Many epithelial tissues contain a luminal space. Paucity of lumen formation results in abnormal organ development, whereas uncontrolled expansion of luminal space is observed in polycystic diseases. Thus it is important to regulate the size of lumen. However, in contrast to the initiation of lumen formation, molecular mechanisms regulating the size of lumen remain largely unknown. In this study, we compared neonatal cholangiocytes with hepatoblasts by microarray and identified genes specifically up-regulated in cholangiocytes. Among those genes, we found that a transcription factor, Grhl2, expands the apical luminal space of cysts derived from liver progenitor cells through up-regulation of Cldn3, Cldn4, and Rab25. Because Cldn3 and Cldn4, whose localization at TJs is regulated by Rab25, induce the maturation of TJs, our results linked the establishment of epithelial junctional complexes with the promotion of morphogenesis, in particular, lumen formation.
Grhl2 is a member of the grainyhead transcription factor family, mammalian homologues of Drosophila
grainyhead. The mammalian grainyhead family consists of three members—Grhl1, 2, and 3. It has been demonstrated that Grhl1 regulates hair anchorage and epidermal differentiation (Wilanowski et al., 2008
), whereas Grhl3 regulates skin permeability by modulating the barrier function of epidermis, urothelial differentiation, and dorsal closure of the neural tube (Yu et al., 2008
). Recently Grhl2 was shown to be a key transcription factor completing neural tube closure (Pyrgaki et al., 2011
; Werth et al., 2010
) and modulating the nature of cell–cell contacts by promoting the formation of the apical junctional complex (Werth et al., 2010
). On the other hand, since mice lacking Grhl2 die around E13.5 before the development of most epithelial organs, including the liver, the roles of Grhl2 in epithelial morphogenesis remain unknown. Our data indicating that Grhl2 enhances epithelial barrier function and morphogenesis of liver progenitor cells in vitro show the possibility that Grhl2 regulates structural and functional differentiation of epithelial tissues.
TJs contain Cldns, as well as occludin and junctional adhesion molecules. Among those TJ proteins, Cldns regulate the paracellular transport of small substances (Tsukita and Furuse, 2002
). Our results indicate that Grhl2 regulates barrier function of bile ducts by up-regulating Cldn3 and 4. Ectopic expression of Cldn3 resulted in the localization of Cldn3 at TJs and the modest expansion of the central lumen. On the other hand, the introduction of Cldn4 alone did not affect the size of lumens, probably because most of Cldn4 was localized at the basolateral domain (). Although it was previously reported that ectopic expression of Cldn4 resulted in its localization at TJs and increased epithelial barrier, epithelial progenitors such as HPPL might lack a transport system required for the proper localization of Cldn4. Our results show that the Grhl2-Rab25 axis plays a key role in barrier function by localizing Cldn4 at TJs.
Rab25 is a member of the Rab11 subfamily consisting of Rab11a, 11b, and 25. Rab11a and Rab25 are implicated in recycling of apical proteins (Casanova et al., 1999
). Rab25 is known to be relatively abundant in gastrointestinal tissues (Goldenring et al., 1993
). Recently it was reported that Rab11a and 25 are involved in the formation of apical lumens (Bryant et al., 2010
). Because HPPL can form small cysts without Rab25 in 3D culture, it may be dispensable for HPPL to form the apical lumen in our system. On the other hand, Rab25 contributes to the formation of mature epithelial structures by localizing Cldn4 at TJs. Given that Rab25-knockout mice did not show any defects in liver (Nam et al., 2010
), Rab11a and Rab25 may have a redundant function in the bile duct development. However, it might be worth examining whether bile ducts without Rab25 are more susceptible to any type of liver injury.
It remains unclear how the Grhl2-Rab25 axis up-regulates Cldn4 and promotes its localization at TJs. Given that Rab25 has been implicated in recycling of apical proteins, it may promote the transport of internalized Cldn4 protein back to TJs, leading to prevention of the traffic of Cldn4 to lysosomes for degradation. Furthermore, a recent article demonstrated that Rab25 but not Rab11a regulates the transcytosis of FcRn from the basolateral to the apical and vice versa (Tzaban et al., 2009
). Given that most of Cldn4 is localized at the basolateral domain, Rab25 might be involved in trafficking Cldn4 from the basolateral domain to TJs by transcytosis, possibly through indirect interactions between Rab25 and Cldn4 (Supplemental Figure S8).
We summarize a molecular network governed by Grhl2 in . Grhl2 enhances epithelial barrier by up-regulating Cldn3 and Cldn4, which leads to lumen expansion. In addition, Grhl2 enhances lumen expansion by up-regulating Rab25, which up-regulates Cldn4 protein and also regulates its localization at TJs. Strong barrier function is probably a physiological requirement for bile duct tubules to prevent any leakage of cytotoxic bile into the liver parenchyma. In fact, it was recently shown in zebrafish that Cldn15 and Cldn15-like b were necessary for normal formation of intestine (Bagnat et al., 2007
) and bile ducts (Cheung et al., 2011
), respectively. Our results reveal a novel mechanism regulating the process by which epithelial cells acquire mature epithelial structures necessary for their physiological functions.
FIGURE 8: A molecular network governed by Grhl2 regulates the lumen size by modulating epithelial barrier function. Our results indicate that Grhl2 transcriptionally regulates Cldn3 and Cldn4, as well as Rab25, for enhancing epithelial morphogenesis, in particular, (more ...)