Search

Hepatoblastoma (HB) is the most frequent malignant liver tumour of childhood. Most HBs develop sporadically but their incidence is highly elevated in patients with familial adenomatous polyposis coli (FAP). These patients carry germline mutations in the adenomatous polyposis coli (APC) tumour suppressor gene. APC forms a multi-protein complex involved in the WNT signalling pathway that controls the stability of beta-catenin, the central effector in this cascade. Whereas APC mutations are rare in sporadic HBs, a high frequency of beta-catenin mutations leading to overactivation of WNT signalling was previously found in these tumours. This pathway is negatively controlled by conductin (axin2), representing a further partner in this signalling complex. To investigate whether alterations in conductin may also be involved in the pathogenesis of sporadic HBs, 37 HBs and five HB cell lines were screened for mutations using single-strand conformation polymorphism (SSCP) analysis, reverse transcription-polymerase chain reaction (RT-PCR), and direct sequencing. In two cases, larger deletions (52 and 1624 bp) leading to frameshifts were found. In addition, one HB carried a somatic point mutation. Expression analysis by competitive RT-PCR in HBs revealed up-regulation of conductin mRNA compared with adjacent liver samples. This mRNA overexpression resulted in increased conductin protein levels demonstrated by western blot analysis. Tumours with activating beta-catenin mutations revealed higher levels of conductin mRNA transcripts. This finding indicates that conductin is a direct target gene of WNT signalling in HBs, as has been demonstrated in other tissues. In summary, conductin mutations may represent an alternative mechanism leading to activation of WNT signalling in HBs. The overexpression of conductin mRNA in HBs reflects activation of the WNT pathway because conductin represents a target gene of WNT signalling in liver tissue., (Copyright (c) 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with fibrofatty replacement of cardiac myocytes, ventricular tachyarrhythmias and sudden cardiac death. In 32 of 120 unrelated individuals with ARVC, we identified heterozygous mutations in PKP2, which encodes plakophilin-2, an essential armadillo-repeat protein of the cardiac desmosome. In two kindreds with ARVC, disease was incompletely penetrant in most carriers of PKP2 mutations.

Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Both beta-catenin and plakoglobin can stimulate the expression of Lef/Tcf target genes in vitro. beta-Catenin is known to associate with Lef/Tcf factors and to participate directly in transactivation in vivo, whereas the role of plakoglobin in transcriptional regulation has been less studied. To analyze the functions of plakoglobin in vivo, we generated transgenic mice expressing in the epidermis N-terminally truncated plakoglobin (DeltaN122-PG) lacking the glycogen synthase kinase 3beta phosphorylation sites and therefore protected against degradation (transgenic line K5-DeltaN122-PG). The expression of DeltaN122-PG led to the formation of additional hair germs, hyperplastic hair follicles, and noninvasive hair follicle tumors, a phenotype reminiscent of that induced by expression of N-terminally truncated beta-catenin. However, if expressed in beta-catenin-null epidermis, DeltaN122-PG did not induce new hair follicle germs and follicular tumors. Thus, DeltaN122-PG cannot substitute for beta-catenin in its signaling functions in vivo and the phenotype observed in K5-DeltaN122-PG mouse skin must be due to the aberrant activation of beta-catenin signaling. On the other hand, the expression of DeltaN122-PG in beta-catenin-null skin significantly increased the survival rate of mutant mice, rescued differentiation, and limited excessive proliferation in the interfollicular epidermis, suggesting that plakoglobin may be involved in the intracellular signaling events essential for epidermal differentiation.

beta-Catenin controls both cadherin-mediated cell adhesion and activation of Wnt target genes. We demonstrate here that the beta-catenin-binding protein BCL9-2, a homolog of the human proto-oncogene product BCL9, induces epithelial-mesenchymal transitions of nontransformed cells and increases beta-catenin-dependent transcription. RNA interference of BCL9-2 in carcinoma cells induces an epithelial phenotype and translocates beta-catenin from the nucleus to the cell membrane. The switch between beta-catenin's adhesive and transcriptional functions is modulated by phosphorylation of Tyr 142 of beta-catenin, which favors BCL9-2 binding and precludes interaction with alpha-catenin. During zebrafish embryogenesis, BCL9-2 acts in the Wnt8-signaling pathway and regulates mesoderm patterning.

In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.

The docking protein Gab1 has been implicated as a mediator of multiple signaling pathways that are activated by a variety of receptor tyrosine kinases and cytokines. We have previously proposed that fibroblast growth factor 1 (FGF1) stimulation of tyrosine phosphorylation of Gab1 and recruitment of phosphatidylinositol (PI) 3-kinase are mediated by an indirect mechanism in which the docking protein fibroblast receptor substrate 2alpha (FRS2alpha) plays a critical role. In this report, we explore the role of Gab1 in FGF1 signaling by using mouse embryo fibroblasts (MEFs) derived from Gab1(-/-) or FRS2alpha(-/-) mice. We demonstrate that Gab1 is essential for FGF1 stimulation of both PI 3-kinase and the antiapoptotic protein kinase Akt, while FGF1-induced mitogen-activated protein kinase (MAPK) stimulation is not affected by Gab1 deficiency. To test the indirect mechanism for FGF1 stimulation of PI 3-kinase and Akt, we use a chimeric docking protein composed of the membrane targeting signal and the phosphotyrosine-binding domain of FRS2alpha fused to the C-terminal portion of Gab1, the region including the binding sites for the complement of signaling proteins that are recruited by Gab1. We demonstrate that expression of the chimeric docking protein in Gab1(-/-) MEFs rescues PI 3-kinase and the Akt responses, while expression of the chimeric docking protein in FRS2alpha(-/-) MEFs rescues stimulation of both Akt and MAPK. These experiments underscore the essential role of Gab1 in FGF1 stimulation of the PI 3-kinase/Akt signaling pathway and provide further support for the indirect mechanism for FGF1 stimulation of PI 3-kinase involving regulated assembly of a multiprotein complex.

Branched hollow tubes form the architectural basis of many mammalian organs. The growth factor HGF/SF and its receptor, the Met receptor tyrosine kinase, stimulate epithelial cells to undergo tubulogenesis in vitro. In this issue of Developmental Cell, O'Brien et al. (2004) look at temporal regulation and the role of two HGF/SF effectors, the ERK 1/2 MAP kinases and matrix metalloproteases, in this process.

T cells encounter two main checkpoints during development in the thymus. These checkpoints are critically dependent on signals derived from the thymic microenvironment as well as from the pre-T cell receptor (pre-TCR) and the alphabeta TCR. Here we show that T cell-specific deletion of beta-catenin impaired T cell development at the beta-selection checkpoint, leading to a substantial decrease in splenic T cells. In addition, beta-catenin also seemed to be a target of TCR-CD3 signals in thymocytes and mature T cells. These data indicate that beta-catenin-mediated signals are required for normal T cell development.

Gene expression profiling of beta-catenin, Cripto and Wnt3 mutant mouse embryos has been used to characterise the genetic networks that regulate early embryonic development. We have defined genes whose expression is regulated by beta-catenin during formation of the anteroposterior axis and the mesoderm, and have identified Cripto, which encodes a Nodal co-receptor, as a primary target of beta-catenin signals both in embryogenesis as well as in colon carcinoma cell lines and tissues. We have also defined groups of genes regulated by Wnt3/beta-catenin signalling during primitive streak and mesoderm formation. Our data assign a key role to beta-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation.

Cadherins and catenins are thought to promote adhesion between pre and postsynaptic elements in the brain. Here we show a role for beta-catenin in localizing the reserved pool of vesicles at presynaptic sites. Deletion of beta-catenin in hippocampal pyramidal neurons in vivo resulted in a reduction in the number of reserved pool vesicles per synapse and an impaired response to prolonged repetitive stimulation. This corresponded to a dispersion of vesicles along the axon in cultured neurons. Interestingly, these effects are not due to beta-catenin's involvement in cadherin-mediated adhesion or wnt signaling. Instead, beta-catenin modulates vesicle localization via its PDZ binding domain to recruit PDZ proteins such as Veli to cadherin at synapses. This study defines a specific role for cadherins and catenins in synapse organization beyond their roles in mediating cell adhesion.

The lungs are divided, both structurally and functionally, into two distinct components, the proximal airways, which conduct air, and the peripheral airways, which mediate gas exchange. The mechanisms that control the specification of these two structures during lung development are currently unknown. Here we show that beta-catenin signaling is required for the formation of the distal, but not the proximal, airways. When the gene for beta-catenin was conditionally excised in epithelial cells of the developing mouse lung prior to embryonic day 14.5, the proximal lung tubules grew and differentiated appropriately. The mice, however, died at birth because of respiratory failure. Analysis of the lungs by in situ hybridization and immunohistochemistry, using molecular markers of the epithelial and mesenchymal components of both proximal and peripheral airways, showed that the lungs were composed primarily of proximal airways. These observations establish, for the first time, both the sites and timing of specification of the proximal and peripheral airways in the developing lung, and that beta-catenin is one of the essential components of this specification.

By conditional gene ablation in mice, we found that beta-catenin, an essential downstream effector of canonical Wnt signaling, is a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal-ventral axis of the limbs. By generation of compound mutants, we also show that beta-catenin acts downstream of the BMP receptor IA in AER induction, but upstream or parallel in dorsal-ventral patterning. Thus, AER formation and dorsal-ventral patterning of limbs are tightly controlled by an intricate interplay between Wnt/beta-catenin and BMP receptor signaling.

beta-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development. We employed here two conditional beta-catenin mutant alleles to alter beta-catenin signaling in the central nervous system of mice: one allele to ablate beta-catenin and the second allele to express a constitutively active beta-catenin. The tissue mass of the spinal cord and brain is reduced after ablation of beta-catenin, and the neuronal precursor population is not maintained. In contrast, the spinal cord and brain of mice that express activated beta-catenin is much enlarged in mass, and the neuronal precursor population is increased in size. beta-Catenin signals are thus essential for the maintenance of proliferation of neuronal progenitors, controlling the size of the progenitor pool, and impinging on the decision of neuronal progenitors to proliferate or to differentiate.

Hepatocyte growth factor/scatter factor (HGF/SF), acting through the receptor tyrosine kinase Met, stimulates cells derived from a variety of different organs to form elongated hollow tubules when grown in three-dimensional gels. In vivo data also indicate a role for HGF/SF and Met in tubule formation during liver and kidney regeneration and mammary gland formation. Activation of Met results in the recruitment of a myriad of signal transducers that regulate dissociation of adherens junctions and the stimulation of cellular motility, survival, proliferation and morphogenesis during tubule formation. Among these many signal transducers, the Gab1 adaptor protein and its effector, the SHP2 tyrosine phosphatase, have been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAP kinase pathway. Here, we discuss the contribution of these and other signaling pathways and the role of HGF/SF and Met in the formation of epithelial cell tubules both in vitro in branching-morphogenesis assays and in vivo during organogenesis.

Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cgamma but not Grb2-associated binder 1 or growth factor receptor-bound protein 2. The H. pylori-induced motogenic response is suppressed and blocked by the inhibition of PLCgamma and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori-infected epithelial cells suggests that CagA could be involved in tumor progression.

Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.

Dendritic cells (DC) are professional antigen-presenting cells that possess both migratory properties and potent T cell stimulatory activity, and that allow the uptake of antigenic material inperipheral tissues and its subsequent presentation in the T cell areas of lymphoid organs. Thus motility represents a central property that is required for DC function. Here we report on the expression of the receptor tyrosine kinase c-met in DC. c-Met is the high affinity receptor for scatter factor (SF)/hepatocyte growth factor, and ligand-activated c-met exhibits mitogenic, morphogenic andmotogenic activity in vivo and in vitro. c-Met is signaling competent in DC since it is effectively tyrosine phosphorylated in response to SF ligand. It is demonstrated here that ligand-activated c-met regulates DC adhesion to the extracellular matrix component laminin but leaves antigen presenting function unaffected. Importantly, in ear sheet explant experiments activationof c-met by ligand induces emigration of cutaneous DC (Langerhans cell, LC) from skin, but SF is not a chemoattractant factor for DC. Our results suggest an important role of the c-met/SF system in DC/LC migration.

Studies of chimeric mice have disclosed that the stem cell hierarchy in the small intestinal epithelium is established during formation of its proliferative units (crypts of Lieberkühn). This process involves a selection among several multipotential progenitors so that ultimately only one survives to supply descendants to the fully formed crypt. In this report, we examine the hypothesis that the level of beta-catenin (beta-cat)-mediated signaling is an important factor regulating this stem cell selection. In the canonical Wnt signaling pathway, beta-catenin can partner with Lef-1/Tcf high mobility group (HMG) box transcription factors to control gene expression. Both Lef-1 and Tcf-4 mRNAs are produced in the fetal mouse small intestine. Tcf-4 expression is sustained, whereas Lef-1 levels fall as crypt formation is completed during the first two postnatal weeks. A Tcf-4 gene knockout is known to block intestinal epithelial proliferation in late fetal life. Therefore, to test the hypothesis, we enhanced beta-catenin signaling in a chimeric mouse model in which the stem cell selection could be monitored. A fusion protein containing the HMG box domain of Lef-1 linked to the trans-activation domain of beta-catenin (Lef-1/beta-cat) was constructed to promote direct stimulation of signaling without being retained in the cytoplasm through interactions with E-cadherin and Apc/Axin. Lef-1/beta-cat was expressed in 129/Sv embryonic stem cell-derived small intestinal epithelial progenitors present in developing B6-ROSA26<-->129/Sv chimeras. Lef-1/beta-cat stimulated expression of a known beta-catenin target (E-cadherin), suppressed expression of Apc and Axin, and induced apoptosis in 129/Sv but not in neighboring B6-ROSA26 epithelial cells. This apoptotic response was not associated with any detectable changes in cell division within the Lef-1/beta-cat-expressing epithelium. By the time crypt development was completed, all 129/Sv epithelial cells were lost. These results indicate that developmental changes in beta-catenin-mediated signaling can play an important role in establishing a stem cell hierarchy during crypt morphogenesis.

In epithelial cells, tyrosine kinases induce the tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which induces endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which we have identified as an E3 ubiquitin-ligase. Hakai contains SH2, RING, zinc-finger and proline-rich domains, and interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, inducing ubiquitination of the E-cadherin complex. Expression of Hakai in epithelial cells disrupts cell--cell contacts and enhances endocytosis of E-cadherin and cell motility. Through dynamic recycling of E-cadherin, Hakai can thus modulate cell adhesion, and could participate in the regulation of epithelial--mesenchymal transitions in development or metastasis.

Activation of Wnt signaling through beta-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of beta-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3beta. Here we show that conductin is upregulated in human tumors that are induced by beta-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of beta-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.

To examine the consequences of repressing beta-catenin/Lef1 signalling in mouse epidermis, we expressed a DeltaNLef1 transgene, which lacks the beta-catenin binding site, under the control of the keratin 14 promoter. No skin abnormalities were detected before the first postnatal hair cycle. However, from 6 weeks of age, mice underwent progressive hair loss which correlated with the development of dermal cysts. The cysts were derived from the base of the hair follicles and expressed morphological and molecular markers of interfollicular epidermis. Adult mice developed spontaneous skin tumours, most of which exhibited sebaceous differentiation, which could be indicative of an origin in the upper part of the hair follicle. The transgene continued to be expressed in the tumours and beta-catenin signalling was still inhibited, as evidenced by absence of cyclin D1 expression. However, patched mRNA expression was upregulated, suggesting that the sonic hedgehog pathway might play a role in tumour formation. Based on our results and previous data on the consequences of activating beta-catenin/Lef1 signalling in postnatal keratinocytes, we conclude that the level of beta-catenin signalling determines whether keratinocytes differentiate into hair or interfollicular epidermis, and that perturbation of the pathway by overexpression of DeltaNLef1 can lead to skin tumour formation.

The development of tissues and organs in embryos is controlled by an interplay of several signaling pathways that cross-talk to provide positional information and induce cell fate specification. One of the major signaling systems is the Wnt pathway which was recently shown to split into several intracellular branches which regulate multiple cellular functions. In the present review, we discuss novel members and their role in the diversification of the Wnt pathway. Many of these components were studied in model organisms such as C.elegans, Drosophila and Xenopus. Here we focus on recent studies of mutant phenotypes in Mouse and Zebrafish which implicate members of the Wnt pathway in processes such as axis and mesoderm formation, initiation of organ development and stem cell differentiation.

Medulloblastoma (MB) represents the most frequent malignant brain tumor in children. Most MBs appear sporadically; however, their incidence is highly elevated in two inherited tumor predisposition syndromes, Gorlin's and Turcot's syndrome. The genetic defects responsible for these diseases have been identified. Whereas Gorlin's syndrome patients carry germ-line mutations in the patched (PTCH) gene, Turcot's syndrome patients with MBs carry germ-line mutations of the adenomatous polyposis coli (APC) gene. The APC gene product is a component of a multiprotein complex controlling beta-catenin degradation. In this complex, Axin plays a major role as scaffold protein. Whereas APC mutations are rare in sporadic MBs, a hot-spot region of beta-catenin (CTNNB1) mutations was identified in a subset of MBs. To find out if Axin is also involved in the pathogenesis of sporadic MBs, we analyzed 86 MBs and 11 MB cell lines for mutations in the AXIN1 gene. Using single-strand conformation polymorphism analysis, screening for large deletions by reverse transcription-PCR, and sequencing analysis, a single somatic point mutation in exon 1 (Pro255Ser) and seven large deletions (12%) of AXIN1 were detected. This indicates that AXIN1 may function as a tumor suppressor gene in MBs.

NF-kappaB/Rel transcription factors and IkappaB kinases (IKK) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has previously been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach we have ubiquitously expressed an NF-kappaB super-repressor to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB revealed defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which resulted in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function.

Docking proteins are substrates of tyrosine kinases and function in the recruitment and assembly of specific signal transduction molecules. Here we found that p62dok family members act as substrates for the c-Ret receptor tyrosine kinase. In addition to dok-1, dok-2, and dok-3, we identified two new family members, dok-4 and dok-5, that can directly associate with Y1062 of c-Ret. Dok-4 and dok-5 constitute a subgroup of dok family members that is coexpressed with c-Ret in various neuronal tissues. Activated c-Ret promotes neurite outgrowth of PC12 cells; for this activity, Y1062 in c-Ret is essential. c-Ret/dok fusion proteins, in which Y1062 of c-Ret is deleted and replaced by the sequences of dok-4 or dok-5, induce ligand-dependent axonal outgrowth of PC12 cells, whereas a c-Ret fusion containing dok-2 sequences does not elicit this response. Dok-4 and dok-5 do not associate with rasGAP or Nck, in contrast to p62dok and dok-2. Moreover, dok-4 and dok-5 enhance c-Ret-dependent activation of mitogen-activated protein kinase. Thus, we have identified a subclass of p62dok proteins that are putative links with downstream effectors of c-Ret in neuronal differentiation.

beta-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of beta-catenin in skin development, we introduced a conditional mutation of the gene in the epidermis and hair follicles using Cre/loxP technology. When beta-catenin is mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. We show that beta-catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in placode formation. If beta-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that beta-catenin is essential for fate decisions of skin stem cells: in the absence of beta-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate.

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

Background & Aims: Inactivation of the adenomatous polyposis coli (APC) gene is observed at early stages of intestinal tumor formation, whereas loss of E-cadherin is usually associated with tumor progression. Because both proteins compete for the binding to beta-catenin, an essential component of the Wnt signaling pathway, reduction of E-cadherin levels in an Apc mouse model could influence both tumor initiation and progression. In addition, loss or haploinsufficiency of E-cadherin may affect tumorigenesis by altering its cell-adhesive and associated functions., Methods: Apc1638N mice were bred with animals carrying a targeted E-cadherin knockout mutation., Results: Double heterozygous animals showed a significant 9-fold and 5-fold increase of intestinal and gastric tumor numbers, respectively, compared with Apc1638N animals. The intestinal tumors of both groups showed no significant differences in grading and staging. Loss of heterozygosity analysis at the Apc and E-cadherin loci in both intestinal and gastric Apc(+/1638N)/E-cad(+/-) tumors revealed loss of the wild-type Apc allele in most cases, whereas the wild-type E-cadherin allele was always retained. This was supported by a positive, although reduced, staining for E-cadherin of intestinal tumor sections., Conclusions: Introduction of the E-cadherin mutation in Apc1638N animals enhances Apc-driven tumor initiation without clearly affecting tumor progression.

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1-/- embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1-/- embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-:c-Met+/- embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.

Interactions between beta-catenin and LEF-1/TCF, APC and conductin/axin are essential for wnt-controlled stabilization of beta-catenin and transcriptional activation. The wnt signal transduction pathway is important in both embryonic development and tumor progression. We identify here amino acid residues in beta-catenin that distinctly affect its binding to LEF-1/TCF, APC and conductin. These residues form separate surface clusters, termed hot spots, along the armadillo superhelix of beta-catenin. We also show that complementary charged and hydrophobic amino acids are required for formation of the bipartite beta-catenin-LEF-1 transcription factor. Moreover, we demonstrate that conductin/axin binding to beta-catenin is essential for beta-catenin degradation, and that APC acts as a cofactor of conductin/axin in this process. Binding of APC to conductin/axin activates the latter and occurs between their SAMP and RGS domains, respectively.

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met-specific branching morphogenesis. It associates directly with c-Met via the c-Met-binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met-binding site is localized to a 13-amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met-binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1-specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Hepatocyte growth factor (HGF; scatter factor) is a multipotent protein with mitogenic, motogenic, and developmental functions. Upon activation, the HGF-receptor c-Met binds and phosphorylates the multisite docking protein Gab1. Besides binding motifs for phosphatidylinositol 3-kinase and Grb2, Gab 1 contains multiple Tyr-X-X-Pro (YXXP) motifs which, when phosphorylated, are potential binding sites for the adapter proteins c-Crk and Crk-like (CRKL). Stimulation of human embryonic kidney cells (HEK293) with HGF leads to Gab1 association with CRKL. The Gab1-CRKL interaction requires both, the SH2 domain of CRKL and the region containing the YXXP motifs in Gab1. CRKL binds via its first SH3 domain to several downstream signal transducers, including C3G an activator of the small GTPase Rap1. Indeed, Rap1 was rapidly activated after HGF stimulation of HEK293 cells. Rap1 activation through HGF was suppressed through transfection of a truncated C3G protein which only contains the SH3-binding motifs of C3G. Transfection of nonmutated Gab1 led to a strong increase of Rap1.GTP in the absence of HGF. In contrast, transfection of the GabDeltaYXXP mutant abolished the elevation of Rap1.GTP by HGF. A replating assay indicated that HGF decreases the adhesion of HEK293 cells. The results presented here delineate a novel signaling pathway from HGF to the GTPase Rap1 which depends on the interaction of the adapter protein CRKL with the exchange factor C3G and could be linked to cell migration.

The anterior-posterior axis of the mouse embryo is defined before formation of the primitive streak, and axis specification and subsequent anterior development involves signaling from both embryonic ectoderm and visceral endoderm. Tauhe Wnt signaling pathway is essential for various developmental processes, but a role in anterior-posterior axis formation in the mouse has not been previously established. Beta-catenin is a central player in the Wnt pathway and in cadherin-mediated cell adhesion. We generated beta-catenin-deficient mouse embryos and observed a defect in anterior-posterior axis formation at embryonic day 5.5, as visualized by the absence of Hex and Hesx1 and the mislocation of cerberus-like and Lim1 expression. Subsequently, no mesoderm and head structures are generated. Intercellular adhesion is maintained since plakoglobin substitutes for beta-catenin. Our data demonstrate that beta-catenin function is essential in anterior-posterior axis formation in the mouse, and experiments with chimeric embryos show that this function is required in the embryonic ectoderm.

Organ culture and transplantation experiments in the early 1960s and 1970s have demonstrated that growth and morphogenesis of the epithelium of the mammary gland are controlled by mesenchymal-epithelial interactions. The identification of molecules that provide the essential signals exchanged in mesenchymal-epithelial interactions is an area of active research. Recent evidence suggests that morphogenic programs of epithelia can be triggered by mesenchymal factors that signal via tyrosine kinase receptors. This review concentrates on the effects of two mesenchymal factors, Hepatocyte Growth Factor/Scatter Factor and neuregulin, on morphogenesis and differentiation of mammary epithelial cells in vitro and signalling pathways involved during morphogenesis of mammary epithelial cells.

Cultured human mammary MCF7 and T47D tumor cell lines were used to test the interference of the partial antiestrogen 4'-hydroxytamoxifen (4-OH-TAM) and the pure antiestrogen ZM 182780 with growth factor (IGF-I, heregulin) signaling pathways. Growth of both cell lines was stimulated by IGF-I (20 ng/ml) or heregulin (3 nM). ZM 182780 effectively blocked growth factor induced as well as basal proliferation of MCF7 cells while the compound was ineffective in interfering with growth factor mitogenic activity in T47D cells. On both cell lines the IGF-I or heregulin- induced proliferation was enhanced further by 4-OH-TAM. This synergism could be inhibited dose-dependently by ZM 182780. When cells were grown in the presence of estradiol plus growth factors, the antiestrogenic potencies of both compounds and the efficacy of ZM 182780 were unaffected, while the efficacy of 4-OH-TAM was reduced. Our data show cell type specific cross-talk between the receptor for estrogen and that for IGF-I or heregulin, which is different in MCF7 and T47D cells, respectively. In MCF7 cells with demonstrable cross-talk, a clear superiority exists for a pure antiestrogen over a partial agonist in interfering with growth factor mitogenic activity.

A major function of the cell-to-cell adhesion molecule E-cadherin is the maintenance of cell adhesion and tissue integrity. E-cadherin deficiency in tumours leads to changes in cell morphology and motility, so that E-cadherin is considered to be a suppressor of invasion. In this study we investigated the functional consequences of three tumour-associated gene mutations that affect the extracellular portion of E-cadherin: in-frame deletions of exons 8 or 9 and a point mutation in exon 8, as they were found in human gastric carcinomas. Human MDA-MB-435S breast carcinoma cells and mouse L fibroblasts were stably transfected with the wild-type and mutant cDNAs, and the resulting changes in localization of E-cadherin, cell morphology, strength of calcium-dependent aggregation as well as cell motility and actin cytoskeleton organization were studied. We found that cells transfected with wild-type E-cadherin showed an epitheloid morphology, while all cell lines expressing mutant E-cadherin exhibited more irregular cell shapes. Cells expressing E-cadherin mutated in exon 8 showed the most scattered appearance, whereas cells with deletion of exon 9 had an intermediate state. Mutant E-cadherins were localized to the lateral regions of cell-to-cell contact sites. Additionally, both exon 8-mutated E-cadherins showed apical and perinuclear localization, and actin filaments were drastically reduced. MDA-MB-435S cells with initial calcium-dependent cell aggregation exhibited decreased aggregation and, remarkably, increased cell motility, when mutant E-cadherin was expressed. Therefore, we conclude that these E-cadherin mutations may not simply affect cell adhesion but may act in a trans-dominant-active manner, i.e. lead to increased cell motility. Our study suggests that E-cadherin mutations affecting exons 8 or 9 are the cause of multiple morphological and functional disorders and could induce the scattered morphology and the invasive behaviour of diffuse type-gastric carcinomas.

Plakoglobin (gamma-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaque of cardiac junctions and is involved in anchorage of cytoskeletal filaments to specific cadherins. Its genetic inactivation leads to an embryonic lethal phenotype due to heart dysfunction related to an impairment in the architecture of intercalated discs and in the stability of the heart tissue. To elucidate the functional consequences of the loss of plakoglobin for myofibrillar function, we monitored passive stress-strain relationship and contractility parameters of demembranated embryonic fibers. Heart fibers obtained from plakoglobin-deficient embryonic mice were significantly less compliant than were fibers from wild-type embryos. This difference was especially pronounced at lower fiber extension levels: at 120% of slack length, compliance was 2.5-fold lower in plakoglobin-deficient mice than in the corresponding wild-type group. Contractile paramenters (force per cross-section; Ca2+ sensitivity of isometric force and shortening velocity at near-zero load) were comparable in all experimental groups. Therefore, we suggest that plakoglobin is important for cardiac compliance but not necessary for the attachment of the myofibrillar apparatus to adherens junctions. Thus, we conclude that the loss of function of desmosomes and the profound disarrangement of junctional components in plakoglobin null embryos is associated with a decreased passive compliance, which may explain the ventricular rupture and consequent pericardial tamponade in embryos lacking plakoglobin.

Intercellular adhesion mediated by the E-cadherin/catenin complex is a prerequisite for epithelial integrity and differentiation. In carcinomas, E-cadherin function is frequently disturbed, and has been suggested to increase invasion and metastasis of tumour cells. beta-catenin has also been implicated in signaling pathways essential for tumour formation. We analysed the E-cadherin/catenin adhesion system of colorectal tumours at different clinical stages. In primary carcinomas (n = 91), there was a frequent reduction in E-cadherin (44%) and alpha-catenin expression (36%). In contrast, beta-catenin and gamma-catenin expression were seldom reduced (4% and 15%, respectively). Similar expression patterns were observed in liver metastases from unrelated colorectal tumours (n = 27). There was a significant relationship between loss of E-cadherin and alpha-catenin expression and poorly differentiated (G3-4) tumours. Our results suggest that reduction of E-cadherin/alpha-catenin expression is a frequent event in primary and metastatic colorectal carcinomas. Furthermore, beta-catenin expression remains normal in colorectal cancer, suggesting the essential role of beta-catenin in signaling pathways.

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met-mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.

Control of stability of beta-catenin is central in the wnt signaling pathway. Here, the protein conductin was found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induced beta-catenin degradation, whereas mutants of conductin that were deficient in complex formation stabilized beta-catenin. Fragments of APC that contained a conductin-binding domain also blocked beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfered with wnt-induced axis formation.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization by B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved by subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response to paracrine interactions with its ligand. C-met expression per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability.

Plakoglobin (γ-catenin), a member of the armadillo family of proteins, is a constituent of the cytoplasmic plaques of desmosomes and adherens junctions and is involved in anchorage of cytoskeletal filaments to specific cadherins. When the plakoglobin gene is ablated in mice, animals die between days 12 and 16 of embryogenesis owing to defects in heart function. Often, their heart ventricles burst, and pericardial tamponade appears to be the immediate cause of death. This tissue instability correlates with the absence of desmosomes and a redistribution of desmosomal proteins in heart, but not in epithelial organs. Plakoglobin is an essential component of cardiac desmosomes and plays a crucial role in the architecture and stabilization of heart tissue., (Copyright © 1998 Elsevier Science Inc. All rights reserved.)

Cadherins are a family of glycoproteins that are associated with cell adhesion mechanisms. They are divided into subclasses. The E- and P-cadherins are regarded as the epithelial subtype. Their expression has been demonstrated in many different carcinoma types. Using immunomorphological techniques, we studied the expression of E-cadherin in a series of 145 human brain tumours with the monoclonal antibody 5H9. Western blot analysis was used to confirm the immunohistochemical data. The tumour types represented were astrocytoma WHO I (n = 7), astrocytoma WHO II (n = 6), astrocytoma WHO III (n = 14), glioblastoma WHO IV (n = 8), oligodendroglioma WHO II (n = 5), ependymoma WHO II (n = 5), choroid plexus papilloma WHO I (n = 5), pineoblastoma WHO IV (n = 5), medulloblastoma WHO IV (n = 5), neurinoma WHO I (n = 5), meningioma WHO I and WHO III (n = 75) and pituitary adenoma WHO I (n = 5). Only choroid plexus papillomas (5/5) and meningiomas showed E-cadherin expression. In benign meningiomas (n = 45; 100%), positive E-cadherin immunoreactivity was found regardless of the histomorphological subtype. E-Cadherin was also expressed in 21 WHO I meningiomas (100%) invading dura, bone, brain, and muscle. In contrast, E-cadherin was absent from the majority of morphologically malignant meningiomas (6/9, 66.6%). In addition, in recurrent meningiomas (n = 9), E-cadherin expression in the recurrent tumours was identical to that in the primary neoplasm except in cases with malignant progression, where the malignant recurrent tumour was E-cadherin negative. In 2 cases of metastasizing meningiomas, no E-cadherin immunoreactivity was found in the primary tumours or their metastases.

Background: Although a number of growth factors bind cell-surface heparan sulphate proteoglycans (HSPGs), the role of this interaction is unclear except for fibroblast growth factor which requires HSPG binding for signalling. Hepatocyte growth factor/scatter factor (HGF/SF) plays important roles in mammalian development and tissue regeneration and acts on target cells through a specific receptor tyrosine kinase encoded by the c-met proto-oncogene. This factor also binds HSPGs with high affinity, but conflicting data have been reported on the role of HSPG binding in HGF/SF signalling., Results: To map the binding sites for HSPG and the Met receptor in HGF/SF, we have engineered a number of HGF/SF mutants in which several clusters of solvent-accessible residues in the hairpin structure of the amino-terminal domain or in kringle 2 have been replaced. Two of the mutants (HP1 and HP2) showed greatly decreased (more than 50-fold) affinity for heparin and HSPGs but retained full mitogenic and motogenic activities on target cells in culture. Furthermore, when compared with wild-type HGF/SF, the HP1 mutant exhibited a delayed clearance from the blood, higher tissue levels and a higher induction of DNA synthesis in normal, adult murine liver., Conclusions: These results establish the following: the binding sites in HGF/SF for Met and for HSPGs can be dissociated by protein engineering; high-affinity binding of HGF/SF to HSPGs is not essential for signalling; one role of HSPG binding in the HGF/SF system appears to be sequestration and degradation of the growth factor; and HGF/SF mutants with decreased affinity for HSPGs exhibit enhanced activity in vivo.

We have analysed the role of the morphogenetic factors hepatocyte growth factor/scatter factor (HGF), neuregulin and E-cadherin in the process of metastasis and morphogenesis of mammary carcinoma cells. The cDNAs for HGF, neuregulin and E-cadherin were stably expressed in anaplastic human MDA MB 435 carcinoma cells. The altered cells were then injected into the mammary fat pads of nude mice, where they form tumors which can spontaneously metastasize to the lungs. We found that expression of HGF or neuregulin promoted metastasis whereas expression of the cell adhesion molecule E-cadherin was inhibitory. Moreover, expression of E-cadherin reconstituted the ability of the cells to form morphogenetic structures in matrigel cultures in response to HGF. These data demonstrate that HGF and neuregulin, which control branching or lobulo-alveolar morphogenesis of normal breast epithelium, do have metastasis-promoting effects on breast carcinoma cells. Moreover, our results suggest that the differential activities of the two factors can be explained by the degree of epithelial differentiation: induction of morphogenesis requires an intact epithelial adhesion and differentiation system, whereas induction of metastasis is observed when the cells have lost their epithelial characteristics.

Expression of E-cadherin, alpha-, beta- and gamma-catenins were studied in 100 patients with primary breast cancer compiled of 57 invasive ductal carcinomas (IDC) and 43 invasive lobular carcinomas (ILC) by means of immunohistochemistry. Loss of E-cadherin was observed in 26 (45.6%), and alpha-, beta- and gamma-catenin expression was lacking in 22 (38.6%), 27 (47.4%) and 22 (38.6%) IDCs, respectively. The expression in ILCs was significantly lower, as compared to IDCs (p<0.001). Immunostaining of both E-cadherin and catenins was completely lacking in 27 (47.4%) IDCs and 30 (93.8%) ILCs. Go-expression of E-cadherin/beta-catenin or E-cadherin/gamma-catenin was preserved more frequently than that of E-cadherin/alpha-catenin complexes. E-cadherin/catenin complex expression showed significant positive correlation with histological differentiation (p=0.037), ER (p=0.017) and PR expression (p=0.052), and negative correlation with c-erbB-2 receptor overexpression (p=0.046). Patients with tumours showing adhesion complexes containing alpha-catenin had an increased overall survival rate compared to other patients. Expression of either E-cadherin or alpha-catenin only, without the formation of entire adhesion complexes, was not correlated with overall survival. Thus, determination of both E-cadherin and catenins is suggested to add further information to estimate the prognosis of breast cancer patients.

We previously isolated the 5' upstream sequences of the mouse P-cadherin gene, in which putative binding sites for several transcription factors were identified between nt-101 and +30. In the study reported here, the promoter activity of the postulated 5' cis-acting sequences of the P-cadherin promoter, and the activity of the proximal E-cadherin promoter were investigated in several murine keratinocyte cell lines showing different levels of P- and E-cadherin expression as well as different morphology and tumorigenic behavior. Cell-type specificity and optimal activity of P-cadherin expression in murine keratinocytes was conferred by 5' sequences located between nt -200 and +30, and the GC-rich region (nt -101 to +80) and a CCAAT box element (nt -65) had a major regulatory role. The cell-type specificity of the E-cadherin promoter, on the other hand, was mediated by a combination of positive regulatory elements, a GC-rich region (nt -58 to -24), and a CCAAT box (nt -65) and repressor elements inside the E-pal sequence. Interestingly, the maximum repressor effect of the E-pal element was observed in non-expressing undifferentiated spindle cells. In vitro binding studies indicated that the GC-rich region of the P-cadherin promoter was mainly recognized by Sp1-related nuclear factors, whereas both AP2- and Sp1-related factors were involved in the interaction of the GC-rich region of the E-cadherin promoter. Common factors (probably related to the CP1 family) seemed also to be involved in the recognition of the CCAAT-box element of both the E- and P-cadherin promoters, but additional specific factors participated in the interaction with the CCAAT box of the E-cadherin promoter. Our studies also support the hypothesis that loss or modification of some of the regulatory factors occurs during mouse skin tumor progression.