Activation Of Various Transcription Factors Research Articles

Abstract 1631: The ribonucleoside analogue, 8-amino-adenosine, inhibits AKT and MAPK signaling in breast cancer cells

Abstract In contrast to DNA-directed, deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-amino-adenosine (8-NH2-Ado) possesses a ribose sugar. This distinct nucleoside analog is RNA-directed and is cytotoxic to a number of primary malignant hematological cells as well as cell lines. The analog's cytotoxicity is associated with a high accumulation of its metabolite, 8-NH2-Ado-triphosphate (8-NH2-ATP), reaching to levels as high as 8 mM with 10 μM 8-NH2-Ado treatment. This accumulation is coupled with a parallel decrease in the level of the endogenous ATP pool by as much as 90%. Unlike DNA-directed nucleoside analogs, in solid tumors the triphosphate of 8-substituted-Ado-analogs accumulates to high levels; thus warrants use of 8-NH2-Ado in these types of malignancies. We report that 10 μM 8-NH2-Ado rapidly induced apoptosis in the breast cancer cell lines, BT-474 and MCF-7 as cleavage of PARP was readily detected in 2 h and 4 h, respectively. To further understand the mechanisms of action of 8-NH2-Ado, we examined its effect on key growth and survival pathways using purified enzymes and cell line models. Because 8-NH2-ATP is an ATP analog, it is feasible that it may alter other processes requiring ATP such as protein kinase activity. To determine if 8-NH2-ATP can directly inhibit the activity of key kinases, we employed Invitrogen's SelectScreen Kinase Profiling Service. Members of the MAPK family were inhibited by 8-NH2-ATP at ratios of 8-NH2-ATP: ATP readily achievable in cells. Specifically, 8-NH2-ATP was a potent, selective inhibitor of the dual specificity mitogen-activated protein kinase (MEK) 1 and 2 were inhibited 100% even at 8-NH2-ATP: ATP ratios of 1.0. Additionally, MEK6 was also inhibited. MEK1 and 2 are kinases that feed the MAPK cascade into the Ras/Raf pathway, and they directly phosphorylate and activate ERK 1/2 which leads to the activation of various transcription factors and cell cycle regulatory proteins to induce growth and survival. MEK6 phosphorylates and activates p38 MAP kinase and includes a p38 isoform that binds ERK 1/2 and regulates ERK nuclear activity. The in vitro inhibition of MEK1, 2, and 6 by 8-NH2-ATP was consistent with the altered phosphorylation status of several important cell-signaling molecules in breast cancer cells. By Western blot analysis, 8-NH2-Ado treatment promoted a dramatic loss of ERK1/2 and AKT phosphorylation in both breast cancer cell lines within 6 h. MEK1, 2, 6 and AKT are attractive targets for cancer therapeutics as during tumor progression these kinases promote numerous processes such as inflammation, growth, survival, and therapeutic resistance. We conclude that 8-NH2-Ado by the accumulation of 8-NH2-ATP can directly inhibit MEK1, 2, and 6. This inhibition may contribute to the cytotoxic actions of 8-NH2-Ado in breast cancer cells and other malignancies dependent on the MEK/ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1631.

Osteopontin selectively regulates p70S6K/mTOR phosphorylation leading to NF-κB dependent AP-1-mediated ICAM-1 expression in breast cancer cells

BackgroundBreast cancer is one of the most frequently diagnosed cancer and accounts for over 400,000 deaths each year worldwide. It causes premature death in women, despite progress in early detection, treatment, and advances in understanding the molecular basis of the disease. Therefore, it is important to understand the in depth mechanism of tumor progression and develop new strategies for the treatment of breast cancer. Thus, this study is aimed at gaining an insight into the molecular mechanism by which osteopontin (OPN), a member of SIBLING (Small Integrin Binding LIgand N-linked Glycoprotein) family of protein regulates tumor progression through activation of various transcription factors and expression of their downstream effector gene(s) in breast cancer.ResultsIn this study, we report that purified native OPN induces ICAM-1 expression in breast cancer cells. The data revealed that OPN induces NF-κB activation and NF-κB dependent ICAM-1 expression. We also observed that OPN-induced NF-κB further controls AP-1 transactivation, suggesting that there is cross talk between NF-κB and AP-1 which is unidirectional towards AP-1 that in turn regulates ICAM-1 expression in these cells. We also delineated the role of mTOR and p70S6 kinase in OPN-induced ICAM-1 expression. The study suggests that inhibition of mTOR by rapamycin augments whereas overexpression of mTOR/p70S6 kinase inhibits OPN-induced ICAM-1 expression. Moreover, overexpression of mTOR inhibits OPN-induced NF-κB and AP-1-DNA binding and transcriptional activity. However, rapamycin further enhanced these OPN-induced effects. We also report that OPN induces p70S6 kinase phosphorylation at Thr-421/Ser-424, but not at Thr-389 or Ser-371 and mTOR phosphorylation at Ser-2448. Overexpression of mTOR has no effect in regulation of OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424. Inhibition of mTOR by rapamycin attenuates Ser-371 phosphorylation but does not have any effect on Thr-389 and Thr-421/Ser-424 phosphorylation of p70S6 kinase. However, OPN-induced phosphorylation of p70S6 kinase at Thr-421/Ser-424 is being controlled by MEK/ERK pathway.ConclusionThese results suggest that blocking of OPN-induced ICAM-1 expression through mTOR/p70S6 kinase signaling pathway may be an important therapeutic strategy for the treatment of breast cancer.

Kaposi Sarcoma-associated herpes virus (KSHV) G protein-coupled receptor (vGPCR) activates the ORF50 lytic switch promoter: A potential positive feedback loop for sustained ORF50 gene expression

KSHV vGPCR, a lytic cycle associated protein, induces several signaling pathways leading to the activation of various transcription factors and consequently the expression of cellular and viral genes. Though the role of vGPCR in KSHV tumorigenicity has been well studied, its function related to the viral life cycle is poorly understood. Reduction in vGPCR by RNA interference also resulted in the reduction in KSHV lytic switch ORF50 gene and protein expression. Induction of vGPCR by doxycycline in BC3.14 cells also resulted in more KSHV production. When this was explored, induction of the ORF50 promoter by vGPCR expression was observed. Further examination of the molecular mechanisms by which vGPCR regulates the ORF50 promoter, using various ORF50 promoter constructs, revealed that induction of ORF50 promoter by vGPCR did not involve AP1 but was dependent on Sp1 and Sp3 transcription factors. vGPCR signaling led to an increase in Sp1 and Sp3 DNA binding activity and a decrease in histone deacetylase (HDAC) activity. These activities were pertussis toxin independent, did not involve Rho and Rac-GTPases and involved the heterotrimeric G protein subunits Galpha12 and Galphaq. Studies using pharmacologic inhibitors and dominant-negative proteins identified phospholipase C, the novel protein kinase C (novel PKC) family and protein kinase D (PKD) as part of the signaling initiated by vGPCR leading to ORF50 promoter activation. Taken together, this study suggests a role for vGPCR in the sustained expression of ORF50 which could lead to a continued activation of lytic cycle genes and ultimately to successful viral progeny formation.

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor kappaB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca(2+)/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cgamma2 activation, influx of extracellular Ca(2+) and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

Control of mammalian gene expression by amino acids, especially glutamine

Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic-leucine zipper factors, activating transcription factors and CCAAT/enhancer-binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient-sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient-sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine-responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor-kappaB in the anti-inflammatory action of glutamine, the stimulatory role of activating protein-1 and the inhibitory role of C/EBP homology binding protein in growth-promotion, and the role of c-myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen-activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene transcription remain elusive.

LAP2 ζ binds BAF and suppresses LAP2 β-mediated transcriptional repression

Proteins of the nuclear envelope have been implicated as participating in gene silencing. BAF, a DNA- and LEM domain-binding protein, has been suggested to link chromatin to the nuclear envelope. We have previously shown that LAP2 β, a LEM-domain inner nuclear membrane protein, represses transcription through binding to HDAC3 and induction of histone H4 deacetylation. We now show that LAP2 ζ, the smallest LAP2 family member, is also involved in regulation of transcription. We show that similar to other LEM-domain proteins LAP2 ζ interacts with BAF. LAP2 ζ-YFP and BAF co-localize in the cytoplasm, and overexpression of LAP2 ζ leads to reduction of nucleoplasmic BAF. Mutations in the LAP2 ζ-YFP LEM domain decrease its interaction with BAF retaining the nucleo-cytoplasmic distribution of BAF. Co-expression of LAP2 β and LAP2 ζ results in inhibition of LAP2 β-induced gene silencing while overexpression of LAP2 ζ alone leads to a small increase in transcriptional activity of various transcription factors. Our results suggest that LAP2 ζ is a transcriptional regulator acting predominantly to inhibit LAP2 β-mediated repression. LAP2 ζ may function by decreasing availability of BAF. These findings could have implications in the study of nuclear lamina-associated diseases and BAF-dependent retroviral integration.

Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T Cell Hyperactivation

Symptoms of T cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-kappaB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-kappaB-responsive genes, a function lost in SIRT1-/- cells. These results support a model where the normal function of SIRT1 as a negative regulator of T cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals.

Hypoxia-inducible Factor-1 Mediates Neuronal Expression of the Receptor for Advanced Glycation End Products following Hypoxia/Ischemia

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

The ePHD protein SPBP interacts with TopBP1 and together they co-operate to stimulate Ets1-mediated transcription

SPBP (Stromelysin-1 PDGF responsive element binding protein) is a ubiquitously expressed 220 kDa nuclear protein shown to enhance or repress the transcriptional activity of various transcription factors. A yeast two-hybrid screen, with the extended plant homeodomain (ePHD) of SPBP as bait, identified TopBP1 (topoisomerase II β-binding protein 1) as a candidate interaction partner of SPBP. TopBP1 has eight BRCA1 carboxy-terminal (BRCT) domains and is involved in DNA replication, DNA damage responses and in the regulation of gene expression. The interaction between SPBP and TopBP1 was confirmed in vitro and in vivo, and was found to be mediated by the ePHD domain of SPBP and the BRCT6 domain of TopBP1. Both SPBP and TopBP1 enhanced the transcriptional activity of Ets1 on the c-myc P1P2- and matrix metalloproteinase-3 (MMP3) promoters. Together they displayed a more than additive effect. Both proteins were associated with these promoters. The involvement of TopBP1 was dependent on the serine 1159 phosphorylation site, known to be important for transcriptional activation. Depletion of endogenous SPBP by siRNA treatment reduced MMP3 secretion by 50% in phorbol ester-stimulated human fibroblasts. Taken together, our results show that TopBP1 and SPBP interact physically and functionally to co-operate as co-activators of Ets1.

Curcumin as “Curecumin”: From kitchen to clinic

Although turmeric (Curcuma longa; an Indian spice) has been described in Ayurveda, as a treatment for inflammatory diseases and is referred by different names in different cultures, the active principle called curcumin or diferuloylmethane, a yellow pigment present in turmeric (curry powder) has been shown to exhibit numerous activities. Extensive research over the last half century has revealed several important functions of curcumin. It binds to a variety of proteins and inhibits the activity of various kinases. By modulating the activation of various transcription factors, curcumin regulates the expression of inflammatory enzymes, cytokines, adhesion molecules, and cell survival proteins. Curcumin also downregulates cyclin D1, cyclin E and MDM2; and upregulates p21, p27, and p53. Various preclinical cell culture and animal studies suggest that curcumin has potential as an antiproliferative, anti-invasive, and antiangiogenic agent; as a mediator of chemoresistance and radioresistance; as a chemopreventive agent; and as a therapeutic agent in wound healing, diabetes, Alzheimer disease, Parkinson disease, cardiovascular disease, pulmonary disease, and arthritis. Pilot phase I clinical trials have shown curcumin to be safe even when consumed at a daily dose of 12g for 3 months. Other clinical trials suggest a potential therapeutic role for curcumin in diseases such as familial adenomatous polyposis, inflammatory bowel disease, ulcerative colitis, colon cancer, pancreatic cancer, hypercholesteremia, atherosclerosis, pancreatitis, psoriasis, chronic anterior uveitis and arthritis. Thus, curcumin, a spice once relegated to the kitchen shelf, has moved into the clinic and may prove to be “Curecumin”.

Protein Inhibitor of Activated STAT 3 Modulates Osteoclastogenesis by Down-Regulation of NFATc1 and Osteoclast-Associated Receptor

Protein inhibitor of activated STAT3 (PIAS3) has been shown to regulate the activity of various transcription factors. In this study, we show that the overexpression of PIAS3 in bone marrow-derived monocyte/macrophage lineage cells attenuates osteoclast formation and down-regulates the expression of NFATc1 and osteoclast-associated receptor (OSCAR), which are important modulators in osteoclastogenesis. PIAS3 has been shown to associate with histone deacetylase 1 as well as with transcription factors, including the microphthalmia transcription factor, NFATc1, and c-Fos. Moreover, overexpression of PIAS3 inhibits the transactivation of target genes such as NFATc1 and OSCAR. This inhibitory effect of PIAS3 is possibly mediated by histone deacetylase 1 recruitment to the promoter regions of NFATc1 and OSCAR. Furthermore, silencing of PIAS3 by RNA interference in osteoclast precursors enhances osteoclast formation as well as gene expression of NFATc1 and OSCAR. Taken together, our results reveal that PIAS3 acts as a modulator in osteoclastogenesis.

Ubiquitin Protein Modification and Signal Transduction: Implications for Inflammatory Bowel Diseases

A dysregulated immune response to luminal antigen(s) is associated with the development of inflammatory bowel diseases (IBDs). A complex network of inflammatory and immune mediators released by immune and nonimmune cells participate in the physiopathology of IBD. At the molecular level, events leading to the improper use of the signaling grid are likely responsible for the dysregulated activation of various transcription factors and subsequent induction of inflammatory genes. The posttranslational modification of signaling proteins by the ubiquitin system is a critical event in activation or repression of transcription factors. Two important transcriptional pathways in which ubiquitin is central are the nuclear factor-kappaB and hypoxia inducible factor-1 (HIF-1) pathways, both of which are important components of intestinal homeostasis. In this review, we discuss the role of ubiquitin modification in relation to nuclear factor-kappaB and HIF-1 signaling and consider its impact on intestinal inflammation. A greater understanding of posttranslational ubiquitin modification may lead to the identification of new therapeutic opportunities for the treatment of IBD.

Characterization of the human zinc finger protein 267 promoter: Essential role of nuclear factor Y

Liver fibrosis results from an excessive deposition of extracellular matrix proteins secreted by activated hepatic stellate cells (HSCs). The activation process is accompanied by an increased activity of various transcription factors, including zinc finger protein 267 (ZNF267). Recently, ZNF267 has been shown to modulate gene expression and to function as a transcriptional repressor. MMP-10 was identified as a target gene; its gene expression and promoter activity are inhibited by ZNF267, which might promote liver fibrogenesis through diminished matrix degradation. However, the transcriptional regulation of the ZNF267 gene is unknown. In the present study, we have cloned and characterized the human ZNF267 promoter containing a 1.5 kb fragment of the 5′-flanking region (−1414/+173). The ZNF267 gene has a TATA-less promoter with multiple transcription initiation sites. Analysis of serial 5′-deletions of luciferase reporter constructs revealed a minimal promoter between −72 and +173 bp. Mutational analysis of putative regulatory elements indicated that a CCAAT box within this region was essential for ZNF267 promoter activity. Electrophoretic mobility shift assays demonstrated that transcription factor nuclear factor Y (NF-Y) bound to the CCAAT box. In co-transfection experiments, NF-YA increased the promoter activity of ZNF267. In conclusion, our results suggest that the binding site for NF-Y is critical for ZNF267 gene regulation and, herewith, the activation of this transcriptional factor may play an important role in the activation process of HSCs and in liver fibrosis.

Inhibition of Transcription Factor Activity by Nuclear Compartment-associated Bcl-2

Using a reporter gene assay in PC12, HEK293, HeLa, and NIH-3T3 cells, we show that the anti-apoptotic protein Bcl-2 significantly inhibits transcriptional activation of various transcription factors, including NF kappa B, AP1, CRE, and NFAT. A Bcl-2 mutant lacking its BH4 domain (Delta BH4) also inhibited transcription, whereas a Bcl-2 mutant lacking its transmembrane domain (Delta TM) was ineffective. Furthermore, Bcl-2 chimeric proteins containing transmembrane domains from the mitochondrial protein monoamine oxidase B (MaoB) or the endoplasmic reticulum protein cytochrome b(5) showed no effect on transcription factor activity. Subcellular localization studies showed that under conditions of transient transfection, the active Bcl-2 forms (wild type and Delta BH4) were predominantly found in the nuclear fraction, whereas the non-active forms (Delta TM, MaoB, and cytochrome b(5)) were in the non-nuclear fraction. Additionally, stably expressed Bcl-2 loses its ability to inhibit transcriptional activation and localizes predominantly to the non-nuclear fraction. Expression of FKBP38 (a chaperone that shuttles Bcl-2 to the mitochondria) removes co-expressed Bcl-2 from the nuclear fraction and reverses its effect on transcription factor activity. Finally, using an inducible gene expression system, we show that nuclear compartment-associated Bcl-2 prevents entry of NF kappa B subunits to the nucleus without affecting NF kappa B release from its cytosolic inhibitory sub-unit I kappa B alpha. These results suggest that (a) Bcl-2 suppresses transcriptional activity of multiple transcription factors; (b) Bcl-2 does not interfere with NF kappa B activation but prevents entrance of its active subunits to the nucleus; (c) membrane anchoring is required for this function of Bcl-2; and (d) association of Bcl-2 with the nuclear compartment is also necessary. We speculate that nuclear compartment-associated Bcl-2 may affect nuclear trafficking of multiple factors necessary for transcriptional activity.

Cyclin D Does Not Provide Essential Cdk4-Independent Functions in Drosophila

The three mammalian D-type cyclins are thought to promote progression through the G1 phase of the cell cycle as regulatory subunits of cyclin-dependent kinase 4 and 6. In addition, they have been proposed to control the activity of various transcription factors without a partner kinase. Here we describe phenotypic consequences of null mutations in Cyclin D, the single D-type cyclin gene in Drosophila. As previously observed with null mutations in the single Drosophila Cdk4 gene, these mutations do not primarily affect progression through the G1 phase. Moreover, the apparently indistinguishable phenotypes of double (CycD and Cdk4) and single mutants (CycD or Cdk4) argue against major independent functions of Cyclin D and Cdk4. The reduced cellular and organismal growth rates observed in both mutants indicate that Cyclin D-Cdk4 acts as a growth driver.

Role of calcium and AMP kinase in the regulation of mitochondrial biogenesis and GLUT4 levels in muscle.

Contractile activity induces mitochondrial biogenesis and increases glucose transport capacity in muscle. There has been much research on the mechanisms responsible for these adaptations. The present paper reviews the evidence, which indicates that the decrease in the levels of high-energy phosphates, leading to activation of AMP kinase (AMPK), and the increase in cytosolic Ca(2+), which activates Ca(2+)/calmodulin-dependent protein kinase (CAMK), are signals that initiate these adaptative responses. Although the events downstream of AMPK and CAMK have not been well characterized, these events lead to activation of various transcription factors, including: nuclear respiratory factors (NRF) 1 and 2, which cause increased expression of proteins of the respiratory chain; PPAR-alpha, which up regulates the levels of enzymes of beta oxidation; mitochondrial transcription factor A, which activates expression of the mitochondrial genome; myocyte-enhancing factor 2A, the transcription factor that regulates GLUT4 expression. The well-orchestrated expression of the multitude of proteins involved in these adaptations is mediated by the rapid activation of PPAR gamma co-activator (PGC) 1, a protein that binds to various transcription factors to maximize transcriptional activity. Activating AMPK using 5-aminoimidizole-4-carboxamide-1-beta-D-riboside (AICAR) and increasing cytoplasmic Ca(2+) using caffeine, W7 or ionomycin in L6 myotubes increases the concentration of mitochondrial enzymes and GLUT4 and enhances the binding of NRF-1 and NRF-2 to DNA. AICAR and Ca-releasing agents also increase the levels of PGC-1, mitochondrial transcription factor A and myocyte-enhancing factors 2A and 2D. These results are similar to the responses seen in muscle during the adaptation to endurance exercise and show that L6 myotubes are a suitable model for studying the mechanisms by which exercise causes the adaptive responses in muscle mitochondria and glucose transport.

B-cell antigen receptor activates transcription factors NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB) via a mechanism that involves diacylglycerol.

Engagement of the B-cell antigen receptor (BCR) induces the activation of various transcription factors, including NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB), which participate in long-term biological responses such as proliferation, survival and differentiation of B-lymphocytes. We addressed the biochemical basis of this process using the DT40 chicken B-cell lymphoma. We discovered that Bruton's tyrosine kinase (BTK) and phospholipase C-gamma2 (PLC-gamma2) are required to activate NFAT and NF-kappaB, and to produce the lipid second messenger diacylglycerol in response to BCR cross-linking. Therefore the functional integrity of the BTK/PLC-gamma2/diacylglycerol signalling axis is crucial for BCR-directed activation of both transcription factors NFAT and NF-kappaB.

Parathyroid hormone inhibits c-Jun N-terminal kinase activity in rat osteoblastic cells by a protein kinase A-dependent pathway.

Treatment of osteoblastic cells with PTH initiates dual signaling cascades resulting in activation of both PKA and PKC. It has been shown that PTH either inhibits or stimulates ERKs depending on dose of the hormone; nevertheless, the ability of PTH to regulate other members of the MAPK family is unknown. Another member of this family, c-Jun-NH(2)-terminal kinase (JNK), is preferentially activated by cytokines and cellular stresses and plays a key role in regulating the activity of various transcription factors. We demonstrate that treatment of UMR 106-01 cells and rat calvarial osteoblasts with PTH (10(-8) M), N-terminal peptides of PTH that selectively activate PKA, or 8-bromo-cAMP (activates PKA) results in the inhibition of JNK activity from high basal levels. Examination of the upstream members of the JNK cascade revealed that both stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4 and MAPK/extracellular signal-related kinase kinase kinase 1 activities were also inhibited after treatment with PTH (10(-8) M). We conclude that treatment of osteoblastic cells with PTH is sufficient to inhibit high basal JNK activity by activation of the PKA signaling cascade.

Low density lipoproteins and mitogenic signal transduction processes: role in the pathogenesis of renal disease.

Abnormalities in lipid and lipoprotein metabolism are commonly observed in patients with chronic renal disease. Specifically, hyperlipidemia and the glomerular deposition of atherogenic lipoproteins (e.g., Low density lipoprotein, LDL; and its oxidized variants) are implicated in key pathobiological processes involved in the development of glomerular disease, including stimulation of monocyte infiltration into the mesangial space, mesangial cell hypercellularity, and mesangial extracellular matrix deposition. This review discusses recent understanding of glomerular mitogenic responses, intracellular signaling events associated with mesangial hypercellularity in renal diseases, and the participation of cholesterol and atherogenic lipoproteins in intracellular signaling pathways involved in mesangial cell proliferation. Generally, the mitogenic intracellular signaling pathways are regulated by the activation of series of transmembrane and cytoplasmic protein tyrosine kinases that converge into the activation of Ras and down-stream mitogen-activated protein kinase (MAP kinase). Activated MAP kinase, through translocating into the nucleus and the activation of various transcription factors and protooncogenes, regulate cell proliferation. The importance of mitogenic intracellular signaling in mesangial proliferative disease has only recently been recognized and showed that the activation of MAP kinase and/or cyclin/cyclin-dependent kinases play crucial role in different phases of cell growth cycle and hypercellularity of glomerular cells in various experimental renal diseases. Using glomerular mesangial cells as an in-vitro model system, studies from our laboratory indicated that the accumulation of LDL and more potently its oxidized forms within the glomerulus, through the activation of membrane receptor tyrosine kinases (e.g., EGF receptor), activate Ras and MAP kinase signaling cascade leading to DNA synthesis and subsequent mesangial cell proliferation. These data suggest that atherogenic lipoproteins may act as one of the major endogenous modulators for mitogenic signaling response and cell proliferation within the glomerulus. It is reasonable to speculate that the correction or reduction of hyperlipidemia, glomerular lipid deposition, and the pro-oxidative milieu within the glomerulus, through the inhibition of mitogenic signaling events, may provide protective environment against mesangial hypercellularity and subsequent matrix deposition, and the progression of renal disease.

Key role of a downstream specificity protein 1 site in cell cycle-regulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells.

Abasic (apurinic/apyrimidinic or AP) sites are a frequent type of DNA damage that threatens genetic stability. The predominant mammalian enzyme initiating repair of AP sites is the Ape1 AP endonuclease (also called Apex or Hap1), which also facilitates DNA binding by several transcription factors (Ref1 activity). We found that expression of the APE1 gene was coordinated with the cell cycle in murine NIH3T3 cells: APE1 mRNA levels rose after the G(1)-S transition and peaked approximately 4-fold higher in early to mid-S phase. The increased APE1 mRNA was the result of transcriptional activation rather than increased mRNA stability. Fusions of various APE1 promoter fragments to the chloramphenicol acetyltransferase CAT reporter gene indicated that APE1 expression depends on two transcription factor Sp1 binding sites within the promoter region. Mutation of these sites or of two CCAAT elements within the APE1 promoter, in conjunction with protein binding studies, demonstrated their specific roles. The Sp1 site upstream of the transcription start, together with an adjacent CCAAT element, establishes a protein-DNA complex required for basal transcription of APE1. The Sp1 site downstream of the transcription start was required for the response to cell growth. Because Ape1 is a dual function enzyme, its cell cycle-dependent expression might affect both DNA repair and the activity of various transcription factors as a function of the cell cycle.