- Research article
- Open Access
Interaction of connexin43 and protein kinase C-delta during FGF2 signaling
© Niger et al; licensee BioMed Central Ltd. 2010
- Received: 7 October 2009
- Accepted: 25 March 2010
- Published: 25 March 2010
We have recently demonstrated that modulation of the gap junction protein, connexin43, can affect the response of osteoblasts to fibroblast growth factor 2 in a protein kinase C-delta-dependent manner. Others have shown that the C-terminal tail of connexin43 serves as a docking platform for signaling complexes. It is unknown whether protein kinase C-delta can physically interact with connexin43.
In the present study, we investigate by immunofluorescent co-detection and biochemical examination the interaction between Cx43 and protein kinase C-delta. We establish that protein kinase C-delta physically interacts with connexin43 during fibroblast growth factor 2 signaling, and that protein kinase C delta preferentially co-precipitates phosphorylated connexin43. Further, we show by pull down assay that protein kinase C-delta associates with the C-terminal tail of connexin43.
Connexin43 can serve as a direct docking platform for the recruitment of protein kinase C-delta in order to affect fibroblast growth factor 2 signaling in osteoblasts. These data expand the list of signal molecules that assemble on the connexin43 C-terminal tail and provide a critical context to understand how gap junctions modify signal transduction cascades in order to impact cell function.
- Cx43 Protein
- MC3T3 Cell
- Plasma Membrane Extract
- Oculodentodigital Dysplasia
Gap junctions are transcellular channels formed by the juxtaposition of two hemichannels, each composed of six connexin monomers, present on adjacent cells. The assembled gap junctions then aggregate to form gap junction plaques. Gap junctional communication maintains metabolic continuity between cells and mediates the rapid and efficient transmission of small molecules, including second messengers among the interconnected cells.
Connexin43 (Cx43) is the principal gap junction protein expressed in osteoblasts and osteocytes, where it has been implicated in transmitting hormonal, mechanical load and growth factor induced signals [1, 2]. Mutation of connexins has been implicated in several diseases [3–5]. Point mutations in GJA1, the gene encoding the gap junction protein Cx43, result in the human pleiotropic disorder oculodentodigital dysplasia, which includes skeletal manifestations . Mouse models of oculodentodigital dysplasia [7, 8] and Cx43 genetic ablation [9–11] have underscored the importance of Cx43 in skeletal function. However, little is known about the molecular mechanisms by which gap junctions regulate the function of skeletal tissues.
Using osteoblast cell lines, we have demonstrated that Cx43 can modulate growth factor responses and signal transduction cascades, leading to altered gene expression and osteoblast function [12–14]. We have recently reported that modulation of Cx43 can affect signal transduction in response to fibroblast growth factor 2 (FGF2) in a protein kinase C-delta (PKCδ)-dependent manner in osteoblasts . PKCδ is a member of the novel PKCs, which, unlike classic PKCs, are calcium-independent but are activated by diacylglycerol and phosphatidylserine. In a previous study, we demonstrated that overexpression of Cx43 in a mouse osteoblast cell line could enhance the transcriptional response of the osteocalcin gene promoter to FGF2 treatment; an effect that could be abrogated by inhibition of PKCδ expression or activity . Conversely, inhibition of Cx43 expression could attenuate the ability of these cells to respond to FGF2. From these data, we hypothesize that Cx43 may recruit PKCδ locally to the gap junction plaque where it can respond to second messengers being propagated through the gap junction channel. Indeed, numerous studies have shown that Cx43 can serve as a docking platform for signal complexes, including β-catenin, src, PKCα and PKCε [15–18]. The novel PKC family member, PKCδ, has not been shown to interact with Cx43. Accordingly, we set out to examine the biochemical interactions between Cx43 and PKCδ as it relates to the effects of FGF2 signaling. By understanding these biochemical interactions, we gain insight into the biologically relevant signals that may be propagated through gap junction channels as well as a greater understanding of how gap junctions regulate signal transduction cascades and, ultimately, cell function.
PKCδ-dependent phosphorylation of Cx43
Immunofluorescent detection of PKCδ and Cx43
Co-immunoprecipitation of Cx43 and PKCδ
Next, we attempted the opposing co-immunoprecipitation, using anti-PKC antibodies to co-immunoprecipitate Cx43. Immunoprecipitations were performed with non-immune IgG, anti-Cx43, or anti-PKC antibodies, and western blots of supernatant (S) and bead (B) fractions from the immunoprecipitations were probed with anti-Cx43 specific antibodies, as indicated in Figure 4C. Non-immune IgG failed to immunoprecipitate PKCs. As expected, anti-Cx43 antibodies were able to immunoprecipitate Cx43 protein. Antibodies against panPKC, PKCδ and PKCε precipitate two products that are detected with Cx43 antibodies, a ~43 kD and a ~46 kD protein. The ~43 kD protein is the non-phosphorylated (NP) form of Cx43, while the ~46 kD protein is a phosphorylated form of Cx43 (p-Cx43). Interestingly, it would appear that phosphorylated Cx43 (~46 kD) is preferentially associated with all three PKCs, as this is the principal product detected in the bead fractions from co-immunoprecipitations with anti-PKC antibodies (Figure 4C, lanes 5,7 and 9), in contrast to the predominance of the ~43 kDa band (NP form) that is observed in the Cx43 direct immunoprecipitation (Figure 4C, lane 3).
To validate that this ~46 kD product corresponds to phosphorylated Cx43, co-immunoprecipitations were performed using non-immune IgG, anti-Cx43 and anti-phospho-PKCδ antibodies, and the immunoprecipitated products were probed by western blotting with anti-Cx43 or anti-phospho-serine specific antibodies. The same noticeable enrichment of the higher molecular weight product (~46 kDa) is observed in the co-immunoprecipitations of Cx43 with anti-PKC antibodies in comparison to the direct Cx43 immunoprecipitation. (Figure 4D). In support of the notion that the 46 kDa protein is a phosphorylated form of Cx43, the anti-phospho-serine antibody strongly reacts with the ~46 kDa product in the co-immunoprecipitations with anti-PKCδ antibodies (Figure 4D). These data reinforce the hypothesis that PKCδ preferentially associates with phospho-Cx43.
GST pulldown of PKCδ with the Cx43 C-terminal tail
Finally, in order to confirm that the association between PKCδ and Cx43 occurs in the C-terminal domain of Cx43, we performed GST-pulldown assays, using a recombinant GST-Cx43 fusion protein that spans amino acids 241-382 of the Cx43 C-terminal tail (GST-Cx43CT). We anticipated association of PKCδ with the C-terminal tail of Cx43 because this domain is commonly associated with the assembly of signal complexes [15–18] and includes characterized PKC phosphorylation sites .
Previously, we have shown that modulation of Cx43 protein levels can affect the impact of FGF2 on osteoblast transcription in a PKCδ-dependent manner . In that study, we reported that over expression of Cx43 potentiates the transcriptional response to FGF2 by enhancing the activation and mobilization of PKCδ in the cell, which ultimately increases transcription of the osteoblast specific gene, osteocalcin. Conversely, we show that inhibition of Cx43 expression or function could inhibit the responsiveness of the osteocalcin gene to FGF2 treatment. These data established the involvement of Cx43 and PKCδ in the osteoblast response to FGF2, however, it remained undetermined how the relative abundance of Cx43 could impact PKCδ function.
Several studies have shown that FGF2 can activate PKCδ [24–29], or that FGF2 can increase phosphorylation of Cx43 [22, 30, 31]. Additionally, the activation of PKC isoforms, by phorbol esters has been shown to result in the inhibition of gap junctional communication [19, 32–35]. However, a physical interaction of PKCδ with Cx43 has never been reported, nor has FGF2 been shown to increase phosphorylation of Cx43 in a PKCδ-dependent manner.
In this study, we demonstrate via immunofluorescence staining and biochemical methods that PKCδ physically interacts with Cx43. While alone, the immunofluorescence does not prove a physical interaction, we think that this data provides strong support of the feasibility of a physical interaction in situ and that the co-precipitation studies are biologically relevant. We go on to characterize the kinetics of the phosphorylation, interaction and cellular translocation following FGF2 treatment, revealing a transient association of PKCδ with Cx43 at the plasma membrane and subsequent accumulation of PKCδ in the nucleus following treatment with FGF2. Our study is the first to show that PKCδ can physically interact with Cx43 in osteoblastic cells, and that there is a PKCδ-dependent phosphorylation of Cx43 at serine 368 that occurs in response to FGF2 administration. It is likely that multiple phosphorylations of Cx43 occur, in addition to the one detected at serine 368, as phosphorylation of serine 368 alone is not sufficient to result in the observed molecular weight shift of Cx43 on SDS-PAGE gels to ~46 kDa [36–38].
A similar interaction between Cx43 and another novel PKC family member, PKCε, has been reported following FGF2 treatment in cardiomyocytes . It is unclear whether the involvement of PKCδ in the FGF2-response and association with Cx43 also occurs in cardiomyocytes, or whether this is an osteoblast-specific interaction. It would not be surprising that PKCδ could also associate with Cx43 in cardiomyocytes, as FGF2, PKCε, PKCδ and Cx43 have all been implicated in cardioprotection following ischemia [39–43]. Notably, in a previously reported study in osteoblasts, we did not detect a translocation of PKCε following FGF2 treatment in these cells, suggesting distinct roles for PKCδ and PKCε in the FGF2-response of bone forming cells .
In this study, we report that PKCδ physically interacts with the C-terminal tail of Cx43. We speculate that the preferential interaction of PKCδ with the phosphorylated form of Cx43 is because PKCδ directly phosphorylates Cx43 (i.e., Cx43 is a substrate for PKCδ). The fact that inhibition of PKCδ activity with rottlerin can prevent phosphorylation of Cx43 at serine 368 indicates that, at a minimum, PKCδ lies upstream of Cx43 phosphorylation, and that Cx43 may be a direct substrate for PKCδ. Alternately, it is possible that the phosphorylation of Cx43 is a prerequisite for the docking of PKCδ with the Cx43 C-terminal tail. Future studies will explore these alternatives. It should also be noted that, while commonly used as an inhibitor of PKCδ, the specificity of rottlerin has been questioned .
In total, these data support our model that Cx43 affects osteoblast function by regulating signal transduction cascades. We predict that intercellular communication via gap junctions results in the sharing of signals/second messengers among coupled osteoblasts, permitting cells to respond more robustly to extracellular stimuli, such as growth factors, hormonal signals or mechanical load. Central to this model is the concept that Cx43 serves as a docking platform for signaling complexes, recruiting them to the gap junction plaque where they can respond to transmitted signals/second messengers. Previously, we had identified PKCδ as a mediator of the Cx43 enhanced response of osteoblast to FGF2 but, it was unknown whether or not PKCδ was recruited to the Cx43 gap junction plaque to participate in signaling . Now, we can add PKCδ to the growing number of signaling complexes that are recruited to and participate in signaling at the gap junction plaque.
By establishing the protein-protein interactions that can occur between gap junctions and signaling complexes, we gain valuable insights into the molecular mechanisms by which gap junctions/connexins can affect cellular signaling to ultimately affect cell function. Additionally, we learn how gap junctions themselves may be regulated. Indeed, phosphorylation of Cx43 can affect its function/transport, including assembly of hemichannels, transport to the plasma membrane, channel gating and turnover [36, 45]. Furthermore, the clarification of these interactions lends insights into the biologically relevant molecules that may be propagated through gap junction channels. The activation of several signaling cascades downstream of the FGF2-FGF receptor complex generates second messengers that may transverse the gap junction channel. Among the signal cascades activated by the FGF2-FGF receptor complex include Ras-ERK MAPK cascade, phospholipase Cγ, phospholipase D and cytosolic phospholipase A2, which produce second messengers like inositol phosphate derivates, diacylglycerol, arachidonic acid, and calcium [46–52]. Of note, several of these second messengers have been shown to regulate PKCδ activation [21, 53–56].
Importantly, it is equally plausible that the role of Cx43 in FGF-signaling is independent of its function as a permeable pore for direct cell-cell communications. Connexins have been widely implicated as functioning as unpaired hemichannels . Furthermore, several studies have revealed important non-channel roles for connexins in cell function [58, 59]. For example, it is possible that Cx43 may affect FGF2-initiated signaling cascades and osteoblast function by sequestering signal complexes to cellular microdomains, altering mitochondrial function, direct nuclear shuttling or affecting signal complex turnover, independent of cell-cell communication.
Future studies are needed to define the need for PKCδ protein docking with the Cx43 gap junction channel. More specifically, if the proximal recruitment of PKCδ to the Cx43-dependent "shared" messenger is required to transmit the amplification of the FGF2 response among osteoblasts. By studying the mechanism of action of gap junctions in transmitting signals among the cells of bone, we hope to be able to understand the role of gap junctions in how skeletal cells coordinate function during bone growth, remodeling and repair.
Cx43 can serve as a direct docking platform for the recruitment of PKCδ in order to affect FGF2 signaling in osteoblasts. PKCδ is found to transiently associate with Cx43 at cell-cell junctions, following stimulation with FGF2. Subsequently, PKCδ accumulates in the nucleus, where we have shown in a previous study that it alters transcription of key osteoblastic genes . These data expand the list of signal molecules that assemble on the Cx43 C-terminal tail and provide a critical context to understand how gap junctions modify signal transduction cascades in order to impact cell function.
Chemicals, Antibodies and Reagents
Unless specified, chemicals were purchased from Sigma (St Louis, MO). Recombinant FGF2 was from R&D Systems (Minneapolis, MN). FGF2 was reconstituted in a sterile vehicle solution (phosphate buffered saline, 0.1% bovine serum albumin, 1 mM dithiothreitol). The sources of antibodies used were Sigma (rabbit anti-Cx43 (#C6219) and mouse anti-phospho-serine (#P5747) antibodies), Santa Cruz Biotechnology (Santa Cruz, CA; rabbit anti-panPKC (#sc-10800), rabbit anti-PKCε (#sc-214) and rabbit anti-PKCδ antibodies (#sc-937)), Cell Signaling Technology (Beverly, MA; rabbit anti-phospho-Cx43 (Ser 368) (#3511) and rabbit anti-phospho-PKCδ (Thr505) (#9374), and Chemicon (Temecula, CA; mouse anti-Cx43 (#MAB3067), anti-GAPDH (#MAB374); and Millipore (Billerica, MA) FITC-conjugated donkey anti-rabbit IgG (#AP182F), Cy3-conjugated donkey anti-mouse IgG antibodies(#AP192C). The following reagents were also used: DAPI (4',6-Diamidine-2'-phenylindole dihydrochloride; Roche, Indianapolis, IN), Protein G PLUS/Protein A-Agarose beads (Calbiochem, La Jolla, CA), non-immune mouse and rabbit IgG antibodies (Calbiochem, San Diego, CA).
Cell Culture and FGF2 Treatments
MC3T3-E1 (clone 4) osteoblasts were purchased from the ATCC (Manassas, VA). These cells were cultured in DMEM (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and a penicillin (50 IU/ml)-streptomycin (50 μg/ml) solution (Cellgro) as described previously . For experiments with FGF2 treatment, cells were serum-starved in DMEM containing 0.1% fetal bovine serum and 0.3% bovine serum albumin for 24 hours. FGF2 was added to the starvation media at 5 ng/ml. The vehicle diluent for FGF2 (phosphate buffered saline, 0.1% bovine serum albumin, 1 mM dithiothreitol) was used as a negative control for FGF2 treatments. Cell viability was routinely monitored using a CCK-8 assay (Alexis Biochemicals, Farmingdale, NY). Cell viability was unaffected by FGF2 or inhibitor treatments when compared to vehicle treated cells. All experiments performed in this study were repeated at least three times, and data from representative experiments are shown.
Whole cell extracts were prepared from confluent cultures of MC3T3 cells using a modified RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM β-glycerophosphate, 1 mM EGTA, 1 mM EDTA, 2 mM sodium vanadate, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1× protease inhibitor cocktail (Sigma)). For western blots using plasma membrane extracts, plasma membrane fractions were obtained and partially purified using a modified sucrose density gradient method . Briefly, FGF2-treated MC3T3 cells from trypsinized cultures were centrifuged at 900 g at 4°C for 5 minutes and the resulting cell pellets were washed twice in cold HBSS (Cellgro). Cells were resuspended in cold hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT) with 1× protease inhibitor cocktail and 1 mM sodium orthovanadate, and incubated on ice for 10 min to allow swelling. Cells were then lysed by adding 1% NP-40. The resulting lysates were centrifuged (1300 g, 10 min., 4°C) to remove unbroken cells and nuclei. The supernatants were then loaded on top of sucrose gradients generated by layering a 10% sucrose (w/v) solution on top of a 60% sucrose solution. Following centrifugation (45,500 g, 30 min., 4°C) enriched plasma membrane fractions were collected in between the 10% and 60% sucrose layers and stored at -80°C until needed. For all western blots, equal amounts of proteins were electrophoresed on 10% SDS-PAGE gels, blotted to PVDF membranes and probed with the indicated antibodies. Membranes were stripped and re-probed with GAPDH antibodies to ensure equal loading of proteins among lanes. All blots were repeated at least three times with independent cell cultures. Representative data are shown for each. Where indicated, the band intensites of western blots were quantitated with ImageJ image analysis software (NIH, Bethesda, MD). Data from multiple blots (a minimum of three) were normalized and averaged. Statistical significance was determined by one-way ANOVA and Tukey's post hoc test. A p-value of <0.05 was used as an indication of statistical significance.
Cells grown to confluence on coverslips in 6-well plates were prepared for immunostaining, following FGF2 treatments for the indicated time. Samples were stained and imaged as previously described . Staining using non-immune rabbit and mouse IgG and anti-rabbit FITC and anti-mouse Cy3 secondary antibodies were used as a negative control. All immunostaining experiments were performed on a minimum of two coverslips per experiment and repeated on at least three separate cultures. Up to four fields of view were imaged per coverslip. Representative images are shown for each experiment.
MC3T3 cells were harvested with IP buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0% Triton-×-100, 10 mM sodium pyrophosphate, 10 mM α-glycerophosphate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate and 1× proteases inhibitor cocktail). Whole cell lysates (500 μg total proteins) were pre-cleared with protein A/G agarose beads at 4°C for 60 minutes. The cleared supernatants were incubated overnight at 4°C with 2 μg of the indicated antibodies, followed by a one-hour incubation at 4°C with protein A/G-agarose beads. After five washes in iced cold-PBS, the protein were eluted from the beads by heating the samples for 5 minutes at 95°C in Laemmli SDS buffer (62.5 mM Tris-HCl, 2%w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). A fraction of the eluted proteins (bead fraction) and supernatant (supe fraction) were then analyzed by western blotting (as described above) with the indicated antibodies.
Glutathione-S-Transferase (GST) Pulldowns
The region of the GJA1 gene corresponding to the C-terminal tail of the Cx43 protein (amino acids 241-382) were PCR amplified with the following forward and reverse primers: mCx43-EcoR1-241-Forward, GGAATTCCGTCTTCTTCAAGGGCGTT and mCx43-XhoI-382-Reverse, CCGCTCGAGTTAAATCTCCAGGTCAGG. The gel purified Cx43 C-terminal tail PCR product was then subcloned into the EcoR1/XhoI sites of the pGEX-5X-2 vector (Amersham, Piscataway, NJ) in-frame with an N-terminal GST tag. The resultant pGEX-Cx43CT (241-382) construct was used to generate purified GST-Cx43CT(241-382) protein, as we have published previously . Empty pGEX-5X-2 vector was used to generate GST alone, to be used as a negative control. Equal amounts of purified GST- or GST-Cx43CT(241-382) protein were covalently crosslinked to Glutathione Sepharose 4B beads (Amersham) with dimethyl pimelimidate. Immobilized GST- or GST-Cx43CT (241-382) beads (25 μl) were incubated with 100 μg of MC3T3 whole cell extracts for 4 hours at 4°C. After being washed five times in pulldown buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 0.1% Tween-20, 0.5 mM DTT, protease inhibitor cocktail), the bound proteins were eluted and analyzed by western blotting.
This work was supported by a grant (R01-AR052719) from the National Institute of Arthritis, Musculoskeletal and Skin Diseases.
- Stains JP, Civitelli R: Gap junctions in skeletal development and function. Biochim Biophys Acta. 2005, 1719 (1-2): 69-81. 10.1016/j.bbamem.2005.10.012.PubMedView ArticleGoogle Scholar
- Jiang JX, Siller-Jackson AJ, Burra S: Roles of gap junctions and hemichannels in bone cell functions and in signal transmission of mechanical stress. Front Biosci. 2007, 12: 1450-1462. 10.2741/2159.PubMedPubMed CentralView ArticleGoogle Scholar
- Laird DW: Life cycle of connexins in health and disease. Biochem J. 2006, 394 (Pt 3): 527-543.PubMedPubMed CentralView ArticleGoogle Scholar
- Wei CJ, Xu X, Lo CW: Connexins and cell signaling in development and disease. Annu Rev Cell Dev Biol. 2004, 20: 811-838. 10.1146/annurev.cellbio.19.111301.144309.PubMedView ArticleGoogle Scholar
- Gerido DA, White TW: Connexin disorders of the ear, skin, and lens. Biochim Biophys Acta. 2004, 1662 (1-2): 159-170. 10.1016/j.bbamem.2003.10.017.PubMedView ArticleGoogle Scholar
- Paznekas WA, Boyadjiev SA, Shapiro RE, Daniels O, Wollnik B, Keegan CE, Innis JW, Dinulos MB, Christian C, Hannibal MC: Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am J Hum Genet. 2003, 72 (2): 408-418. 10.1086/346090.PubMedPubMed CentralView ArticleGoogle Scholar
- Flenniken AM, Osborne LR, Anderson N, Ciliberti N, Fleming C, Gittens JE, Gong XQ, Kelsey LB, Lounsbury C, Moreno L: A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia. Development. 2005, 132 (19): 4375-4386. 10.1242/dev.02011.PubMedView ArticleGoogle Scholar
- Dobrowolski R, Sasse P, Schrickel JW, Watkins M, Kim JS, Rackauskas M, Troatz C, Ghanem A, Tiemann K, Degen J: The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans. Hum Mol Genet. 2008, 17 (4): 539-554. 10.1093/hmg/ddm329.PubMedPubMed CentralView ArticleGoogle Scholar
- Chung DJ, Castro CH, Watkins M, Stains JP, Chung MY, Szejnfeld VL, Willecke K, Theis M, Civitelli R: Low peak bone mass and attenuated anabolic response to parathyroid hormone in mice with an osteoblast-specific deletion of connexin43. J Cell Sci. 2006, 119 (Pt 20): 4187-4198. 10.1242/jcs.03162.PubMedView ArticleGoogle Scholar
- Lecanda F, Warlow PM, Sheikh S, Furlan F, Steinberg TH, Civitelli R: Connexin43 deficiency causes delayed ossification, craniofacial abnormalities, and osteoblast dysfunction. J Cell Biol. 2000, 151 (4): 931-944. 10.1083/jcb.151.4.931.PubMedPubMed CentralView ArticleGoogle Scholar
- Grimston SK, Brodt MD, Silva MJ, Civitelli R: Attenuated response to in vivo mechanical loading in mice with conditional osteoblast ablation of the connexin43 gene (Gja1). J Bone Miner Res. 2008, 23 (6): 879-886. 10.1359/jbmr.080222.PubMedPubMed CentralView ArticleGoogle Scholar
- Stains JP, Civitelli R: Gap junctions regulate extracellular signal-regulated kinase signaling to affect gene transcription. Mol Biol Cell. 2005, 16 (1): 64-72. 10.1091/mbc.E04-04-0339.PubMedPubMed CentralView ArticleGoogle Scholar
- Stains JP, Lecanda F, Screen J, Towler DA, Civitelli R: Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters. J Biol Chem. 2003, 278 (27): 24377-24387. 10.1074/jbc.M212554200.PubMedView ArticleGoogle Scholar
- Lima F, Niger C, Hebert C, Stains JP: Connexin43 potentiates osteoblast responsiveness to fibroblast growth factor 2 via a protein kinase C-delta/Runx2-dependent mechanism. Mol Biol Cell. 2009, 20 (11): 2697-2708. 10.1091/mbc.E08-10-1079.PubMedPubMed CentralView ArticleGoogle Scholar
- Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC: Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev. 2003, 83 (4): 1359-1400.PubMedView ArticleGoogle Scholar
- Giepmans BN: Gap junctions and connexin-interacting proteins. Cardiovasc Res. 2004, 62 (2): 233-245. 10.1016/j.cardiores.2003.12.009.PubMedView ArticleGoogle Scholar
- Herve JC, Bourmeyster N, Sarrouilhe D: Diversity in protein-protein interactions of connexins: emerging roles. Biochim Biophys Acta. 2004, 1662 (1-2): 22-41. 10.1016/j.bbamem.2003.10.022.PubMedView ArticleGoogle Scholar
- Herve JC, Bourmeyster N, Sarrouilhe D, Duffy HS: Gap junctional complexes: from partners to functions. Prog Biophys Mol Biol. 2007, 94 (1-2): 29-65. 10.1016/j.pbiomolbio.2007.03.010.PubMedView ArticleGoogle Scholar
- Lampe PD, TenBroek EM, Burt JM, Kurata WE, Johnson RG, Lau AF: Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol. 2000, 149 (7): 1503-1512. 10.1083/jcb.149.7.1503.PubMedPubMed CentralView ArticleGoogle Scholar
- Martelli AM, Evangelisti C, Nyakern M, Manzoli FA: Nuclear protein kinase C. Biochim Biophys Acta. 2006, 1761 (5-6): 542-551.PubMedView ArticleGoogle Scholar
- Steinberg SF: Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J. 2004, 384 (Pt 3): 449-459.PubMedPubMed CentralView ArticleGoogle Scholar
- Doble BW, Ping P, Kardami E: The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation. Circ Res. 2000, 86 (3): 293-301.PubMedView ArticleGoogle Scholar
- Bowling N, Huang X, Sandusky GE, Fouts RL, Mintze K, Esterman M, Allen PD, Maddi R, McCall E, Vlahos CJ: Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart. J Mol Cell Cardiol. 2001, 33 (4): 789-798. 10.1006/jmcc.2000.1349.PubMedView ArticleGoogle Scholar
- Im HJ, Muddasani P, Natarajan V, Schmid TM, Block JA, Davis F, van Wijnen AJ, Loeser RF: Basic fibroblast growth factor stimulates matrix metalloproteinase-13 via the molecular cross-talk between the mitogen-activated protein kinases and protein kinase Cdelta pathways in human adult articular chondrocytes. J Biol Chem. 2007, 282 (15): 11110-11121. 10.1074/jbc.M609040200.PubMedPubMed CentralView ArticleGoogle Scholar
- Peluso JJ: Basic fibroblast growth factor (bFGF) regulation of the plasma membrane calcium ATPase (PMCA) as part of an anti-apoptotic mechanism of action. Biochem Pharmacol. 2003, 66 (8): 1363-1369. 10.1016/S0006-2952(03)00486-6.PubMedView ArticleGoogle Scholar
- Peluso JJ, Pappalardo A, Fernandez G: Basic fibroblast growth factor maintains calcium homeostasis and granulosa cell viability by stimulating calcium efflux via a PKC delta-dependent pathway. Endocrinology. 2001, 142 (10): 4203-4211. 10.1210/en.142.10.4203.PubMedGoogle Scholar
- Wert MM, Palfrey HC: Divergence in the anti-apoptotic signalling pathways used by nerve growth factor and basic fibroblast growth factor (bFGF) in PC12 cells: rescue by bFGF involves protein kinase C delta. Biochem J. 2000, 352 (Pt 1): 175-182. 10.1042/0264-6021:3520175.PubMedPubMed CentralView ArticleGoogle Scholar
- Kim BG, Kim HJ, Park HJ, Kim YJ, Yoon WJ, Lee SJ, Ryoo HM, Cho JY: Runx2 phosphorylation induced by fibroblast growth factor-2/protein kinase C pathways. Proteomics. 2006, 6 (4): 1166-1174. 10.1002/pmic.200500289.PubMedView ArticleGoogle Scholar
- Kim HJ, Kim JH, Bae SC, Choi JY, Ryoo HM: The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem. 2003, 278 (1): 319-326. 10.1074/jbc.M203750200.PubMedView ArticleGoogle Scholar
- Srisakuldee W, Jeyaraman MM, Nickel BE, Tanguy S, Jiang ZS, Kardami E: Phosphorylation of connexin-43 at serine 262 promotes a cardiac injury-resistant state. Cardiovasc Res. 2009, 83 (4): 672-681. 10.1093/cvr/cvp142.PubMedView ArticleGoogle Scholar
- Srisakuldee W, Nickel BE, Fandrich RR, Jiang ZS, Kardami E: Administration of FGF-2 to the heart stimulates connexin-43 phosphorylation at protein kinase C target sites. Cell Commun Adhes. 2006, 13 (1-2): 13-19. 10.1080/15419060600631326.PubMedView ArticleGoogle Scholar
- Hawat G, Baroudi G: Differential modulation of unapposed connexin 43 hemichannel electrical conductance by protein kinase C isoforms. Pflugers Arch. 2008, 456 (3): 519-527. 10.1007/s00424-007-0426-9.PubMedView ArticleGoogle Scholar
- Kwak BR, van Veen TA, Analbers LJ, Jongsma HJ: TPA increases conductance but decreases permeability in neonatal rat cardiomyocyte gap junction channels. Exp Cell Res. 1995, 220 (2): 456-463. 10.1006/excr.1995.1337.PubMedView ArticleGoogle Scholar
- Liu TF, Johnson RG: Effects of TPA on dye transfer and dye leakage in fibroblasts transfected with a connexin 43 mutation at ser368. Methods Find Exp Clin Pharmacol. 1999, 21 (6): 387-390. 10.1358/mf.1922.214.171.1241917.PubMedView ArticleGoogle Scholar
- Bao X, Reuss L, Altenberg GA: Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of Serine 368. J Biol Chem. 2004, 279 (19): 20058-20066. 10.1074/jbc.M311137200.PubMedView ArticleGoogle Scholar
- Solan JL, Lampe PD: Connexin43 phosphorylation: structural changes and biological effects. Biochem J. 2009, 419 (2): 261-272. 10.1042/BJ20082319.PubMedPubMed CentralView ArticleGoogle Scholar
- Musil LS, Cunningham BA, Edelman GM, Goodenough DA: Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol. 1990, 111 (5 Pt 1): 2077-2088. 10.1083/jcb.111.5.2077.PubMedView ArticleGoogle Scholar
- Solan JL, Fry MD, TenBroek EM, Lampe PD: Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation. J Cell Sci. 2003, 116 (Pt 11): 2203-2211. 10.1242/jcs.00428.PubMedView ArticleGoogle Scholar
- Boengler K, Schulz R, Heusch G: Connexin 43 signalling and cardioprotection. Heart. 2006, 92 (12): 1724-1727. 10.1136/hrt.2005.066878.PubMedPubMed CentralView ArticleGoogle Scholar
- Barnett ME, Madgwick DK, Takemoto DJ: Protein kinase C as a stress sensor. Cell Signal. 2007, 19 (9): 1820-1829. 10.1016/j.cellsig.2007.05.014.PubMedPubMed CentralView ArticleGoogle Scholar
- Saffitz JE, Hames KY, Kanno S: Remodeling of gap junctions in ischemic and nonischemic forms of heart disease. J Membr Biol. 2007, 218 (1-3): 65-71. 10.1007/s00232-007-9031-2.PubMedView ArticleGoogle Scholar
- Churchill EN, Mochly-Rosen D: The roles of PKCdelta and epsilon isoenzymes in the regulation of myocardial ischaemia/reperfusion injury. Biochem Soc Trans. 2007, 35 (Pt 5): 1040-1042.PubMedView ArticleGoogle Scholar
- Kardami E, Detillieux K, Ma X, Jiang Z, Santiago JJ, Jimenez SK, Cattini PA: Fibroblast growth factor-2 and cardioprotection. Heart Fail Rev. 2007, 12 (3-4): 267-277. 10.1007/s10741-007-9027-0.PubMedView ArticleGoogle Scholar
- Davies SP, Reddy H, Caivano M, Cohen P: Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000, 351 (Pt 1): 95-105. 10.1042/0264-6021:3510095.PubMedPubMed CentralView ArticleGoogle Scholar
- Solan JL, Lampe PD: Key connexin 43 phosphorylation events regulate the gap junction life cycle. J Membr Biol. 2007, 217 (1-3): 35-41. 10.1007/s00232-007-9035-y.PubMedPubMed CentralView ArticleGoogle Scholar
- Mohammadi M, Honegger AM, Rotin D, Fischer R, Bellot F, Li W, Dionne CA, Jaye M, Rubinstein M, Schlessinger J: A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-gamma 1. Mol Cell Biol. 1991, 11 (10): 5068-5078.PubMedPubMed CentralView ArticleGoogle Scholar
- Gotoh N: Regulation of growth factor signaling by FRS2 family docking/scaffold adaptor proteins. Cancer Sci. 2008, 99 (7): 1319-1325. 10.1111/j.1349-7006.2008.00840.x.PubMedView ArticleGoogle Scholar
- Ahmed A, Plevin R, Shoaibi MA, Fountain SA, Ferriani RA, Smith SK: Basic FGF activates phospholipase D in endothelial cells in the absence of inositol-lipid hydrolysis. Am J Physiol. 1994, 266 (1 Pt 1): C206-212.PubMedGoogle Scholar
- Sa G, Das T: Basic fibroblast growth factor stimulates cytosolic phospholipase A2, phospholipase C-gamma1 and phospholipase D through distinguishable signaling mechanisms. Mol Cell Biochem. 1999, 198 (1-2): 19-30. 10.1023/A:1006970710298.PubMedView ArticleGoogle Scholar
- Oh DY, Park SY, Cho JH, Lee KS, Min do S, Han JS: Phospholipase D1 activation through Src and Ras is involved in basic fibroblast growth factor-induced neurite outgrowth of H19-7 cells. J Cell Biochem. 2007, 101 (1): 221-234. 10.1002/jcb.21166.PubMedView ArticleGoogle Scholar
- Dailey L, Ambrosetti D, Mansukhani A, Basilico C: Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 2005, 16 (2): 233-247. 10.1016/j.cytogfr.2005.01.007.PubMedView ArticleGoogle Scholar
- Raucci A, Bellosta P, Grassi R, Basilico C, Mansukhani A: Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways. J Cell Physiol. 2008, 215 (2): 442-451. 10.1002/jcp.21323.PubMedView ArticleGoogle Scholar
- Gschwendt M: Protein kinase C delta. Eur J Biochem. 1999, 259 (3): 555-564. 10.1046/j.1432-1327.1999.00120.x.PubMedView ArticleGoogle Scholar
- Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, Messing RO: Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol. 2000, 279 (3): L429-438.PubMedGoogle Scholar
- O'Flaherty JT, Chadwell BA, Kearns MW, Sergeant S, Daniel LW: Protein kinases C translocation responses to low concentrations of arachidonic acid. J Biol Chem. 2001, 276 (27): 24743-24750. 10.1074/jbc.M101093200.PubMedView ArticleGoogle Scholar
- Vucenik I, Ramakrishna G, Tantivejkul K, Anderson LM, Ramljak D: Inositol hexaphosphate (IP6) blocks proliferation of human breast cancer cells through a PKCdelta-dependent increase in p27Kip1 and decrease in retinoblastoma protein (pRb) phosphorylation. Breast Cancer Res Treat. 2005, 91 (1): 35-45. 10.1007/s10549-004-6456-5.PubMedView ArticleGoogle Scholar
- Goodenough DA, Paul DL: Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol. 2003, 4 (4): 285-294. 10.1038/nrm1072.PubMedView ArticleGoogle Scholar
- Jiang JX, Gu S: Gap junction- and hemichannel-independent actions of connexins. Biochim Biophys Acta. 2005, 1711 (2): 208-214. 10.1016/j.bbamem.2004.10.001.PubMedPubMed CentralView ArticleGoogle Scholar
- Rodriguez-Sinovas A, Cabestrero A, Lopez D, Torre I, Morente M, Abellan A, Miro E, Ruiz-Meana M, Garcia-Dorado D: The modulatory effects of connexin 43 on cell death/survival beyond cell coupling. Prog Biophys Mol Biol. 2007, 94 (1-2): 219-232. 10.1016/j.pbiomolbio.2007.03.003.PubMedView ArticleGoogle Scholar
- Adam PJ, Boyd R, Tyson KL, Fletcher GC, Stamps A, Hudson L, Poyser HR, Redpath N, Griffiths M, Steers G: Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer. J Biol Chem. 2003, 278 (8): 6482-6489. 10.1074/jbc.M210184200.PubMedView ArticleGoogle Scholar
- Stains JP, Lecanda F, Towler DA, Civitelli R: Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter. Biochem J. 2005, 385 (Pt 2): 613-623.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.