The purpose of this paper is to provide a brief overview of current thinking on the role of connexins, in particular Cx43, in growth regulation, and a more detailed discussion as to potential mechanisms involved with an emphasis on gene expression. While the precise molecular mechanism by which connexins can affect the growth of normal or tumor cells remains elusive, a number of exciting reports have expanded our understanding and are presented in some detail. Thus, we will discuss (Section 2): the role of protein-protein interactions in integrating connexins into multiple signal transduction pathways; phosphorylation at specific sites and reversal of growth inhibition; the role of the carboxy-terminal regulatory domain as a signaling molecule. Some of our latest work on the potential functions of endogenously produced carboxy-terminal fragments of Cx43 are also presented (Section 3). Finally, Section 4 will pay tribute to the rapidly emerging realization that connexins such as Cx43 and Cx32 exert important and extensive effects on gene expression, particularly those genes linked to growth regulation.

BackgroundThe content and composition of cerebrospinal fluid (CSF) is determined in large part by the choroid plexus (CP) and specifically, a specialized epithelial cell (CPe) layer that responds to, synthesizes, and transports peptide hormones into and out of CSF. Together with ventricular ependymal cells, these CPe relay homeostatic signals throughout the central nervous system (CNS) and regulate CSF hydrodynamics. One new candidate signal is augurin, a newly recognized 14 kDa protein that is encoded by esophageal cancer related gene-4 (Ecrg4), a putative tumor suppressor gene whose presence and function in normal tissues remains unexplored and enigmatic. The aim of this study was to explore whether Ecrg4 and its product augurin, can be implicated in CNS development and the response to CNS injury.MethodsEcrg4 gene expression in CNS and peripheral tissues was studied by in situ hybridization and quantitative RT-PCR. Augurin, the protein encoded by Ecrg4, was detected by immunoblotting, immunohistochemistry and ELISA. The biological consequence of augurin over-expression was studied in a cortical stab model of rat CNS injury by intra-cerebro-ventricular injection of an adenovirus vector containing the Ecrg4 cDNA. The biological consequences of reduced augurin expression were evaluated by characterizing the CNS phenotype caused by Ecrg4 gene knockdown in developing zebrafish embryos.ResultsGene expression and immunohistochemical analyses revealed that, the CP is a major source of Ecrg4 in the CNS and that Ecrg4 mRNA is predominantly localized to choroid plexus epithelial (CPe), ventricular and central canal cells of the spinal cord. After a stab injury into the brain however, both augurin staining and Ecrg4 gene expression decreased precipitously. If the loss of augurin was circumvented by over-expressing Ecrg4 in vivo, BrdU incorporation by cells in the subependymal zone decreased. Inversely, gene knockdown of Ecrg4 in developing zebrafish embryos caused increased proliferation of GFAP-positive cells and induced a dose-dependent hydrocephalus-like phenotype that could be rescued by co-injection of antisense morpholinos with Ecrg4 mRNA.ConclusionAn unusually elevated expression of the Ecrg4 gene in the CP implies that its product, augurin, plays a role in CP-CSF-CNS function. The results are all consistent with a model whereby an injury-induced decrease in augurin dysinhibits target cells at the ependymal-subependymal interface. We speculate that the ability of CP and ependymal epithelium to alter the progenitor cell response to CNS injury may be mediated, in part by Ecrg4. If so, the canonic control of its promoter by DNA methylation may implicate epigenetic mechanisms in neuroprogenitor fate and function in the CNS.

Mitogenic stimulation of cardiomyocytes is associated with decreased gap junction coupling and protein kinase C (PKC)-mediated phosphorylation of the gap junction protein connexin43 (Cx43). Identification of and interference with the amino acid(s) that becomes phosphorylated in response to stimulation are important steps towards defining the relationship between Cx43 phosphorylation and cell cycle. Using immunoblotting and phosphospecific antibodies we were able to show that serine-262 (S262) on Cx43 becomes phosphorylated in response to growth factor or PKC stimulation of cardiomyocytes. To examine the effect of Cx43, S262 phosphorylation and cell-cell contact (and/or coupling) on DNA synthesis, we overexpressed wild-type (wt) or mutant Cx43, carrying a S262-to-alanine (S262A, simulating the unphosphorylated state) or a S262-to-aspartate (S262D, simulating constitutive phosphorylation) substitutions in cultures of cell-cell contact forming or isolated cardiomyocytes. Overexpression of wt-Cx43 caused a significant decrease in DNA synthesis irrespective of the presence of cell-cell contact. In cell-cell contact forming cultures, the S262D mutation reversed while the S262A mutation increased the inhibitory effect of Cx43. In the absence of cell-cell contact, the S262-Cx43 mutations had no significant effect on Cx43 inhibition of DNA synthesis. Dye-coupling, evaluated by scrape-loading, indicated increased gap junction permeability in S262A (compared to wt or S262D) overexpressing myocytes. We conclude that Cx43 inhibits cardiomyocyte DNA synthesis irrespectively of cell-cell contact or coupling. Cell-cell contact, and possibly gap junction-mediated communication is required, however, in order to reverse Cx43 inhibition of DNA synthesis by S262 phosphorylation.

Gate leakage currents in AlGaN/GaN heterostructure field-effect transistor ͑HFET͒ structures with conventional and polarization-enhanced barriers have been studied. Comparisons of extensive gate leakage current measurements with two-dimensional simulations show that vertical tunneling is the dominant mechanism for gate leakage current in the standard-barrier HFET and that the enhanced-barrier structure suppresses this mechanism in order to achieve a reduced leakage current. An analytical model of vertical tunneling in a reverse-biased HFET gate-drain diode is developed to evaluate the plausibility of this conclusion. The model can be fit to the measured data, but suggests that additional leakage mechanisms such as lateral tunneling from the edge of the gate to the drain or defect-assisted tunneling also contribute to the total leakage current. The vertical tunneling current mechanism is shown to be more significant to the gate leakage current in III-V nitride HFETs than in HFETs fabricated in other III-V material systems, in which the lateral tunneling current component generally dominates the gate leakage current.

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We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4, an epigenetically regulated gene, which unlike other tumor suppressor genes, encodes an orphan-secreted, ligand-like protein. In human cell lines, Ecrg4 gene expression was low, Ecrg4 protein undetectable, and Ecrg4 promoter hypermethylation high (45-90%) and reversible by the methylation inhibitor 5-AzaC. In contrast, Ecrg4 gene expression in fresh, normal human PBMCs and PMNs was 600-800 times higher than in cultured cell lines, methylation of the Ecrg4 promoter was low (<3%), and protein levels were readily detectable in lysates and on the cell surface. Flow cytometry, immunofluorescent staining, and cell surface biotinylation established that full-length, 14-kDa Ecrg4 was localized on PMN and monocyte cell surfaces, establishing that Ecrg4 is a membrane-anchored protein. LPS treatment induced processing and release of Ecrg4, as detected by flow and immunoblotting, whereas an effect of fMLF treatment on Ecrg4 on the PMN cell surface was detected on the polarized R2 subpopulation of cells. This loss of cell surface Ecrg4 was associated with the detection of intact and processed Ecrg4 in the conditioned media of fresh leukocytes and was shown to be associated with the inflammatory response that follows severe, cutaneous burn injury. Furthermore, incubation of macrophages with a soluble Ecrg4-derived peptide increased the P-p65, suggesting that processing of an intact sentinel Ecrg4 on quiescent circulating leukocytes leads to processing from the cell surface following injury and macrophage activation.

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The role of spontaneous and piezoelectric polarization in III-V nitride heterostructures is investigated. Polarization effects and crystal polarity are reviewed in the context of nitride heterostructure materials and device design, and a detailed analysis of their influence in nitride heterostructure field-effect transistors is presented. The combined effects of spontaneous and piezoelectric polarization are found to account well for carrier concentrations observed in AlGaN/ GaN transistor structures with low to moderate Al concentrations, while the data for higher Al concentrations are consistent with defect formation in the AlGaN barrier. Theoretical analysis suggests that incorporation of In into the barrier and/or channel layers can substantially increase polarization charge at the heterojunction interface. The use of polarization effects to engineer Schottky barrier structures with large enhancements in barrier height is also discussed, and electrical characteristics of transistors with conventional and polarization-enhanced Schottky barrier gates are presented. The polarization-enhanced barrier is found to yield a marked reduction in gate leakage current, but to have little effect on transistor breakdown voltage.