A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer.

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein-protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.

Rank-Rankl-Opg Axis in Multiple Sclerosis: The Contribution of Placenta.

Women with multiple sclerosis (MS) can safely become pregnant and give birth, with no side effects or impediments. Pregnancy is generally accepted as a period of well-being in which relapses have a softer evolution, particularly in the third trimester. Herein, we hypothesized that the placenta, via its "secretome", could contribute to the recognized beneficial effects of pregnancy on MS activity. We focused on a well-known receptor/ligand/decoy receptor system, such as the one composed by the receptor activator of nuclear factor-kB (RANK), its ligand (RANKL), and the decoy receptor osteoprotegerin (OPG), which have never been investigated in an integrated way in MS, pregnancy, and placenta. We reported that pregnancy at the term of gestation influences the balance between circulating RANKL and its endogenous inhibitor OPG in MS women. We demonstrated that the placenta at term is an invaluable source of homodimeric OPG. By functional studies on astrocytes, we showed that placental OPG suppresses the mRNA expression of the CCL20, a chemokine responsible for Th17 cell recruitment. We propose placental OPG as a crucial molecule for the recognized beneficial effect of late pregnancy on MS and its potential utility for the development of new and more effective therapeutic approaches.

The Estrogen Receptor α Signaling Pathway Controls Alternative Splicing in the Absence of Ligands in Breast Cancer Cells.

BACKGROUND: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. METHODS: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. RESULTS: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3'end processing. ApoERα depletion results in 758 isoform switching events with effects on 3'end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional regulation is part of the mechanisms by which apoERα controls epithelial-to-mesenchymal transition and BC cell proliferation. In primary BCs, the inclusion levels of the experimentally identified alternatively spliced exons are associated with overall and disease-free survival. CONCLUSION: Our data supports the role of apoERα in maintaining the luminal phenotype of BC cells by extensively regulating gene expression at the alternative splicing level.

The transcription factor Nurr1 is upregulated in amyotrophic lateral sclerosis patients and SOD1-G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons (MNs) in the central nervous system. ALS etiology is highly multifactorial and multifarious, and an effective treatment is still lacking. Neuroinflammation is a hallmark of ALS and could be targeted to develop new therapeutic approaches. Interestingly, the transcription factor Nurr1 has been demonstrated to have an important role in the inflammatory process in several neurological disorders, such as Parkinson's disease and multiple sclerosis. In the present paper, we demonstrate for the first time that Nurr1 expression levels are upregulated in the peripheral blood of ALS patients. Moreover, we investigated Nurr1 function in the SOD1-G93A mouse model of ALS. Nurr1 was strongly upregulated in the spinal cord during the asymptomatic and early symptomatic phases of the disease, where it promoted the expression of brain-derived neurotrophic factor mRNA and the repression of NFκB pro-inflammatory targets, such as inducible nitric oxide synthase. Therefore, we hypothesize that Nurr1 is activated in an early phase of the disease as a protective endogenous anti-inflammatory mechanism, although not sufficient to reverse disease progression. On the basis of these observations, Nurr1 could represent a potential biomarker for ALS and a promising target for future therapies.

A computational approach based on the colored Petri net formalism for studying multiple sclerosis.

BACKGROUND: Multiple Sclerosis (MS) is an immune-mediated inflammatory disease of the Central Nervous System (CNS) which damages the myelin sheath enveloping nerve cells thus causing severe physical disability in patients. Relapsing Remitting Multiple Sclerosis (RRMS) is one of the most common form of MS in adults and is characterized by a series of neurologic symptoms, followed by periods of remission. Recently, many treatments were proposed and studied to contrast the RRMS progression. Among these drugs, daclizumab (commercial name Zinbryta), an antibody tailored against the Interleukin-2 receptor of T cells, exhibited promising results, but its efficacy was accompanied by an increased frequency of serious adverse events. Manifested side effects consisted of infections, encephalitis, and liver damages. Therefore daclizumab has been withdrawn from the market worldwide. Another interesting case of RRMS regards its progression in pregnant women where a smaller incidence of relapses until the delivery has been observed. RESULTS: In this paper we propose a new methodology for studying RRMS, which we implemented in GreatSPN, a state-of-the-art open-source suite for modelling and analyzing complex systems through the Petri Net (PN) formalism. This methodology exploits: (a) an extended Colored PN formalism to provide a compact graphical description of the system and to automatically derive a set of ODEs encoding the system dynamics and (b) the Latin Hypercube Sampling with PRCC index to calibrate ODE parameters for reproducing the real behaviours in healthy and MS subjects.To show the effectiveness of such methodology a model of RRMS has been constructed and studied. Two different scenarios of RRMS were thus considered. In the former scenario the effect of the daclizumab administration is investigated, while in the latter one RRMS was studied in pregnant women. CONCLUSIONS: We propose a new computational methodology to study RRMS disease. Moreover, we show that model generated and calibrated according to this methodology is able to reproduce the expected behaviours.

The Adaptive Immune System in Multiple Sclerosis: An Estrogen-Mediated Point of View.

Multiple sclerosis (MS) is a chronic central nervous system inflammatory disease that leads to demyelination and neurodegeneration. The third trimester of pregnancy, which is characterized by high levels of estrogens, has been shown to be associated with reduced relapse rates compared with the rates before pregnancy. These effects could be related to the anti-inflammatory properties of estrogens, which orchestrate the reshuffling of the immune system toward immunotolerance to allow for fetal growth. The action of these hormones is mediated by the transcriptional regulation activity of estrogen receptors (ERs). Estrogen levels and ER expression define a specific balance of immune cell types. In this review, we explore the role of estradiol (E2) and ERs in the adaptive immune system, with a focus on estrogen-mediated cellular, molecular, and epigenetic mechanisms related to immune tolerance and neuroprotection in MS. The epigenome dynamics of immune systems are described as key molecular mechanisms that act on the regulation of immune cell identity. This is a completely unexplored field, suggesting a future path for more extensive research on estrogen-induced coregulatory complexes and molecular circuitry as targets for therapeutics in MS.

Depletion of tumor-associated macrophages switches the epigenetic profile of pancreatic cancer infiltrating T cells and restores their anti-tumor phenotype.

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by a complex tumor microenvironment that supports its progression, aggressiveness and resistance to therapies. The delicate interplay between cancer and immune cells creates the conditions for PDA development, particularly due to the functional suppression of T cell anti-tumor effector activity. However, some of the mechanisms involved in this process are still poorly understood. In this study, we analyze whether the functional and epigenetic profile of T cells that infiltrate PDA is modulated by the microenvironment, and in particular by tumor-associated macrophages (TAMs). CD4 and CD8 T cells obtained from mice orthotopically injected with syngeneic PDA cells, and untreated or treated with Trabectedin, a cytotoxic drug that specifically targets TAMs, were sorted and analyzed by flow cytometry and characterized for their epigenetic profile. Assessment of cytokine production and the epigenetic profile of genes coding for IL10, T-bet and PD1 revealed that T cells that infiltrated PDA displayed activated Il10 promoter and repressed T-bet activity, in agreement with their regulatory phenotype (IL10high/IFNγlow, PD1high). By contrast, in Trabectedin-treated mice, PDA-infiltrating T cells displayed repressed Il10 and Pdcd1 and activated T-bet promoter activity, in accordance with their anti-tumor effector phenotype (IL10low/IFNγhigh), indicating a key role of TAMs in orchestrating functions of PDA-infiltrating T cells by modulating their epigenetic profile towards a pro-tumoral phenotype. These results suggest the targeting of TAMs as an efficient strategy to obtain an appropriate T cell anti-tumor immune response and open new potential combinations for PDA treatment.

Pregnancy Epigenetic Signature in T Helper 17 and T Regulatory Cells in Multiple Sclerosis.

Increasing evidence supports the anti-inflammatory role of estrogens in Multiple Sclerosis (MS), originating from the observation of reduction in relapse rates among women with MS during pregnancy, but the molecular mechanisms are still not completely understood. Using an integrative data analysis, we identified T helper (Th) 17 and T regulatory (Treg) cell-type-specific regulatory regions (CSR) regulated by estrogen receptor alpha (ERα). These CSRs were validated in polarized Th17 from healthy donors (HD) and in peripheral blood mononuclear cells, Th17 and Treg cells from relapsing remitting (RR) MS patients and HD during pregnancy. 17ß-estradiol induces active histone marks enrichment at Forkhead Box P3 (FOXP3)-CSRs and repressive histone marks enrichment at RAR related orphan receptor C (RORC)-CSRs in polarized Th17 cells. A disease-associated epigenetic profile was found in RRMS patients during pregnancy, suggesting a FOXP3 positive regulation and a RORC negative regulation in the third trimester of pregnancy. Altogether, these data indicate that estrogens act as immunomodulatory factors on the epigenomes of CD4+ T cells in RRMS; the identified CSRs may represent potential biomarkers for monitoring disease progression or new potential therapeutic targets.

A Novel Functional Domain of Tab2 Involved in the Interaction with Estrogen Receptor Alpha in Breast Cancer Cells.

Tab2, originally described as a component of the inflammatory pathway, has been implicated in phenomena of gene de-repression in several contexts, due to its ability to interact with the NCoR corepressor. Tab2 interacts also with steroid receptors and dismisses NCoR from antagonist-bound Estrogen and Androgen Receptors on gene regulatory regions, thus modifying their transcriptional activity and leading to pharmacological resistance in breast and prostate cancer cells. We demonstrated previously that either Tab2 knock-down, or a peptide mimicking the Estrogen Receptor alpha domain interacting with Tab2, restore the antiproliferative response to Tamoxifen in Tamoxifen-resistant breast cancer cells. In this work, we map the domain of Tab2 responsible of Estrogen Receptor alpha interaction. First, using both co-immunoprecipitation and pull-down with recombinant proteins, we found that the central part of Tab2 is primarily responsible for this interaction, and that this region also interacts with Androgen Receptor. Then, we narrowed down the essential interaction region by means of competition assays using recombinant protein pull-down. The interaction motif was finally identified as a small region adjacent to, but not overlapping, the Tab2 MEKK1 phosphorylation sites. A synthetic peptide mimicking this motif efficiently displaced Tab2 from interacting with recombinant Estrogen Receptor alpha in vitro, prompting us to test its efficacy using derivatives of the MCF7 breast carcinoma cell lines that are spontaneously resistant to Tamoxifen. Indeed, we observed that this mimic peptide, made cell-permeable by addition of the TAT minimal carrier domain, reduced the growth of Tamoxifen-resistant MCF7 cells in the presence of Tamoxifen. These data indicate a novel functional domain of the Tab2 protein with potential application in drug design.

E2 Regulates Epigenetic Signature on Neuroglobin Enhancer-Promoter in Neuronal Cells.

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERß) that bind to specific regulatory genomic regions. We focused our attention on 17ß-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Luminal long non-coding RNAs regulated by estrogen receptor alpha in a ligand-independent manner show functional roles in breast cancer.

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

Genome-wide activity of unliganded estrogen receptor-α in breast cancer cells.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

Genomic lens on neuroglobin transcription.

Neuroglobin is a brain globin with neuroprotective effects against ischemia and related pathological processes, acting as O2 sensor and antiapoptotic pathway transducer. Here, we survey data on neuroanatomical coexpression of transcription factors, epigenetic signature and predictive transcription factor binding sites at the neuroglobin gene locus to find hints of pathways to neuroglobin transcriptional regulation. These data provide a glimpse of how neuroglobin expression may translate into neuronal diversity and function, as well as disease.

The role of Transposable Elements in shaping the combinatorial interaction of Transcription Factors.

BACKGROUND: In the last few years several studies have shown that Transposable Elements (TEs) in the human genome are significantly associated with Transcription Factor Binding Sites (TFBSs) and that in several cases their expansion within the genome led to a substantial rewiring of the regulatory network. Another important feature of the regulatory network which has been thoroughly studied is the combinatorial organization of transcriptional regulation. In this paper we combine these two observations and suggest that TEs, besides rewiring the network, also played a central role in the evolution of particular patterns of combinatorial gene regulation. RESULTS: To address this issue we searched for TEs overlapping Estrogen Receptor α (ERα) binding peaks in two publicly available ChIP-seq datasets from the MCF7 cell line corresponding to different modalities of exposure to estrogen. We found a remarkable enrichment of a few specific classes of Transposons. Among these a prominent role was played by MIR (Mammalian Interspersed Repeats) transposons. These TEs underwent a dramatic expansion at the beginning of the mammalian radiation and then stabilized. We conjecture that the special affinity of ERα for the MIR class of TEs could be at the origin of the important role assumed by ERα in Mammalians. We then searched for TFBSs within the TEs overlapping ChIP-seq peaks. We found a strong enrichment of a few precise combinations of TFBS. In several cases the corresponding Transcription Factors (TFs) were known cofactors of ERα, thus supporting the idea of a co-regulatory role of TFBS within the same TE. Moreover, most of these correlations turned out to be strictly associated to specific classes of TEs thus suggesting the presence of a well-defined "transposon code" within the regulatory network. CONCLUSIONS: In this work we tried to shed light into the role of Transposable Elements (TEs) in shaping the regulatory network of higher eukaryotes. To test this idea we focused on a particular transcription factor: the Estrogen Receptor α (ERα) and we found that ERα preferentially targets a well defined set of TEs and that these TEs host combinations of transcriptional regulators involving several of known co-regulators of ERα. Moreover, a significant number of these TEs turned out to be conserved between human and mouse and located in the vicinity (and thus candidate to be regulators) of important estrogen-related genes.

Dysregulation of SREBP2 induces BACE1 expression.

ß-Amyloid hyperproduction has been observed in response to alterations in neuronal intracellular cholesterol storage, efflux, and synthesis, induced in rats by a high-fat diet. It has been suggested that cholesterol homeostasis is altered in Alzheimer's disease resulting in higher ß- and γ-secretase activity. In the current study the neuronal activation status of sterol regulatory element binding protein 2 (SREBP2) as well as its involvement in ß-secretase BACE1 activity was investigated in high-fat fed rats (26% fat and 4% cholesterol for 20 weeks), and in SK-N-BE neuroblastoma cells exposed to 20 µM cholesterol. This work demonstrates that in the brain a hyperlipidic diet is able to induce a hyper-expression of BACE1 and determine an unbalance in cerebral cholesterol homeostasis so that SREBP2 is activated. In addition, we show for the first time the involvement of SREBP2 on expression of BACE1 in SK-N-BE cells exposed to high cholesterol. Although the enhanced risk of Alzheimer's disease in metabolic syndrome is related to several factors, our results suggest that SREBP2, which can be modulated by the impairment of cerebral cholesterol homeostasis, has a direct role on BACE1 expression and may be involved in Alzheimer's disease progression.

Diacylglycerol kinase-alpha mediates hepatocyte growth factor-induced epithelial cell scatter by regulating Rac activation and membrane ruffling.

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.

Diacylglycerol kinase is required for HGF-induced invasiveness and anchorage-independent growth of MDA-MB-231 breast cancer cells.

BACKGROUND: Estrogen receptor (ER)-negative breast cancers have a worse prognosis than ER-positive cancers, being more aggressive and overexposed to stimuli leading to their progression. Hepatocyte growth factor (HGF) has been associated with proliferation, migration and invasion of tumor cells, and several tumors, including those of breast cancer, produce HGF and overexpress its receptor. Diacylglycerol kinases (Dgks), which phosphorylate diacylglycerol to phosphatidic acid, are key regulators of cell signaling. Our research was focused on their role in HGF-induced invasion of MDA-MB-231 cells, a model of ER-negative breast cancer. MATERIALS AND METHODS: Dgk activity was evaluated with a kinase assay, MDA-MB-231 cell invasion via culturing of cells in matrigel-coated transwells, and anchorage-independent growth was assessed using a soft agar assay. RESULTS: HGF induces Dgk activation in MDA-MB-231 cells that is required for cell invasiveness. Moreover, Dgks are involved in MDA-MB-231 anchorage-independent growth. CONCLUSION: Dgks could be a target for ER-negative breast cancer therapy.

Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.

ErbB4 expression in neural progenitor cells (ST14A) is necessary to mediate neuregulin-1beta1-induced migration.

Activation of the receptor tyrosine kinase ErbB4 leads to various cellular responses such as proliferation, survival, differentiation, and chemotaxis. Two pairs of naturally occurring ErbB4 isoforms differing in their juxtamembrane (JMa/JMb) and C termini (cyt1/cyt2) have been described. To examine the role of ErbB4 in neuron migration, we cloned and stably transfected each of the four ErbB4 isoforms in ST14A cells (a neural progenitor cell line derived from the striatum of embryonic day 14 rats) endogenously expressing the other members of the ErbB family: ErbB1, ErbB2, and ErbB3. Using immunoprecipitation assays, we showed that the neuregulin-1beta1 (NRG1beta1) stimulus induced ErbB4 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) recruitment and activation (as demonstrated by Akt phosphorylation) either directly (ErbB4 cyt1 isoform) or indirectly (ErbB4 cyt2 isoform). We examined the ability of the four ErbB4 isoforms to induce chemotaxis and cell proliferation in response to NRG1beta1 stimulation. Using migration assays, we observed that only ErbB4-expressing cells stimulated with NRG1beta1 showed a significant increase in migration, whereas the growth rate remained unchanged. Additional assays showed that inhibition of PI3K (but not of phospholipase Cgamma) dramatically reduced migratory activity. Our data show that ErbB4 signaling via PI3K activation plays a fundamental role in controlling NRG1beta1-induced migration.

Activation of diacylglycerol kinase alpha is required for VEGF-induced angiogenic signaling in vitro.

Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.