrosine kinases.
Functional GJIC has been shown in a variety of cell types of the immune system, such as T and B lymphocytes, dendritic cells, microglia, monocytes, macrophages, neutrophils and mast cells [32–38]. In vitro experiments have demonstrated Cx43 mediated functional GJIC between thymic epithelial cells [39, 40]. In addition, data obtained from Cx43-/- mice revealed that this protein is important to normal T cell lymphopoiesis .
Despite the multiple possibilities of regulation of thymic physiology by diverse neuroendocrine products , few previous studies have evaluated GJIC modulation in thymic epithelial cells. Head et al. [43, 44] demonstrated, by dye injection, that treatment of thymic epithelial cells with soluble factors such as interleukin-1 (IL-1), growth hormone (GH), adrenocorticotrophic hormone (ACTH), steroid hormones and neuropeptides induced a partial inhibition of coupling and in some cases it diminished thymulin secretion.
The modulation of GJIC may also be evaluated through the activation of different intracellular signaling pathways by specific second messenger analogs, as well as agonists or antagonists of relevant signaling molecules. The importance of cAMP and PKC in mediating intracellular signaling of diverse extracellular messengers is widely recognized [45, 46]. cAMP activates cAMP-dependent protein kinase (PKA) ; and PKC is activated by diacylglycerol and/or calcium (or neither depending on its isoform), as a result of phospholipid signaling pathways . The effects of cAMP on GJIC have been investigated in systems such as hepatocyte primary cultures, cardiac myocytes, ovarian follicles, myometrium, carotid body and retina [15, 49–53]; and in cell lines derived from ovarian granulosa cells, endometrial and colonic epithelium, endothelium, osteoblasts, and mammary tumor cells [54–57].
The effects of PKC activation on GJIC have also been investigated . In general, cAMP acts by enhancing GJIC while PKC inhibits it [49, 51, 57–59]. However, contrasting results have been reported for both signaling pathways [15, 50, 53, 56, 60]. Nevertheless, to our knowledge the effects of cAMP elevation or PKC activation on GJIC have not been studied in the immune system. Herein we investigated the putative modulatory effects of cAMP and PKC on GJIC between thymic epithelial cells (TEC). Our results demonstrate that inter-TEC GJIC is upregulated by cAMP and downregulated by PKC.
Evaluation of functional GJIC-mediated dye transfer by flow cytometry
cAMP elevation enhances inter-TEC GJIC
We observed the same effect when the intracellular dye microinjection assay was performed on IT76M1 cells.
cAMP elevation upregulates Cx43 at different levels
Phorbol ester induced PKC activation and down-regulates inter-TEC GJIC
Since the phospholipid signaling frequently induces both PKC activation and [Ca2+]i elevation, we also treated the mouse TEC line with both PMA and the calcium ionophore ionomycin (1 μg/ml). In these conditions, a partial but statistically significant inhibition of dye coupling was still seen (Figure 5G).
Inter-TEC GJIC is not modulated by the presence of thymocytes
Since thymocytes play an important role in TEC differentiation and organization during ontogeny of the thymus [70, 71], we investigated if the contact with thymocytes could modulate GJIC in the thymic epithelium.
In the present study we demonstrated, by various experimental approaches, that cAMP and PKC are involved in the modulation of inter-TEC GJIC: the cAMP agonist 8-Br-cAMP enhanced inter-TEC coupling whereas PMA-induced PKC activation triggered an opposite effect.
Using flow cytometry we first detected in a mouse TEC line, that up to 90% of DiIc18(3)+ cells co-cultured in 1:1 ratio with calcein+ cells became double positive, a phenomenon which was readily inhibited by the gap junction inhibitors 18-β-glycyrrhetinic acid and carbenoxolone. This result clearly demonstrates that the thymic epithelium spontaneously forms a GJIC-dependent functional syncytium in vitro. Under these conditions, 8-Br-cAMP and forskolin did not significantly modify this spontaneous percentage of coupled cells. However, both compounds enhanced up to 3 fold the calcein fluorescence intensity of the double positive cells, indicating an increase in the rate of dye transfer among coupled cells. Importantly, 8-Br-cAMP also induced an increment in GJIC in TNC-derived primary cultures of human TEC. These data clearly show that elevation of cAMP upregulates inter-TEC GJIC, similar to what has been reported for effects on cell types in other systems [51, 55].
Experiments performed with the mouse TEC line revealed that 8-Br-cAMP and forskolin treatments induced an accumulation of Cx43 protein at cell-to-cell contact regions, seen as punctate clusters. Furthermore, 8-Br-cAMP induced an increase in Cx43 gene transcription, suggesting that the protein accumulation may be a consequence of altered Cx43 protein translation. Such an alteration in connexin synthesis and accumulation in the cell membrane is in accordance with previous description of modulation of gap junctions by cAMP elevating agonists after long-term evaluations (2-24 hrs, and 7 days) [51, 52, 54, 57, 72–74]. However, short-term modulation also has been demonstrated, with description of changes in gap junctional conductance within minutes of treatment with 8-Br-cAMP [75–77]; in some cell types, changes in dye coupling were also detected within 2-10 minutes after this treatment [55, 56, 77]. In cases of both short- and long-term evaluations alteration in the degree of connexin phosphorylation has also been demonstrated [73, 76]. Interestingly, some of these reports have demonstrated that connexin isoforms other than Cx43 also may be regulated by cAMP analogs, such as Cx26, Cx40 and possibly Cx32 [49, 56, 76, 77]. Thus, we cannot exclude the possibility that other connexin isoforms, not yet characterized in TEC, might also be regulated by 8-Br-cAMP and forskolin in IT-76M1 cells.
We also investigated whether physiological stimuli, could signal through cyclic nucleotides, might be involved in modulation of inter-TEC GJIC. When we evaluated VIP and adenosine, we did not observe any change in the degree of cell coupling in the mouse TEC preparation in any concentration of each molecule used. Similar results were seen despite the various concentrations applied for each molecule.
We should mention however, that our finding on VIP is at variance with the data reported by Head and co-workers . These authors reported an inhibition of GJIC in a rat TEC. Although by now we cannot explain such a difference, it may be related to cell line variations or presumably due to receptor signaling dynamics that cannot be merely mimicked by a single agonist , or may be related to differences in methodological approaches given that the authors treated the cells before GJIC formation.
In any case, adenosine, which may also signal through cyclic nucleotides depending on its concentration and the activated P1 receptor , did not modify the basal level of inter-TEC GJIC as well. These results demonstrate that VIP and adenosine possibly are not physiological modulators of GJIC among TECs. A recent review indicates that adrenoreceptor agonists mediate thymus homeostasis and local T cell development . In addition, reports have suggested a role for catecholamines on thymic epithelial cells controlling proliferation and cytokine secretion [68, 80]. Our data demonstrate that epinephrine, and activation of cAMP, lead to an increasing dye coupling in TEC cells suggesting that this mediator could endogenously control communication between thymic epithelial cells and contribute to thymus physiology. We are currently investigating other endogenous molecules which also signal through cAMP, in order to evaluate its potential to modulate inter-TEC GJIC.
In a second set of experiments, we demonstrated that PKC activation (induced herein by the phorbol ester, PMA) significantly inhibits the dye coupling in both mouse and human TEC in vitro models.
The fact that the same inhibitory effect was seen in a mouse TEC line as well as in primary cultures of human TEC deserve further discussion. The study by Chanson et al.  demonstrated that GJIC inhibition was induced by phorbol esters in a liver-derived cell line but not in differentiated primarily cultures of pancreatic exocrine cells. Similarly, the inhibitory effect of phorbol esters on GJIC was observed in primary cultured developing lens cells, which express Cx43 and Cx49, but not in lentoid cells (differentiated lens cells), which express Cx46 and Cx49, demonstrating that the effects of these compounds might be dependent on the cellular differentiation stage and its pattern of Cx expression .
By contrast, the presence of thymocytes apparently is not involved in the control of inter-TEC GJIC, at least in the co-culture experimental conditions that we used. Nevertheless, further studies are still necessary in order to completely discard a role for thymocytes in the control of inter-TEC communication mediated by gap junctions.
In summary, our data strongly indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. Importantly, this control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations.
In a second vein, we showed that two other extracellular messenger molecules, which also signal through cyclic nucleotides, VIP and adenosine, did not mimic the positive action of 8-Br-cAMP, but epinephrine was able to reproduce this effect, suggesting a specificity control of cAMP in the mechanism of inter-TEC GJIC.
Overall, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.
Lucifer yellow, triton X-100, 18-β-glycyrrhetinic acid (GRA), forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), phorbol 12- myristate 13-acetate (PMA) and ionomycin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Calcein-AM and DiIc18(3) dyes were obtained from Molecular Probes (Eugene, OR, USA), and heptanol was from Merk (Darmstadt, Germany). Collagenase A, dispase II and DNAse grade II were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN, USA), whereas fetal calf serum was from Hyclone Laboratories (South Logan, UT, USA).
Thymic epithelial cultures
The mouse TEC line, IT-76M1, was obtained from BALB/c-derived thymic stromal cells after continuous culture. The epithelial nature of this line was ascertained by the presence of desmosomes and cytokeratin filaments [82–84]. These cells were routinely maintained in culture with RPMI 1640 medium supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere.
In addition to the mouse TEC line, we used in some experiments, primary cultures of human TEC, obtained after isolation of thymic nurse cells complexes. For that, fragments of human thymus were obtained from children subjected to cardiac surgery, following the guidelines of the Oswaldo Cruz Foundation's ethics committee. Thymic nurse cells (TNC) are thymic lymphoepithelial complexes that harbor a variable number of thymocytes . When settled in culture, TNCs gradually release thymocytes, and after 3-5 days, a thymocyte-free primary culture of epithelial cells is established. The TNC isolation was performed according to the procedures currently done in our laboratory . In brief, human thymic fragments were minced (~1 mm3) and gently agitated for 20 min in RPMI 1640 medium. Released thymocytes were discarded and the thymic fragments were suspended in collagenase A solution (0.2 mg/mL) and further agitated at room temperature (RT) for 20 min. The supernatant was again discarded and the remaining fragments were dissociated enzymatically with a CDD solution (collagenase A - 0.2 mg/mL; dispase II - 0.2 mg/mL; DNAse grade II - 5 μg/ml) for 20 min at 37°C. The digestion product was centrifuged and the pellet suspended in PBS. These cell suspensions were carefully layered above 10 mL of fetal calf serum (Cultilab, Campinas, Brazil) placed in 15 mL tubes. The typically heavier TNCs were allowed to sediment. The TNCs obtained by this process were cultured, and after complete thymocyte release, resulting epithelial cultures were used in all experiments.
To evaluate connexin expression, TEC were cultured on glass coverslips until confluence. The cells were fixed in cold (-20°C) methanol and further permeabilized by a solution containing 0.2% Triton X-100. The cells were then incubated overnight at 4°C with anti-connexin 43 rabbit polyclonal antibody (Zymed Laboratories, South San Francisco, CA, USA). After washing, the cells were incubated for 1 hour at room temperature with appropriate alexa 488-conjugated secondary antibody (Molecular Probes, Eugene, OR, USA) to reveal the specific labeling. The cells were washed twice, and the coverslips were mounted in PBS-glycerol (3:1) containing 0.1% para-phenylene diamine, an anti-oxidation agent. Cells were analyzed in a Nikon Eclipse TE-300 microscope with phase-contrast and epifluorescence optics, and photographed using a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, Michigan, USA).
Dye microinjection assay
The evaluation of GJIC by intracellular dye microinjection assay was performed as previously described . Human TNC-derived epithelial cells and IT76 M1 cells were cultured in small Petri dishes until confluence. Visualization of cells was performed with an inverted microscope equipped with epifluorescence optics (Axiovert 100, Carl Zeiss, Oberkochen, Germain). The glass microelectrodes were pulled from borosilicate glass (World Precision Instruments, New Haven, CT, USA) using a pipette puller (model PC-10, Narishige, Tokyo, Japan), filled with lucifer yellow (50 mg/ml in 150 mM LiCl) and positioned using a three-dimensional micromanipulator model (MMO-203, Narishige, Tokyo, Japan). Cells were then iontophoretically microinjected with lucifer yellow through brief current pulses. After 1 minute, the number of adjacent cells that acquired the lucifer yellow was quantified. To ascertain that the dye coupling was mediated by GJIC, in some experiments human TEC were also treated with heptanol (5 mM), a gap junction inhibitor.
GJIC was evaluated using flow cytometry as previously described [61, 87]. After confluence, one sample of the mouse TEC line was loaded with calcein-AM (0.5 M) for 30 minutes, while the other was labeled with the lipophilic molecule Dilc18(3) (10 μM) for 1 hour. Cells were then washed 5 times with PBS and enzymatically dissociated. Calcein+DiIc18(3)- cells and calcein-DiIc18(3)+ cells were co-cultured at 1:1 ratio, being treated (during the co-culture period of 6 hours) or not with drugs of interest. Thereafter, cells were dissociated and the double positive cells [calcein+ DiIc18(3)+] were quantified by flow cytometry, using a FacsCalibur device (Becton-Dickinson, Mountain View, CA, USA). Double-positive cells thus corresponded the functional calcein transfer from calcein+DiIc18(3)- to calcein-DiIc18(3)+ cells. At least 104 cells were acquired in each experimental condition. In some experiments, TEC were also co-cultured with freshly isolated thymocytes. For this purpose, thymuses were obtained from BALC/c mice, maintained at the Oswaldo Cruz Foundation Animal Facilities (Rio de Janeiro, Brazil).
RNA extraction and Northern Blotting
Total RNA was extracted from the mouse TEC line, using TRIzol Reagent (Gibco/BRL, Grand Island, NY, USA). For northern blots, 10-20 μg of total RNA was loaded in 1.2% agarose gel containing 0.12 mg/L ethidium bromide. Gels were blotted onto a nylon membrane and fixed by ultraviolet light. For detection of Cx43 mRNA, membranes were pre-hybridized for 1 hr at 65°C in rapid hybridization buffer. Then the membrane was hybridized for 2 hrs at 65°C with rapid hybridization buffer with a 32P-labeled cDNA probe for Cx43 or GAPDH. Membranes were washed once in 2× standard saline citrate buffer containing 0.1% SDS at room temperature, then exposed to X-ray film.
For statistical comparisons we applied the two- tail paired Student's t test, implemented by the GraphPad Prism software. Differences with p < 0.05 were considered significant.
This study and authors was supported by grants from Conselho Nacional de Desenvolvimento Científico and Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Fundação Oswaldo Cruz (Fiocruz).
- Caveney S: The role of gap junctions in development. Annu Rev Physiol. 1985, 47: 319-335. 10.1146/annurev.ph.47.030185.001535.View ArticlePubMedGoogle Scholar
- Decrock E, Vinken M, De VE, Krysko DV, D'Herde K, Vanhaecke T, Vandenabeele P, Rogiers V, Leybaert L: Connexin-related signaling in cell death: to live or let die?. Cell Death Differ. 2009, 16: 524-536. 10.1038/cdd.2008.196.View ArticlePubMedGoogle Scholar
- Hertzberg EL, Lawrence TS, Gilula NB: Gap junctional communication. Annu Rev Physiol. 1981, 43: 479-491. 10.1146/annurev.ph.43.030181.002403.View ArticlePubMedGoogle Scholar
- Kardami E, Dang X, Iacobas DA, Nickel BE, Jeyaraman M, Srisakuldee W, Makazan J, Tanguy S, Spray DC: The role of connexins in controlling cell growth and gene expression. Prog Biophys Mol Biol. 2007, 94: 245-264. 10.1016/j.pbiomolbio.2007.03.009.View ArticlePubMedGoogle Scholar
- Loewenstein WR: Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev. 1981, 61: 829-913.PubMedGoogle Scholar
- Vinken M, Vanhaecke T, Papeleu P, Snykers S, Henkens T, Rogiers V: Connexins and their channels in cell growth and cell death. Cell Signal. 2006, 18: 592-600. 10.1016/j.cellsig.2005.08.012.View ArticlePubMedGoogle Scholar
- Vinken M, Vanhaecke T, Papeleu P, Snykers S, Henkens T, Rogiers V: Connexins and their channels in cell growth and cell death. Cell Signal. 2006, 18: 592-600. 10.1016/j.cellsig.2005.08.012.View ArticlePubMedGoogle Scholar
- Unger VM, Kumar NM, Gilula NB, Yeager M: Three-dimensional structure of a recombinant gap junction membrane channel. Science. 1999, 283: 1176-1180. 10.1126/science.283.5405.1176.View ArticlePubMedGoogle Scholar
- Perkins GA, Goodenough DA, Sosinsky GE: Formation of the gap junction intercellular channel requires a 30 degree rotation for interdigitating two apposing connexons. J Mol Biol. 1998, 277: 171-177. 10.1006/jmbi.1997.1580.View ArticlePubMedGoogle Scholar
- Unwin PN, Zampighi G: Structure of the junction between communicating cells. Nature. 1980, 283: 545-549. 10.1038/283545a0.View ArticlePubMedGoogle Scholar
- Shibata Y, Kumai M, Nishii K, Nakamura K: Diversity and molecular anatomy of gap junctions. Med Electron Microsc. 2001, 34: 153-159. 10.1007/s007950100008.View ArticlePubMedGoogle Scholar
- Sohl G, Willecke K: Gap junctions and the connexin protein family. Cardiovasc Res. 2004, 62: 228-232. 10.1016/j.cardiores.2003.11.013.View ArticlePubMedGoogle Scholar
- Yeager M, Nicholson BJ: Structure of gap junction intercellular channels. Curr Opin Struct Biol. 1996, 6: 183-192. 10.1016/S0959-440X(96)80073-X.View ArticlePubMedGoogle Scholar
- Bevans CG, Kordel M, Rhee SK, Harris AL: Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules. J Biol Chem. 1998, 273: 2808-2816. 10.1074/jbc.273.5.2808.View ArticlePubMedGoogle Scholar
- Cole WC, Garfield RE: Evidence for physiological regulation of myometrial gap junction permeability. Am J Physiol. 1986, 251: C411-C420.PubMedGoogle Scholar
- Imanaga I: Cell-to-cell coupling studied by diffusional methods in myocardial cells. Experientia. 1987, 43: 1080-1083. 10.1007/BF01956043.View ArticlePubMedGoogle Scholar
- Saez JC, Connor JA, Spray DC, Bennett MV: Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions. Proc Natl Acad Sci USA. 1989, 86: 2708-2712. 10.1073/pnas.86.8.2708.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsien RW, Weingart R: Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut end method. J Physiol. 1976, 260: 117-141.PubMed CentralView ArticlePubMedGoogle Scholar
- Krysko DV, Leybaert L, Vandenabeele P, D'Herde K: Gap junctions and the propagation of cell survival and cell death signals. Apoptosis. 2005, 10: 459-469. 10.1007/s10495-005-1875-2.View ArticlePubMedGoogle Scholar
- Sanderson MJ, Charles AC, Boitano S, Dirksen ER: Mechanisms and function of intercellular calcium signaling. Mol Cell Endocrinol. 1994, 98: 173-187. 10.1016/0303-7207(94)90136-8.View ArticlePubMedGoogle Scholar
- Sheridan JD, Atkinson MM: Physiological roles of permeable junctions: some possibilities. Annu Rev Physiol. 1985, 47: 337-353. 10.1146/annurev.ph.47.030185.002005.View ArticlePubMedGoogle Scholar
- Simon AM, Goodenough DA: Diverse functions of vertebrate gap junctions. Trends Cell Biol. 1998, 8: 477-483. 10.1016/S0962-8924(98)01372-5.View ArticlePubMedGoogle Scholar
- Alldredge B: Clinical connexions. J Clin Pathol. 2008, 61: 885-890. 10.1136/jcp.2008.055699.View ArticlePubMedGoogle Scholar
- Cronier L, Crespin S, Strale PO, Defamie N, Mesnil M: Gap junctions and cancer: new functions for an old story. Antioxid Redox Signal. 2009, 11: 323-338. 10.1089/ars.2008.2153.View ArticlePubMedGoogle Scholar
- Kelsell DP, Dunlop J, Hodgins MB: Human diseases: clues to cracking the connexin code?. Trends Cell Biol. 2001, 11: 2-6. 10.1016/S0962-8924(00)01866-3.View ArticlePubMedGoogle Scholar
- Laird DW: Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta. 2005, 1711: 172-182. 10.1016/j.bbamem.2004.09.009.View ArticlePubMedGoogle Scholar
- Simon AM: Gap junctions: more roles and new structural data. Trends Cell Biol. 1999, 9: 169-170. 10.1016/S0962-8924(99)01547-0.View ArticlePubMedGoogle Scholar
- White TW, Paul DL: Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol. 1999, 61: 283-310. 10.1146/annurev.physiol.61.1.283.View ArticlePubMedGoogle Scholar
- Spray DC, Scemes E: Effects of intracellular pH (and Ca2+) on gap junction channels. PH and Brain function. Edited by: Kalai K, Ransom BR. 1998, New York, Wiley-Liss, Inc, 477-489.Google Scholar
- Spray DC, Bennett MV: Physiology and pharmacology of gap junctions. Annu Rev Physiol. 1985, 47: 281-303. 10.1146/annurev.ph.47.030185.001433.View ArticlePubMedGoogle Scholar
- Lampe PD, Lau AF: Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys. 2000, 384: 205-215. 10.1006/abbi.2000.2131.View ArticlePubMedGoogle Scholar
- Branes MC, Contreras JE, Saez JC: Activation of human polymorphonuclear cells induces formation of functional gap junctions and expression of connexins. Med Sci Monit. 2002, 8: BR313-BR323.PubMedGoogle Scholar
- Eugenin EA, Eckardt D, Theis M, Willecke K, Bennett MV, Saez JC: Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-gamma and tumor necrosis factor-alpha. Proc Natl Acad Sci USA. 2001, 98: 4190-4195. 10.1073/pnas.051634298.PubMed CentralView ArticlePubMedGoogle Scholar
- Eugenin EA, Branes MC, Berman JW, Saez JC: TNF-alpha plus IFN-gamma induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses. J Immunol. 2003, 170: 1320-1328.View ArticlePubMedGoogle Scholar
- Inagaki-Ohara K, Sawaguchi A, Suganuma T, Matsuzaki G, Nawa Y: Intraepithelial lymphocytes express junctional molecules in murine small intestine. Biochem Biophys Res Commun. 2005, 331: 977-983. 10.1016/j.bbrc.2005.04.025.View ArticlePubMedGoogle Scholar
- Krenacs T, van DM, Lindhout E, Rosendaal M: Direct cell/cell communication in the lymphoid germinal center: connexin43 gap junctions functionally couple follicular dendritic cells to each other and to B lymphocytes. Eur J Immunol. 1997, 27: 1489-1497. 10.1002/eji.1830270627.View ArticlePubMedGoogle Scholar
- Matsue H, Yao J, Matsue K, Nagasaka A, Sugiyama H, Aoki R, Kitamura M, Shimada S: Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs. J Immunol. 2006, 176: 181-190.View ArticlePubMedGoogle Scholar
- Vliagoftis H, Hutson AM, Mahmudi-Azer S, Kim H, Rumsaeng V, Oh CK, Moqbel R, Metcalfe DD: Mast cells express connexins on their cytoplasmic membrane. J Allergy Clin Immunol. 1999, 103: 656-662. 10.1016/S0091-6749(99)70239-3.View ArticlePubMedGoogle Scholar
- Alves LA, Campos de Carvalho AC, Cirne Lima EO, Rocha e Souza CM, Dardenne M, Spray DC, Savino W: Functional gap junctions in thymic epithelial cells are formed by connexin 43. Eur J Immunol. 1995, 25: 431-437. 10.1002/eji.1830250219.View ArticlePubMedGoogle Scholar
- Ehmann UK, Calderwood SK, Stevenson MA: Gap-junctional communication between feeder cells and recipient normal epithelial cells correlates with growth stimulation. In Vitro Cell Dev Biol Anim. 2001, 37: 100-110. 10.1290/1071-2690(2001)037<0100:GJCBFC>2.0.CO;2.View ArticlePubMedGoogle Scholar
- Montecino-Rodriguez E, Leathers H, Dorshkind K: Expression of connexin 43 (Cx43) is critical for normal hematopoiesis. Blood. 2000, 96: 917-924.PubMedGoogle Scholar
- Savino W, Dardenne M: Neuroendocrine control of thymus physiology. Endocr Rev. 2000, 21: 412-443. 10.1210/er.21.4.412.PubMedGoogle Scholar
- Head GM, Mentlein R, Kranz A, Downing JE, Kendall MD: Modulation of dye-coupling and proliferation in cultured rat thymic epithelium by factors involved in thymulin secretion. J Anat. 1997, 191 (Pt 3): 355-365. 10.1046/j.1469-7580.1997.19130355.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Head GM, Mentlein R, von PB, Downing JE, Kendall MD: Neuropeptides exert direct effects on rat thymic epithelial cells in culture. Dev Immunol. 1998, 6: 95-104. 10.1155/1998/41349.PubMed CentralView ArticlePubMedGoogle Scholar
- Hanoune J, Defer N: Regulation and role of adenylyl cyclase isoforms. Annu Rev Pharmacol Toxicol. 2001, 41: 145-174. 10.1146/annurev.pharmtox.41.1.145.View ArticlePubMedGoogle Scholar
- Nishizuka Y: Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992, 258: 607-614. 10.1126/science.1411571.View ArticlePubMedGoogle Scholar
- Francis SH, Corbin JD: Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol. 1994, 56: 237-272. 10.1146/annurev.ph.56.030194.001321.View ArticlePubMedGoogle Scholar
- Divecha N, Irvine RF: Phospholipid signaling. Cell. 1995, 80: 269-278. 10.1016/0092-8674(95)90409-3.View ArticlePubMedGoogle Scholar
- Saez JC, Gregory WA, Watanabe T, Dermietzel R, Hertzberg EL, Reid L, Bennett MV, Spray DC: cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture. Am J Physiol. 1989, 257: C1-11.PubMedGoogle Scholar
- Granot I, Dekel N: Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem. 1994, 269: 30502-30509.PubMedGoogle Scholar
- Darrow BJ, Fast VG, Kleber AG, Beyer EC, Saffitz JE: Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res. 1996, 79: 174-183.View ArticlePubMedGoogle Scholar
- Abudara V, Garces G, Saez JC: Cells of the carotid body express connexin43 which is up-regulated by cAMP. Brain Res. 1999, 849: 25-33. 10.1016/S0006-8993(99)01946-0.View ArticlePubMedGoogle Scholar
- Kojima A, Nakahama K, Ohno-Matsui K, Shimada N, Mori K, Iseki S, Sato T, Mochizuki M, Morita I: Connexin 43 contributes to differentiation of retinal pigment epithelial cells via cyclic AMP signaling. Biochem Biophys Res Commun. 2008, 366: 532-538. 10.1016/j.bbrc.2007.11.159.View ArticlePubMedGoogle Scholar
- Atkinson MM, Lampe PD, Lin HH, Kollander R, Li XR, Kiang DT: Cyclic AMP modifies the cellular distribution of connexin43 and induces a persistent increase in the junctional permeability of mouse mammary tumor cells. J Cell Sci. 1995, 108 (Pt 9): 3079-3090.PubMedGoogle Scholar
- Burghardt RC, Barhoumi R, Sewall TC, Bowen JA: Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43. J Membr Biol. 1995, 148: 243-253.View ArticlePubMedGoogle Scholar
- Chanson M, Bruzzone R, Spray DC, Regazzi R, Meda P: Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue. Am J Physiol. 1988, 255: C699-C704.PubMedGoogle Scholar
- Romanello M, Moro L, Pirulli D, Crovella S, D'Andrea P: Effects of cAMP on intercellular coupling and osteoblast differentiation. Biochem Biophys Res Commun. 2001, 282: 1138-1144. 10.1006/bbrc.2001.4710.View ArticlePubMedGoogle Scholar
- Lampe PD: Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly. J Cell Biol. 1994, 127: 1895-1905. 10.1083/jcb.127.6.1895.View ArticlePubMedGoogle Scholar
- Sirnes S, Kjenseth A, Leithe E, Rivedal E: Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication. Biochem Biophys Res Commun. 2009, 382: 41-45. 10.1016/j.bbrc.2009.02.141.View ArticlePubMedGoogle Scholar
- Sakai N, Blennerhassett MG, Garfield RE: Intracellular cyclic AMP concentration modulates gap junction permeability in parturient rat myometrium. Can J Physiol Pharmacol. 1992, 70: 358-364.View ArticlePubMedGoogle Scholar
- Fonseca PC, Nihei OK, Savino W, Spray DC, Alves LA: Flow cytometry analysis of gap junction-mediated cell-cell communication: advantages and pitfalls. Cytometry A. 2006, 69: 487-493.View ArticlePubMedGoogle Scholar
- Nihei OK, Campos de Carvalho AC, Spray DC, Savino W, Alves LA: A novel form of cellular communication among thymic epithelial cells: intercellular calcium wave propagation. Am J Physiol Cell Physiol. 2003, 285: C1304-C1313.View ArticlePubMedGoogle Scholar
- Simonds WF: G protein regulation of adenylate cyclase. Trends Pharmacol Sci. 1999, 20: 66-73. 10.1016/S0165-6147(99)01307-3.View ArticlePubMedGoogle Scholar
- Bellinger DL, Lorton D, Horn L, Brouxhon S, Felten SY, Felten DL: Vasoactive intestinal polypeptide (VIP) innervation of rat spleen thymus, and lymph nodes. Peptides. 1997, 18: 1139-1149. 10.1016/S0196-9781(97)00075-2.View ArticlePubMedGoogle Scholar
- Reubi JC, Horisberger U, Kappeler A, Laissue JA: Localization of receptors for vasoactive intestinal peptide, somatostatin, and substance P in distinct compartments of human lymphoid organs. Blood. 1998, 92: 191-197.PubMedGoogle Scholar
- Apasov SG, Koshiba M, Chused TM, Sitkovsky MV: Effects of extracellular ATP and adenosine on different thymocyte subsets: possible role of ATP-gated channels and G protein-coupled purinergic receptor. J Immunol. 1997, 158: 5095-5105.PubMedGoogle Scholar
- Ralevic V, Burnstock G: Receptors for purines and pyrimidines. Pharmacol Rev. 1998, 50: 413-492.PubMedGoogle Scholar
- von PB, Kurz B, Mentlein R: Effect of transmitters and co-transmitters of the sympathetic nervous system on interleukin-6 synthesis in thymic epithelial cells. Neuroimmunomodulation. 1999, 6: 45-50. 10.1159/000026363.View ArticleGoogle Scholar
- Pahujaa M, Anikin M, Goldberg GS: Phosphorylation of connexin43 induced by Src: regulation of gap junctional communication between transformed cells. Exp Cell Res. 2007, 313: 4083-4090. 10.1016/j.yexcr.2007.09.010.View ArticlePubMedGoogle Scholar
- Manley NR: Thymus organogenesis and molecular mechanisms of thymic epithelial cell differentiation. Semin Immunol. 2000, 12: 421-428. 10.1006/smim.2000.0263.View ArticlePubMedGoogle Scholar
- Naquet P, Naspetti M, Boyd R: Development, organization and function of the thymic medulla in normal, immunodeficient or autoimmune mice. Semin Immunol. 1999, 11: 47-55. 10.1006/smim.1998.0149.View ArticlePubMedGoogle Scholar
- Azarnia R, Dahl G, Loewenstein WR: Cell junction and cycle AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type. J Membr Biol. 1981, 63: 133-146. 10.1007/BF01969454.View ArticlePubMedGoogle Scholar
- Traub O, Look J, Paul D, Willecke K: Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol. 1987, 43: 48-54.PubMedGoogle Scholar
- Wang Y, Rose B: Clustering of Cx43 cell-to-cell channels into gap junction plaques: regulation by cAMP and microfilaments. J Cell Sci. 1995, 108 (Pt 11): 3501-3508.PubMedGoogle Scholar
- Burt JM, Spray DC: Inotropic agents modulate gap junctional conductance between cardiac myocytes. Am J Physiol. 1988, 254: H1206-H1210.PubMedGoogle Scholar
- Saez JC, Spray DC, Nairn AC, Hertzberg E, Greengard P, Bennett MV: cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. Proc Natl Acad Sci USA. 1986, 83: 2473-2477. 10.1073/pnas.83.8.2473.PubMed CentralView ArticlePubMedGoogle Scholar
- Van Rijen HV, van Veen TA, Hermans MM, Jongsma HJ: Human connexin40 gap junction channels are modulated by cAMP. Cardiovasc Res. 2000, 45: 941-951. 10.1016/S0008-6363(99)00373-9.View ArticlePubMedGoogle Scholar
- Marinissen MJ, Gutkind JS: G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol Sci. 2001, 22: 368-376. 10.1016/S0165-6147(00)01678-3.View ArticlePubMedGoogle Scholar
- Leposavic G, Pilipovic I, Radojevic K, Pesic V, Perisic M, Kosec D: Catecholamines as immunomodulators: a role for adrenoceptor-mediated mechanisms in fine tuning of T-cell development. Auton Neurosci. 2008, 144: 1-12. 10.1016/j.autneu.2008.09.003.View ArticlePubMedGoogle Scholar
- Kurz B, Feindt J, von GB, Kranz A, Loppnow H, Mentlein R: Beta-adrenoceptor-mediated effects in rat cultured thymic epithelial cells. Br J Pharmacol. 1997, 120: 1401-1408. 10.1038/sj.bjp.0701045.PubMed CentralView ArticlePubMedGoogle Scholar
- Tenbroek EM, Louis CF, Johnson R: The differential effects of 12-O-tetradecanoylphorbol-13-acetate on the gap junctions and connexins of the developing mammalian lens. Dev Biol. 1997, 191: 88-102. 10.1006/dbio.1997.8703.View ArticlePubMedGoogle Scholar
- Cirne-Lima EO, van EW, Savino W: Cortical and medullary phenotypes within a mouse thymic epithelial cell line. In Vitro Cell Dev Biol Anim. 1993, 29A: 443-445. 10.1007/BF02639375.View ArticlePubMedGoogle Scholar
- Dardenne M, Savino W, Gagnerault MC, Itoh T, Bach JF: Neuroendocrine control of thymic hormonal production. I. Prolactin stimulates in vivo and in vitro the production of thymulin by human and murine thymic epithelial cells. Endocrinology. 1989, 125: 3-12. 10.1210/endo-125-1-3.View ArticlePubMedGoogle Scholar
- Itoh T, Doi H, Chin S, Nishimura T, Kasahara S: Establishment of mouse thymic nurse cell clones from a spontaneous BALB/c thymic tumor. Eur J Immunol. 1988, 18: 821-824. 10.1002/eji.1830180525.View ArticlePubMedGoogle Scholar
- Guyden JC, Pezzano M: Thymic nurse cells: a microenvironment for thymocyte development and selection. Int Rev Cytol. 2003, 223: 1-37. 10.1016/S0074-7696(05)23001-2.View ArticlePubMedGoogle Scholar
- Smaniotto S, de Mello-Coelho V, Villa-Verde DM, Pleau JM, Postel-Vinay MC, Dardenne M, Savino W: Growth hormone modulates thymocyte development in vivo through a combined action of laminin and CXC chemokine ligand 12. Endocrinology. 2005, 146: 3005-3017. 10.1210/en.2004-0709.View ArticlePubMedGoogle Scholar
- Kiang DT, Kollander R, Lin HH, LaVilla S, Atkinson MM: Measurement of gap junctional communication by fluorescence activated cell sorting. In Vitro Cell Dev Biol Anim. 1994, 30A: 796-802. 10.1007/BF02631304.View ArticlePubMedGoogle Scholar
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