Martin Villalba

Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21WAF1/Cip1, p27Kip1, p57Kip2 and p16INK4a expression. Zac1‐mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.

miR-143 interferes with ERK5 signaling, and abrogates prostate cancer progression in mice.

Background Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions miR-143 is as a new target for prostate cancer treatment.

Translocation of PKCθ in T cells is mediated by a nonconventional, PI3-K– and Vav-dependent pathway, but does not absolutely require phospholipase C

PKCθ plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-κB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCθ to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCθ, we addressed here the question of whether the translocation and activation of PKCθ in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCγ1-deficient T cell line, or a dominant negative PLCγ1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCθ are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCα. Membrane or lipid raft recruitment of PKCθ (but not PKCα) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCθ, but did not associate with it. These results provide evidence that a nonconventional PI3-K– and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCθ in T cells.

The NF-κB member p65 controls glutamine metabolism through miR-23a.

Cancer cells have elevated aerobic glycolysis that is termed the Warburg effect. But several tumor cells, including leukemic cells, also increase glutamine metabolism, which is initiated by glutaminase (GLS). The microRNA (miRNA) miR-23 targets GLS mRNA and inhibits expression of GLS protein. Here we show that in human leukemic Jurkat cells the NF-κB p65 subunit binds to miR-23a promoter and inhibits miR-23a expression. Histone deacetylase (HDAC) inhibitors release p65-induced inhibition. Jurkat cells growing in glutamine decrease proliferation due to cell accumulation in G0/G1 phase. Nevertheless, cells get used to this new source of energy by increasing GLS expression, which correlates with an increase in p65 expression and its translocation to the nucleus, leading to a higher basal NF-κB activity. Jurkat cells overexpressing p65 show increase basal GLS expression and proliferate faster than control cells in glutamine medium. Overexpressing miR-23a in leukemic cells impaired glutamine use and induces mitochondrial dysfunction leading to cell death. Therefore, p65 activation decreases miR-23a expression, which facilitates glutamine consumption allowing leukemic cells to use this alternative source of carbon and favoring their adaptation to the metabolic environment.

Positive feedback regulation of PLCγ1/Ca2+ signaling by PKCθ in restimulated T cells via a Tec kinase-dependent pathway

PKCθ plays an essential role in activation of mature T cells. Here, we report that the TCR/CD28‐induced tyrosine phosphorylation and activation of PLCγ1 was significantly impaired in PKCθ –/– primary, restimulated T cells. Consistent with this finding, receptor‐induced Ca2+ mobilization, NF‐AT DNA‐binding activity and the membrane translocation of PKCα, a PLCγ1‐dependent conventional PKC, were also markedly reduced in the same cells. Moreover, a dominant‐negative PLCγ1 mutant blocked the PKCθ‐induced activation of an AP‐1 reporter gene in Jurkat and primary cells. Regulation of PLCγ1 signaling by PKCθ required the tyrosine kinase Tec since a dominant‐negative Tec mutant blocked PKCθ‐induced AP‐1 (but not NF‐κB) activation. In addition, wild‐type Tec, but not Itk or Rlk, potently activated AP‐1. Furthermore, Tec was found to constitutively associate with PKCθ, an interaction that like AP‐1 activation required the pleckstrin‐homology domain of Tec. These findings define a novel PKCθ‐initiated pathway that regulates Ca2+ signaling and AP‐1 activation via Tec and PLCγ1. Moreover, they identify Tec as a key point downstream of PKCθ, where TCR‐ and PKCθ‐induced signaling pathways, leading to AP‐1 versus NF‐κB activation, diverge in T cells.

ERK5 Activates NF-κB in Leukemic T Cells and Is Essential for Their Growth In Vivo

MAPK cascades play a central role in the cellular response to the environment. The pathway involving the MAPK ERK5 mediates growth factor- and stress-induced intracellular signaling that controls proliferation or survival depending upon the cell context. In this study, we show that reducing ERK5 levels with a specific small hairpin RNA 5 (shERK5) reduced cell viability, sensitized cells to death receptor-induced apoptosis, and blocked the palliative effects of phorbol ester in anti-Fas Ab-treated cells. shERK5 decreased nuclear accumulation of the NF-κB p65 subunit, and conversely, ectopic activation of ERK5 led to constitutive nuclear localization of p65 and increased its ability to trans activate specific reporter genes. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferated in vitro, but failed to induce s.c. tumors in mice. Our results suggest that ERK5 is essential for survival of leukemic T cells in vivo, and thus represents a promising target for therapeutic intervention in this type of malignancy.

Concomitant induction of apoptosis and necrosis in cerebellar granule cells following serum and potassium withdrawal

SERUM and potassium withdrawal-induced cell death of cerebellar granule cells is one of the most popular models used for studying neuronal apoptosis in vitro, and it is generally assumed that compounds preventing cell death in this model prevent apoptosis. In the present study we demonstrate that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. Each of these death mechanisms could be blocked by activation of different pathways, such as raising cAMP production or stimulation of the IGF-1 receptor. Studies on serum and potassium withdrawalinduced cell death of cerebellar granule cells should therefore carefully assess whether a given compound is preventing apoptosis or necrosis using appropriate techniques.

SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes

The AP-1 family member JunB is a critical regulator of T cell function. JunB is a transcriptional activator of various cytokine genes, such as IL-2, IL-4, and IL-10; however, the post-translational modifications that regulate JunB activity in T cells are poorly characterized. We show here that JunB is conjugated with small ubiquitin-like modifier (SUMO) on lysine 237 in resting and activated primary T cells and T cell lines. Sumoylated JunB associated with the chromatin-containing insoluble fraction of cells, whereas nonsumoylated JunB was also in the soluble fraction. Blocking JunB sumoylation by mutation or use of a dominant-negative form of the SUMO-E2 Ubc-9 diminished its ability to transactivate IL-2 and IL-4 reporter genes. In contrast, nonsumoylable JunB mutants showed unimpaired activity with reporter genes controlled by either synthetic 12-O-tetradecanoylphorbol-13-acetate response elements or NF-AT/AP-1 and CD28RE sites derived from the IL-2 promoter. Ectopic expression of JunB in activated human primary CD4+ T cells induced activation of the endogenous IL-2 promoter, whereas the nonsumoylable JunB mutant did not. Thus, our work demonstrates that sumoylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation.

Guanine Exchange-Dependent and -Independent Effects of Vav1 on Integrin-Induced T Cell Spreading

Vav1 is a 95-kDa member of the Dbl family of guanine exchange factors and a prominent hemopoietic cell-specific protein tyrosine kinase substrate, the involvement of which in cytoskeletal rearrangements has been linked to its ability to activate Rho family small GTPases. β1 integrin ligation by fibronectin induced Vav1 phosphorylation in peripheral blood lymphocytes and in two different T cell lines. Vav1 overexpression led to massive T cell spreading on β1 integrin ligands, and, conversely, two dominant negative mutants blocked integrin-induced spreading. Vav1 and β1 integrin ligation synergistically activated Pak, but not Rac, Cdc42, or c-Jun N-terminal kinase. In addition, Vav1 cooperated with constitutively active V12Rac mutant, but not with V12Cdc42, to induce T cell spreading after integrin occupancy. More importantly, a Vav1 mutant that lacked guanine exchange factor activity still cooperated with V12Rac. In contrast, a point mutation in the SH2 domain of Vav1 abolished this synergistic effect. Therefore, our results suggest a new regulatory effect of Vav1 in T cell spreading, which is independent of its guanine exchange factor activity.

PACAP‐38 Protects Cerebellar Granule Cells from Apoptosis

Abstract: Pituitary adenylate cyclase‐activating polypeptides (PACAP‐27 and ‐38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions including the cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP‐38 on apoptosis induced by potassium deprivation. We demonstrated that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. We showed that PACAP‐38 increased survival of cerebellar neurons in a dose‐dependent manner by specifically decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP‐38 induced activation of the extracellular signal‐regulated kinase (ERK)‐type of MAP kinase through a cAMP‐dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the anti‐apoptotic effect of PACAP‐38, suggesting that MAP kinase pathway activation is necessary for PACAP‐38 effect.