Current Drug Targets - Volume 3, Issue 6, 2002

The synthesis and the degradation of gap junctions involve multiple steps that may provide targets for the modulation of intercellular communication. Many studies using cultured cells have examined the effects of inhibitors of protein synthesis, trafficking, or degradation upon connexins. Similarly, activators or inhibitors of various protein kinases have been shown to affect connexin assembly or proteolysis. These studies have helped to elucidate the connexin lifecycle. But, because of their lack of specificity for gap junction proteins, these agents would be expected to have limited therapeutic utility and to produce several deleterious side effects. However, more selective agents are being developed based on specific features of the connexin sequences. Molecular genetic approaches have been used to introduce wild-type connexins to increase intercellular communication in otherwise poorly coupled cells. Decreased intercellular communication may be obtained by application of peptides that mimic the extracellular loops and may prevent docking of hemi-channels. Alternatively, introducing mutant connexins that interfere with the oligomerization / export of endogenous connexins or with channel function by formation of non-functional heteromeric hemi-channels can also reduce intercellular communication.

Gap junction channel selectivity, open probability, and recruitment are properties that affect the transit of solutes from cell to cell. Selectivity is the property of an open channel that acts as a discriminator, allowing the passage of specific solutes while denying transit to others. Open probability is another parameter that can affect the efficiency of cell to cell transit. Channel recruitment is another facilitating mechanism able to affect transit. Rapid alterations in any of these properties suggests that dynamic effects are possible for gap junction channel mediated cell to cell coupling. Suspect modulators presumably capable of effecting dynamic change would be the kinases. The properties of gap junction channels in the context of selectivity, open probability and recruitment are discussed here along with the ability of kinases to affect them. These processes and their regulation are also targets for regulating cell-to-cell communication through therapeutics. Clearly these same properties are ones that disease states might also opportunistically alter to affect physiological processes.

Phenotypic variability in smooth muscle cells accounts, in large part, for the incredible functional diversity required of the involuntary hollow organs of the body (i.e., respiratory passages, blood vessels, gastrointestinal tract, urogenital tract, etc.). In all instances coordination of smooth muscle cell responses, that is, contraction and relaxation, is critical to normal organ function. While numerous biological mechanisms exist for coordinating smooth muscle cell responses, intercellular communication through gap junctions represents a common denominator present in all organ systems. In this report, we review the evidence documenting the presence and functional significance of myocyte gap junctions to physiologically distinct organ systems, and furthermore, provide some examples of their putative roles in organ pathology. Finally, we advance the thesis that despite their ubiquity and heterogeneous expression, gap junctions are nonetheless potentially attractive therapeutic targets for the treatment of certain smooth muscle disorders. Their therapeutic efficacy will necessarily hinge on the existence of connexin isoformselective junctional effects. The overall rationale for targeting the intercellular pathway is therefore analogous to strategies that target other ubiquitously expressed ion channels, such as calcium or potassium channels. Such strategies have proved efficacious for the treatment of a wide range of human smooth muscle disorders including hypertension, urinary incontinence and sexual function.

Intercellular communication through gap junctions is essential for the regulation of normal cellular processes. In the diseased state, however, gap junctions may be decreased, inappropriately expressed, or constitutively expressed in either the open or closed state. Thus, it may prove important to develop therapeutic agents to either induce or prevent channel closure. To address this dilemma, the mechanisms that cause channel gating as well as the structure-function and permeability determinants of connexins provide useful information. Residues in the C-terminal tail of Connexin 43 are implicated as sites for phosphorylation by kinases that directly mediate channel gating as well as binding sites that influence gating properties. Gating of gap junctions by pH, insulin, and other growth factors has also been associated with the C-terminal domain. The rational design of inhibitors to channel gating may prove useful for the development of therapeutic agents to maintain Connexin 43 in the open state, with potential benefits in diseases such as cancer, arrhythmias, and the diabetic lens. Alternatively, modeling approaches to obtain gap junctions that are constitutively closed might be targeted to designing compounds that could potentially occlude the pore. In this case, knowledge of the pore-lining residues, as well as permeability determinants, would be useful for developing connexin-specific inhibitors that may have future therapeutic potential for tumor invasiveness and stroke treatment. Thus, information from existing and future studies may lead to the development of site-directed, specific modulators of gap junction communication with potential implications in the therapeutic treatment of disease.

Connexin-null mice and human genetic gap junction diseases illustrate the important roles that gap junction channels play under normal conditions, and the neuro- and cardioprotective effects of gap junction blocking agents demonstrate that closure of these channels may be beneficial in certain pathological situations. This overview summarizes studies in which gap junction modifying reagents have been characterized, highlighting examples of agents for which selectivity for gap junction subtypes has been demonstrated. In addition, strategies for targeting connexin domains through peptide inhibitors are outlined, which may ultimately provide agents that are not only connexin-selective in their actions, but also affect only a subset of a gap junction channel's gating responses.

The development of the most efficacious strategy for the prevention and treatment of cancers is based on understanding the underlying mechanisms of carcinogenesis. This includes the knowledge that the carcinogenic process is a multi-step, multi-mechanism process and that no two cancers are alike, in spite of some apparent universal characteristics, such as their inability to have growth control, to terminally differentiate, to apoptose abnormally and to have an apparent extended or immortalized life span. The multi-step process, involving the “initiation” of a single cell via some irreversible process, with the clonal expansion of this initiated cell by suppressing growth control and inhibiting apoptosis (promotion step), leads to a situation whereby additional genetic and epigenetic events can take place (progression step) to confer the necessary phenotypes of invasiveness, and metasis (neoplastic stage). While it is clear that, in principle, prevention of each of these three steps is possible, in practical terms, while it would make sense to minimize the initiation step, one can never reduce this step to zero. On the other hand, since the promotion step is the ratelimiting step of carcinogenesis, intervening to block this step makes the most sense. Also, by understanding the ultimate biological function that confers growth control, terminal differentiation or apoptosis for cells, there is even some hope of treating some, but not all, malignant cells such that they can regain some ability to perform these vital cellular functions.Gap junctional intercellular communication (GJIC) has been speculated to be a necessary, if not sufficient, biological function of metazoan cells for the regulation of growth control, differentiation and apoptosis of normal progenitor cells. Normal, contact-inhibited fibroblast and epithelial cells have functional GJIC, while most, if not all, tumor cells have dysfunctional homologous or heterologous GJIC. Cancer cells are characterized by the lack of growth control, inability to terminally differentiate or apoptose under normal conditions and have extended or immortalized life spans. Chemical tumor promoters, growth factors and hormones have been shown to inhibit GJIC. Several oncogenes and anti-sense connexin genes have been shown to down-regulate GJIC function. Anti-oncogene drugs, anti-tumor promoting natural and synthetic chemicals, as well as GJIC-deficient neoplastic cells, transfected with various connexin genes, have been shown to re-gain GJIC and growth control with the loss of tumorigenicity. Therefore, the hypothesis for a rational approach to identify anti-tumor promoting chemopreventive drugs and anti-carcinogenic treatments is to use the prevention of the down regulation of GJIC by the tumor promoters and the restoration of GJIC in neoplastic cells.While previous and many current strategies for chemoprevention and therapy have been based on treating specific oncogene products or cell signalling mechanisms, as well as advance molecular modifications of older strategies, none have specifically used the prevention of GJIC by agents during the rate limiting step of carcinogenesis or the restoration of GJIC in neoplastic cells which are deficient in GJIC. Since there are multiple mechanisms by which GJIC is down regulated during the tumor promotion phase and since stable GJIC deficiencies in neoplastic cells can be the result of transcriptional, translational or posttranslational mechanisms, it should be clear there would not be one “golden bullet” approach to resolve either the chemoprevention or therapeutic approach. Even with the hypothesis that GJIC, which depends on the transcription of normal connexin genes, their normal translation, trafficking, assembly and function, it should be clear that cells with normal connexin genes and potentially normal GJIC might not have functional GJIC because of dysfunction of other defects in cancer cells, namely cell-adhesion or cell-matrix problems (both of which are necessary for GJIC to occur). In essence, if dietary or chemopreventive / therapy is to be effective, the strategy must either ameliorate the growth stimulatory effects of exogenous chemicals, growth factors or hormones, that trigger various mitogenic / anti-apoptotic signal transducing systems that inhibit GJIC.

Important roles for connexins have emerged from studies linking connexin mutations to human disease. Use of connexins tagged with GFP have provided a clearer picture of the mechanisms that govern connexin channel function and it is now evident that functional forms of connexin channel include cell-cell channels and unapposed hemichannels. Clustering appears to be a requirement for opening of cell-cell channels and suggests that dynamic changes occur in plaques (clusters) as they form and grow that are critical for channel function. In particular, recruitment or generation of ‘silent’ channels has gained support as a mechanism by which coupling can be dynamically regulated within formed plaques. Two distinct voltage sensitive gating mechanisms appear to be built-into each hemichannel, one putatively located at the cytoplasmic entrance and the other at the extracellular end, each differing in sensitivity, kinetics and degree of channel / hemichannel closure. The extracellular gate may also be that which opens unapposed hemichannels in the plasma membrane and be the final target of many known chemical agents that act as uncouplers of cell-cell communication. An understanding of the structural requirements for regulation via gating and clustering represents an important preclinical step in the design of therapeutic agents to treat disorders arising from connexin channel and hemichannel dysfunction.