**Gap Junction Intercellular Communication and Connexin Expression Profile in Normal Liver Cells and Hepatocarcinoma**

Glaucia M. Machado-Santelli1 and Marisa Ionta2

*1Department of Cell and Developmental Biology, Institute of Biomedical Sciences,University of Sao Paulo, Sao Paulo 2Institute of Biomedical Sciences, Federal University of Alfenas, Minas Gerais Brazil*

## **1. Introduction**

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> Gap junction intercellular communication (GJIC) is considered to play a relevant role in homeostasis of multicellular organisms by regulating processes such as cell proliferation and cell differentiation. Specialized membrane structures mediate cell-to-cell communication, the gap junctional channels, which allow the transfer of molecules less than 1000Daltons (Da) between adjacent cells such as ions, aminoacids, nucleotides, metabolites and second messengers. Gap junctional channels consist of the two juxtaposed hemichannels called connexons, each of them constituted of six proteic subunits composed of connexin (Cx). These proteins are codified by multigene family with at least 21 members and their expression is tissue specific. The different isoforms are named according to their molecular mass (kilo Dalton) and they share a similar structure of four membrane-spanning domains, two extracellular loops, one cytoplasmic loop, one cytosolic N-terminal tail and Cterminal region. The transmembrane domains and the extracellular loops are highly conserved among the family members. N-terminus is also conserved, while the cytoplasmic loop and C-terminus show great variation in terms of sequence and length. Furthermore cytoplasmic tail and loop are susceptible to various post-translational modifications (including phosphorylation), which are important to modulate functional activity of connexin (Figure 1).

> The liver represents an interesting system to study gap junction intercellular communication (GJIC) and through the years, a wealth of knowledge is available about functional GJIC and connexin expression profile in different physiological conditions which include cell proliferation, cell differentiation and cell death and disruption of GJIC has been associated with hepatocarcinogenesis process.

#### **2. Gap junction channels biogenesis and degradation mechanisms**

The synthesis, assembly and turnover of GJ channels follow the general secretory roles for membrane proteins. Connexins are synthesized by membrane-bound ribosomes and they are cotranslationally integrated into the endoplasmic reticulum membrane. The

Gap Junction Intercellular Communication and Connexin

(reviewed by Mese et al., 2007).

Expression Profile in Normal Liver Cells and Hepatocarcinoma 277

liver, connexin 26 is able to form heteromeric connexon with Cx32 but did not form heteromeric connexon with connexin 43 once the isoforms need to be compatible. It is important to notice that homomeric channels formed by Cx32 or Cx26 present differences in permeability when compared to heteromeric channels formed by just Cxs 26 or Cx32

Docking of connexons implies their insertion into gap junction plaques which comprise punctuated regions at membrane juxtaposed area (Figure 2). The formation of functional

Fig. 2. Gap junctional channels clustered in plaques: A) laser scanning confocal microscopy image of Cx43/GFP-transfected HTC cells showing gap junctional plaques (arrows); B) transmission electron microphotography of gap junctional plaque (asterisk) (courtesy of

Prof. Dr.Victor Arana-Chavez, FOUSP).

oligomerization of connexins into connexon (hemichannel) occurs in a progressive fashion starting in the endoplasmic reticulum and ending in the trans-Golgi network, however the exact localization of oligomerization depends on the connexin type. It is thought that both Cx26 and Cx32 oligomerize in the endoplasmic reticulum, whereas Cx43 oligomerizes in the *trans*-Golgi network (Musil and Goodenough, 1993; Martin et al., 2001).

Connexons are then delivery to the cell surface via vesicles transported through microtubules, which fuse to plasma membrane. Upon arrival at the cell membrane, connexons can either reside in nonjunctional regions or docking with an opposing connexon to form fully functional channels.

Connexons can be **homomeric** (formed by a single type of connexin) or **heteromeric** (formed by more than one type of connexin). Functional channels are **homotypic** when formed by identical connexons (homomeric or heteromeric) or **heterotypic**, formed by the interaction between different connexons (Figure 1).

Fig. 1. A) The connexin structure consists of four membrane-spanning domains, two extracellular and one intra-cellular loops, one N-terminal tail and one C-terminal tail; B) Six connexins ( represented by cylinder) organize the connexon which can be homomeric (with a single connexin isoform) or heteromeric ( formed by more than one type of connexin); C)Two connexons dock together to form a gap junctional channel that can be homotypic if they are identical or heterotypic, if they are different.

The ability to form homotypic and heterotypic channels with homomeric and heteromeric connexons adds even greater versatility to the functional modulation of GJ channels. In

oligomerization of connexins into connexon (hemichannel) occurs in a progressive fashion starting in the endoplasmic reticulum and ending in the trans-Golgi network, however the exact localization of oligomerization depends on the connexin type. It is thought that both Cx26 and Cx32 oligomerize in the endoplasmic reticulum, whereas Cx43 oligomerizes in the

Connexons are then delivery to the cell surface via vesicles transported through microtubules, which fuse to plasma membrane. Upon arrival at the cell membrane, connexons can either reside in nonjunctional regions or docking with an opposing connexon

Connexons can be **homomeric** (formed by a single type of connexin) or **heteromeric** (formed by more than one type of connexin). Functional channels are **homotypic** when formed by identical connexons (homomeric or heteromeric) or **heterotypic**, formed by the interaction

Fig. 1. A) The connexin structure consists of four membrane-spanning domains, two extracellular and one intra-cellular loops, one N-terminal tail and one C-terminal tail; B) Six connexins ( represented by cylinder) organize the connexon which can be homomeric (with a single connexin isoform) or heteromeric ( formed by more than one type of connexin); C)Two connexons dock together to form a gap junctional channel that can be homotypic if

The ability to form homotypic and heterotypic channels with homomeric and heteromeric connexons adds even greater versatility to the functional modulation of GJ channels. In

they are identical or heterotypic, if they are different.

*trans*-Golgi network (Musil and Goodenough, 1993; Martin et al., 2001).

to form fully functional channels.

between different connexons (Figure 1).

liver, connexin 26 is able to form heteromeric connexon with Cx32 but did not form heteromeric connexon with connexin 43 once the isoforms need to be compatible. It is important to notice that homomeric channels formed by Cx32 or Cx26 present differences in permeability when compared to heteromeric channels formed by just Cxs 26 or Cx32 (reviewed by Mese et al., 2007).

Docking of connexons implies their insertion into gap junction plaques which comprise punctuated regions at membrane juxtaposed area (Figure 2). The formation of functional

Fig. 2. Gap junctional channels clustered in plaques: A) laser scanning confocal microscopy image of Cx43/GFP-transfected HTC cells showing gap junctional plaques (arrows); B) transmission electron microphotography of gap junctional plaque (asterisk) (courtesy of Prof. Dr.Victor Arana-Chavez, FOUSP).

Gap Junction Intercellular Communication and Connexin

contributes to lose of the differentiated phenotype.

Expression Profile in Normal Liver Cells and Hepatocarcinoma 279

and/or Cx26 (Zhang et al., 1994). Hepatocytes cultured *in vitro* commonly increase Cx43 expression with the concomitant decrease of Cx32 expression (Figure 3). Thus, the immortalized cell lines derived from liver express Cx43 instead of Cx32. Furthermore, hepatocarcinogenesis process leads the hepatocytes to express again Cx43, event that

Fig. 3. Laser scanning confocal microscope images of BRL3A (Normal liver cells) submitted to immunofluorescence reaction with anti-connexin43 antibody: A) TRITC-phalloidin stained microfilaments; B) Cx43 is presented in the cytoplasm and gap junctions (green); C)

nuclei in blue and D) merged channels .

gap junction requires the appropriate cell-to-cell adhesion. There is evidence that interaction of Cx43 with the tight junction protein (ZO1) may play a role in regulating the size of the gap junction plaque (Hunter et al., 2003).

In general, the turnover rate of connexin is very fast in relation to other plasma membrane proteins. According to studies performed *in vivo* and *in vitro,* the half-lives of Cx26 and Cx32 are respectively 2 and 3 hours (Traub et al., 1983; 1987). The removal of gap junction from the plasma membrane occurs by endocytosis. During this process, both membranes of the gap junction are internalized into one of the adjacent cells and thereby form a doublemembrane vacuole called annular gap junction. These structures are further degraded by both lysosomes and proteasomes. The preferential degradation pathway is associated to both cell and connexin type ( Laird et al., 2005; 2006). The degradation of Cx32 in the liver occurs mainly via the lysosomal pathway (Rahman et al., 1993). Furthermore the phosphorylation status of connexin is important to regulate its internalization and degradation. *In vitro* studies have been done to understand the mechanism involved in Cx43 internalization and degradation. They showed that the internalization and degradation of Cx43 gap junction is closely related to its hyperphosphorylation (via Mek/Erk pathway) and ubiquitination (Leithe and Rivedal, 2004).
