Although ZO-1 and ZO-2 are expressed not only in endothelia and epithelia but also in a wide variety of tissues, ZO-3 is exclusively expressed in epithelial cells. Thus, ZO-3 was suggested to have some important roles specifically at the TJs of epithelial cells. It was therefore surprising that the ZO-3 deficiency did not result in any significant abnormalities at the whole body or the cellular level. ZO-3−/− mice were viable and fertile, and their epithelia possessed well-organized TJs, indicating that ZO-3 is not required for the normal development or survival of mice in the laboratory environment. Moreover, ZO-3−/− F9 cells differentiated into epithelial cells normally in the presence of retinoic acid, and these epithelial cells bore TJs with a normal appearance and molecular architecture. A Ca2+ switch experiment has shown that ZO-3 is not responsible for the process of TJ formation either.
What, then, is the physiological function of ZO-3? It is possible that its real function has been overlooked in mice grown in the laboratory environment. For example, the possibility cannot be excluded that ZO-3 is indispensable for some pathological events such as certain defense mechanisms of epithelial cells against microorganisms. Another possibility is, of course, that the TJ MAGUKs—ZO-1, ZO-2, and ZO-3—are functionally redundant and that ZO-1 and/or ZO-2 functionally compensate for the lack of ZO-3 not only at the whole body but also at the cellular level. Indeed, these three proteins shared significant structural similarities, which is the reason why they are collectively called MAGUKs. Thus, it is important to closely consider the molecular relationships between these three TJ MAGUKs. ZO-1, ZO-2, and ZO-3 were all reported to directly bind to the COOH termini of claudins at their PDZ1 domains (13
). At least in vitro, ZO-1 forms a heterodimeric complex with ZO-2 or ZO-3 through PDZ2-PDZ2 interaction (21
), but ZO-2 does not bind to ZO-3. If this is the case also in vivo, it is speculated that ZO-1 has its own specific function and that ZO-2 and ZO-3 are functionally redundant. Consistent with such a contention, in ZO-3−/−
visceral endodermal cells the concentration of ZO-2 was increased slightly but significantly at TJs. Furthermore, our studies with ZO-1
-deficient Eph4 epithelial cells revealed that ZO-1 and ZO-2 are not necessarily functionally redundant. Thus, in ZO-1−/−
Eph4 cells, the formation of TJs was retarded after a Ca2+
switch, and cingulin disappeared from TJs, and these phenotypic changes were rescued by the exogenous expression of ZO-1 but not by that of ZO-2, indicating that ZO-1 has its own specific functions (52
). Therefore, it can be speculated that the lack of abnormalities in TJs of ZO-3−/−
mice and F9 cells is due to the functional compensation of ZO-2 for the ZO-3 deficiency. However, we found that suppression of ZO-2 expression by RNA interference did not affect the TJ architecture in ZO-3−/−
F9 cells. It might be that a trace amount of residual ZO-2 was sufficient to rescue ZO-3 function. More likely, however, is that ZO-3 indeed has no particular molecular functions at least in the normal laboratory environment. Several observations support this assumption. (i) A Ca2+
switch experiment has shown that the loss of ZO-3 did not even affect the process of TJ formation. (ii) Cultured mouse Eph4 epithelial cells originally express only a negligible amount of ZO-3 but have a normal epithelial morphology with well-established TJs (20
). (iii) In both Eph4 cells and F9 cells, the concomitant suppression of ZO-1 and ZO-2 sufficiently disrupted the TJ structure (51
). (iv) Exogenous expression of either ZO-1 or ZO-2 in ZO-1
-deficient Eph4 cells, where all three TJ MAGUKs are substantially lost, successively rescued the TJ structure, whereas that of ZO-3 did not (51
). Thus, among three homologous TJ MAGUKs, ZO-3 is the only molecule that does not have an ability to polymerize TJ strands by itself. Taken together, these observations show that, unlike ZO-1 and ZO-2, ZO-3 appears to have no physiological functions that are apparent under normal laboratory conditions.
Finally, we should discuss two TJ plaque proteins, cingulin and Patj, that were reported to be localized at TJs and to directly bind to ZO-3 (9
). Our observations here clearly showed that the localization of these proteins is not dependent on ZO-2 or ZO-3. Taken together, with our previous analyses of ZO-1−/−
Eph4 cells (52
), we can conclude that cingulin is recruited to TJs through its binding to ZO-1, not to ZO-2 or ZO-3. Patj, which is thought to be directly involved in the epithelial polarization, was reported to directly bind to the COOH terminus of ZO-3 through its PDZ6 domain, and Patj was shown to lose its ability to localize at TJs when its PDZ6 domain was deleted (40
). It has not been shown that Patj binds to ZO-1 or ZO-2 directly. Through these findings, it was assumed that ZO-3 is indispensable for the localization of Patj at TJs, but the present findings clearly do not support this assumption. Since Patj binds to several TJ-associated molecules other than ZO-3 such as Pals1 and Par6 (35
), it would be reasonable to speculate that in the absence of ZO-3, Patj is recruited to TJs through its binding to these proteins. Indeed, both Pals1 and Par6 were normally concentrated at TJs in ZO-3−/−
visceral endodermal cells (not shown).
The present study did not clarify the specific functions of ZO-3 at either the whole-body level or the cellular level. Further generation of mutant mice lacking the TJ MAGUKs ZO-1, -2, and -3, both singly and in combination, will lead to a better understanding of the physiological relevance of the occurrence of these three closely related proteins at TJs.