It has been long recognized that α and γ isoforms of adducin are enriched at intercellular junctions in cultured epithelial cell monolayers and simple mucosal epithelia in vivo.24,31
Despite this junctional affiliation, the involvement of adducin in regulation of epithelial AJs and TJs remains poorly understood. Recently, we have examined the role of this membrane skeleton protein in the dynamics of epithelial junctions by using siRNA-mediated knockdown of α and γ adducin isoforms in SK-CO15 human intestinal epithelial cells.32
Remodeling of epithelial junctions was induced by a so called ‘calcium switch'. This model involves removal of extracellular calcium to trigger disassembly of preformed AJs and TJs followed by calcium re-addition to the culture medium (calcium repletion) to induce orchestrated recovery of junctional structure and functions.14–16,33
We observed that knockdown of either α- or γ-adducins in SK-CO15 cells attenuated reassembly of apical junctions and development of the paracellular barrier triggered by calcium repletion. Interestingly, loss of adducin expression delayed reformation of both AJs and TJs, although α- or γ-adducins consistently colocalized with AJ, but not TJ proteins in newly-assembled and mature intercellular contacts.32
Since AJ assembly represents an early step of epithelial differentiation that is required for the subsequent formation of TJs, we believe that adducin depletion directly impairs the establishment of epithelial AJs, which in turn attenuates TJ reassembly. Eventually, epithelial cells were able to assemble morphologically-normal cell-cell contacts even in the absence of adducin, however, such contacts appear to be less stable comparing to those of normal cells. This notion of contact instability is based on the observed collapse of the lateral plasma membrane and the increased long-range intramembrane mobility of E-cadherin in adducin-depleted cells.27
Given our findings that adducin promotes the establishment of epithelial AJs and TJs one can suggest that this membrane skeleton protein should antagonize junctional disassembly. Indeed, we observed such antagonisms while examining the effects of adducin isoforms knockdown on disruption of AJs and TJs in HPAF II human pancreatic epithelial cells exposed to protein kinase C (PKC)-activating phorbol ester. PKC activation is known to potently disrupt cell-cell contacts in several types of epithelia by stimulating remodeling of the peri-junctional F-actin and triggering internalization of AJ/TJ proteins.34,35
On the other hand, PKC phosphorylates adducin at several serine residues (Ser726, Ser712 and Ser660) in their C-terminal MARKS domain.36,37
This phosphorylation has been shown to inhibit adducin functions by decreasing its associations with actin filaments and spectrin.36,37
We found that phorbol ester induced rapid phosphorylation of α- and γ-adducins which was accompanied by their disappearance from the intercellular junctions.32
Loss of adducin from cell-cell contacts appears to be an early event of the phorbol ester signaling that preceded AJ and TJ disassembly. Furthermore, depletion of either α- or γ-adducins significantly accelerated disruption of AJs and TJs induced by PKC activation.32
These results suggest that PKC-dependent phosphorylation of adducin triggers its early release from complexes with spectrin and actin filaments, thereby enhancing remodeling of the cortical cytoskeleton and destabilizing epithelial junctions. Importantly, protein kinase A and Rho-dependent kinase, as well as cytokines such as pleiotropin are known to phosphorylate adducin and alter cellular distribution and activity of this scaffolding protein.37,38
Therefore, adducin can be important down-stream effector of different signaling cascades that regulate stability and remodeling of epithelial junctions.
Our study also provides an important insight into the mechanisms that mediate the effects of adducin on epithelial junctions. These mechanisms involve organization of the spectrin network and assembly of actin filaments at the intercellular contacts. For example, we found that depletion of adducin isoforms decreased expression of βII-spectrin in intestinal epithelial cells and delayed recruitment of this protein to newly-forming AJs.32
This observation suggests that loss of adducin impairs formation of the highly-ordered spectrin lattice at the plasma membrane of contacting epithelial cells, which is likely to be responsible for the attenuated junctional assembly. Another mechanism that can mediate destabilization of apical junctions in adducin-depleted epithelia involves impaired formation of the perijunctional F-actin belt. Previous biochemical studies described the ability of α-adducin to cap28
actin filaments in cell-free systems. However, we demonstrated for the first time that adducin regulates assembly of the F-actin cytoskeleton in epithelial cells. This conclusion is supported by findings that siRNA-mediated depletion of α and γ adducins increased the G/F actin ratio, which indicates either impaired polymerization or enhanced depolymerization of actin filaments. Furthermore, adducin downregulation attenuated formation of the perijunctional F-actin bundles during reestablishment of epithelial AJs.32
Given the crucial role of the circumferential F-actin belt in supporting structure of epithelial junctions, this defective F-actin assembly should underline the impaired formation of AJs and TJs in adducin-depleted epithelial cells. What type of adducin-F-actin interactions (filament capping or cross-linking) are involved in organization of the perijunctional cytoskeleton and whether spectrin is essential for these events remain to be determined. It is also unclear whether the effects of adducin depletion on the remodeling of actin filaments and spectrin assembly at intercellular junctions represent two distinct mechanisms or if they are mutually dependent. However, based on a classical model of adducin action, it is likely that loss of this scaffolding protein breaks a physical link between spectrin oligomers and actin filaments which is important for proper organization of both cytoskeletal structures at the areas of cell-cell contacts.