Concetta Ambrosino

Cell cycle regulation by p38 MAP kinases

MAP kinases are signaling intermediaries that alloweukaryotic cells to interpret and respond to manydifferent stimuli. Three major MAP kinase pathwayshave been identified. These include the extracellularsignal-regulated kinases (ERK1 and ERK2), which aremainly activated by mitogens and serum stimulation.In contrast, the c-Jun N-terminal kinases (JNK) and thep38 MAP kinases are strongly activated by environ-mental and genotoxic stresses.The p38 MAP kinases are also implicated in severalaspects of the immune response. The first member ofthis family to be isolated was a protein (p38), whichwas rapidly phosphorylated on tyrosine residues uponlipopolysaccharide stimulation (Han et al., 1994). Itwas also found as an stress-activated protein kinase(Reactivating Kinase or RK) that stimulates MAPKAPkinase-2 and phosphorylation of the small heat shockprotein Hsp27 (Rouse et al., 1994). Finally, the sameprotein was identified as a target of pyridinyl-imidazole drugs (Cytokine-Suppressive antiinflamma-tory drug Binding Protein or CSBP) that inhibit theproduction of the pro-inflammatory cytokinesinterleukin-1 and tumor necrosis factor (Lee et al.,1994). Analysis of the cDNA sequence revealed thatthis protein (p38/RK/CSBP) was the vertebrate homo-logue of the

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer

Estrogen effects on mammary epithelial and breast cancer (BC) cells are mediated by the nuclear receptors ERα and ERβ, transcription factors that display functional antagonism with each other, with ERβ acting as oncosuppressor and interfering with the effects of ERα on cell proliferation, tumor promotion and progression. Indeed, hormone-responsive, ERα+ BC cells often lack ERβ, which when present associates with a less aggressive clinical phenotype of the disease. Recent evidences point to a significant role of microRNAs (miRNAs) in BC, where specific miRNA expression profiles associate with distinct clinical and biological phenotypes of the lesion. Considering the possibility that ERβ might influence BC cell behavior via miRNAs, we compared miRNome expression in ERβ+ vs ERβ− hormone-responsive BC cells and found a widespread effect of this ER subtype on the expression pattern of these non-coding RNAs. More importantly, the expression pattern of 67 miRNAs, including 10 regulated by ERβ in BC cells, clearly distinguishes ERβ+, node-negative, from ERβ−, metastatic, mammary tumors. Molecular dissection of miRNA biogenesis revealed multiple mechanisms for direct regulation of this process by ERβ+ in BC cell nuclei. In particular, ERβ downregulates miR-30a by binding to two specific sites proximal to the gene and thereby inhibiting pri-miR synthesis. On the other hand, the receptor promotes miR-23b, -27b and 24-1 accumulation in the cell by binding in close proximity of the corresponding gene cluster and preventing in situ the inhibitory effects of ERα on pri-miR maturation by the p68/DDX5-Drosha microprocessor complex. These results indicate that cell autonomous regulation of miRNA expression is part of the mechanism of action of ERβ in BC cells and could contribute to establishment or maintenance of a less aggressive tumor phenotype mediated by this nuclear receptor.

Negative Feedback Regulation of MKK6 mRNA Stability by p38α Mitogen-Activated Protein Kinase

ABSTRACT p38 mitogen-activated protein (MAP) kinases play an important role in the regulation of cellular responses to all kinds of stresses. The most abundant and broadly expressed p38 MAP kinase is p38α, which can also control the proliferation, differentiation, and survival of several cell types. Here we show that the absence of p38α correlates with the up-regulation of one of its upstream activators, the MAP kinase kinase MKK6, in p38α−/− knockout mice and in cultured cells derived from them. In contrast, the expression levels of the p38 activators MKK3 and MKK4 are not affected in p38α-deficient cells. The increase in MKK6 protein concentration correlates with increased amounts of MKK6 mRNA in the p38α−/− cells. Pharmacological inhibition of p38α also up-regulates MKK6 mRNA levels in HEK293 cells. Conversely, reintroduction of p38α into p38α−/− cells reduces the levels of MKK6 protein and mRNA to the normal levels found in wild-type cells. Moreover, we show that the MKK6 mRNA is more stable in p38α−/− cells and that the 3′untranslated region of this mRNA can differentially regulate the stability of the lacZ reporter gene in a p38α-dependent manner. Our data indicate that p38α can negatively regulate the stability of the MKK6 mRNA and thus control the steady-state concentration of one of its upstream activators.

Identification of a hormone-regulated dynamic nuclear actin network associated with estrogen receptor alpha in human breast cancer cell nuclei

Estrogen receptor α (ERα) is a modular protein of the steroid/nuclear receptor family of transcriptional regulators that upon binding to the hormone undergoes structural changes, resulting in its nuclear translocation and docking to specific chromatin sites. In the nucleus, ERα assembles in multiprotein complexes that act as final effectors of estrogen signaling to the genome through chromatin remodeling and epigenetic modifications, leading to dynamic and coordinated regulation of hormone-responsive genes. Identification of the molecular partners of ERα and understanding their combinatory interactions within functional complexes is a prerequisite to define the molecular basis of estrogen control of cell functions. To this end, affinity purification was applied to map and characterize the ERα interactome in hormone-responsive human breast cancer cell nuclei. MCF-7 cell clones expressing human ERα fused to a tandem affinity purification tag were generated and used to purify native nuclear ER-containing complexes by IgG-Sepharose affinity chromatography and glycerol gradient centrifugation. Purified complexes were analyzed by two-dimensional DIGE and mass spectrometry, leading to the identification of a ligand-dependent multiprotein complex comprising β-actin, myosins, and several proteins involved in actin filament organization and dynamics and/or known to participate in actin-mediated regulation of gene transcription, chromatin dynamics, and ribosome biogenesis. Time course analyses indicated that complexes containing ERα and actin are assembled in the nucleus early after receptor activation by ligands, and gene knockdown experiments showed that gelsolin and the nuclear isoform of myosin 1c are key determinants for assembly and/or stability of these complexes. Based on these results, we propose that the actin network plays a role in nuclear ERα actions in breast cancer cells, including coordinated regulation of target gene activity, spatial and functional reorganization of chromatin, and ribosome biogenesis.

An NF-kappaB site in the 5'-untranslated leader region of the human immunodeficiency virus type 1 enhances the viral expression in response to NF-kappaB-activating stimuli.

The 5′-untranslated leader region of human immunodeficiency virus, type 1 (HIV-1), includes a complex array of putative regulatory elements whose role in the viral expression is not completely understood. Here we demonstrate the presence of an NF-κB-responsive element in the trans-activation response (TAR) region of HIV-1 that confers the full induction of HIV-1 long terminal repeat (LTR) in response to NF-κB-activating stimuli, such as DNA alkylating agents, phorbol 12-myristate 13-acetate, and tumor necrosis factor-α. The TAR NF-κB site GGGAGCTCTC spans from positions +31 to +40 and cooperates with the NF-κB enhancer upstream of the TATA box in the NF-κB-mediated induction of HIV-1 LTR. The conclusion stems from the following observations: (i) deletion of the two NF-κB sites upstream of the TATA box reduces, but does not abolish, the HIV-1 LTR activation by NF-κB inducers; (ii) deletion or base pair substitutions of the TAR NF-κB site significantly reduce the HIV-1 LTR activation by NF-κB inducers; (iii) deletions of both the NF-κB sites upstream of the TATA box and the TAR NF-κB site abolish the activation of HIV-1 LTR in response to NF-κB inducers. Moreover, the p50·p65 NF-κB complex binds to the TAR NF-κB sequence and trans-activates the TAR NF-κB-directed expression. The identification of an additional NF-κB site in the HIV-1 LTR points to the relevance of NF-κB factors in the HIV-1 life cycle.

Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.

A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei.

Estrogen receptors α (ER‐α) and β (ER‐β) play distinct biological roles in onset and progression of hormone‐responsive breast cancer, with ER‐β exerting a modulatory activity on ER‐α‐mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER‐β fused to a tandem affinity purification‐tag in estrogen‐responsive MCF‐7 cells and applied tandem affinity purification and nanoLC‐MS/MS to identify the ER‐β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co‐purify with ER‐β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.

Identification of proteins associated with ligand-activated estrogen receptor α in human breast cancer cell nuclei by tandem affinity purification and nano LC-MS/MS

Estrogen receptor α (ER‐α) is a key mediator of estrogen actions in breast cancer (BC) cells. Understanding the effects of ligand‐activated ER‐α in target cells requires identification of the molecular partners acting in concert with this nuclear receptor to transduce the hormonal signal. We applied tandem affinity purification (TAP), glycerol gradient centrifugation and MS analysis to isolate and identify proteins interacting with ligand‐activated ER‐α in MCF‐7 cell nuclei. This led to the identification of 264 ER‐associated proteins, whose functions highlight the hinge role of ER‐α in the coordination of multiple hormone‐regulated nuclear processes in BC cells.

Molecular mechanisms of selective estrogen receptor modulator activity in human breast cancer cells: identification of novel nuclear cofactors of antiestrogen-ERα complexes by interaction proteomics.

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.

Localization of BDNF expression in the developing brain of zebrafish

The brain‐derived neurotrophic factor (BDNF) gene is expressed in differentiating and post‐mitotic neurons of the zebrafish embryo, where it has been implicated in Huntingtons disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real‐time quantitative polymerase chain reaction (RT‐qPCR) and whole‐mount in situ hybridization (WISH). RT‐qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co‐staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF‐positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease.