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1.
Mol Cell Biol ; 21(14): 4713-24, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11416147

ABSTRACT

Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.


Subject(s)
Apoptosis , Leucine Zippers , MAP Kinase Kinase 4 , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neurons/cytology , Protein Serine-Threonine Kinases , Animals , Caspase Inhibitors , Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Activation , Gene Expression , Humans , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 7 , MAP Kinase Kinase Kinase 5 , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/physiology , Mitogen-Activated Protein Kinase Kinases/metabolism , Myristic Acid/metabolism , Nerve Growth Factor/metabolism , Neurons/metabolism , PC12 Cells , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rats , Signal Transduction , Sympathetic Nervous System/cytology , Ultraviolet Rays , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , Mitogen-Activated Protein Kinase Kinase Kinase 11
2.
J Neurochem ; 77(3): 849-63, 2001 May.
Article in English | MEDLINE | ID: mdl-11331414

ABSTRACT

Although the mechanism of neuronal death in Alzheimer's disease (AD) has yet to be elucidated, a putative role for c-jun in this process has emerged. Thus, it was of interest to delineate signal transduction pathway(s) which regulate the transcriptional activity of c-jun, and relate these to alternate gene inductions and biochemical processes associated with beta-amyloid (Abeta) treatment. In this regard, the survival promoting activity of CEP-1347, an inhibitor of the stress-activated/c-jun N-terminal (SAPK/JNK) kinase pathway, was evaluated against Abeta-induced cortical neuron death in vitro. Moreover, CEP-1347 was used as a pharmacologic probe to associate multiple biochemical events with Abeta-induced activation of the SAPK/JNK pathway. CEP-1347 promoted survival and blocked Abeta-induced activation of JNK kinase (MKK4, also known as MEK-4, JNKK and SEK1) as well as other downstream events associated with JNK pathway activation. CEP-1347 also blocked Abeta-induction of cyclin D1 and DP5 genes and blocked Abeta-induced increases in cytoplasmic cytochrome c, caspase 3-like activity and calpain activation. The critical time window for cell death blockade by CEP-1347 resided within the peak of Abeta-induced MKK4 activation, thus defining this point as the most upstream event correlated to its survival-promoting activity. Together, these data link the SAPK/JNK pathway and multiple biochemical events associated with Abeta-induced neuronal death and further delineate the point of CEP-1347 interception within this signal transduction cascade.


Subject(s)
Amyloid beta-Peptides/pharmacology , Apoptosis/drug effects , Carbazoles/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neurons/physiology , Amyloid beta-Peptides/genetics , Animals , Calpain/metabolism , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Cytochrome c Group/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Enzyme Activation/drug effects , Female , Gene Expression/drug effects , L-Lactate Dehydrogenase/metabolism , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/metabolism , Neurons/ultrastructure , Pregnancy , Rats , Rats, Sprague-Dawley
3.
Brain Res ; 780(2): 294-303, 1998 Jan 12.
Article in English | MEDLINE | ID: mdl-9507169

ABSTRACT

Inflammatory processes may play a critical role in the pathogenesis of the degenerative changes and cognitive impairments associated with Alzheimer's disease (AD). In the present study, lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria was used to produce chronic, global inflammation within the brain of young rats. Chronic infusion of LPS (0.25 microgram/h) into the 4th ventricle for four weeks produced (1) an increase in the number of glial fibrillary acidic protein-positive activated astrocytes and OX-6-positive reactive microglia distributed throughout the brain, with the greatest increase occurring within the temporal lobe, particularly the hippocampus, (2) an induction in interleukin-1 beta, tumor necrosis factor-alpha and beta-amyloid precursor protein mRNA levels within the basal forebrain region and hippocampus, (3) the degeneration of hippocampal CA3 pyramidal neurons, and (4) a significant impairment in spatial memory as determined by decreased spontaneous alternation behavior on a T-maze.


Subject(s)
Alzheimer Disease/immunology , Neuritis/immunology , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Astrocytes/metabolism , Choline O-Acetyltransferase/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/immunology , Glial Fibrillary Acidic Protein/genetics , Glutamate Decarboxylase/genetics , Interleukin-1/genetics , Interleukin-6/genetics , Lipopolysaccharides , Maze Learning , Microglia/metabolism , Neuritis/chemically induced , Neuritis/metabolism , Neurons/chemistry , Neurons/enzymology , Prosencephalon/immunology , Prosencephalon/metabolism , RNA, Messenger/analysis , Rats , Rats, Inbred F344 , Tumor Necrosis Factor-alpha/genetics
4.
J Neurosci ; 16(13): 4146-54, 1996 Jul 01.
Article in English | MEDLINE | ID: mdl-8753876

ABSTRACT

Although the interleukin-1beta converting enzyme (ICE)/CED-3 family of proteases has been implicated recently in neuronal cell death in vitro and in ovo, the role of specific genes belonging to this family in cell death in the nervous system remains unknown. To address this question, we examined the in vivo expression of one of these genes, Ice, after global forebrain ischemia in gerbils. Using RT-PCR and Western immunoblot techniques, we detected an increase in the mRNA and protein expression of ICE in hippocampus during a period of 4 d after ischemia. Chromatin condensation was observed in CA1 neurons within 2 d after ischemia. Internucleosomal DNA fragmentation and apoptotic bodies were observed between 3 and 4 d after ischemia, a period during which CA1 neuronal death is maximal. In nonischemic brains, ICE-like immunoreactivity was relatively low in CA1 pyramidal neurons but high in scattered hippocampal interneurons. After ischemia, ICE-like immunoreactivity was not altered in these neurons. ICE-like immunoreactivity, however, was observed in microglial cells in the regions adjacent to the CA1 layer as early as 2 d after ischemic insult. The increase in ICE-like immunoreactivity was robust at 4 d after ischemia, a period that correlates with the DNA fragmentation observed in hippocampal homogenates of ischemic brains. These results provide the first evidence for the localization and induction of ICE expression in vivo after ischemia and suggest an indirect role for ICE in ischemic damage through mediation of an inflammatory response.


Subject(s)
Brain Ischemia/enzymology , Cysteine Endopeptidases/metabolism , Hippocampus/enzymology , Animals , Apoptosis , Base Sequence , Caspase 1 , Cysteine Endopeptidases/genetics , Gerbillinae , Hippocampus/pathology , Immunohistochemistry , Male , Microglia/enzymology , Molecular Probes/genetics , Molecular Sequence Data , Neurons/pathology , RNA, Messenger/metabolism , Time Factors , Tissue Distribution
5.
Oncogene ; 10(5): 1003-7, 1995 Mar 02.
Article in English | MEDLINE | ID: mdl-7898917

ABSTRACT

The Rel/NF-kappa B family transcriptional factors plays an important role in the regulation of immune and acute phase responses. The activity of Rel/NF-kappa B complexes is regulated by their interactions with members of the I kappa B family of inhibitors. We have previously shown that the RelB/p52 heterodimer is not effectively inhibited by any of the known I kappa B molecules: I kappa B alpha, I kappa B gamma and Bcl3. Here we report that the C-terminal domain of p100 (the putative I kappa B delta) functions as a strong inhibitor of RelB/p52 transcriptional activity. In vivo interaction with I kappa B delta leads to the cytoplasmic retention and decreased DNA binding activity of RelB/p52 complexes. Thus, I kappa B delta is the only I kappa B molecule able to efficiently modulate the activity of RelB/p52 heterodimer. In Daudi cells, a 46 kD protein, probably representing the C-terminal product of the proteolytic processing of p100, remains associated with Rel/NF-kappa B complexes and might have a transient regulatory function. Our results indicate a specific role for the putative I kappa B delta and suggest a possible mechanism of how the truncation of the ankyrin domain of p100, found in a number of lymphoid neoplasias, might contribute to tumorigenesis.


Subject(s)
NF-kappa B/physiology , Proto-Oncogene Proteins , Transcription Factors/antagonists & inhibitors , Cell Line , DNA/metabolism , Humans , NF-kappa B/antagonists & inhibitors , NF-kappa B p52 Subunit , Transcription Factor RelB , Transcription, Genetic
6.
EMBO J ; 13(19): 4608-16, 1994 Oct 03.
Article in English | MEDLINE | ID: mdl-7925301

ABSTRACT

The Rel-NF-kappa B family of transcription factors plays a crucial role in the regulation of genes involved in inflammatory and immune responses. We demonstrate that in vivo, in contrast to the other members of the family, RelB associates efficiently only with NF-kappa B1 (p105-p50) and NF-kappa B2 (p100-p52), but not with cRel or p65. The RelB-p52 heterodimers display a much lower affinity for I kappa B alpha than RelB-p50 heterodimers or p65 complexes. However, similarly to the other Rel-NF-kappa B complexes, RelB-p52 can upregulate the synthesis of I kappa B alpha leading to the cytoplasmic trapping of dimers which have a higher affinity for the inhibitor. We suggest that a hierarchy of interactions between I kappa B alpha and the different Rel-NF-kappa B complexes governs their cellular distribution. This results in the presence of two distinct pools of NF-kappa B activity which differ in their composition: one a constitutive nuclear and the other an inducible cytoplasmic activity.


Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Proteins , NF-kappa B/metabolism , Proto-Oncogene Proteins , Transcription Factors/metabolism , B-Lymphocytes/metabolism , Biopolymers , Cell Line , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/physiology , Gene Expression Regulation/physiology , Humans , NF-KappaB Inhibitor alpha , NF-kappa B/physiology , Protein Binding , Transcription Factor RelB , Transcription Factors/physiology
7.
Mol Cell Biol ; 13(3): 1572-82, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8441398

ABSTRACT

RelB, a member of the Rel family of transcription factors, can stimulate promoter activity in the presence of p50-NF-kappa B or p50B/p49-NF-kappa B in mammalian cells. Transcriptional activation analysis reveals that the N and C termini of RelB are required for full transactivation in the presence of p50-NF-kappa B. RelB/p50-NF-kappa B hybrid molecules containing the Rel homology domain of p50-NF-kappa B and the N and C termini of RelB have high transcriptional activity compared with wild-type p50-NF-kappa B. The N and C termini of RelB cooperate in transactivation in cis or trans configuration. Alterations in the structure of the leucine zipper-like motif present in the N terminus of RelB significantly decrease the transcriptional capacity of RelB and of different RelB/p50-NF-kappa B hybrid molecules.


Subject(s)
Leucine Zippers , Retroviridae Proteins, Oncogenic/genetics , Trans-Activators/genetics , Transcription, Genetic , Transcriptional Activation/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell-Free System , Cells, Cultured , DNA-Binding Proteins , Humans , Mice , Molecular Sequence Data , Oncogene Proteins v-rel , Structure-Activity Relationship , Transfection
8.
Mol Cell Biol ; 12(6): 2898-908, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1588976

ABSTRACT

The liver is one of the few adult tissues that has the capacity to regenerate following hepatectomy or toxic damage. In examining the early growth response during hepatic regeneration, we found that a highly induced immediate-early gene in regenerating liver encodes RL/IF-1 (regenerating liver inhibitory factor) and is the rat homolog of human MAD-3 and probably of chicken pp40. RL/IF-1 has I kappa B activity of broad specificity in that it inhibits the binding of p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50, but not p50 homodimeric NF-kappa B, to kappa B sites. Like RL/IF-1, several members of the NF-kappa B and rel family of transcription factors are immediate-early genes in regenerating liver and mitogen-treated cells. We examined changes in kappa B site binding activity during liver regeneration and discovered a rapidly induced novel kappa B site-binding complex designated PHF [posthepatectomy factor(s)]. PHF is induced over 1,000-fold within minutes posthepatectomy in a protein synthesis-independent manner, with peak activity at 30 min, and is not induced by sham operation. PHF is distinct from p50-p65 NF-kappa B, which is present only in the inactive form in liver posthepatectomy. Although early PHF complexes do not interact strongly with anti-p50 antibodies, PHF complexes present later (3 to 5 h) posthepatectomy react strongly, suggesting that they contain a p50 NF-kappa B subunit. Unlike p50-p65 NF-kappa B, c-Rel-p50, and RelB-p50 complexes, PHF binding to kappa B sites is not inhibited by RL/IF-1. One role of RL/IF-1 in liver regeneration may be to inhibit p50-p65 NF-kappa B activity present in hepatic cells, allowing for the preferential binding of PHF to kappa B sites. Because PHF is induced immediately posthepatectomy in the absence of de novo protein synthesis, PHF could have a role in the regulation of liver-specific immediate-early genes in regenerating liver.


Subject(s)
DNA-Binding Proteins/metabolism , Liver Regeneration , NF-kappa B/antagonists & inhibitors , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Female , Gene Expression , I-kappa B Proteins , Macromolecular Substances , Molecular Sequence Data , NF-KappaB Inhibitor alpha , NF-kappa B/metabolism , Oligodeoxyribonucleotides/chemistry , Proto-Oncogene Proteins c-rel , RNA, Messenger/genetics , Rats , Sequence Alignment , Time Factors
9.
Mol Cell Biol ; 12(2): 674-84, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1732739

ABSTRACT

We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family. Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain. The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity. RelB does not bind with high affinity to NF-kappa B sites, but heterodimers between RelB and p50-NF-kappa B do bind to different NF-kappa B-binding sites with a similar affinity to that shown by p50-NF-kappa B homodimers. However, RelB/p50-NF-kappa B heterodimers, in contrast to p50-NF-kappa B homodimers, transactivate transcription of a promoter containing a kappa B-binding site.


Subject(s)
NF-kappa B/metabolism , Oncogenes/genetics , Retroviridae Proteins, Oncogenic/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , DNA-Binding Proteins , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation/physiology , Mice , Molecular Sequence Data , Multigene Family/genetics , Oncogene Proteins v-rel , Precipitin Tests , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retroviridae Proteins, Oncogenic/chemistry , Retroviridae Proteins, Oncogenic/genetics , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
10.
Cell Biophys ; 15(1-2): 21-7, 1989.
Article in English | MEDLINE | ID: mdl-2476224

ABSTRACT

In studies on adenovirus promoters, predominantly on the late E2A promoter of adenovirus type 2 (Ad2), we have demonstrated by a number of experimental approaches that the sequence-specific methylation of three 5'-CCGG-3' sequences inactivates this promoter. Recently, we have developed a cell-free transcription system in which the methylation-inactivation of eukaryotic promoters can be studied in detail. It has also been shown that methylation-caused promoter inactivation can be reversed by the 289 amino acid E1A protein of Ad2 or of adenovirus type 5. In the presence of this protein with a transactivating effect, transcription is initiated at the authentic cap site of the methylated late E2A promoter. A similar reactivation of the methylated late E2A promoter can also be effected by a cis-acting genetic element, i.e., the strong enhancer of human cytomegalovirus. Further studies will be directed toward the biochemical mechanisms of promoter silencing by sequence-specific methylations.


Subject(s)
Adenoviruses, Human/genetics , DNA, Viral/metabolism , Gene Expression Regulation , Promoter Regions, Genetic , Base Sequence , Cytomegalovirus/genetics , DNA, Viral/genetics , Enhancer Elements, Genetic , Genes, Viral , Humans , Methylation , Transcription, Genetic
12.
J Virol ; 62(11): 3941-6, 1988 Nov.
Article in English | MEDLINE | ID: mdl-2459407

ABSTRACT

Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific promoter methylations necessitated the development of a cell-free transcription system that responded to in vitro promoter methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, the activities of two adenovirus type 2 promoters in the nonmethylated and methylated forms were compared. The late E2A promoter in vitro methylated at three 5'-CCGG-3' (HpaII) sequences at nucleotides -215, +6, and +24, or the major late promoter in vitro methylated at nucleotide -52 in the 5'-CCGG-3' sequence or at nucleotide -13 in the 5'-GCGC-3' (HhaI) sequence exhibited strikingly lower activities than did the nonmethylated constructs or exhibited no activity at all. The designated nucleotide positions were calculated relative to the cap sites of the two promoters. Upon in vitro transcription, the methylation pattern of the E2A late promoter was shown to be stable. For the inhibitory effects by sequence-specific methylations to be elicited, circular templates had to be used, the DNA titers had to be at critical levels for each extract, and high protein concentrations had to be maintained. When a template mixture of the nonmethylated major late promoter and the 5'-CCGG-3' methylated late E2A promoter of adenovirus type 2 DNA was used, the major late promoter was active and the methylated late E2A promoter was inhibited or inactivated. Activity levels of the two different promoters could be assessed simultaneously in the same assay due to differences in lengths between the products of transcription from the late E2A and major late promoters. Thus inhibition in the cell-free system could be proven to be specific for the methylated promoter. We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and somehow participate in their inactivation.


Subject(s)
Adenoviridae/genetics , DNA, Viral/metabolism , Gene Expression Regulation , Promoter Regions, Genetic , Adenoviridae/metabolism , Base Sequence , Cell-Free System , DNA-Binding Proteins/metabolism , Endonucleases , HeLa Cells , Humans , Methylation , RNA/analysis , Single-Strand Specific DNA and RNA Endonucleases , Templates, Genetic
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