Pierre D. McCrea

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.

Telomerase modulates Wnt signalling by association with target gene chromatin

Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/β-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/β-catenin signalling by serving as a cofactor in a β-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior–posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert-/- mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/β-catenin signalling pathway.

Catenins as mediators of the cytoplasmic functions of cadherins

SUMMARY The catenins are polypeptides that bind to the conserved cytoplasmic tail of cadherins and are required for cadherin function. α-Catenin is related to vinculin and seems to be required for the interaction of cadherins with the actin cytoskeleton. β-Catenin is homologous to armadillo, a segment polarity gene in Drosophila that participates in developmental signaling. Recent findings indicate that β-catenin also participates in developmental signaling and embryonic patterning in Xenopus laevis. At least a portion of the electrophoretic band migrating at the position of γ-catenin consists of plakoglobin, a desmosomal and zonula adherens protein that has high sequence similarity to β-catenin and armadillo. The catenins may be involved in the regulation of cadherin function during tissue morphogenesis and tumorigenesis.

Non-canonical Wnt signals are modulated by the Kaiso transcriptional repressor and p120-catenin

Gastrulation movements are critical for establishing the three principal germ layers and the basic architecture of vertebrate embryos. Although the individual molecules and pathways involved are not clearly understood, non-canonical Wnt signals are known to participate in developmental processes, including planar cell polarity and directed cell rearrangements. Here we demonstrate that the dual-specificity transcriptional repressor Kaiso, first identified in association with p120-catenin, is required for Xenopus gastrulation movements. In addition, depletion of xKaiso results in increased expression of the non-canonical xWnt11, which contributes to the xKaiso knockdown phenotype as it is significantly rescued by dominant-negative Wnt11. We further demonstrate that xWnt11 is a direct gene target of xKaiso and that p120-catenin association relieves xKaiso repression in vivo. Our results indicate that p120-catenin and Kaiso are essential components of a new developmental gene regulatory pathway that controls vertebrate morphogenesis.

Inhibition of Wnt signaling by the osteoblast-specific transcription factor Osterix

The recent identification of the genes responsible for several human genetic diseases affecting bone homeostasis and the characterization of mouse models for these diseases indicated that canonical Wnt signaling plays a critical role in the control of bone mass. Here, we report that the osteoblast-specific transcription factor Osterix (Osx), which is required for osteoblast differentiation, inhibits Wnt pathway activity. First, in calvarial cells of embryonic day (E)18.5 Osx-null embryos, expression of the Wnt antagonist Dkk1 was abolished, and that of Wnt target genes c-Myc and cyclin D1 was increased. Moreover, our studies demonstrated that Osx bound to and activated the Dkk1 promoter. In addition, Osx inhibited β-catenin-induced Topflash reporter activity and β-catenin-induced secondary axis formation in Xenopus embryos. Importantly, in calvaria of E18.5 Osx-null embryos harboring the TOPGAL reporter transgene, β-galactosidase activity was increased, suggesting that Osx inhibited the Wnt pathway in osteoblasts in vivo. Our data further showed that Osx disrupted binding of Tcf to DNA, providing a likely mechanism for the inhibition by Osx of β-catenin transcriptional activity. We also showed that Osx decreased osteoblast proliferation. Indeed, E18.5 Osx-null calvaria showed greater BrdU incorporation than wild-type calvaria and that Osx overexpression in C2C12 mesenchymal cells inhibited cell growth. Because Wnt signaling has a major role in stimulating osteoblast proliferation, we speculate that Osx-mediated inhibition of osteoblast proliferation is a consequence of the Osx-mediated control of Wnt/β-catenin activity. Our results add a layer of control to Wnt/β-catenin signaling in bone.

Fascin, an actin-bundling protein associated with cell motility, is upregulated in hormone receptor negative breast cancer.

Loss of hormone receptor (HR) status in breast carcinomas is associated with increased tumour cell motility and invasiveness. In an immunohistological study of 58 primary breast cancers, oestrogen (ER) and progesterone (PR) receptor levels were inversely correlated with the expression of fascin, an actin-bundling protein associated with cell motility (P< 0.0001 and P = 0.0019, respectively). In addition, fascin was preferentially expressed in non-diploid tumours (P = 0.03). In summary, the upregulation of fascin in HR-negative breast cancers may contribute to their more aggressive behaviour.

A role for Kaiso–p120ctn complexes in cancer?

Kaiso belongs to the zinc finger and broad-complex, tramtrack and bric-a-brac/poxvirus and zinc finger (BTB/POZ) protein family that has been implicated in tumorigenesis. Kaiso was first discovered in a complex with the armadillo-domain protein p120ctn and later shown to function as a transcriptional repressor. As p120ctn seems to relieve Kaiso-mediated repression, its altered intracellular localization in some cancer cells might result in aberrant Kaiso nuclear activity. Intriguingly, Kaisos target genes include both methylated and sequence-specific recognition sites. The latter include genes that are modulated by the canonical Wnt (β-catenin–T-cell factor) signalling pathway. Further interest in Kaiso stems from findings that its cytoplasmic versus nuclear localization is modulated by complex cues from the microenvironment.

C-erbB-2/ HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines

The over-expression of c-erbB-2/ HER-2, a receptor tyrosine kinase, correlates with poor prognosis in patients with breast and ovarian cancer. In the human breast cancer cell line, MDA-MB-435, c-erbB-2 over-expression results in increased chemoinvasion and higher metastatic properties in nude mice. However, the mechanisms by which c-erbB-2 increases the malignant potential of cells remains unclear. We have determined that over-expression of c-erbB-2 in MDA-MB-435 cells, and in some additional breast cancer cell lines, is associated with graphic increases in mRNA and protein levels of the actin bundling protein fascin. Heightened fascin expression has been observed in other systems to result in greatly increased cell motility, and indeed, our work employing semi-automated time-lapse microscopy demonstrates that MDA-MB-435 cells over-expressing c-erbB-2 exhibit significantly heightened cellular dynamics and locomotion, while visualization of bundled microfilaments within fixed cells revealed enhanced formation of dendritic-like processes, microspikes and other dynamic actin based structures. To address the means by which c-erbB-2 over-expression might result in elevated fascin levels, we identified multiple perfect match TCF and NF-κB consensus sites in fascin’s promoter and first intron, which appeared consistent with the greater endogenous transcriptional activities of TCF and NF-κB in c-erbB-2 over-expressing MDA-MB-435 cells. While such transcriptional modulation may occur in the context of the intact gene/chromatin, subsequent tests using reporter constructs did not support involvement of these signaling pathways. In conclusion, highly increased fascin levels were observed in MDA-MB-435 over-expressing c-erbB-2, likely contributing to these cells’ altered actin dynamics, and increased cell motility and malignancy. Studies in progress aim to discern the means by which c-erbB-2 over-expression leads to transcriptional activation of the fascin gene.

Vertebrate development requires ARVCF and p120 catenins and their interplay with RhoA and Rac

Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context.

Lung epithelial branching program antagonizes alveolar differentiation

Significance Mammalian organs, including the lung and the kidney, often use a branched design to maximize their functional capacity and efficiency. Lung formation requires two developmental processes: branching morphogenesis to build a treelike tubular network, and alveolar differentiation to generate specialized epithelial cells for gas exchange. Although each process has been extensively studied, much less is known about whether and how the two processes are coordinated. We show that an epithelial branching morphogenesis program antagonizes alveolar differentiation in the mouse lung. Our findings may provide fresh insights to lung immaturity in preterm neonates and the increase in organ complexity during evolution. Mammalian organs, including the lung and kidney, often adopt a branched structure to achieve high efficiency and capacity of their physiological functions. Formation of a functional lung requires two developmental processes: branching morphogenesis, which builds a tree-like tubular network, and alveolar differentiation, which generates specialized epithelial cells for gas exchange. Much progress has been made to understand each of the two processes individually; however, it is not clear whether the two processes are coordinated and how they are deployed at the correct time and location. Here we show that an epithelial branching morphogenesis program antagonizes alveolar differentiation in the mouse lung. We find a negative correlation between branching morphogenesis and alveolar differentiation temporally, spatially, and evolutionarily. Gain-of-function experiments show that hyperactive small GTPase Kras expands the branching program and also suppresses molecular and cellular differentiation of alveolar cells. Loss-of-function experiments show that SRY-box containing gene 9 (Sox9) functions downstream of Fibroblast growth factor (Fgf)/Kras to promote branching and also suppresses premature initiation of alveolar differentiation. We thus propose that lung epithelial progenitors continuously balance between branching morphogenesis and alveolar differentiation, and such a balance is mediated by dual-function regulators, including Kras and Sox9. The resulting temporal delay of differentiation by the branching program may provide new insights to lung immaturity in preterm neonates and the increase in organ complexity during evolution.