armadillo

EVOLUTIONARY HOMOLOGS

Table of contents

Protein interactions of Armadillo homologs: Miscellanous interactions

Wnt signaling plays a crucial role in a number of developmental processes and in tumorigenesis. beta-Catenin is stabilized by Wnt signaling and associates with the TCF/LEF family of transcription factors, thereby activating transcription of Wnt target genes. Constitutive activation of beta-catenin-TCF-mediated transcription resulting from mutations in adenomatous polyposis coli (APC), beta-catenin, or Axin is believed to be a critical step in tumorigenesis among divergent types of cancers. The transactivation potential of the beta-catenin-TCF complex is enhanced by its interaction with a BCL9-like protein, B9L, in addition to BCL9 (see Drosophila Legless). B9L is required for enhanced beta-catenin-TCF-mediated transcription in colorectal tumor cells and for beta-catenin-induced transformation of RK3E cells. Furthermore, expression of B9L is aberrantly elevated in about 43% of colorectal tumors, relative to the corresponding noncancerous tissues. These results suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling (Adachi, 2004).

Fascin, the Actin bundling protein coded for by singed in Drosophila, binds to ß-catenin's Armadillo repeat domain. In vitro competition and domain-mapping experiments demonstrate that Fascin and E-cadherin utilize a similar binding site within ß-catenin, such that they form mutually exclusive complexes with ß-catenin. Fascin and ß-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins colocalize with Fascin and ß-catenin at cell leading edges. It is likely that ß-catenin participates in modulating cytoskeletal dynamics in association with Fascin, perhaps in a coordinate manner with its functions in Cadherin and APC complexes. Whatever the biological role of the Fascin-ß-catenin complex, Fascin itself does not appear to be required for the function of all bundled filaments. For example, Singed mutants evidence a variety of apparently normal cell activities even in embryos harboring strong singed alleles, leaving open the possibility that other bundling proteins effectively assume the role of Fascin in various contexts (Tao, 1996).

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, beta-catenin, with several molecular partners have been shown to be important for cell signaling and cell-cell adhesion. To examine if beta-catenin has a role in tubulogenesis, the effect of expressing NH2-terminal deleted beta-catenins was tested in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiate tubulogenesis by forming many long cell extensions. Expression of NH2-terminal deleted beta-catenins inhibits formation of these cell extensions. Both DeltaN90 beta-catenin, which binds to alpha-catenin, and DeltaN131 beta-catenin, which does not bind to alpha-catenin, inhibit formation of cell extensions and tubule development, indicating that a function of beta-catenin distinct from its role in cadherin-mediated cell-cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein is localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlates with the colocalization and accumulation of NH2-terminal deleted beta-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts results in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH2-terminal deleted beta-catenins results in cells that proliferate but form cell aggregates (polyps) within the cyst rather than tubules. These results demonstrate an unexpected role for beta-catenin in cell migration and indicate that dynamic beta-catenin-APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis (Pollack, 1997).

To study the relationship between plakoglobin expression and the level of beta-catenin, and the localization of these proteins in the same cell, two different tumor cell lines were used that express N-cadherin, and alpha- and beta-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression results in a dose-dependent decrease in the level of beta-catenin in each clone. Induction of plakoglobin expression increases the turnover of beta-catenin without affecting RNA levels, suggesting posttranslational regulation of beta-catenin. In plakoglobin overexpressing cells, both beta-catenin and plakoglobin are localized at cell-cell junctions. Stable transfection of mutant plakoglobin molecules shows that deletion of the N-cadherin binding domain, but not the alpha-catenin binding domain, abolishes beta-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocks the decrease in beta-catenin levels and results in accumulation of both beta-catenin and plakoglobin in the nucleus. These results suggest that (1) plakoglobin substitutes effectively with beta-catenin for association with N-cadherin in adherens junctions, (2) extrajunctional beta-catenin is rapidly degraded by the proteasome-ubiquitin system but, (3) excess beta-catenin and plakoglobin translocate into the nucleus (Salomon, 1997).

What function of APC protein is regulated by beta-catenin? It is noteworthy that in actively migrating epithelial cells, bundles of microtubules invade cell extentions and coalesce at clusters of APC protein that are localized at the leading edge of cell protrusions. In vitro, APC protein binds to and bundles microtubules; in transfected cells, exogenous APC protein codistributes along the length of microtubules. Addition of nocodazole to cells results in disruption of both microtubules and APC protein localization to the tips of membrane extensions and inhibition of direct cell migration. An interesting corollary to these observations is that during the formation of stable extensions in growth cone outgrowth, individual microtubules actively invade cell protrusions and are subsequently organized into bundles that stabilize the direction of migration. Formation of cell extensions during epithelial tubulogenesis may involve similar processes in which establishing and stabilizing the direction of migration involves the reorganization and stabilization of microtubules. It is suggested that beta-catenin regulates a function of APC protein in organizing microtubules that are required for the formation and/or stabilization of cell extensions during tubulogenesis (Pollack, 1997).

Human LAR, the homolog of DLAR, a Drosophila transmembrane protein tyrosine phosphatase, associates with the cadherin-catenin complex. This association requires the amino-terminal domain of ß-catenin but does not require the armadillo repeats, which mediate association with cadherins. The association is not mediated by alpha-catenin or by cadherins. LAR-protein tyrosine phosphatases are phosphorylated on tyrosine in a TrkA-dependent manner, and their association with the cadherin-catenin complex is reduced in cells treated with NGF. It is proposed that changes in tyrosine phosphorylation of ß-catenin, mediated by TrkA and LAR-PTPs control cadherin adhesive function during processes such as neurite outgrowth (Kypta, 1996).

One approach to understanding the function of presenilin 1 (PS1) is to identify those proteins with which it interacts. Evidence for a function in developmental patterning comes from C. elegans, in which a PS homolog has been identified by screening for suppressors of a mutation in Notch/lin-12, a gene that specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, the two hybrid system and confirmatory co-immunoprecipitations were used to identify a novel catenin, termed delta-catenin, that interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles: they are components of cell-cell adherens junctions, and may also serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacts with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene (Zhou, 1997).

Mutations of the presenilin-1 gene are a major cause of familial early-onset Alzheimer's disease. Presenilin-1 can associate with members of the catenin family of signaling proteins, but the significance of this association is unknown. Presenilin-1 is shown to form a complex with beta-catenin in vivo that increases beta-catenin stability. Pathogenic mutations in the presenilin-1 gene reduce the ability of presenilin-1 to stabilize beta-catenin, and lead to increased degradation of beta-catenin in the brains of transgenic mice. Moreover, beta-catenin levels are markedly reduced in the brains of Alzheimer's disease patients with presenilin-1 mutations. Loss of beta-catenin signaling increases neuronal vulnerability to apoptosis induced by amyloid-beta protein. Thus, mutations in presenilin-1 may increase neuronal apoptosis by altering the stability of beta-catenin, predisposing individuals to early-onset Alzheimer's disease (Zhang, 1998).

Families bearing mutations in the presenilin-1 (PSI) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co-immunoprecipitation with PS1 in transfected COS-7 cells indicates that PSI directly interacts with endogenous beta-catenin, and the interaction requires residues 322-450 of PSI and 445-676 of beta-catenin. Both proteins are co-localized in the endoplasmic reticulum. Over-expression of PS1 reduces the level of cytoplasmic beta-catenin, and inhibits beta-catenin-T cell factor-regulated transcription. These results indicate that PSI plays a role as inhibitor of the beta-catenin signal, which may be connected with the AD dysfunction (Murayama, 1998).

Catenin pp120 is the prototype of a subfamily of Armadillo proteins, comprising ARVCF, p0071, delta-catenin/NPRAP, and plakophilins 1 and 2. Characterization of the nonreceptor tyrosine kinase FER has identified a tight physical association with catenin pp120 and has led to the suggestion that FER may be involved in cell-cell signaling. The majority of FER is localized to the cytoplasmic fraction where it forms a complex with the actin-binding protein cortactin. The Src homology 2 sequence of FER is required for directly binding cortactin, and phosphorylation of the FER-cortactin complex is up-regulated in cells treated with peptide growth factors. Using a dominant-negative mutant of FER, evidence is provided that FER kinase activity is required for the growth factor-dependent phosphorylation of cortactin. These data suggest that cortactin is likely to be a direct substrate of FER. These observations provide additional support for a role for FER in mediating signaling from the cell surface, via growth factor receptors, to the cytoskeleton. The nature of the FER-cortactin interaction, and their putative enzyme-substrate relationship, support the previous proposal that one of the functions of the Src homology 2 sequences of nonreceptor tyrosine kinases is to provide a binding site for their preferred substrates (Kim, 1998).

The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins that undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF), which co-localize in intracellular membranes but are tightly regulated in their stoichiometry and abundance. Linear glycerol velocity and discontinuous sucrose gradient analysis were used to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of an approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein (APP). In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of an approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF (Yu, 1998).

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. Although the integrity of cell-cell contacts, such as adherens junctions, is essential for the maintenance of functional epithelia, they need to be rapidly disassembled during migration. The transmembrane cell adhesion protein E-cadherin and the cytoplasmic catenins are molecular elements of these structures. Epithelial cell migration is accompanied by tyrosine phosphorylation of beta-catenin and an increase of its free cytoplasmic pool. The protein-tyrosine phosphatase LAR (leukocyte common antigen related) colocalizes with the cadherin-catenin complex in epithelial cells and associates with beta-catenin and plakoglobin (see Drosophila Dlar). Interestingly, ectopic expression of LAR inhibits epithelial cell migration by preventing phosphorylation and the increase in the free pool of beta-catenin; moreover, it inhibits tumor formation in nude mice. These data support a function for LAR in the regulation of epithelial cell-cell contacts at adherens junctions as well as in the control of beta-catenin signaling functions. Thus PTP-LAR appears to play an important role in the maintenance of epithelial integrity, and a loss of its regulatory function may contribute to malignant progression and metastasis (Muller, 1999).

The Alzheimer's disease-linked gene presenilin 1 (PS1) is required for intramembrane proteolysis of APP and Notch. In addition, recent observations strongly implicate PS1 as a negative regulator of the Wnt/ß-catenin signaling pathway, although the mechanism underlying this activity is unknown. Presenilin has been shown to function as a scaffold that rapidly couples ß-catenin phosphorylation through two sequential kinase activities independent of the Wnt-regulated Axin/CK1alpha complex. Thus, presenilin deficiency results in increased ß-catenin stability in vitro and in vivo by disconnecting the stepwise phosphorylation of ß-catenin, both in the presence and absence of Wnt stimulation. These findings highlight an aspect of ß-catenin regulation outside of the canonical Wnt-regulated pathway and a function of presenilin separate from intramembrane proteolysis (Kang, 2002).

beta-catenin, the vertebrate homolog of the Drosophila Armadillo protein, has been shown to have dual cellular functions, as a component of both the cadherin-catenin cell adhesion complex and the Wnt signaling pathway. Upon receipt of the Wnt signal, beta-catenin becomes stabilized in the cytoplasm and subsequently is made available for interaction with transcription factors of the lymphocyte enhancer factor-1/T-cell factor family, resulting in a nuclear localization of beta-catenin. Although beta-catenin does not bind DNA directly, its carboxyl- and amino-terminal regions exhibit a transactivating activity still not well understood molecularly. An interaction partner of beta-catenin is reported: a nuclear protein designated Pontin52 (also known as TIP49), a protein 79% homologous to the uncharacterized Drosophila CG4003. Pontin52 binds beta-catenin in the region of Armadillo repeats 2-5 and, more importantly, also binds the TATA box binding protein. Evidence is presented for a multiprotein complex composed of Pontin52, beta-catenin, and lymphocyte enhancer factor-1/T-cell factor. These results suggest involvement of Pontin52 in the nuclear function of beta-catenin (Bauer, 1998).

The p35-Cdk5 kinase (see Drosophila Cdk5) has been implicated in a variety of functions in the central nervous system (CNS), including axon outgrowth, axon guidance, fasciculation, and neuronal migration during cortical development. In p35(-/-) mice, embryonic cortical neurons are unable to migrate past their predecessors, leading to an inversion of cortical layers in the adult cortex. In order to identify molecules important for p35-Cdk5-dependent function in the cortex, a screen was undertaken for p35-interacting proteins using the two-hybrid system. In this study, the identification of a novel interaction between p35 and the versatile cell adhesion signaling molecule beta-catenin is reported. The p35 and beta-catenin proteins interact in vitro and colocalize in transfected COS cells. In addition, the p35-Cdk5 kinase is associated with a beta-catenin-N-cadherin complex in the cortex. In N-cadherin-mediated aggregation assays, inhibition of Cdk5 kinase activity using the Cdk5 inhibitor roscovitine leads to the formation of larger aggregates of embryonic cortical neurons. This finding was recapitulated in p35(-/-) cortical neurons, which aggregate to a greater degree than wild-type neurons. In addition, introduction of active p35-Cdk5 kinase into COS cells leads to a decreased beta-catenin-N-cadherin interaction and loss of cell adhesion. The association between p35-Cdk5 and an N-cadherin adhesion complex in cortical neurons and the modulation of N-cadherin-mediated aggregation by p35-Cdk5 suggests that the p35-Cdk5 kinase is involved in the regulation of N-cadherin-mediated adhesion in cortical neurons (Kwon, 2000).

Several models can be proposed to account for the regulation of cadherin-mediated adhesion by the p35-Cdk5 kinase. As p35-Cdk5 is a protein serine/threonine kinase, it may phosphorylate one or more components of the cadherin-adhesion complex, ultimately leading to decreased cell adhesion. Indeed, beta-catenin contains three minimal consensus sites for phosphorylation by Cdks. However, there is little evidence to support a role for serine/threonine phosphorylation as a modulator of cadherin-mediated adhesion in cortical neurons. On the other hand, evidence of regulation of tyrosine phosphorylation of beta-catenin and the cadherins by the EGF receptor, the kinase Src, and the phosphatases PTP1B and LAR suggests that tyrosine phosphorylation may serve as an important mechanism to regulate cadherin-mediated adhesion. The role of tyrosine phosphorylation in cadherin-mediated adhesion is interesting in light of the recent identification of a Cdk5-interacting protein, Cables, which bridges Cdk5 and the non-receptor tyrosine kinase Abl (L. Zukerberg, G. Patrick, M. Nicolic, S. Humbert, L. Lanier, F. Gertler, et al., unpublished observations cited in Kwon, 2000). Additionally, the p35-Cdk5 kinase may function as a scaffold to assemble molecules that act to destabilize N-cadherin-mediated adhesion. For instance, p35-Cdk5 interacts with the active form of the small GTPase Rac, and modulates Pak1 kinase activity. As Rac activity is necessary for cadherin-mediated adhesion, it is possible that the p35-Cdk5 kinase may regulate cadherin-mediated adhesion by regulating a Rac-dependent signaling pathway (Kwon, 2000).

Only about 1% of endogenous beta-catenin binds to p35, whereas more than 10% of total p35 binds to beta-catenin in the embryonic brain lysates. This observation suggests that whereas the beta-catenin-N-cadherin complex may be one of the major targets for the p35-Cdk5 kinase, only a small fraction of the beta-catenin-N-cadherin complex in the developing cortex is actually regulated by p35-Cdk5. Indeed, no difference was detected in the overall levels and association of beta-catenin and N-cadherin in membrane extracts derived from the embryonic and adult cortices of p35^-/- mice. Thus, it is possible that p35-Cdk5 regulation of N-cadherin mediated adhesion is only crucial in a very specific population of migrating cortical neurons. In fact, it may be that p35-Cdk5 regulation of N-cadherin-mediated adhesion is relevant to neurons only when they traverse the intermediate zone of the developing cortex (Kwon, 2000).

ß-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of ß-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in ß-catenin itself. Another tumor suppressor, PTEN, is also involved in the regulation of nuclear ß-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. Nuclear ß-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a ß-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of ß-catenin and enhances its rate of degradation. A pathway is defined that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of ß-catenin stability, nuclear ß-catenin expression, and transcriptional activity. These data indicate that ß-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression (Persad, 2001).

Mechanistically, these results indicate that PTEN induces an increase in the phosphorylation of ß-catenin, thereby increasing its relative rate of degradation in PTEN-transfected PC3 cells compared with the control cells. It is likely that this increased phosphorylation is a direct result of the observed increase in GSK-3 activity induced by PTEN. It is well known that ß-catenin stability is regulated by phosphorylation of the protein at Ser 33/37/45 and Thr 41 by GSK-3 at its NH2 terminus, followed by ubiquitination proteasome-mediated degradation. This increased degradation of ß-catenin may effectively lower cellular concentration of the protein and prevent its further accumulation in the nucleus, leading to decreased nuclear ß-catenin. It is also possible that PTEN may regulate the translocation of ß-catenin from the nucleus and subsequently induce its degradation in the cytosol. However, further studies are required to explore this latter hypothesis (Persad, 2001).

PTEN-transfected PC3 cells also appear to exhibit a more prominent membranal localization of ß-catenin. This may be related to the fact that PTEN appears to induce the transcription of E-cadherin in PC3 cells, which do not normally express E-cadherin. Also, it is possible that the reexpression of E-cadherin in PC3 cells may contribute to the observed decrease in nuclear ß-catenin. ß-Catenin is known to interact with the cytoplasmic domains of E-cadherin, linking it to the actin cytoskeleton. Thus, the more prominent localization of ß-catenin to the cell surface may be related to the reappearance of its cell membrane anchor, E-cadherin, in the PTEN-reexpressing cells. However, no E-cadherin-ß-catenin complexes could be detected in PTEN-transfected PC3 cells. Also, the lack of interaction between ß-catenin and E-cadherin demonstrates that stimulation of E-cadherin expression is unlikely to play a significant role in the observed decreased expression of nuclear ß-catenin. However, it should be noted that PC3 cells do express N-cadherin and this expression is unaffected by reexpression of PTEN. Loss of E-cadherin expression has been linked to the acquisition of an invasive and/or metastatic phenotype and it is this antiinvasive and/or antimetastatic property of E-cadherin that may be of significance in relation to the tumor suppressor PTEN. In support of this, reexpression of E-cadherin by transfection has been shown to suppress the invasive phenotype in E-cadherin-negative prostate tumor cell clones. Overexpression or constitutive activation of integrin-linked kinase (ILK) has been shown to result in an invasive phenotype concomitant with downregulation of E-cadherin expression, translocation of ß-catenin to the nucleus, and formation of the LEF-1-ß-catenin bipartite complex. Also, ILK has been shown to be regulated by the tumor suppressor PTEN (Persad, 2001).

Cyclin D1 is known to be one of the oncogenic targets of ß-catenin. In PC3 cells, the expression level of cyclin D1 is upregulated in a constitutive, serum-independent manner. More importantly, replacement of PTEN or inhibition of ILK results in dramatic suppression of the expression levels of cyclin D1. Furthermore, transient overexpression of GSK-3-WT also suppresses cyclin D1 expression. These results are supported by the observation that overexpression of ILK stimulates cyclin D1 expression. Also, ILK has been shown recently to regulate cyclin D1 transcription via a pathway involving GSK-3 and the cyclic AMP response element binding protein transcription factor. Cyclin D1 expression in PC3 cells changes in a parallel manner to ß-catenin expression in response to reexpression of PTEN or expression of ILK-KD and GSK-3. Therefore, it is proposed that the alterations in cyclin D1 expression very likely represent the physiological end result of the regulation of ß-catenin by PTEN and ILK via GSK-3. It should be pointed out that although nuclear ß-catenin and cyclin D1 expressions undergo parallel alterations due to reexpression of PTEN or inhibition of ILK, the expression of the CDK inhibitors p27Kip and p21Cip remain unchanged. This illustrates the specificity of the alterations induced upon ß-catenin and cyclin D1. PTEN, ILK-KD, and GSK-3-WT all dramatically reduce cyclin D1 promoter activity. Furthermore, Northern blot analysis demonstrates that ILK-KD, PTEN-WT, and GSK-3 induce dramatic inhibitory effects upon cyclin D1 transcriptional expression. This supports the working hypothesis that PTEN and ILK can regulate nuclear ß-catenin through GSK-3. This is in agreement with the fact that ß-catenin is known to be regulated by GSK-3 and recent studies have identified GSK-3 as a critical regulatory component for the transcriptional activity and binding of the TCF-LEF-1-ß-catenin complex transcription factors (Persad, 2001).

In conclusion, a novel pathway involving PI-3 kinase/PTEN, ILK, and GSK-3 has been demonstrated that maintains tight control over the levels and localization of ß-catenin. In prostate cancer cells, as well in other malignancies where PTEN is either lost or inactive, this control may be eliminated, resulting in elevated ß-catenin levels, its accumulation in the nucleus, and increased transcription of its oncogenic targets. Cyclin D1 is a known target of ß-catenin and it is also the first participant of the chain of cyclins and CDKs that control progression through the G1 and S phase of the cell cycle. Therefore, it is conceivable that PTEN and ILK, by virtue of their capacity to regulate ß-catenin and subsequently cyclin D1, may ultimately regulate the progression of cells through the cell cycle. By virtue of their ability to regulate the expression of E-cadherin, PTEN/PI-3 kinase and ILK may also control the metastatic potential of cancer cells. Therefore, the inhibition of a potent regulator such as ILK may present a feasible alternative means of treating the numerous forms of tumors where the PI-3 kinase-dependent signal transduction pathway is dysregulated due to mutations of the tumor suppressor PTEN (Persad, 2001).

Renal dysplasia, the most frequent cause of childhood renal failure in humans, arises from perturbations in a complex series of morphogenetic events during embryonic renal development. The molecular pathogenesis of renal dysplasia is largely undefined. While investigating the role of a BMP-dependent pathway that inhibits branching morphogenesis in vitro, a novel model of renal dysplasia was generated in a transgenic (Tg) model of ALK3 (activin-like kinase 3; BMPR1A) receptor signaling. This study reports the renal phenotype, and the discovery of molecular interactions between effectors in the BMP and WNT signaling pathways in dysplastic kidney tissue. Expression of the constitutively active ALK3 receptor ALK3^QD, in two independent transgenic lines causes renal aplasia/severe dysgenesis in 1.5% and 8.4% of hemizygous and homozygous Tg mice, respectively, and renal medullary cystic dysplasia in 49% and 74% of hemizygous and homozygous Tg mice, respectively. The dysplastic phenotype, which included a decreased number of medullary collecting ducts, increased medullary mesenchyme, collecting duct cysts and decreased cortical thickness, is apparent by E18.5. The pathogenesis of dysplasia in these mice was investigated, and a 30% decrease in branching morphogenesis was demonstrated at E13.5 before the appearance of histopathogical features of dysplasia. The formation of ß-catenin/SMAD1/SMAD4 molecular complexes was also demonstrated in dysplastic renal tissue. Increased transcriptional activity of a ß-catenin reporter gene in ALK3^QD;Tcf-gal mice demonstrates functional cooperativity between the ALK3 and ß-catenin-dependent signaling pathways in kidney tissue. Together with the results in the dysplastic mouse kidney, the findings that phospho-SMAD1 and ß-catenin are overexpressed in human fetal dysplastic renal tissue suggest that dysregulation of these signaling effectors is pathogenic in human renal dysplasia. This work provides novel insights into the role that crucial developmental signaling pathways may play during the genesis of malformed renal tissue elements (Hu, 2003).

The cerebellum provides an excellent system for understanding how afferent and target neurons coordinate sequential intercellular signals and cell-autonomous genetic programs in development. Mutations in the orphan nuclear receptor RORalpha block Purkinje cell differentiation with a secondary loss of afferent granule cells. Early transcriptional targets of RORalpha include both mitogenic signals for afferent progenitors and signal transduction genes required to process their subsequent synaptic input. RORalpha acts through recruitment of gene-specific sets of transcriptional cofactors, including ß-catenin, p300, and Tip60, but appears independent of CBP. One target promoter is Sonic hedgehog, and recombinant Sonic hedgehog restores granule precursor proliferation in RORalpha-deficient cerebellum. These results suggest a link between RORalpha and ß-catenin pathways, confirm that a nuclear receptor employs distinct coactivator complexes at different target genes, and provide a logic for early RORalpha expression in coordinating expression of genes required for reciprocal signals in cerebellar development (Gold, 2003).

Recent studies have led to a model of the molecular pathway that specifies the endoderm during vertebrate gastrulation. The HMG box transcription factor Sox17 is a key component of this pathway and is essential for endoderm formation; however, the molecular events controlled by Sox17 are largely unknown. Several direct transcriptional targets of Sox17, including Foxa1 and Foxa2, have been identified. ß-catenin, a component of Wnt signaling pathway, physically interacts with Sox17 and potentiates its transcriptional activation of target genes. A motif has been identified in the C terminus of Sox17, which is conserved in all the SoxF subfamily of Sox proteins, and this motif is required for the ability of Sox17 to both transactivate target genes and bind ß-catenin. Nuclear ß-catenin is present in endoderm cells of the gastrula, and depletion of ß-catenin from embryos results in a repression of Sox17 target genes. These data suggest that in a mechanism analogous to Tcf/Lef interacting with ß-catenin, Sox17 and ß-catenin interact to transcribe endodermal target genes (Sinner, 2004).

Chondrogenesis is a multistep process that is essential for endochondral bone formation. Previous results have indicated a role for ß-catenin and Wnt signaling in this pathway. This study shows the existence of physical and functional interactions between ß-catenin and Sox9, a transcription factor that is required in successive steps of chondrogenesis. In vivo, either overexpression of Sox9 or inactivation of ß-catenin in chondrocytes of mouse embryos produces a similar phenotype of dwarfism with decreased chondrocyte proliferation, delayed hypertrophic chondrocyte differentiation, and endochondral bone formation. Furthermore, either inactivation of Sox9 or stabilization of ß-catenin in chondrocytes also produces a similar phenotype of severe chondrodysplasia. Sox9 markedly inhibits activation of ß-catenin-dependent promoters and stimulates degradation of ß-catenin by the ubiquitination/proteasome pathway. Likewise, Sox9 inhibits ß-catenin-mediated secondary axis induction in Xenopus embryos. ß-Catenin physically interacts through its Armadillo repeats with the C-terminal transactivation domain of Sox9. It is hypothesized that the inhibitory activity of Sox9 is caused by its ability to compete with Tcf/Lef for binding to ß-catenin, followed by degradation of ß-catenin. These results strongly suggest that chondrogenesis is controlled by interactions between Sox9 and the Wnt/ß-catenin signaling pathway (Akiyama, 2004).

The Hedgehog (Hh) and Wingless (Wnt) families of secreted signaling molecules have key roles in embryonic development and adult tissue homeostasis. In the developing neural tube, Wnt and Shh, emanating from dorsal and ventral regions, respectively, have been proposed to govern the proliferation and survival of neural progenitors. Surprisingly, Shh is required for the growth and survival of cells in both ventral and dorsal neural tube. This study demonstrates that inhibition of Shh signaling causes a reduction in Wnt-mediated transcriptional activation. This reduction requires Gli3. Assays in embryos and cell lines indicate that repressor forms of the Hh-regulated transcription factor, Gli3 (Gli3R), which are generated in the absence of Hh signaling, inhibit canonical Wnt signaling. Gli3R acts by antagonizing active forms of the Wnt transcriptional effector, β-catenin. Consistent with this, Gli3R appears to physically interact with the carboxy-terminal domain of β-catenin, a region that includes the transactivation domain. These data offer an explanation for the proliferative defects in Shh null embryos and suggest a novel mechanism for crosstalk between the Hh and Wnt pathways (Ulloa, 2007).

The canonical Wnt-β-catenin signaling pathway is important for a variety of developmental phenomena as well as for carcinogenesis. In hippocampal neurons, NMDA-receptor-dependent activation of calpain induces the cleavage of β-catenin at the N terminus, generating stable, truncated forms. These β-catenin fragments accumulate in the nucleus and induced Tcf/Lef-dependent gene transcription. Fosl1, one of the immediate-early genes, was identified as a target of this signaling pathway. In addition, exploratory behavior by mice resulted in a similar cleavage of β-catenin, as well as activation of the Tcf signaling pathway, in hippocampal neurons. Both β-catenin cleavage and Tcf-dependent gene transcription are suppressed by calpain inhibitors. These findings reveal another pathway for β-catenin-dependent signaling, in addition to the canonical Wnt-β-catenin pathway, and suggest that this other pathway could play an important role in activity-dependent gene expression (Abe, 2007).

Wnt ligands have pleiotropic and context-specific roles in embryogenesis and adult tissues. Among other effects, certain Wnts stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T-cell factor (TCF)-mediated transcription. Mutations resulting in constitutive beta-catenin stabilization underlie development of several human cancers. Genetic studies in Drosophila highlighted the split ends (spen) gene as a positive regulator of Wnt-dependent signaling. This study has assessed the role of SHARP, a human homologue of spen, in Wnt/beta-catenin/TCF function in mammalian cells. SHARP gene and protein expression were found to be elevated in human colon and ovarian endometrioid adenocarcinomas and mouse colon adenomas and carcinomas carrying gene defects leading to beta-catenin dysregulation. When ectopically expressed, the silencing mediator for retinoid and thyroid receptors/histone deacetylase 1-associated repressor protein (SHARP) protein potently enhances beta-catenin/TCF transcription of a model reporter gene and cellular target genes. Inhibition of endogenous SHARP function via RNA inhibitory (RNAi) approaches antagonized beta-catenin/TCF-mediated activation of target genes. The effect of SHARP on beta-catenin/TCF-regulated genes is mediated via a functional interaction between SHARP and TCF. beta-Catenin-dependent neoplastic transformation of RK3E cells is enhanced by ectopic expression of SHARP, and RNAi-mediated inhibition of endogenous SHARP in colon cancer cells inhibits their transformed growth. These findings implicate SHARP as an important positive regulator of Wnt signaling in cancers with beta-catenin dysregulation (Feng, 2007).

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