Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a “gatekeeper” function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.
In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)--a new copper compound exhibiting antineoplastic activity--on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-L-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas.
Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in essentially all tissues. There are 21 connexin genes in the human genome and different tissues express different connexin genes. Most connexins are known to be phosphoproteins. Phosphorylation can regulate connexin assembly into gap junctions, gap junction turnover and channel gating. Given the importance of gap junctions in development, proliferation and carcinogenesis, regulation of gap junction phosphorylation in response to wounding, hypoxia and other tissue insults is proving to be critical for cellular response and return to homeostasis. Connexin43 (Cx43) is the most widely and highly expressed gap junction protein, both in cell culture models and in humans, thus more research has been done on it and more reagents to it are available. In particular, antibodies that can report Cx43 phosphorylation status have been created allowing temporal examination of specific phosphorylation events in vivo. This review is focused on the use of these antibodies in tissue in situ, predominantly looking at Cx43 phosphorylation in brain, heart, endothelium and epithelium with reference to other connexins where data is available. These data allow us to begin to correlate specific phosphorylation events with changes in cell and tissue function.
Abstract-Pressure overload is a common pathological insult to the heart and the resulting hypertrophy is an independent risk factor for sudden cardiac death. Gap junction remodeling (GJR) has been described in hypertrophied hearts; however, a detailed understanding of the remodeling process and its effects on impulse propagation is lacking. Moreover, there has been little progress developing therapeutic strategies to diminish GJR. Accordingly, transverse aortic banding (TAC) was performed in mice to determine the effects of progressive pathological hypertrophy on connexin (Cx)43 expression, posttranslational phosphorylation, gap junction assembly, and impulse propagation. Within 2 weeks after TAC, total and phospho-Cx43 abundance was reduced and incorporation of Cx43 into gap junctional plaques was markedly diminished. These molecular changes were associated with progressive slowing of impulse propagation, as determined by optical mapping with voltage-sensitive dyes. Treatment with the aldosterone receptor antagonist spironolactone, which has been shown to diminish sudden arrhythmic death in clinical trials, was examined for its effects on GJR. We found that spironolactone blunted the development of GJR and also potently reversed established GJR, both at the molecular and functional levels, without diminishing the extent of hypertrophy. These data suggest a potential mechanism for some of the salutary electrophysiological and clinical effects of mineralocorticoid antagonists in myopathic hearts.
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