Connexin43 and development of primary bone tumors: osteosarcoma and Ewing's sarcoma

Gap junction channels

According to Schleiden's theory, cells are autonomous units that are circumscribed by a diffusion barrier that is able to prevent any exchange with the surrounding cells [ ]. However, in the late 1960s, Kanno and Loewenstein demonstrated that the fluorescein, a molecule of 376 Da, can pass freely from one cell to another [ ]. In this context, Loewenstein and coll. have proposed that this passage was due to channels that cross the membranes of the two cells in contact. These channels would be composed of two hemichannels able to form an intercellular channel [ , ]. In 1975, the term “communicating junctions” appeared in reference to the function of these structures [ ].

It is now accepted that gap junctions are membrane structures that allow for the direct transfer of small molecules between adjacent cells. Their presence is observed in almost all types of cells, with a few exceptions such as circulating blood cells, certain neurons, mature adult skeletal muscle cells, and spermatozoids. Studies that have used electron microscopy or optical diffraction approaches have demonstrated that each intercellular channel consists of two hemichannels (connexons), both composed of six gap junction proteins (connexins). These connexons align head-to-head on opposite cell surfaces to form an intercellular canal [ ]. Connexons thus form a central pore of 2 nm (nm) in diameter, allowing the diffusion of ions and hydrophilic molecules of molecular mass lower than 1200 Da, like second cytoplasmic messengers (for example, calcium, inositol triphosphate, adenosine monophosphate, glucose, glutamate, etc.) [ , ] and even anticancer drugs [ ]. More recently, studies have demonstrated that siRNA and miRNA can also pass through Gj [ , ] ( Fig. 22.1A ). Homotypic or heterotypic intercellular channels can be functional [ ]. The presence of hemichannels at the membrane has also been demonstrated in various cells [ ].

Connexins

To date, 21 gene codings for various connexins have been identified in the human genome, and 20 have been found in the mouse genome [ ]. Connexins are currently classified according to their molecular weight in kDa. For example, connexin43 and connexin32 represent connexin with a molecular weight of 43 and 32 kDa, respectively. In mammals, connexins have a similar topology that is characterized by four hydrophobic transmembrane domains (M1 to M4) connected by an intracellular loop (IL) and two extracellular loops (EL-1 and EL-2).

The NH2- and COOH-terminal domains are located in the cytoplasm [ , ] ( Fig. 22.1B ). Connexins of different species exhibit a well-conserved structure in the four transmembrane domains (M1 to M4) and the extracellular loops (EL-1 and EL-2). The transmembrane regions M1, M2, and M4 contain hydrophobic residues, whereas the M3 domain has polar residues that allow the formation of the aqueous pore of the channel. The EL-1 and EL-2 extracellular loops contain three cysteine residues that form intramolecular disulfide bonds that play a crucial role in the formation of the channel [ , ]. Connexins differ in their IL and their COOH-terminal domains. The sequences and the lengths of these domains are highly variable between different connexins.

Regulation of intercellular communication

Communication via intercellular channels is regulated at multiple levels. Schematically, the overall conductance between two cells depends mainly on the unitary conductance of each channel and on the total number of functional channels.

Regulation of unitary conductance

The unitary conductance of each channel can be regulated by various factors such as Ca ²⁺ and H ⁺ concentration, the phosphorylation levels of connexin, or by various compounds such as lipophilic compounds.

Ca ²⁺ was the first compound for which a functional regulation of intercellular communication has been demonstrated [ ]. This original work has demonstrated that in a localized lesion of the heart tissue, the damaged cells are quickly isolated from neighboring cells by establishing a high electrical resistance barrier due to an increase in the level of intercellular Ca ²⁺ . Similarly, acidification of the intracellular medium reduces junctional communication [ ]. It is established that an increase in intracellular Ca ²⁺ or H ⁺ concentration allows the closure of gap junctions in many tissues [ ].

Posttranslational modifications such as nitrosylation, ubiquitination, sumoylation, and phosphorylation regulate Gj [ ]. For example, connexins may be phosphorylated at their COOH-terminal domain by several protein kinases, thereby regulating the unitary conductance of intercellular channels [ ]. Chemical phosphatase approaches have demonstrated that the connexin43 phosphorylation level plays an important role in the regulation of Gj in rat cardiac cells [ , ]. The main effectors of these regulations are protein kinase A (PKA) and protein kinase C (PKC). Several phosphorylation domains are thus located on the COOH-terminal end of various connexins. For connexin43, junctional communication increases in response to phosphorylation by PKA and reduces in response to phosphorylation by PKC. These processes of connexins phosphorylation are important for channel opening and the formation and removal of gap junction channels [ , ].

Alcohols (heptanol and octanol), fatty acids, and some steroids can change the conformational structure of the connexins and thus close the channels; this mainly occurs through inserting into the cytoplasmic membrane near the channels [ ].

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