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.     

Acute internalization of gap junctions in vascular endothelial cells in response to inflammatory mediator-induced G-protein coupled receptor activation

During the inflammatory response, activation of G-protein coupled receptors (GPCRs) by inflammatory mediators rapidly leads to inhibition of gap junction intercellular communication (GJIC); however, the steps that lead to this inhibition are not known. Combining high-resolution fluorescence microscopy and functional assays, we found that activation of the GPCRs PAR-1 and ET_A/B by their natural inflammatory mediator agonists, thrombin and endothelin-1, resulted in rapid and acute internalization of gap junctions (GJs) that coincided with the inhibition of GJIC followed by increased vascular permeability. The endocytosis protein clathrin and the scaffold protein ZO-1 appeared to be involved in GJ internalization, and ZO-1 was partially displaced from GJs during the internalization process. These findings demonstrate that GJ internalization is an efficient mechanism for modulating GJIC in inflammatory response.     

Development of a prototype reporter-based assay for the evaluation of gap junctional intercellular communication

A new reporter-based assay for the evaluation of gap junctional intercellular communication (GJIC) is presented. This assay was applied to the study of endogenous GJIC as well as to the evaluation of cell-to-cell communication exogenously induced in non-coupling cells by transfection with connexin 32. The results obtained with 18--glycyrrhetinic acid indicate that this assay system can be used to monitor the GJIC induced by transport of cAMP induced by activation of the dopamine 1 receptor cascade.     

Evidence for the Presence of a Free C-Terminal Fragment of Cx43 in Cultured Cells

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete® and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.     

A strategy for the suppression of tumorigenesis induced by biomaterials: Restoration of transformed phenotype of polyetherurethane-induced tumor cells by Cx43 transfection

Biomaterials such as polyetherurethans (PEUs) are the scaffolding, which is indispensable for the development of the bio-artificial organs. However, PEUs can induce tumors in subcutaneous implantation sites in rat. We have shown that the different inhibitory potential of gap junctional intercellular communication (GJIC) on the surface of the biomaterials, including PEUs, is a key step in determining the tumorigenic potential. Here we show that suppression of a gap junctional protein connexin 43 (Cx43) plays an important role in in vivo tumorigenesis induced by PEUs for the first time and that Cx43 transfection may be an effective strategy for preventing tumorigenesis induced by biomaterials. Rat tumor cell line U41 is derived from tumors in the subcutaneous implantation of PEU films. The GJIC and the expression of Cx43 were suppressed in U41. The restoration of normal phenotype, such as reduction of growth rate, recovery of contact inhibition and loss of colony formation ability in soft agar, was achieved by Cx43 transfection. These results strongly suggest that suppression of Cx43 expression plays an important role in the development of rat malignant fibrous histiocytoma (MFHC) caused by PEUs and that Cx43 transfection is effective for prevention of tumorigenesis induced by PEUs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

Summary Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels     

Intercellular communication and the control of growth: XII. Alteration of junctional permeability by simian virus 40. roles of the large and smallT antigens

Summary We studied the action of temperature-sensitive mutant simian virus 40—a transformation-inducing DNA virus—on the junctional permeability to mono-, di- and triglutamate in rat embryo-, pancreas islet (epithelia)-, and 10T1/2 cell cultures. Junctional permeability was reduced (reversibly) in the transformed state. To dissect the genetics of this alteration, we used two kinds of mutant virus DNA. One kind had a temperature-sensitive mutation on theA gene, rendering the largeT antigen (the gene product) thermolabile (T ⁺ T ^–). The other had a deletion on theF gene, in addition, abolishing (permanently) the expression of the littlet antigen (t ^–). The junctional alteration occurred in the conditionT ⁺ t ⁺, but not in the conditionsT ^– t ⁺,T ⁺ t ^– orT ^– t ^–. Both antigens, thus, are necessary for this junctional alteration—a genetic requirement identical to that for decontrol of growth (but distinct from that of the cytoskeletal alteration).     

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by thesrc gene in a revertant cell with normal cytoskeleton

Summary To learn whether the reduction of cell-to-cell communication in transformation is a possible primary effect of pp60^src phosphorylation or secondary to a cytoskeletal alteration, we examined the junctional permeability in transformed cells with normal cytoskeleton. The permeability to fluorescentlabelled mono- and diglutamate was compared in clones of Faras' vole cells—clones transformed by Rous sarcoma virus and reverted from that transformation. One revertant clone (partial revertant), had the high levels of pp60^src kinase activity and tumorigenicity of the fully transformed parent clone, but had lost the cytoskeletal alterations of that clone. Another revertant clone (full revertant) had lost the tumorigenicity and most of the pp60^src kinase activity, in addition (J.F. Nawrocki et al., 1984,Mol. Cell Biol.4:212). The junctional permeability of thepartial revertant with normal cytoskeleton was similar to that of the fully transformed parent clone with abnormal cytoskeleton. The permeabilities of both were lower than those of thefull revertant and the normal uninfected cell, demonstrating that the junctional change by thesrc gene is independent of the cytoskeletal one.     

The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

EGF rapidly translocates tight junction proteins from the cytoplasm to the cell-cell contact via protein kinase C activation in TMK-1 gastric cancer cells

Tight junctions are commonly disrupted in cancer cells, including gastric cancer. Various growth factors have been reported to affect the localization of tight junction-associated proteins such as ZO-1 and occludin. We investigated the effect of epidermal growth factor (EGF), a growth factor that is often overexpressed in gastric cancer, and fetal bovine serum (FBS) on the localization of ZO-1 and occludin in a gastric cancer cell line. In the poorly differentiated gastric cancer cell line TMK-1, immunohistochemistry demonstrated that ZO-1 and occludin were predominantly localized to the cytoplasm, although there was some weak expression at the cell-cell contact. When the medium was replaced with fresh medium containing 10% FBS, ZO-1 and occludin were rapidly translocated from the cytosol to the cell-cell contact. A similar effect was seen in EGF exposure. These effects induced by FBS or EGF were attenuated in the presence of protein kinase C (PKC) inhibitors calphostin C and bisindolylmaleimide I, but not another PKC inhibitor G?6976, PD98059 (MAPK inhibitor), LY294002 (PI3 kinase inhibitor) or KT5720 (protein kinase A inhibitor). These results suggest that serum-derived factors, including EGF, can rapidly alter the localization of ZO-1 and occludin via a protein kinase C signaling pathway in TMK-1 gastric cancer cells.     

Modulation of prostate cancer growth in bone microenvironments

Bone remains one of the major sites, and most lethal host organs, for prostate cancer metastasis. Prostate cell spread and establishment in bone depends on multiple reciprocal modifications of bone stromal and epithelial cancer cell behaviors. This review focuses on recent advances in the characterization of cell-cell and cell-matrix interplay, effects on cell growth, adhesion and invasion, and several therapeutic possibilities for co-targeting prostate cancer cells and bone stroma. We address the topic from three main perspectives: (1) the normal and aging bone stromal environment, (2) the "reactive" bone stromal environment, and (3) the cancerous prostate epithelial cells themselves. First, normal, and especially aging, bones provide uniquely rich and "fertile soil" for roaming cancer cells. The interactions between prostate cancer cells and insoluble extracellular matrices, soluble growth factors, and/or sex steroid hormones trigger bone remodeling, through increased osteoclastogenesis and furthur matrix metalloproteinase activity. Second, after cancer cell arrival and establishment in the bone, host stromal cells respond, becoming "reactive" in a process again involving extracellular matrix remodeling, together with growth factor and steroid receptor signaling this process ultimately enhances cancer cell migration, stromal transdifferentiation, and invasion of the cancer tissues by stromal, inflammatory, and immune-responsive cells. Third, prostate cancer cells also respond to supportive bone microenvironments, where soluble and matrix-associated molecules affect cancer cell growth and gene expression, especially altering cancer cell surface receptor and integrin-mediated cell signaling. We discuss both integrin cell-matrix and gap junctional cell-cell communication between cancer cells and their microenvironments during prostate cancer progression.     

Cx40.8, a Cx43-like protein, forms gap junction channels inefficiently and may require Cx43 for its association at the plasma membrane

In addition to having a Cx43 ortholog, the zebrafish genome also contains a Cx43-like gene, Cx40.8. Here, we investigate the expression of cx40.8 in zebrafish fins and the function of Cx40.8 in HeLa cells. We find that cx40.8 is present in the same population of dividing cells as cx43. Unlike Cx43, dye coupling assays suggest that Cx40.8 only inefficiently forms functional gap junction channels. However, co-transfection reveals that Cx40.8 can co-localize with Cx43 in gap junction plaques, and that the resulting plaques contain functional gap junction channels. Together, these data suggest the possibility that Cx40.8 may functionally interact with Cx43 to regulate cell proliferation in vivo.

Structured summary

MINT-7266123: cx40.8 (genbank_protein_gi:68354404) and cx43 (uniprotkb:O57474) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells

The G-protein-mediated coupling of a glucagon receptor to ATP-dependent K channels—K_ATP—has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches, K_ATP channel activity was inhibited by low concentrations of glucagon (IC₅₀ = 2.4 nm); the inhibitory effect vanished at concentrations greater than 50 nm. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently divergent results. In excised inside-out patches, K_ATP channel activity was inhibited by addition of subunits purified from either erythrocyte or retina (IC₅₀ = 50 pm and 1 nm, respectively). Subsequent exposure of the patch to _i or _o reversed this effect. In excised inside-out patches, increasing Mg²⁺ in the bath stimulated the channel activity between 0 and 0.5 mm, but blocked it at higher concentrations (IC₅₀ = 2.55 mm). In most cases (70%), GTP had a stimulatory effect at concentrations up to 100 m. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified _i or _o . These results are compatible with a model whereby channel inhibition by activated G _S -coupled receptors occurs, at least in part, via association of the subunits of G _S with _i / _o subunits and deactivation of the _i / _o -dependent stimulatory pathway. On the basis of this hypothesis, a model is developed to describe the effects of G proteins on the K_ATP channel, as well as to account for the concentration-dependent stimulation and inhibition of K_ATP channel by Mg²⁺. An interpretation of the ability of glucagon to potentiate, but not initiate, insulin release is also given in terms of this model and the effects of ATP on K_ATP channels.This work was supported by grant DCB-89 19368 from the National Science Foundation and a research grant (W-P 880513) from the American Diabetes Association to B.R.The authors would like to thank Dr. A.E. Boyd, III for supplying the RINm5F and HIT cells, Drs. J. Codina and L. Birnbaumer for supplying the G protein and subunits from erythrocyte, Dr. R.A. Cerione for supplying the G protein subunit from retina, and Mrs. Satoko Hagiwara for preparing and maintaining the cell cultures.     

Progression to metastatic stage in a cellular model of prostate cancer is associated with methylation of the androgen receptor gene and transcriptional suppression of the insulin-like growth factor-I receptor gene

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF1R) expression. DNA methylation of CpG islands is an epigenetic mechanism associated with gene silencing. Recent studies have demonstrated that methylation occurs early in prostate carcinogenesis and, furthermore, may contribute to androgen independence. The methylation status of the AR and IGF1R genes was evaluated in a series of prostate cancer cell lines corresponding to early (benign) and advanced (metastatic) stages of the disease. Results of 5-Aza-2′-deoxycytidine (5-Aza) experiments, methylation-specific PCR, and sodium bisulfite-direct DNA sequencing revealed that the AR promoter is hypermethylated in metastatic M12, but not in benign P69, cells. On the other hand, no methylation was seen in the IGF1R promoter at any stage of the disease. We show, however, that 5-Aza treatment, which caused demethylation of the AR promoter, led to a significant increase in IGF1R mRNA levels, whereas addition of the AR inhibitor flutamide decreased the IGF1R mRNA levels to basal values measured prior to the 5-Aza treatment. Given that the IGF1R gene has been identified as a downstream target for AR action, our data is consistent with a model in which the AR gene undergoes methylation during progression of the disease, leading to dysregulation of AR targets, including the IGF1R gene, at advanced metastatic stages.     

A robust protocol for site-directed mutagenesis of the D1 protein inChlamydomonas reinhardtii: A PCR-splicedpsbA gene in a plasmid conferring spectinomycin resistance was introduced into apsbA deletion strain

In this paper, we describe a protocol to obtain a site-directed mutants in thepsbA gene ofChlamydomonas reinhardtii, which overcomes several drawbacks of previous protocols, and makes it possible to generate a mutant within a month. Since the large size of the gene, and the presence of four large introns has made molecular genetics of thepsbA gene rather unwieldy, we have spliced all of the exons of thepsbA gene by PCR to facilitate genetic manipulation and sequencing of the gene. The resultant construct (plasmid pBA153, with several unique restriction sites introduced at exon boundaries) carried 1.2 and 1.8 kb intact sequences from the 5- and 3-flanking regions, respectively. The plasmid was used to transform a D1-deletion mutant and was found to complement the deletion and restore photosynthetic activity. In addition, a bacterialaadA gene conferring spectinomycin resistance (spe ^r) was inserted downstream of the intron-freepsbA gene, to give construct pBA155. This allowed selection of mutant strains deficient in photosynthesis by using spectinomycin resistance, and eliminated the possibility of selection for revertant strains which is a consequence of having to use photosynthetic activity as a selection pressure. Finally, pBA155 was used to construct pBA157, in which additional restriction sites were inserted to facilitate cassette mutagenesis for generation of mutations in spans thought to be involved in donor-side interactions. AllpsbA deletion strains transformed with intron-freepsbA-aadA constructs encoding the wild-type D1 sequence, and screened on spectinomycin plates for thespe ^r phenotype, were able to grow photosynthetically, and all showed identical kinetics for electron transfer from primary (Q_A) to secondary quinone (Q_B) in Photosystem II, as assayed by the decay of the high fluorescence yield on oxidation of the reduced primary acceptor (Q_A ^–).     

Hex-1, a gene unique to filamentous fungi, encodes the major protein of the Woronin body and functions as a plug for septal pores

We have identified a gene, named hex-1, that encodes the major protein in the hexagonal crystals, or Woronin bodies, of Neurospora crassa. Analysis of a strain with a null mutation in the hex-1 gene showed that the septal pores in this organism were not plugged when hyphae were damaged, leading to extensive loss of cytoplasm. When grown on agar plates containing sorbose, the hex-1(-) strain showed extensive lysis of hyphal tips. The HEX-1 protein was predicted to be 19,125 Da. Analysis of the N-terminus of the purified protein indicated that 16 residues are cleaved, yielding a protein of 17,377 Da. A polyclonal antibody raised to the HEX-1 protein recognized multiple forms of the protein, apparently dimers and tetramers that were resistant to solubilization by sodium dodecyl sulfate and reducing reagents. Treatment of the protein with phosphatase caused dissociation of these oligomers. Preparations enriched in Woronin bodies contained catalase activity, which was not detected in comparable fractions from the hex-1(-) mutant strain. These results support the hypothesis that the Woronin body is a specialized peroxisome that functions as a plug for septal pores.     

Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.