A semaphorin-plexin-Rasal1 signaling pathway inhibits gastrin expression and protects against peptic ulcers.

Peptic ulcer disease is a frequent clinical problem with potentially serious complications such as bleeding or perforation. A decisive factor in the pathogenesis of peptic ulcers is gastric acid, the secretion of which is controlled by the hormone gastrin released from gastric G cells. However, the molecular mechanisms regulating gastrin plasma concentrations are poorly understood. Here, we identified a semaphorin-plexin signaling pathway that operates in gastric G cells to inhibit gastrin expression on a transcriptional level, thereby limiting food-stimulated gastrin release and gastric acid secretion. Using a systematic siRNA screening approach combined with biochemical, cell biology, and in vivo mouse experiments, we found that the RasGAP protein Rasal1 is a central mediator of plexin signal transduction, which suppresses gastrin expression through inactivation of the small GTPase R-Ras. Moreover, we show that Rasal1 is pathophysiologically relevant for the pathogenesis of peptic ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs), a main risk factor of peptic ulcers in humans. Last, we show that application of recombinant semaphorin 4D alleviates peptic ulcer disease in mice in vivo, demonstrating that this signaling pathway can be harnessed pharmacologically. This study unravels a mode of G cell regulation that is functionally important in gastric homeostasis and disease.

Characterization of a L136P mutation in Formin-like 2 (FMNL2) from a patient with chronic inflammatory bowel disease.

Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.

Mechanochemical control of epidermal stem cell divisions by B-plexins.

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Postmitotic expansion of cell nuclei requires nuclear actin filament bundling by α-actinin 4.

The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, and membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle α-actinin 4 (ACTN4) as a critical regulator to facilitate F-actin reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filament structures, and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin-binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei, we find that ACTN4 is necessary for postmitotic nuclear actin reorganization and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size and chromatin organization during mitotic cell division.

Cell type-selective pathways and clinical associations of lysophosphatidic acid biosynthesis and signaling in the ovarian cancer microenvironment.

The peritoneal fluid of ovarian carcinoma patients promotes cancer cell invasion and metastatic spread with lysophosphatidic acid (LPA) as a potentially crucial mediator. However, the origin of LPA in ascites and the clinical relevance of individual LPA species have not been addressed. Here, we show that the levels of multiple acyl-LPA species are strongly elevated in ascites versus plasma and are associated with short relapse-free survival. Data derived from transcriptome and secretome analyses of primary ascite-derived cells indicate that (a) the major route of LPA synthesis is the consecutive action of a secretory phospholipase A2 (PLA2 ) and autotaxin, (b) that the components of this pathway are coordinately upregulated in ascites, and (c) that CD163+CD206+ tumor-associated macrophages play an essential role as main producers of PLA2 G7 and autotaxin. The latter conclusion is consistent with mass spectrometry-based metabolomic analyses of conditioned medium from ascites cells, which showed that tumor-associated macrophages, but not tumor cells, are able to produce 20:4 acyl-LPA in lipid-free medium. Furthermore, our transcriptomic data revealed that LPA receptor (LPAR) genes are expressed in a clearly cell type-selective manner: While tumor cells express predominantly LPAR1-3, macrophages and T cells also express LPAR5 and LPAR6 at high levels, pointing to cell type-selective LPA signaling pathways. RNA profiling identified cytokines linked to cell motility and migration as the most conspicuous class of LPA-induced genes in macrophages, suggesting that LPA exerts protumorigenic properties at least in part via the tumor secretome.

Mutant p53 promotes tumor progression and metastasis by the endoplasmic reticulum UDPase ENTPD5.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5'-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.

SCAI promotes DNA double-strand break repair in distinct chromosomal contexts.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.

Functional interaction of SCAI with the SWI/SNF complex for transcription and tumor cell invasion.

We have recently characterized SCAI (Suppressor of Cancer Cell Invasion), a transcriptional modulator regulating cancer cell motility through suppression of MAL/SRF dependent gene transcription. We show here that SCAI is expressed in a wide range of normal human tissues and its expression is diminished in a large array of primary human breast cancer samples indicating that SCAI expression might be linked to the etiology of human cancer. To establish a functional link between SCAI and tumorigenesis we performed affinity columns to identify SCAI-interacting proteins. Our data show that SCAI interacts with the tumor suppressing SWI/SNF chromatin remodeling complex to promote changes in gene expression and the invasive capacities of human tumor cells. Moreover our data implicate a functional hierarchy between SCAI and BRM, since SCAI function is abrogated in the absence of BRM expression.

Regulation of myocardin-related transcriptional coactivators through cofactor interactions in differentiation and cancer.

Transcriptional signaling networks are orchestrated and fine-tuned through multiple interactions of transcription factors with subsets of cofactors thereby assembling multiprotein complexes to negatively or positively balance transcriptional output. These mechanisms account for the large diversity of target genes but also for time and tissue specific gene regulations through single transcription factors such as SRF. One family of SRF coactivators that has attracted much attention is represented by the myocardin-related transcription factors (MRTFs). MRTFs themselves are controlled through interactions with a growing number of cofactors and transcriptional regulators. We recently identified SCAI (suppressor of cancer cell invasion), which can associate with MAL (MRTF-A) to modulate invasive cancer cell migration through regulation of beta1-integrin expression and function. However, SCAI is likely to have additional functions depending on the tissue environment and signaling program. Interestingly, SCAI not only inhibits MRTF-A but can also regulate the activities of other MRTFs such as myocardin, or the oncogenic OTT-MAL fusion protein. Thus, SCAI may act in very different conditions such as during cancer progression, development or cell differentiation.

SCAI acts as a suppressor of cancer cell invasion through the transcriptional control of beta1-integrin.

Gene expression reprogramming governs cellular processes such as proliferation, differentiation and cell migration through the complex and tightly regulated control of transcriptional cofactors that exist in multiprotein complexes. Here we describe SCAI (suppressor of cancer cell invasion), a novel and highly conserved protein that regulates invasive cell migration through three-dimensional matrices. SCAI acts on the RhoA-Dia1 signal transduction pathway and localizes in the nucleus, where it binds and inhibits the myocardin-related transcription factor MAL by forming a ternary complex with serum response factor (SRF). Genome-wide expression analysis surprisingly reveals that one of the strongest upregulated genes after suppression of SCAI is beta1-integrin. Decreased levels of SCAI are tightly correlated with increased invasive cell migration, and SCAI is downregulated in several human tumours. Functional analysis of the beta1-integrin gene strongly argues that SCAI is a novel transcriptional cofactor that controls gene expression downstream of Dia1 to dictate changes in cell invasive behaviour.

Tyrosine phosphatase SHP-2 is a regulator of p27(Kip1) tyrosine phosphorylation.

Tyrosine phosphorylation of the cell cycle regulator p27(Kip1) plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27(Kip1) and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27(Kip1) and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27(Kip1). Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27(Kip1) in vitro. Most importantly, we could illustrate that SHP-2 modulates p27(Kip1) stability and contributes to p27(Kip1)-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27(Kip1) tyrosine phosphorylation.

Identification of Neurochondrin as a new interaction partner of the FH3 domain of the Diaphanous-related formin Dia1.

Mammalian Diaphanous (Dia)-related formins initiate the assembly of filamentous actin downstream of Rho GTPases to regulate cellular processes such as cytokinesis, cell polarity, cell motility and adhesion. In this work, we show that Neurochondrin (NC) is a novel Dia1 interacting protein. NC specifically binds to the formin homology 3 (FH3), but not to the FH1 or FH2 domain of Dia1. Both proteins show a partial co-localization in dissociated primary rat hippocampal neurons. Ectopic expression of both proteins induced neurite outgrowth in Neuro2A cells. Using a series of deletion mutants of NC we could show that the first 100 amino acids were responsible for its effect on neurite outgrowth, whereas the C-terminal part of NC had no neurite outgrowth promoting activity. Moreover, co-expression of the C terminus of NC with Dia1DeltaDAD resulted in a dramatic reduction of Dia1-induced neurite outgrowth. On the basis of actin fractionation assays, SRF-activity assays as well as microtubule stabilization assays, we could demonstrate that the C terminus of NC does not influence the actin polymerizing activity of Dia1, indicating a more specific function of NC in the modulation of Dia1 activity.

Get to grips: steering local actin dynamics with IQGAPs.

IQGAPs are actin-binding proteins that scaffold numerous interaction partners, transmitting extracellular signals that influence mitogenic, morphological and migratory cell behaviour. However, the precise mechanisms by which IQGAP proteins influence actin dynamics and actin filament structures have been elusive. Now that IQGAP1 has emerged as a potential key regulator of actin-cytoskeletal dynamics by recruiting both the actin related protein (Arp)2/3 complex and/or formin-dependent actin polymerizing machineries, we propose that IQGAP1 might coordinate the function of mechanistically different actin nucleators for cooperative localized actin filament production in various cellular processes.

Dia1 and IQGAP1 interact in cell migration and phagocytic cup formation.

The Diaphanous-related formin Dia1 nucleates actin polymerization, thereby regulating cell shape and motility. Mechanisms that control the cellular location of Dia1 to spatially define actin polymerization are largely unknown. In this study, we identify the cytoskeletal scaffold protein IQGAP1 as a Dia1-binding protein that is necessary for its subcellular location. IQGAP1 interacts with Dia1 through a region within the Diaphanous inhibitory domain after the RhoA-mediated release of Dia1 autoinhibition. Both proteins colocalize at the front of migrating cells but also at the actin-rich phagocytic cup in macrophages. We show that IQGAP1 interaction with Dia1 is required for phagocytosis and phagocytic cup formation. Thus, we identify IQGAP1 as a novel component involved in the regulation of phagocytosis by mediating the localization of the actin filament nucleator Dia1.

Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion.

The RhoA-effector Dia1 controls actin-dependent processes such as cytokinesis, SRF transcriptional activity, and cell motility. Dia1 polymerizes actin through its formin homology (FH) 2 domain. Here we show that Dia1 acts upstream of RhoA independently of its effects on actin assembly. Dia1 binds to the leukemia-associated Rho-GEF (LARG) through RhoA-dependent release of Dia1 autoinhibition. The FH2 domain stimulates the guanine nucleotide exchange activity of LARG in vitro. Our results reveal that Dia1 is necessary for LPA-stimulated Rho/ROCK signaling and bleb-associated cancer cell invasion. Thus, Dia1-dependent RhoA activation constitutes a positive feedback mechanism to modulate cell behavior.

Galpha12/13 is essential for directed cell migration and localized Rho-Dia1 function.

Scratch-wound assays are frequently used to study directed cell migration, a process critical for embryogenesis, invasion, and tissue repair. The function and identity of trimeric G-proteins in cell behavior during wound healing is not known. Here we show that Galpha12/13, but not Galphaq/11 or Galphai, is indispensable for coordinated and directed cell migration. In mouse embryonic fibroblasts endogenous Rho activity is present at the rear of migrating cells but also at the leading edge, whereas it is undetectable at the cell front of Galpha12/13-deficient mouse embryonic fibroblasts. Spatial activation of Rho at the wound edge can be stimulated by lysophosphatidic acid. Active Rho colocalizes with the diaphanous-related formin Dia1 at the cell front. Galpha12/13-deficient cells lack Dia1 localization to the wound edge and are unable to form orientated, stable microtubules during wound healing. Knock down of Dia1 reveals its requirement for microtubule stabilization as well as polarized cell migration. Thus, we identified Galpha12/13-proteins as essential components linking extracellular signals to localized Rho-Dia1 function during directed cell movement.

Down-regulation of Rap1 activity is involved in ephrinB1-induced cell contraction.

Ephrins are cell surface ligands that activate Eph receptor tyrosine kinases. This ligand-receptor interaction plays a central role in the sorting of cells. We have previously shown that the ephrinB-EphB signalling pathway is also involved in the migration of intestinal precursor cells along the crypts. Using the colon cell line DLD1 expressing the EphB2 receptor, we showed that stimulation of these cells with soluble ephrinB1 results in a rapid retraction of cell extensions and a detachment of cells. On ephrinB1 stimulation, the small GTPases Rho and Ras are activated and Rap1 is inactivated. Importantly, when a constitutively active Rap1 mutant was introduced into these cells, ephrinB1-induced retraction was inhibited. From these results, we conclude that down-regulation of Rap1 is a prerequisite for ephrin-induced cell retraction in colon cells.

Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha.

Previously we have shown that protein kinase C (PKC)-mediated reorganization of the actin cytoskeleton in smooth muscle cells is transmitted by the non-receptor tyrosine kinase, Src. Several authors have described how 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of cells results in an increase of Src activity, but the mechanism of the PKC-mediated Src activation is unknown. Using PKC isozymes purified from Spodoptera frugiperda insect cells, we show here that PKC is not able to activate Src directly. Our data reveal that the PKC-dependent Src activation occurs via the activation of the protein tyrosine phosphatase (PTP) PTP alpha. PTP alpha becomes activated in vivo after TPA stimulation. Further, we show that PKC delta phosphorylates and activates only PTP alpha in vitro but not any other of the TPA-responsive PKC isozymes that are expressed in A7r5 rat aortic smooth muscle cells. To further substantiate our data, we show that cells lacking PKC delta have a markedly reduced PTP alpha and Src activity after 12-O-tetradecanoylphorbol-13-acetate stimulation. These data support a model in which the main mechanism of 12-O-tetradecanoylphorbol-13-acetate-induced Src activation is the direct phosphorylation and activation of PTP alpha by PKC delta, which in turn dephosphorylates and activates Src.