Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking.

Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.

Xarvcf, Xenopus member of the p120 catenin subfamily associating with cadherin juxtamembrane region.

The catenin ARVCF is a member of the p120(ctn) subfamily of Armadillo proteins. A number of catenins directly bind cadherin cytoplasmic tails, contributing to the modulation of cell-cell adhesion and motility processes. Some catenins, such as beta-catenin (and likely p120(ctn)), have additional roles within signaling pathways regulating gene transcription. We have isolated the Xenopus homolog of human ARVCF. Utilizing the cadherin membrane proximal region known to bind p120(ctn) and delta-catenin, coimmunoprecipitation experiments demonstrate that Xarvcf, likewise, binds cadherin in this region and that corresponding point mutations within conserved residues abrogate the Xarvcf-cadherin association. Western blot analysis of Xarvcf protein across a series of developmental stages reveals changes in protein mobility, likely due to changes in phosphorylation. Xarvcf is a maternally provided transcript and expressed in the embryo throughout all stages of development. Interestingly, Xarvcf mRNA is differentially spliced to produce several isoforms, one of which is developmentally regulated. In common with the putative post-translational modifications of the Xarvcf protein, the presence of alternative splice isoforms suggests that Xarvcf possesses the capacity to effect developmental functions in a regulatable manner.

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification.

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.

Inhibitory effects of 12-O-tetradecanoylphorbol-13-acetate on dye leakage from single Novikoff cells and on dye transfer between reaggregated cell pairs.

The inhibitory effects of 12-O-tetradeconylphorbol-13-acetate (TPA) on the Lucifer Yellow leakage in single (nondissociated and dissociated) Novikoff cells in the presence of ethyleneglycotetraacetic acid (EGTA) and transfer between reaggregated cell pairs were investigated. Under treatment with TPA, single Novikoff cells showed inhibition of the dye leakage in the presence of EGTA, especially after 60-min treatment. There were only slight differences between nondissociated and dissociated cells in these experiments. The dye transfer in reaggregated cell pairs were significantly inhibited after 30-min treatment with TPA. In pretreatment with TPA during the recovery period of reaggregation the dye transfer in cell pairs was completely blocked. It was suggested that TPA blocks the assembly of gap junction, while it had no remarkable effect on gating mechanism of the hemichannels.

Intercellular communication is reduced by TPA and Ki-ras p21 in quiescent, but not proliferating, NRK cells.

We investigated whether the growth state of NRK cells (proliferating or quiescent by serum deprivation) affected the ability of oncogenic Ki-ras p21 and the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), to alter gap junctional communication. We evaluated gap junctional permeance by rate analysis of the transfer of a fluorescent dye, Lucifer Yellow, between cell pairs. We found that while the gap junctions of proliferating NRK cells were unresponsive to both TPA and to Ki-ras p21, junctional communication in quiescent cells was significantly inhibited by brief exposures to 100 ng/ml TPA. Furthermore, activity of Ki-ras p21 2 h prior to TPA exposure enhanced the inhibitory effect of TPA in quiescent cells. Junctional sensitivity to TPA was transient, with inhibition of junctional communication detected at 10 min and refractory after 60 min of continuous exposure. The suppression of junctional communication by TPA was completely prevented if the oncogenic p21 had been active for a longer period of time (48 h). The application of a phorbol ester derivative (4 alpha-PDD), which does not activate protein kinase C, did not affect the ability of quiescent cells to communicate. From these results we conclude that there is a cell-state dependence of junctional sensitivity to TPA in NRK cells and that ras p21 activity potentiates the junctional response to TPA. One interesting possibility is that this involved a cell-cycle effect.