Recently, mutations in human GLIS3 have been linked to NDH (45
). In addition to neonatal diabetes and congenital hypothyroidism, NDH is associated with facial anomalies, PKD, congenital glaucoma, and liver fibrosis. To further study the role of Glis3 in this disease, we generated for the first time mutant mice that are impaired in Glis3 function. Glis3 mutant mice develop polycystic kidneys and neonatal diabetes and exhibit a very short life span. The development of hyperglycemia and hypoinsulinemia, caused by an insufficiency of pancreatic β cells (Kang et al., submitted), rather than the development of polycystic kidneys, which at PND3 is rather moderate in severity, may be the major cause of the shortened life span in Glis3zf/zf
mice. Whether these mice develop hypothyroidism, glaucoma, and liver fibrosis has yet to be determined. Development of polycystic kidneys was reported only for NDH patients with the most severe abnormalities (NDH1 patients) (45
). As with Glis3zf/zf
mice, NDH1 patients have a very short life span (10 days to 16 months) and neonatal diabetes. NDH1 patients have a frameshift mutation that results in a loss of the C-terminal activation domain of GLIS3 (3
). Thus, the phenotype of Glis3zf/zf
mutant mice appears to be the most similar to the abnormalities observed in NDH1 patients. This analogy suggests that Glis3zf/zf
mutant mice will provide an excellent model to study this syndrome at a molecular and mechanistic level.
Development of renal cysts in Glis3zf/zf
mice was observed as early as E15.5 and increased with age. Cyst formation originated from glomeruli, tubules, and collecting ducts, corresponding to the observed expression of Glis3 in kidney in parietal cells and epithelial cells lining the tubules and collecting ducts. Thus, Glis3 expression overlaps that of many other genes implicated in PKD. Study of cystic renal diseases in humans and in transgenic mice revealed that many proteins implicated in these diseases localize to the primary cilium and are either a structural component of the primary cilium or function as part of a signal transduction pathway associated with the primary cilium (36
). These observations led to the hypothesis that ciliary dysfunction plays a key role in the etiology of cystic renal disease. This link raised the possibility that the development of renal cysts in Glis3zf/zf
mice may also have a connection to the primary cilium. This hypothesis was supported by our data showing that Glis3 is associated with the primary cilium in renal tubules and in confluent TKPTS renal tubule epithelial cells expressing EGFP-Glis3. Interestingly, the family member Glis2, which has been implicated in nephronophthisis, an autosomal recessive cystic kidney disease (2
), was also found to be associated with the primary cilium. Moreover, members of the closely related Gli family have been reported to localize to the primary cilium as well (14
). The primary cilium plays a critical role in the activation of the sonic hedgehog (Shh)/Gli signal transduction pathway. In the absence of Shh, its receptor Patched-1 (Ptch1) prevents the accumulation of smoothened (Smo) in the primary cilium. Binding of Shh to Ptch1 inactivates the receptor and results in the activation of Smo, which then accumulates within the cilium and activates Gli. Subsequently, Gli translocates to the nucleus, where it regulates the transcription of target genes. In addition to Shh/Gli, the primary cilium plays a critical role in several other signal transduction pathways (10
). Its association with the primary cilium suggests that Glis3 may be part of a cilium-mediated signal transduction pathway and requires activation before it is translocated to the cytoplasm and nucleus (Fig. ). Because Shh does not affect Glis3-mediated transactivation (our unpublished observations), regulation of Glis3 activity likely involves another signal.
FIG. 8. Model illustrating the links between Glis3, the primary cilium, Wwtr1, and cystic renal disease. We demonstrate that Glis3 is associated with the primary cilium, where it may be activated by a yet-unidentified signal (e.g., flow, G-protein-linked receptor). (more ...)
Although many proteins associated with the primary cilium have been implicated in the development of cystic renal disease, the precise molecular mechanisms underlying these diseases have not yet been fully established. Planar cell polarity and oriented cell division have been reported to play an important role in the postnatal development of nephrons, while misoriented cell division appears to be part of the mechanism leading to cystogenesis (6
). It has been proposed that the primary cilium may guide oriented cell division (11
). Our observations indicate that Glis3 dysfunction or Glis3 knockdown does not prevent the formation of the primary cilium, suggesting that Glis3 does not regulate the expression of an essential structural component of the cilium. However, dilated and cystic tubules in Glis3zf/zf
mice contain relatively fewer cells with a primary cilium. Because cell proliferation negatively affects the formation of primary cilia, this decrease might involve an indirect mechanism and be due to increased proliferation, as shown by the increase in BrdU-positive cells and the reduced level of cdkn1a observed in renal cysts of Glis3zf/zf
mice. In addition to increased proliferation, renal tubule epithelial cells in Glis3zf/zf
mice exhibit changes in cell-cell and cell-matrix interactions, as indicated by alterations in the distribution of β-catenin, ZO-1, and Na+
ATPase α. Changes in the distribution patterns of EGFR, ZO-1, and E-cadherin were also observed in mouse proximal tubule TKPTS cells in which Glis3 was downregulated. Interestingly, the effects of Glis3 on junctional proteins resemble those of PC-1. Expression of PC-1 (Pkd1) in MDCK cells induces cell migration and reabsorption of ZO-1, E-cadherin, and β-catenin at the leading edge of migrating cells, while Pkd1−/−
mouse embryo fibroblasts exhibit a reduced migratory capability (8
). It was concluded that PC-1 might be a regulator of epithelial plasticity by controlling cell polarity, cell migration, and cell-cell and cell-matrix interactions, functions important in the formation, elongation, and maintenance of renal tubules. Such a mechanism may also play a role in the control of renal functions by Glis3. Interestingly, the observation that Dctn5 and Pkd1 expression were decreased in the kidneys of Glis3zf/zf
mice and in Glis3 downregulated TKPTS cells would be consistent with this hypothesis (Fig. ). Also notable was that in Glis3zf/zf
mice, pancreatic tubules were also found to be dilated, suggesting a common mechanism for renal and pancreatic cyst formation (Kang et al., submitted). Whether such a common mechanism is responsible for the insufficiency in pancreatic β cells observed in Glis3zf/zf
mice needs further study.
Because of the significant similarities between different cystic renal phenotypes and the connection between ciliary proteins and cystic renal diseases (49
), it is not surprising that functional links are being found between some of the proteins implicated in these diseases. For example, PKHD1 is a target gene of hepatocyte nuclear factor HNF-1β, and mutations in both genes have been implicated in cystic renal disease (5
). In this study, we identify a novel link between Glis3 and the transcriptional modulator Wwtr1. In both Glis3 mutant mice and Wwtr1 null mice, the development of glomerulocysts are prominent (20
) and both Glis3 and Wwtr1 promote osteogenesis and repress adipogenesis (4
). Wwtr1 can function as a corepressor as well as a coactivator (19
). It represses peroxisome proliferator-activated receptor γ (PPARγ)-mediated transcription while it enhances transcriptional activation by Cbfa1/Runx2, T-box transcription factor 5 (TBX5), paired box homeotic gene 3 (Pax3), and thyroid transcription factor 1 (TTF1) (9
). Wwtr1 interacts through its WW domain directly with these transcription factors by recognizing a P/LPXY motif. Glis3, which functions as a positive regulator of Glis-BS-dependent transcription (3
), contains four putative P/LPXY motifs (Fig. ). These observations raised the possibility that Wwtr1 and Glis3 might interact with each other and that Wwtr1 might function as a coactivator of Glis3-mediated transcription. Coimmunoprecipitation and mammalian two-hybrid analyses demonstrated that Wwtr1 and Glis3 are part of the same protein complex, while in vitro pull-down analysis indicated that Glis3 interacts with Wwtr1 directly. Deletion of its N terminus affected the interaction of Glis3 with Wwtr1 to some extent, suggesting that the N terminus may contribute to the interaction either through direct binding or through the mediation of other proteins within the Glis3-Wwtr1 complex. Deletion of the C terminus, containing the fourth P/LPXY motif, or mutation of the C-terminal motif (Glis3M4) abolished the interaction of Glis3 with Wwtr1 almost completely. These data indicate that the P/LPXY motif is a major requirement for the interaction and are consistent with the conclusion that Wwtr1 recognizes Glis3 directly via this motif. We further showed that increased expression of Wwtr1 significantly enhanced Glis3-mediated transcriptional activation, consistent with the concept that it acts as a coactivator of Glis3. Although the family member Glis1 contains two putative P/LPXY motifs, Wwtr1 did not interact with Glis1 and did not significantly enhance Glis1 transcriptional activity. Glis2, which does not contain any P/LPXY motif, also did not interact with Wwtr1. These observations indicate that the interaction of Glis3 with Wwtr1 is specific and that Wwtr1 functions as an effective coactivator of Glis3 but not of Glis1 or Glis2. We previously showed that the transcriptional activation domain is located in the C-terminal region of Glis3, a region that includes the fourth P/LPXY motif (3
). This, together with the observation that this motif is required for the interaction with the coactivator Wwtr1, suggested that this motif may be part of the transactivation function of Glis3. This conclusion was supported by data showing that mutations in this motif (PPHY to PAHA) greatly diminished the transcriptional activity of Glis3 (Fig. ). This is consistent with the finding that a base insertion in the GLIS3
gene of NDH1 patients results in a frameshift and that deletion of the C terminus, including the fourth P/LPXY motif, causes loss of Glis3 transcriptional activity (3
). Because Glis3ΔZF5
, which matches the mutation in Glis3zf/zf
mice, can interact with Wwtr1 but is unable to bind Glis-BS, the development of the renal and pancreatic phenotype in Glis3zf/zf
mice is related to its inability to induce GLIS-BS-dependent transactivation and not due to lack of Wwtr1 interaction.
The interaction between Wwtr1 and Glis3 was further supported by observations showing that coexpression of Glis3 with Wwtr1 promoted the nuclear localization of Wwtr1. Previous studies reported that Wwtr1 shuttles in and out of the nucleus (22
). This shuttling seems to be controlled at several levels. In the cytoplasm, phosphorylation of Wwtr1 by the Hippo kinase signaling cascade promotes interaction with 14-3-3 proteins and retention in the cytoplasm. In the nucleus, Wwtr1 can interact with several transcription factors (9
). It has been suggested that competition may exist between the transcriptional machinery and 14-3-3 proteins for Wwtr1 binding. Interaction of Glis3 with Wwtr1 may result in nuclear retention of Wwtr1 (Fig. ), as has been proposed for the interaction between Wwtr1 and the transcription cofactor ARC105 (51
). However, we cannot rule out that Glis3 forms a complex with Wwtr1 in the cytoplasm and then translocates to the nucleus.
In summary, this study demonstrates that Glis3 mutant mice have a very short life span and develop polycystic kidneys and neonatal diabetes, abnormalities that are very similar to those observed in patients with NDH1. Therefore, these mice provide an excellent model to study the molecular mechanisms underlying this syndrome. We further identify two important elements of the Glis3 signal transduction pathway. We show that Glis3 localizes to the primary cilium and propose that upon activation of Glis3 by a primary cilium-associated signaling pathway, Glis3 is transported by intraflagellar transport into the cytoplasm and subsequently into the nucleus (Fig. ). In the nucleus, Glis3 interacts with several coactivators, including Wwtr1, that mediate the transcriptional activation of Glis3 target genes. Thus, Glis3 and Wwtr1 are part of overlapping transcription regulatory networks that are critical in maintaining normal renal structure and homeostasis.