Recent studies using glycogen synthase kinase-3B (GSK-3B)-deficient mouse embryonic fibroblasts suggest that GSK-3B positively regulates nuclear factor KB (NFKB)-mediated gene transcription. Because NFKB is suggested to participate in cell proliferation and survival pathways in pancreatic cancer, we investigated the role of GSK-3B in regulating these cellular processes. Herein, we show that pancreatic cancer cells contain a pool of active GSK-3B and that pharmacologic inhibition of GSK-3 kinase activity using small molecule inhibitors or genetic depletion of GSK-3B by RNA interference leads to decreased cancer cell proliferation and survival. Mechanistically, we show that GSK-3B influences NFKBmediated gene transcription at a point distal to the IK kinase complex, as only ectopic expression of the NFKB subunits p65/p50, but not an IK kinase B constitutively active mutant, could rescue the decreased cellular proliferation and survival associated with GSK-3B inhibition. Taken together, our results simultaneously identify a previously unrecognized role for GSK-3B in cancer cell survival and proliferation and suggest GSK-3B as a potential therapeutic target in the treatment of pancreatic cancer. (Cancer Res 2005; 65(6): 2076-81)
Herein, we show that the hematopoietic-specific GEF VAV1 is ectopically expressed in primary pancreatic adenocarcinomas due to demethylation of the gene promoter. Interestingly, VAV1-positive tumors had a worse survival rate compared to VAV1-negative tumors. Surprisingly, even in the presence of oncogenic KRAS, VAV1 RNAi abrogates neoplastic cellular proliferation in vitro and in vivo, thus identifying Vav1 as a growth-stimulatory protein in this disease. Vav1 acts synergistically with the EGF receptor to stimulate pancreatic tumor cell proliferation. Mechanistically, the effects of Vav1 require its GEF activity and the activation of Rac1, PAK1, and NF-kappaB and involve cyclin D1 upregulation. Thus, the discovery of prooncogenic pathways regulated by Vav1 makes it an attractive target for therapeutic intervention.
Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.
The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present results on the modification of the CAP region of a set of triazolylphenyl-based HDACIs, and show that the nature of substitution on the phenyl ring plays a role in their selectivity for HDAC1 versus HDAC6, with low to moderate selectivity (2-51-fold) being achieved. In light of the valuable selectivity and potency that were identified for the triazolylphenyl ligand 6b in the inhibition of HDAC6 (IC50 = 1.9 nM), this compound represents a valuable research tool and a candidate for further chemical modifications. Lastly, these new HDACIs were studied for both their anticancer and antimalarial activity, which serve to validate the superior activity of the HDACI 10c.
NF-B is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-B activation, an IB kinase (IKK)-dependentprocess. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKC␣, but not PKC1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca 2؉ influx, the catalytically active PKC␣A25E induces IKK activity and NF-B-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca 2؉ binding to PKC␣ and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKC␣ dependent and that a later phase (5 to 25 min) of IKK activation is PKC dependent. Activation of IKK-and NF-B-dependent transcription by PKC␣A25E is abrogated by the PKC inhibitor rottlerin or the expression of the kinase-inactive form of PKC. Taken together, our results suggest that PKC␣ acts upstream of PKC to activate the IKK complex and NF-B in T lymphocytes following TCR activation.Identification of the molecular events regulating T-cell activation is paramount to understanding the regulation of the immune response. The signal transduction pathways triggered by antigen presentation lead to the immediate activation of multiple transcription factors that further amplify the process of lymphocyte activation, ultimately leading to cellular proliferation and division. T-cell receptor (TCR) engagement, together with the second costimulatory signal derived from engagement of the CD28 receptor, results in NF-B activation (13,31,36). When occurring together with NF-AT, AP-1, and octomer, NF-B activation leads to interleukin-2 (IL-2) expression (17,27,30).NF-B is a heterodimer of transcription factors that belong to the Rel family of proteins. The canonical NF-B is a heterodimer of p65 (RelA) with p50 or p52 (35,50,54). This heterodimer is anchored by a group of proteins named IB, which function to retain NF-B in the cytosol by masking its nuclear localization signal (1,4,45,60). IB␣ is the prototype IB molecule known to control the subcellular localization of NF-B (p50/p65). Following activation of certain signal transduction pathways, a site-specific hyperphosphorylation of IB␣ at S32 and S36 renders the inhibitor molecule susceptible to site-specific ubiquitination and subsequent degradation by the proteasome complex (8,9,18,62,68). This releases NF-B, allowing it to undergo nuclear translocation. Two IB␣ kinases, IKK␣ (19,52,70) and IKK (46), which are contained within a high-molecular-weight complex, target the phosphorylation of S32 and S36 of IB␣ following stimulation by various stimuli. While...
RhoG, a member of the Rho family of GTPases, has been implicated as a regulator of the actin cytoskeleton. In this study, we show a novel function for the small GTPase RhoG on the regulation of the interferon-c promoter and nuclear factor of activated T cells (NFAT) gene transcription in lymphocytes. Optimal function of RhoG for the expression of these genes requires a calcium signal, normally provided by the antigen receptor. In addition, RhoG potentiation of NFAT requires the indirect activity of Rac and Cdc42; however, pathways distinct from those activated by Rac and Cdc42 mediate RhoG activation of NFAT-dependent transcription. Using effector domain mutants of RhoG we found that its ability to potentiate NFAT-dependent transcription correlates with its capacity to increase actin polymerization, supporting the suggestion that NFAT-dependent transcription is an actin-dependent process. RhoG also promotes T-cell spreading on fibronectin, a property that is independent of its ability to enhance NFAT-dependent transcription. Hence, these results implicate RhoG in leukocyte trafficking and the control of gene expression induced in response to antigen encounter.
The histone deacetylases (HDACs) are able to regulate gene expression and inhibitors of the HDACs (HDACIs) hold promise in the treatment of cancer as well as a variety of neurodegenerative diseases. To investigate the possibility to achieve some measure of isoform selectivity in the inhibition of the HDACs, we prepared a small series of 2,4′-diaminobiphenyl ligands functionalized at the para-amino group with an appendage containing either a hydroxamate or a mercaptoacetamide group and coupled to an amino acid residue at the ortho-amino group. A smaller series of substituted phenylthiazoles was also explored. Some of these newly synthesized ligands show low nM potency in the HDAC inhibition assays and display micromolar to low nanomolar IC 50 values when tested against five pancreatic cancer cell lines. The isoform selectivity of these ligands for the Class I HDACs (HDAC1-3 and 8) and Class IIb HDACs (HDAC6 and HDAC10) together with QSAR studies of their correlation with the lipophilicity are presented. Of particular interest is the HDAC6 selectivity of the mercaptoacetamides.
Polarization of lipid rafts and granules to the site of target contact is required for the development of cell-mediated killing by cytotoxic lymphocytes. We have previously shown that these events require the activation of proximal protein tyrosine kinases. However, the downstream intracellular signaling molecules involved in the development of cell-mediated cytotoxicity remain poorly defined. We report here that a RhoA/ROCK/LIM-kinase axis couples the receptor-initiated protein tyrosine kinase activation to the reorganization of the actin cytoskeleton required for the polarization of lipid rafts and the subsequent generation of cell-mediated cytotoxicity. Pharmacologic and genetic interruption of any element of this RhoA/ROCK/LIM-kinase pathway inhibits both the accumulation of F-actin and lipid raft polarization to the site of target contact and the subsequent delivery of the lethal hit. These data define a specialized role for a RhoA→ROCK→LIM-kinase pathway in cytotoxic lymphocyte activation.
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