W. Birchmeier Lab

As shown by conditional mutagenesis in mice, we found that beta-catenin regulates precursor and stem cells in skin, nervous system, hair and heart. Shp2 control precursors in nerves, liver, limbs, and kidney. Activating mutations of beta-catenin and Met induce cancer stem cells in head and neck and mammary gland tumors. We have also shown that Shp2/Mapk controls goblet/paneth cells of the intestine and regulates hair stem cells in the hair cycle. 

In cooperation with the Technical University Dresden and the University Clinic Düsseldorf, we have recently investigated the role of Wnt/beta-catenin and the histone methyl transferase Mll1 in cancer stem cells of salivary gland, head and neck and colon cancers. In cooperation with Nikolaus Rajewsky`s group at BIMSB, we have performed Single Cell Sequencing of our salivary gland tumors in mice. We found that the tumors originate in basal cells by EMT, then become Wnt-dependent before the end luminal-like.

We have also identified cancer stem cells, which are responsible for the most common form of human kidney cancer. In cooperation with the Charité Urology department, the company EPO on campus and the FMP Screening unit, we have identified Wnt and Notch inhibitors, which strongly reduce the growth of kidney cancer stem cells in three model systems: self-renewing sphere cultures, 3D tumor organoids and xenografts in immune-deficient mice. These latter projects are directed towards clinical trials in humans. Together with the TU München, we have studied the role of Shp2 in cancer drug resistance.

In cooperation with the FMP, the Leibniz Institute of Molecular Pharmacology, we develop improved and new small molecule inhibitors of beta-catenin/Tcf4 and Shp2.

2019

The Wnt-driven Mll1 Epigenome Regulates Salivary Gland and Head and Neck Cancer

Qionghua Zhu, Liang Fang, Julian Heuberger, Andrea Kranz, Jörg Schipper, Kathrin Scheckenbach, Ramon Oliveira Vidal, Daniele Yumi Sunaga-Franze, Marion Müller, Annika Wulf-Goldenberg, Sascha Sauer, Walter Birchmeier.

https://www.sciencedirect.com/science/article/pii/S2211124718320047

We identified a regulatory system that acts downstream of Wnt/β-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/β-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activity and genome-wide increased H3K4 tri-methylation at promoters. Null mutations of Mll1 in tumor mice and in xeno-transplanted human head and neck tumors resulted in loss of self-renewal of tumor-propagating cells and in block of tumor formation, but did not alter normal tissue homeostasis. CRISPR/Cas9 mutagenesis and pharmacological interference of Mll1 at sequences that inhibit essential protein-protein interactions or the SET enzyme active site also blocked the self-renewal of mouse and human tumor-propagating cells. Our work provides strong genetic evidence for a crucial role of Mll1 in solid tumors. Moreover, inhibitors targeting specific Mll1 interactions might offer additional directions for therapies to treat these aggressive tumors.

2018

Mutant KRAS-driven cancers depend on  PTPN11/SHP2 phosphatase

Ruess DA, Heynen GJ, Ciecielski KJ, Ai J, Berninger A, Kabacaoglu D, Görgülü K, Dantes Z, Wörmann SM, Diakopoulos KN, Karpathaki AF, Kowalska M, Kaya-Aksoy E, Song L,  Zeeuw van der Laan EA, López-Alberca MP, Nazaré M, Reichert M, Saur D, Erkan MM, Hopt UT, Sainz Jr B, Birchmeier W, Schmid RM, Lesina M, Algül H.

Nat Med. 2018 Jul;24(7):954-960. doi: 10.1038/s41591-018-0024-8

The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors¹. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways^1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro⁸. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers.

2016

Orchestrating Wnt signaling for metabolic liver zonation.

Birchmeier W.

Nat Cell Biol. 2016 Apr 27;18(5):463-5. doi: 10.1038/ncb3349.

Wnt/β-catenin signalling is an important regulator of liver development, zonation and regeneration. The cell surface complex RSPO-LGR4/5-ZNF3/RNF43 is now shown to direct Wnt/β-catenin signalling in orchestrating the division of the liver into functionally distinct metabolic zones, providing insights into the mechanisms that influence organ development and regeneration.

A Small-Molecule Antagonist of the β-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis.

Fang L, Zhu Q, Neuenschwander M, Specker E, Wulf-Goldenberg A, Weis WI, Von Kries JP, Birchmeier W.

Cancer Res. 2016 Feb 15;76(4):891-901. doi: 10.1158/0008-5472.CAN-15-1519. Epub 2015 Dec 8.

Wnt/β-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/β-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/β-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell-cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy.

New Wnt/β-catenin target genes promote experimental metastasis and migration of colorectal cancer cells through different signals.

Qi J, Yu Y, Akilli Öztürk Ö, Holland JD, Besser D, Fritzmann J, Wulf-Goldenberg A, Eckert K, Fichtner I, Birchmeier W.

Gut. 2016 Oct;65(10):1690-701. doi: 10.1136/gutjnl-2014-307900. Epub 2015 Jul 8.

OBJECTIVES:

We have previously identified a 115-gene signature that characterises the metastatic potential of human primary colon cancers. The signature included the canonical Wnt target gene BAMBI, which promoted experimental metastasis in mice. Here, we identified three new direct Wnt target genes from the signature, and studied their functions in epithelial-mesenchymal transition (EMT), cell migration and experimental metastasis.

DESIGN:

We examined experimental liver metastases following injection of selected tumour cells into spleens of NOD/SCID mice. Molecular and cellular techniques were used to identify direct transcription target genes of Wnt/β-catenin signals. Microarray analyses and experiments that interfered with cell migration through inhibitors were performed to characterise downstream signalling systems.

RESULTS:

Three new genes from the colorectal cancer (CRC) metastasis signature, BOP1, CKS2 and NFIL3, were identified as direct transcription targets of β-catenin/TCF4. Overexpression and knocking down of these genes in CRC cells promoted and inhibited, respectively, experimental metastasis in mice, EMT and cell motility in culture. Cell migration was repressed by interfering with distinct signalling systems through inhibitors of PI3K, JNK, p38 mitogen-activated protein kinase and/or mTOR. Gene expression profiling identified a series of migration-promoting genes, which were induced by BOP1, CKS2 and NFIL3, and could be repressed by inhibitors that are specific to these pathways.

CONCLUSIONS:

We identified new direct Wnt/β-catenin target genes, BOP1, CKS2 and NFIL3, which induced EMT, cell migration and experimental metastasis of CRC cells. These genes crosstalk with different downstream signalling systems, and activate migration-promoting genes. These pathways and downstream genes may serve as therapeutic targets in the treatment of CRC metastasis.

2015

Gab1 and Mapk Signaling Are Essential in the Hair Cycle and Hair Follicle Stem Cell Quiescence.

Akilli Öztürk Ö, Pakula H, Chmielowiec J, Qi J, Stein S, Lan L, Sasaki Y, Rajewsky K, Birchmeier W.

Cell Rep. 2015 Oct 20;13(3):561-72. doi: 10.1016/j.celrep.2015.09.015. Epub 2015 Oct 8.

Gab1 is a scaffold protein that acts downstream of receptor tyrosine kinases. Here, we produced conditional Gab1 mutant mice (by K14- and Krox20-cre) and show that Gab1 mediates crucial signals in the control of both the hair cycle and the self-renewal of hair follicle stem cells. Remarkably, mutant hair follicles do not enter catagen, the destructive phase of the hair cycle. Instead, hair follicle stem cells lose quiescence and become exhausted, and thus no stem cell niches are established in the bulges. Moreover, conditional sustained activation of Mapk signaling by expression of a gain-of-function Mek1(DD) allele (by Krox20-cre) rescues hair cycle deficits and restores quiescence of the stem cells. Our data thus demonstrate an essential role of Gab1 downstream of receptor tyrosine kinases and upstream of Shp2 and Mapk in the regulation of the hair cycle and the self-renewal of hair follicle stem cells.

Shp2 signaling suppresses senescence in PyMT-induced mammary gland cancer in mice.

Lan L, Holland JD, Qi J, Grosskopf S, Vogel R, Györffy B, Wulf-Goldenberg A, Birchmeier W.

EMBO J. 2015 Jun 3;34(11):1493-508. doi: 10.15252/embj.201489004. Epub 2015 Mar 3.

In this study, we have used techniques from cell biology, biochemistry, and genetics to investigate the role of the tyrosine phosphatase Shp2 in tumor cells of MMTV-PyMT mouse mammary glands. Genetic ablation or pharmacological inhibition of Shp2 induces senescence, as determined by the activation of senescence-associated β-gal (SA-β-gal), cyclin-dependent kinase inhibitor 1B (p27), p53, and histone 3 trimethylated lysine 9 (H3K9me3). Senescence induction leads to the inhibition of self-renewal of tumor cells and blockage of tumor formation and growth. A signaling cascade was identified that acts downstream of Shp2 to counter senescence: Src, focal adhesion kinase, and Map kinase inhibit senescence by activating the expression of S-phase kinase-associated protein 2 (Skp2), Aurora kinase A (Aurka), and the Notch ligand Delta-like 1 (Dll1), which block p27 and p53. Remarkably, the expression of Shp2 and of selected target genes predicts human breast cancer outcome. We conclude that therapies, which rely on senescence induction by inhibiting Shp2 or controlling its target gene products, may be useful in blocking breast cancer.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo.

Grosskopf S, Eckert C, Arkona C, Radetzki S, Böhm K, Heinemann U, Wolber G, Von Kries JP, Birchmeier W, Rademann J.

ChemMedChem. 2015 May;10(5):815-26. doi: 10.1002/cmdc.201500015. Epub 2015 Apr 15.

Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-{(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazino}benzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71±15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

2014

Shp2/MAPK signaling controls goblet/paneth cell fate decisions in the intestine.

Heuberger J, Kosel F, Qi J, Grossmann KS, Rajewsky K, Birchmeier W.

Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3472-7. doi: 10.1073/pnas.1309342111. Epub 2014 Feb 18.

In the development of the mammalian intestine, Notch and Wnt/β-catenin signals control stem cell maintenance and their differentiation into absorptive and secretory cells. Mechanisms that regulate differentiation of progenitors into the three secretory lineages, goblet, paneth, or enteroendocrine cells, are not fully understood. Using conditional mutagenesis in mice, we observed that Shp2-mediated MAPK signaling determines the choice between paneth and goblet cell fates and also affects stem cells, which express the leucine-rich repeat-containing receptor 5 (Lgr5). Ablation of the tyrosine phosphatase Shp2 in the intestinal epithelium reduced MAPK signaling and led to a reduction of goblet cells while promoting paneth cell development. Conversely, conditional mitogen-activated protein kinase kinase 1 (Mek1) activation rescued the Shp2 phenotype, promoted goblet cell and inhibited paneth cell generation. The Shp2 mutation also expanded Lgr5+ stem cell niches, which could be restricted by activated Mek1 signaling. Changes of Lgr5+ stem cell quantities were accompanied by alterations of paneth cells, indicating that Shp2/MAPK signaling might affect stem cell niches directly or via paneth cells. Remarkably, inhibition of MAPK signaling in intestinal organoids and cultured cells changed the relative abundance of Tcf4 isoforms and by this, promoted Wnt/β-catenin activity. The data thus show that Shp2-mediated MAPK signaling controls the choice between goblet and paneth cell fates by regulating Wnt/β-catenin activity.

Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination.

Sheean ME, McShane E, Cheret C, Walcher J, Müller T, Wulf-Goldenberg A, Hoelper S, Garratt AN, Krüger M, Rajewsky K, Meijer D, Birchmeier W, Lewin GR, Selbach M, Birchmeier C.

Genes Dev. 2014 Feb 1;28(3):290-303. doi: 10.1101/gad.230045.113.

Myelination depends on the synthesis of large amounts of myelin transcripts and proteins and is controlled by Nrg1/ErbB/Shp2 signaling. We developed a novel pulse labeling strategy based on stable isotope labeling with amino acids in cell culture (SILAC) to measure the dynamics of myelin protein production in mice. We found that protein synthesis is dampened in the maturing postnatal peripheral nervous system, and myelination then slows down. Remarkably, sustained activation of MAPK signaling by expression of the Mek1DD allele in mice overcomes the signals that end myelination, resulting in continuous myelin growth. MAPK activation leads to minor changes in transcript levels but massively up-regulates protein production. Pharmacological interference in vivo demonstrates that the effects of activated MAPK signaling on translation are mediated by mTOR-independent mechanisms but in part also by mTOR-dependent mechanisms. Previous work demonstrated that loss of ErbB3/Shp2 signaling impairs Schwann cell development and disrupts the myelination program. We found that activated MAPK signaling strikingly compensates for the absence of ErbB3 or Shp2 during Schwann cell development and myelination.

2013

Combined Wnt/β-Catenin, Met, and CXCL12/CXCR4 Signals Characterize Basal Breast cancer and Predict Disease Outcome.

Holland JD, Györffy B, Vogel R, Eckert K, Valenti G, Fang L, Lohneis P, Elezkurtaj S, Ziebold U, and Birchmeier W.

Cell Rep. 2013 Nov 23. pii: S2211-1247(13)00649-9.

Prognosis for patients with estrogen-receptor (ER)-negative basal breast cancer is poor, and chemotherapy is currently the best therapeutic option. We have generated a compound-mutant mouse model combining the activation of β-catenin and HGF (Wnt-Met signaling), which produced rapidly growing basal mammary gland tumors. We identified the chemokine system CXCL12/CXCR4 as a crucial driver of Wnt-Met tumors, given that compound-mutant mice also deficient in the CXCR4 gene were tumor resistant. Wnt-Met activation rapidly expanded a population of cancer-propagating cells, in which the two signaling systems control different functions, self-renewal and differentiation. Molecular therapy targeting Wnt, Met, and CXCR4 in mice significantly delayed tumor development. The expression of a Wnt-Met 322 gene signature was found to be predictive of poor survival of human patients with ER-negative breast cancers. Thus, targeting CXCR4 and its upstream activators, Wnt and Met, might provide an efficient strategy for breast cancer treatment.

Wnt/Rspondin/β-catenin signals control axonal sorting and lineage progression in Schwann cell development.

Grigoryan T, Stein S, Qi J, Wende H, Garratt AN, Nave KA, Birchmeier C, and Birchmeier W.

Proc Natl Acad Sci U S A. 2013 Nov 5;110(45):18174-9. Epub 2013 Oct 22.

During late Schwann cell development, immature Schwann cells segregate large axons from bundles, a process called "axonal radial sorting." Here we demonstrate that canonical Wnt signals play a critical role in radial sorting and assign a role to Wnt and Rspondin ligands in this process. Mice carrying β-catenin loss-of-function mutations show a delay in axonal sorting; conversely, gain-of-function mutations result in accelerated sorting. Sorting deficits are accompanied by abnormal process extension, differentiation, and aberrant cell cycle exit of the Schwann cells. Using primary cultured Schwann cells, we analyze the upstream effectors, Wnt and Rspondin ligands that initiate signaling, and downstream genetic programs that mediate the Wnt response. Our analysis contributes to a better understanding of the mechanisms of Schwann cell development and fate decisions.

Wnt/β-catenin signalling induces MLL to create epigenetic changes in salivary gland tumours.

Wend P, Fang L, Zhu Q, Schipper JH, Loddenkemper C, Kosel F, Brinkmann V, Eckert K, Hindersin S, Holland JD, Lehr S, Kahn M, Ziebold U, and Birchmeier W.

EMBO J. 2013 Jul 17;32(14):1977-89. Epub 2013 Jun 4.

We show that activation of Wnt/β-catenin and attenuation of Bmp signals, by combined gain- and loss-of-function mutations of β-catenin and Bmpr1a, respectively, results in rapidly growing, aggressive squamous cell carcinomas (SCC) in the salivary glands of mice. Tumours contain transplantable and hyperproliferative tumour propagating cells, which can be enriched by fluorescence activated cell sorting (FACS). Single mutations stimulate stem cells, but tumours are not formed. We show that β-catenin, CBP and Mll promote self-renewal and H3K4 tri-methylation in tumour propagating cells. Blocking β-catenin-CBP interaction with the small molecule ICG-001 and small-interfering RNAs against β-catenin, CBP or Mll abrogate hyperproliferation and H3K4 tri-methylation, and induce differentiation of cultured tumour propagating cells into acini-like structures. ICG-001 decreases H3K4me3 at promoters of stem cell-associated genes in vitro and reduces tumour growth in vivo. Remarkably, high Wnt/β-catenin and low Bmp signalling also characterize human salivary gland SCC and head and neck SCC in general. Our work defines mechanisms by which β-catenin signals remodel chromatin and control induction and maintenance of tumour propagating cells. Further, it supports new strategies for the therapy of solid tumours.

2012

Neocortical dendritic complexity is controlled during development by NOMA-GAP-dependent inhibition of Cdc42 and activation of cofilin.

Rosário M, Schuster S, Jüttner R, Parthasarathy S, Tarabykin V, and Birchmeier W.

Genes Dev. 2012 Aug 1;26(15):1743-57. Epub 2012 Jul 18.

Neocortical neurons have highly branched dendritic trees that are essential for their function. Indeed, defects in dendritic arborization are associated with human neurodevelopmental disorders. The molecular mechanisms regulating dendritic arbor complexity, however, are still poorly understood. Here, we uncover the molecular basis for the regulation of dendritic branching during cortical development. We show that during development, dendritic branching requires post-mitotic suppression of the RhoGTPase Cdc42. By generating genetically modified mice, we demonstrate that this is catalyzed in vivo by the novel Cdc42-GAP NOMA-GAP. Loss of NOMA-GAP leads to decreased neocortical volume, associated specifically with profound oversimplification of cortical dendritic arborization and hyperactivation of Cdc42. Remarkably, dendritic complexity and cortical thickness can be partially restored by genetic reduction of post-mitotic Cdc42 levels. Furthermore, we identify the actin regulator cofilin as a key regulator of dendritic complexity in vivo. Cofilin activation during late cortical development depends on NOMA-GAP expression and subsequent inhibition of Cdc42. Strikingly, in utero expression of active cofilin is sufficient to restore postnatal dendritic complexity in NOMA-GAP-deficient animals. Our findings define a novel cell-intrinsic mechanism to regulate dendritic branching and thus neuronal complexity in the cerebral cortex.

Wnt/β-catenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells.

Klaus A, Müller M, Schulz H, Saga Y, Martin JF, and Birchmeier W.

Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10921-6. Epub 2012 Jun 18.

Progenitor cells of the first and second heart fields depend on cardiac-specific transcription factors for their differentiation. Using conditional mutagenesis of mouse embryos, we define the hierarchy of signaling events that controls the expression of cardiac-specific transcription factors during differentiation of cardiac progenitors at embryonic day 9.0. Wnt/β-catenin and Bmp act downstream of Notch/RBPJ at this developmental stage. Mutation of Axin2, the negative regulator of canonical Wnt signaling, enhances Wnt and Bmp4 signals and suffices to rescue the arrest of cardiac differentiation caused by loss of RBPJ. Using FACS enrichment of cardiac progenitors in RBPJ and RBPJ/Axin2 mutants, embryo cultures in the presence of the Bmp inhibitor Noggin, and by crossing a Bmp4 mutation into the RBPJ/Axin2 mutant background, we show that Wnt and Bmp4 signaling activate specific and nonoverlapping cardiac-specific genes in the cardiac progenitors: Nkx2-5, Isl1 and Baf60c are controlled by Wnt/β-catenin, and Gata4, SRF, and Mef2c are controlled by Bmp signaling. Our study contributes to the understanding of the regulatory hierarchies of cardiac progenitor differentiation and outflow tract development and has implications for understanding and modeling heart development.

2011

The tyrosine phosphatase Shp2 acts downstream of GDNF/Ret in branching morphogenesis of the developing mouse kidney.

Willecke R, Heuberger J, Grossmann K, Michos O, Schmidt-Ott K, Walentin K, Costantini F, and Birchmeier W.

Dev Biol. 2011 Dec 15;360(2):310-7. Epub 2011 Oct 8.

The tyrosine phosphatase Shp2 acts downstream of various growth factors, hormones or cytokine receptors. Mutations of the Shp2 gene are associated with several human diseases. Here we have ablated Shp2 in the developing kidneys of mice, using the ureteric bud epithelium-specific Hoxb7/Cre. Mutant mice produced a phenotype that is similar to mutations of the genes of the GDNF/Ret receptor system, that is: strongly reduced ureteric bud branching and downregulation of the Ret target genes Etv4 and Etv5. Shp2 mutant embryonic kidneys also displayed reduced cell proliferation at the branch tips and branching defects, which could not be overcome by GDNF in organ culture. We also examined compound mutants of Shp2 and Sprouty1, which is an inhibitor of receptor tyrosine kinase signaling in the kidney. Sprouty1 single mutants produce supernumerary ureteric buds, which branch excessively. Sprouty1 mutants rescued branching deficits in Ret(-/-) and GDNF(-/-) kidneys. Sprouty1; Shp2 double mutants showed no rescue of kidney branching. Our data thus indicate an intricate interplay of Shp2 and Sprouty1 in signaling downstream of receptor tyrosine kinases during kidney development. Apparently, Shp2 mediates not only GDNF/Ret but also signaling by other receptor tyrosine kinases in branching morphogenesis of the embryonic kidney.

BCL9-2 promotes early stages of intestinal tumor progression.

Brembeck FH, Wiese M, Zatula N, Grigoryan T, Dai Y, Fritzmann J, and Birchmeier W.

Gastroenterology. 2011 Oct;141(4):1359-70, 1370.e1-3. Epub 2011 Jun 23.

BACKGROUND & AIMS: The roles of the 2 BCL9 and 2 Pygopus genes in Wnt to β-catenin signaling are not clear in vertebrates. We examined their expression and function in normal and tumor intestinal epithelia in mice and humans. METHODS: Specific antibodies were generated to characterize the BCL9 and Pygopus proteins in normal intestine and in colon tumors. Targets of BCL9 and Pygopus in colon cancer cells were analyzed using small interfering RNA analysis. Transgenic mice were created that overexpressed BCL9-2 in intestine; these were crossed with APCMin/+ mice to create BCL9-2;APCMin/+ mice. RESULTS: BCL9 and Pygopus2 were expressed in all normal intestinal and colon cancer cells. BCL9-2 was detectable only in the villi, not in the crypts of normal intestine. BCL9-2 was up-regulated in adenomas and in almost all colon tumors, with a concomitant increase of Pygopus2, whereas levels of BCL9 were similar between normal and cancer cells. Transgenic overexpression of BCL9-2 in the intestine of BCL9-2; APCMin/+ mice increased formation of adenomas that progressed to invasive tumors, resulting in reduced survival time. Using small interfering RNA analysis, we found that BCL9s and Pygopus are not targets of Wnt in colon cancer cells, but Wnt signaling correlated with levels of BCL9-2. BCL9-2 regulated expression of β-catenin-dependent and -independent target genes that have been associated with early stages of intestinal tumorigenesis. CONCLUSIONS: BCL9-2 promotes early phases of intestinal tumor progression in humans and in transgenic mice. BCL9-2 increases the expression of a subset of canonical Wnt target genes but also regulates genes that are required for early stages of tumor progression.

E-cadherin is crucial for embryonic stem cell pluripotency and can replace OCT4 during somatic cell reprogramming.

Redmer T, Diecke S, Grigoryan T, Quiroga-Negreira A, Birchmeier W, and Besser D.

EMBO Rep. 2011 Jul 1;12(7):720-6. doi: 10.1038/embor.2011.88.

We report new functions of the cell-adhesion molecule E-cadherin in murine pluripotent cells. E-cadherin is highly expressed in mouse embryonic stem cells, and interference with E-cadherin causes differentiation. During cellular reprogramming of mouse fibroblasts by OCT4, SOX2, KLF4 and c-MYC, fully reprogrammed cells were exclusively observed in the E-cadherin-positive cell population and could not be obtained in the absence of E-cadherin. Moreover, reprogrammed cells could be established by viral E-cadherin in the absence of exogenous OCT4. Thus, reprogramming requires spatial cues that cross-talk with essential transcription factors. The cell-adhesion molecule E-cadherin has important functions in pluripotency and reprogramming.

2009

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development.

Grossmann KS, Wende H, Paul FE, Cheret C, Garratt AN, Zurborg S, Feinberg K, Besser D, Schulz H, Peles E, Selbach M, Birchmeier W, and Birchmeier C.

Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16704-9. Epub 2009 Sep 11.

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.

A colorectal cancer expression profile that includes transforming growth factor ß inhibitor BAMBI predicts metastatic potential.

Fritzmann J, Morkel M, Besser D, Budczies J, Kosel F, Brembeck FH, Stein U, Fichtner I, Schlag PM, and Birchmeier W.

Gastroenterology. 2009 Jul;137(1):165-75. Epub 2009 Mar 26.

Background & Aims: Much is known about the genes and mutations that cause colorectal cancer (CRC), yet only a few have been associated with CRC metastasis. We performed expression profiling experiments to identify genetic markers of risk and to elucidate the molecular mechanisms of CRC metastasis. Methods: We compared gene expression patterns between metastatic and non-metastatic, stage-matched human colorectal carcinomas by microarray analysis. Correlations between BAMBI and metastasis-free survival were examined by quantitative real-time PCR using an independent set of human colon carcinomas. Human colon cancer cell lines were analyzed for BAMBI regulation, cell motility and experimental metastasis. Results: We established a signature of 115 genes that differentiated metastatic from non-metastatic primary tumors. Among these, the transforming growth factor (TGF) ßinhibitor BAMBI was highly expressed in about half of metastatic primary tumors and metastases but not in non-metastatic tumors. BAMBI is a target of canonical Wnt signaling that involves the ß-catenin co-activator BCL9-2. We observed an inverse correlation between level of BAMBI expression and metastasis-free survival time of patients. BAMBI inhibits TGFß signaling and increases migration in colon cancer cells. In mice, overexpression of BAMBI caused colon cancer cells to form tumors that metastasized more frequently to liver and lymph nodes than control cancer cells. Conclusion: BAMBI regulates colorectal cancer metastasis by connecting the Wnt/ß -catenin and TGFß signaling pathways. The metastatic expression signature we describe, along with BAMBI levels, can be used in prognosis. Developmental signaling pathways appear to act in hierarchies and cooperate in tumor cell migration, invasion and metastasis.

2008

Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking.

Hellmuth K, Grosskopf S, Lum CT, Würtele M, Röder N, von Kries JP, Rosario M, Rademann J, and Birchmeier W.

Proc Natl Acad Sci U S A. 2008 May 14. [Epub ahead of print]

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent  growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.

2007

Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis.

Klaus A, Saga Y, Taketo MM, Tzahor E, and Birchmeier W.

Proc Natl Acad Sci U S A. 2007 Nov 20;104(47):18531-6.

Heart formation requires the coordinated recruitment of multiple cardiac progenitor cell populations derived from both the first and second heart fields. In this study, we have ablated the Bmp receptor 1a and the Wnt effector beta-catenin in the developing heart of mice by using MesP1-cre, which acts in early mesoderm progenitors that contribute to both first and second heart fields. Remarkably, the entire cardiac crescent and later the primitive ventricle were absent in MesP1-cre; BmpR1a(lox/lox) mutants. Although myocardial progenitor markers such as Nkx2-5 and Isl1 and the differentiation marker MLC2a were detected in the small, remaining cardiac field in these mutants, the first heart field markers, eHand and Tbx-5, were not expressed. We conclude from these results that Bmp receptor signaling is crucial for the specification of the first heart field. In MesP1-cre; beta-catenin(lox/lox) mutants, cardiac crescent formation, as well as first heart field markers, were not affected, although cardiac looping and right ventricle formation were blocked. Expression of Isl1 and Bmp4 in second heart field progenitors was strongly reduced. In contrast, in a gain-of-function mutation of beta-catenin using MesP1-cre, we revealed an expansion of Isl1 and Bmp4 expressing cells, although the heart tube was not formed. We conclude from these results that Wnt/beta-catenin signaling regulates second heart-field development, and that a precise amount and/or timing of Wnt/beta-catenin signaling is required for proper heart tube formation and cardiac looping.

Distinct requirements for Gab1 in Met and EGF receptor signaling in vivo.

Schaeper U, Vogel R, Chmielowiec J, Huelsken J, Rosário M, and Birchmeier W.

Proc Natl Acad Sci U S A. 2007 Sep 25;104(39):15376-81.

Gab1 is a multiadaptor protein that has been shown to be required for multiple processes in embryonic development and oncogenic transformation. Gab1 functions by amplifying signal transduction downstream of various receptor tyrosine kinases through recruitment of multiple signaling effectors, including phosphatidylinositol 3-kinase and Shp2. Until now, the functional significance of individual interactions in vivo was not known. Here we have generated knockin mice that carry point mutations in either the P13K or Shp2 binding sites of Gab1. We show that different effector interactions with Gab1 play distinct biological roles downstream of Gab1 during the development of different organs. Recruitment of phosphatidylinositol 3-kinase by Gab1 is essential for EGF receptor-mediated embryonic eyelid closure and keratinocyte migration, and the Gab1–Shp2 interaction is crucial for Met receptor-directed placental development and muscle progenitor cell migration to the limbs. Furthermore, we investigate the dual association of Gab1 with the Met receptor. By analyzing knockin mice with mutations in the Grb2 or Met binding site of Gab1, we show that the requirements for Gab1 recruitment to Met varies in different biological contexts. Either the direct or the indirect interaction of Gab1 with Met is sufficient for Met-dependent muscle precursor cell migration, whereas both modes of interaction are required and neither is sufficient for placenta development, liver growth, and palatal shelf closure. These data demonstrate that Gab1 induces different biological responses through the recruitment of distinct effectors and that different modes of recruitment for Gab1 are required in different organs.

The neurite outgrowth multiadaptor RhoGAP, NOMA-GAP, regulates neurite extension through SHP2 and Cdc42.

Rosário M, Franke R, Bednarski C, and Birchmeier W.

J Cell Biol 178(3): 503-516. (2007-07-30)

Neuronal differentiation involves the formation and extension of neuronal processes. We have identified a novel regulator of neurite formation and extension, the neurite outgrowth multiadaptor, NOMA-GAP, which belongs to a new family of multiadaptor proteins with RhoGAP activity. We show that NOMA-GAP is essential for NGF-stimulated neuronal differentiation and for the regulation of the ERK5 MAP kinase and the Cdc42 signaling pathways downstream of NGF. NOMA-GAP binds directly to the NGF receptor, TrkA, and becomes tyrosine phosphorylated upon receptor activation, thus enabling recruitment and activation of the  tyrosine phosphatase SHP2. Recruitment of SHP2 is required for the stimulation of neuronal process extension and for sustained activation of ERK5 downstream of NOMA-GAP. In addition, we show that NOMA-GAP promotes neurite outgrowth by tempering activation of the Cdc42/PAK signaling pathway in response to NGF. NOMA-GAP, through its dual function as a multiadaptor and RhoGAP protein, thus plays an essential role downstream of NGF in promoting neurite outgrowth and extension.

c-Met is essential for wound healing in the skin.

Chmielowiec J, Borowiak M, Morkel M, Stradal T, Munz B, Werner S, Wehland J, Birchmeier C, and Birchmeier W.

J Cell Biol. 177: 151-162 (2007-04-09)

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.

Bmp and Wnt/beta-catenin signals control expression of the transcription factor Olig3 and the specification of spinal cord neurons.

Zechner D, Muller T, Wende H, Walther I, Taketo MM, Crenshaw EB 3rd, Treier M, Birchmeier W, and Birchmeier C.

Dev Biol. 2007 Mar 1;303(1):181-90.

In the developing spinal cord, signals of the roof plate pattern the dorsal progenitor domain and control the specification of three neuron types, dorsal interneurons dI1, dI2, and dI3. Bmp and Wnt/beta-catenin signals as well as transcription factors like Olig3 or Ngn1/2 are essential in this process. We have studied the epistatic relationship between Bmp and Wnt/beta-catenin signals and the transcription factor Olig3 in dorsal spinal cord patterning. Using beta-catenin gain-of-function and compound beta-catenin gain-of-function/Olig3 loss-of-function mutations in mice, we could show that Wnt/beta-catenin signals act upstream of Olig3 in the specification of dI2 and dI3 neurons. The analysis of such compound mutant mice allowed us to distinguish between the two functions of Wnt/beta-catenin signaling in proliferation and patterning of dorsal progenitors. Using electroporation of chick spinal cords, we further demonstrate that Bmp signals act upstream of Wnt/beta-catenin in the regulation of Olig3 and that Wnt/beta-catenin signals play an instructive role in controlling Olig3 expression. We conclude that Wnt/beta-catenin and BMP signals coordinately control the specification of dorsal neurons in the spinal cord.

2006

Diversin regulates heart formation and gastrulation movements in development.

Moeller H, Jenny A, Schaeffer HJ, Schwarz-Romond T, Mlodzik , Hammerschmidt M, and Birchmeier W.

Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15900-5.

Canonical and non-canonical Wnt signaling regulate crucial events in the development of vertebrates and invertebrates. In this work we show that vertebrate Diversin, a potential orthologue of Drosophila Diego, controls fusion of heart precursors and gastrulation movements in zebrafish embryogenesis. These events are regulated by non-canonical Wnt signaling, which is independent of ß-catenin. We found that Diversin directly interacts with Dishevelled, and that this interaction is necessary and sufficient to mediate signals of the non-canonical Wnt pathway to downstream effectors like Rho family GTPases and Jun N-terminal kinase. The ankyrin repeats of Diversin are required for the interaction with Dishevelled, for the activation of non-canonical Wnt signaling and for the biological responses. The mutation K446M in the DEP domain of vertebrate Dishevelled, which mimics a classical Drosophila loss of function mutation, prevents functional interaction with Diversin's ankyrin repeats. Diversin also affects planar cell polarity in Drosophila, which is controlled by the non-canonical Wnt signaling pathway. Our data thus demonstrate that Diversin and Dishevelled function together in a mutually dependent fashion in zebrafish gastrulation and organ formation.

2004

The neuronal scaffold protein Shank3 mediates signaling and biological function of the receptor tyrosine kinase Ret in epithelial cells.

Schuetz G, Rosário M, Grimm J, Boeckers TM, Gundelfinger ED, and Birchmeier W.

J Cell Biol 167: 945-952. (2004-12-06)

Shank proteins, initially also described as ProSAP proteins, are scaffolding adaptors that have been previously shown to integrate neurotransmitter receptors into the cortical cytoskeleton at postsynaptic densities. We show here that Shank proteins are also crucial in receptor tyrosine kinase signaling. The PDZ domain–containing Shank3 protein was found to represent a novel interaction partner of the receptor tyrosine kinase Ret, which binds specifically to a PDZ-binding motif present in the Ret9 but not in the Ret51 isoform. Furthermore, we show that Ret9 but not Ret51 induces epithelial cells to form branched tubular structures in three-dimensional cultures in a Shank3-dependent manner. Ret9 but not Ret51 has been previously shown to be required for kidney development. Shank3 protein mediates sustained Erk–MAPK and PI3K signaling, which is crucial for tubule formation, through recruitment of the adaptor protein Grb2. These results demonstrate that the Shank3 adaptor protein can mediate cellular signaling, and provide a molecular mechanism for the biological divergence between the Ret9 and Ret51 isoform.

Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, and Birchmeier W.

J Cell Biol 167: 149-160. (2004-10-11)

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.

Essential role of BCL9-2 in the switch between beta-catenin's adhesive and transcriptional functions.

Brembeck FH, Schwarz-Romond T, Bakkers J, Wilhelm S, Hammerschmidt M, and Birchmeier W.

Genes Dev 18: 2225-2230. (2004-09-15)

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.

2003

Beta-Catenin regulates Cripto- and Wnt3-dependent gene expression programs in mouse axis and mesoderm formation.

Morkel M, Huelsken J, Wakamiya M, Ding J, Van De Wetering M, Clevers H, Taketo MM, Behringer RR, Shen MM, and Birchmeier W.

Development. 2003 Dec;130(25):6283-6294.

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.

Beta-Catenin mediated signalling is essential for AER induction and dorsal-ventral patterning of limbs.

Natalia Sochnikova, Dietmar Zechner, Joerg Hülsken1, Makoto M. Taketo, E. Bryan Crenshaw III and Walter Birchmeier

Genes Dev 17: 1963-1968. (2003-01-01)

Distinct signalling centres direct the formation and outgrowth of vertebrate limbs. The apical ectodermal ridge (AER) controls proximal-distal outgrowth and patterning of limbs, and FGFs are key molecules that regulate these events. Dorsal-ventral patterning is controlled by ectodermally derived signals such as Wnt-7a and BMPs. We found that beta-catenin, an essential downstream effector of the canonical Wnt signalling pathway, is a key regulator of both AER induction and dorsal-ventral patterning of the limbs. Conditional inactivation of beta-catenin in the limb ectoderm of mice using Brn4Cre results in severe limb abnormalities. No AER is formed, and expression of signalling molecules like Bmp2, Bmp4 and Fgf8 is blocked. Conversely, dominant-active beta-catenin induces ectopic expression of these genes. These findings suggest that ectodermal beta-catenin acts genetically upstream of BMPs to initiate AER formation and to activate Fgf8 expression. Moreover, ablation of beta-catenin results in a loss of En-1 expression in the ventral ectoderm, and leads to a dorsalisation of distal limb structures. Taken together, our data demonstrate that beta-catenin-mediated signalling in the ectoderm of limbs is essential for both, AER induction and dorsal-ventral patterning.

2002

The ankyrin repeat protein Diversin recruits Casein kinase Ie to the ß-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.

Thomas Schwarz-Romond, Christian Asbrand, Jeroen Bakkers, Michael Kühl, Hans-Joerg Schaeffer, Jörg Huelsken, Jürgen Behrens, Matthias Hammerschmidt, and Walter Birchmeier

Genes Dev 16: 2073-2084. (2002-01-01)

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 b-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway employs 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 Ie (CKIe) and Axin/Conductin. Diversin recruits CKIe to the b-catenin degradation complex that consists of Axin/Conductin and GSK3b and allows efficient phosphorylation of b-catenin, thereby inhibiting b-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish demonstrates that Diversin is crucial for axis formation, which depends on b-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, that functions only in the pathway that controls planar cell polarity. Our data demonstrate 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 b-catenin pathway and stimulates signaling via JNK (Schwarz-Romond et al., 2002)

Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex.

Yasuyuki Fujita, Gerd Krause, Martin Scheffner, Dietmar Zechner, Hugo E. Molina Leddy, Jürgen Behrens, Thomas Sommer, and Walter Birchmeier

Nature Cell Biology 4: 222-231. (2002-01-01)

In epithelial cells, tyrosine kinases induce 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 (Fujita et al., 2002)

2001

beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin.

Jörg Hülsken, Regina Vogel, Bettina Erdmann, George Cotsarelis, and Walter Birchmeier

Cell 105: 533-545. (2001-05-18)

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.

Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation.

Jan Grimm, Martin Sachs, Stefan Britsch, S. Di Cesare, Thomas Schwarz-Romond, Kari Alitalo, and Walter Birchmeier

J Cell Biol 154: 345-354. (2001-01-01)

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. 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 (Grimm et al., 2001).

2000

Requirement for beta-catenin in anterior-posterior axis formation in mice.

Jörg Hülsken, Regina Vogel, Volker Brinkmann, Bettina Erdmann, Carmen Birchmeier, and Walter Birchmeier

J Cell Biol 148: 567-578. (2000-02-07)

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. 

Coupling of Gab1 to c-Met and downstream effectors mediate biological responses.

Martin Sachs, Henning Brohmann, Dietmar Zechner, Müller Thomas, Joerg Huelsken, Ingrid Walther. Ingrid Walther, Ute Schaeper. Carmen Birchmeier, and Walter Birchmeier

J Cell Biol 149: 1419-1432. (2000-01-01)

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 non-receptor tyrosine kinases (Schaeper et al., 2000). We performed a functional analysis of Gab1 by targeted mutagenesis in the mouse, and compared 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 embryonic day 13.5 and 16.5, 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 Gab1, c-Met and SF/HGF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-: c-Met+/- compound mutants. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin and IGF receptors, which require IRS1 and IRS2 as specific docking proteins (Sachs et al., 2000).