Biomedical subjects

Michael Dragunow

Publications and source records attributed to Michael Dragunow.

9 recordsLinked to original sources

PubMed2006

Activation of activating transcription factor 2 by p38 MAP kinase during apoptosis induced by human amylin in cultured pancreatic beta-cells.

Amylin-mediated islet beta-cell death is implicated in diabetogenesis. We previously reported that fibrillogenic human amylin (hA) evokes beta-cell apoptosis through linked activation of Jun N-terminal kinase 1 (JNK 1) and a caspase cascade. Here we show that p38 kinase [p38 mitogen-activated protein (MAP) kinase] became activated by hA treatment of cultured beta-cells whereas extracellular signal-regulated kinase (ERK) did not; by contrast, nonfibrillogenic rat amylin (rA) altered neither. Pretreatment with the p38 kinase-inhibitor SB203580 decreased hA-induced apoptosis and caspase-3 activation by approximately 30%; as did combined SB203580 and JNK inhibitor I, by about 70%; and the combination of SB203580, the JNK inhibitor I and a caspase-8 inhibitor, by 100%. These findings demonstrate the requirement for concurrent activation of the p38 kinase, JNK and caspase-8 pathways. We further showed that hA elicits time-dependent activation of activating transcription factor 2 (ATF-2), which was largely suppressed by SB203580, indicating that this activation is catalyzed mainly by p38 kinase. Furthermore, hA-induced apoptosis was suppressed by specific antisense ATF-2, and increased phospho-ATF-2 (p-ATF-2) was associated with increased CRE (cAMP-response element) DNA binding and CRE-mediated transcriptional activity, as well as enhancement of c-jun promoter activation. We also detected changes in the phosphorylation status and composition of the CRE complex that may play important roles in regulation of distinct downstream target genes. These studies establish p38 MAP kinase-mediated activation of ATF-2 as a significant mechanism in hA-evoked beta-cell death, which may serve as a target for pharmaceutical intervention and effective suppression of beta-cell failure in type-2 diabetes.

Activating Transcription Factor 2↗

PubMed2004

Over expression of ATF-3 protects rat hippocampal neurons from in vivo injection of kainic acid.

ATF-3 is a member of the ATF superfamily of transcription factors and is strongly associated with episodes of cellular stress. We demonstrate an association between increases in ATF-3 protein and resistance to exitotoxic cell death in vivo. Intra-hippocampal injection of kainic acid elicited a robust increase in endogenous ATF-3 within kainate-resistant cells of the dentate gyrus, while overexpression of exogenous ATF-3 was found to protect vulnerable CA3 neurons from the same insult. These results suggest a positive contribution to neuronal survival in the context of stress-induced death, and support an anti-apoptotic role for ATF-3 in the brain.

Activating Transcription Factor 3↗

PubMed2003

ATF3 enhances c-Jun-mediated neurite sprouting.

The AP-1 transcription factor c-Jun is induced in axotomized neurons of the peripheral and central nervous systems, and in both cases upregulation of c-Jun expression has been correlated with axonal regeneration. More recently there has been interest in the c-Jun-related bZIP transcription factor, ATF3, and its function in neurons. ATF3 is also induced in nerve cells in response to axotomy and there is a correlation between increased ATF3 expression and upregulation of c-Jun in surviving neurons. Moreover, c-Jun is able to induce expression of ATF3. We investigated the effect of co-expressing c-Jun and ATF3 in two neuronal-like cell lines to model transcriptional events occurring in axotomized neurons undergoing regeneration. We show that expression of ATF3 with c-Jun significantly enhances c-Jun-mediated neurite sprouting, and that this phenotype is most likely mediated by a physical association of these two transcription factors. Our results suggest that a program of axonal regeneration is initiated when both c-Jun and ATF3 are upregulated in neurons in response to axotomy.

Activating Transcription Factor 3↗

PubMed2003

Fibrillogenic amylin evokes islet beta-cell apoptosis through linked activation of a caspase cascade and JNK1.

Fibrillogenic human amylin elicits pancreatic beta-cell apoptosis that may contribute to development of type-2 diabetes. Here, we demonstrated that activation of a caspase cascade is necessary for induction of apoptosis by fibrillogenic amylin variants in two pancreatic beta-cell lines. Human amylin, as well as truncated 8-37human amylin, evoked sequential activation of caspases-8 and -3, and apoptosis, whereas non-beta-sheet forming and non-fibrillogenic homologs, such as [25,28,29triprolyl]human amylin, did not, implying that the beta-sheet conformer is required for human amylin-induced caspase activation. Significant inhibition of apoptosis was evoked by a selective caspase-1 inhibitor, indicating that caspase-1 is also essential for activation of the caspase cascade. Furthermore, we showed that specific jnk1 antisense oligonucleotides, which suppress phospho-JNK1 expression, effectively decreased human amylin-induced activation of c-Jun. Studies of the interplay between the caspase cascade and the JNK pathway showed that both apoptosis and caspase-3 activation were suppressed by treatment with a JNK inhibitor and by transfection of antisense jnk1 oligonucleotides or antisense-c-jun, whereas a selective inhibitor of caspases-1 and -3 prevented apoptosis but not c-Jun activation. Thus, the JNK1 activation preceded activation of caspases-1 and -3. However, selective JNK inhibition had no effect on caspase-8 activation, and selective caspase-8 inhibition only partially suppressed apoptosis and c-Jun activation, indicating that caspase-8 may partially act upstream of the JNK pathway. Our studies demonstrate a functional interaction of a caspase cascade and JNK1. Fibrillogenic amylin can evoke a JNK1-mediated apoptotic pathway, which is partially dependent and partially independent of caspase-8, and in which caspase-3 acts as a common downstream effector.

Amyloid↗

PubMed2003

Increased cell proliferation and neurogenesis in the adult human Huntington's disease brain.

Neurogenesis has recently been observed in the adult human brain, suggesting the possibility of endogenous neural repair. However, the augmentation of neurogenesis in the adult human brain in response to neuronal cell loss has not been demonstrated. This study was undertaken to investigate whether neurogenesis occurs in the subependymal layer (SEL) adjacent to the caudate nucleus in the human brain in response to neurodegeneration of the caudate nucleus in Huntington's disease (HD). Postmortem control and HD human brain tissue were examined by using the cell cycle marker proliferating cell nuclear antigen (PCNA), the neuronal marker beta III-tubulin, and the glial cell marker glial fibrillary acidic protein (GFAP). We observed a significant increase in cell proliferation in the SEL in HD compared with control brains. Within the HD group, the degree of cell proliferation increased with pathological severity and increasing CAG repeats in the HD gene. Most importantly, PCNA+ cells were shown to coexpress beta III-tubulin or GFAP, demonstrating the generation of neurons and glial cells in the SEL of the diseased human brain. Our results provide evidence of increased progenitor cell proliferation and neurogenesis in the diseased adult human brain and further indicate the regenerative potential of the human brain.

Adult↗

PubMed2002

Increased expression and activation of c-Jun contributes to human amylin-induced apoptosis in pancreatic islet beta-cells.

The role of c-Jun in apoptosis evoked by human amylin was investigated using human and rat insulinoma beta-cell lines. Two transient increases in the levels of c-jun mRNA were detected at 30 minutes and eight hours after treatment with human amylin. The level of c-Jun protein was also up-regulated in a time-dependent manner, reaching maximal levels after eight hours of exposure. However, no c-Jun induction was detected in cells treated with vehicle only or with rat amylin, indicating that the amyloidogenic feature of the human peptide may be important for c-Jun induction. We found that c-Jun was activated by phosphorylation specifically at Ser63 at four hours, but not at Ser73, after treatment with human amylin, preceding increased c-Jun protein. Furthermore, expression of an antisense c-jun (AS-c-jun), which suppressed protein levels of both c-Jun and phosphorylated-c-Jun, caused a marked reduction in apoptotic cell death, whereas the corresponding sense c-jun (S-c-jun) had no effect on changes of either c-Jun production or apoptosis. This indicated that increased expression and activation of c-Jun is required for human amylin-induced apoptosis. Immunocytochemical studies showed a significant increase in nuclear staining for c-Jun, phosphorylated-c-Jun (Ser63) and phosphorylated-JNK, suggesting that c-Jun may be activated through activation of JNK. In addition, electrophoretic mobility-shift assays showed that the increase in expression and phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays demonstrated that c-Jun, c-Fos and ATF-2 are part of the AP-1 complex, indicating that activated c-Jun is dimerized with c-Fos or ATF-2 for control of its target gene expression. Finally, we showed that human amylin triggers AP-1-mediated transcriptional activation. Our results suggest strongly that human amylin induces apoptosis through stimulation of expression and activation of c-Jun, and that co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process.

Amyloid↗

PubMed2002

Induction and experience-dependent consolidation of stable long-term potentiation lasting months in the hippocampus.

Long-term potentiation (LTP) is widely regarded as a memory mechanism, but it is not known whether it can last long enough to underlie very long-term memory. We report that high-frequency stimulation (HFS) paradigms applied to the rat dentate gyrus can elicit stable LTP lasting months and up to at least 1 year. The induction of stable LTP was sensitive to stimulation variables on the day of HFS and was associated with phosphorylation of cAMP response element-binding protein. The maintenance of stable LTP was also experience-dependent, because it was reversed when animals were exposed repeatedly to an enriched environment beginning 14 d post-HFS. However, stable LTP eventually consolidated over time and became resistant to reversal, because exposure to enriched environments 90 d post-HFS failed to influence stable LTP maintenance. Thus, LTP can be shown to meet one of the principal criteria for a very long-term memory storage mechanism. However, under naturalistic environmental conditions, LTP may normally be retained in the hippocampus for only short periods of time.

Animals↗

PubMed2002

Muscarinic receptor-mediated phosphorylation of cyclic AMP response element binding protein in human neuroblastoma cells.

This study describes the effect of signalling through muscarinic acetylcholine receptors on two transcription factors implicated in long-term synaptic plasticity and memory formation, EGR1 and the cyclic AMP response element binding protein (CREB). In SK-N-SH neuroblastoma cells, treatment with the cholinergic agonist carbachol led to maximal induction of EGR1 1 h after stimulation. This was preceded by the phosphorylation of CREB, which peaked as early as 5 minutes after carbachol treatment. The levels of both EGR1 and phosphorylated CREB (pCREB) slowly decayed over 4-8 h. CREB phosphorylation and EGR1 induction showed similar sensitivity to carbachol concentration, with EC(50) values in the range of 1-10 microM, and the changes in both transcription factors were blocked by the muscarinic antagonist atropine. As has been described elsewhere, EGR1 induction was dependent on activation of p42/44 MAP kinase, as it was blocked by the MEK inhibitor U0126. However, CREB phosphorylation by carbachol was largely unaffected by MAP kinase blockade. As both CREB phosphorylation and EGR1 induction have been linked to long-term potentiation and some forms of memory consolidation, these results may implicate CREB and EGR1 in independent or partially independent cholinergic signalling pathways involved in memory processes.

Atropine↗

PubMed2002

Selective down-regulation of c-jun gene expression by pentoxifylline and c-jun antisense interrupts platelet-derived growth factor signaling: pentoxifylline inhibits phosphorylation of c-Jun on serine 73.

Platelet-derived growth factor (PDGF) signals through several pathways, including mitogen-activated protein (MAP) kinase, Jun kinase, and C kinase, and stimulates proliferation of fibroblasts. Pentoxifylline inhibits PDGF-driven proliferation of fibroblasts. We have reported that pentoxifylline did not inhibit binding of PDGF to its specific cell-surface receptors or PDGF receptor phosphorylation. In this study, we investigated the effect of PDGF on the expression of c-fos and c-jun, because c-fos and c-jun form activator protein-1 complexes that stimulate genes involved in proliferation. We determined whether pentoxifylline would alter the expression of c-fos and c-jun. Our results indicate that PDGF induced the expression of both c-fos and c-jun. Pentoxifylline effectively reduced c-jun gene expression, which had been up-regulated by PDGF, but did not alter c-fos gene expression. The lack of effect on c-fos supports other studies from this laboratory, which indicate that pentoxifylline did not inhibit PDGF activation of MAP kinase. Treatment of fibroblasts with a phosphothioate c-jun antisense oligodeoxynucleotide reduced the levels of c-Jun protein and blocked PDGF-stimulated proliferation, suggesting a critical role for c-jun in PDGF-mediated proliferation. Combination of pentoxifylline and c-jun antisense suggested that they were likely inhibiting PDGF-stimulated proliferation at a single site in the PDGF signaling pathway. These results suggest that pentoxifylline inhibits PDGF-stimulated proliferation by selectively decreasing c-jun expression. To further define the mechanism of action of pentoxifylline, we assessed the effect of pentoxifylline on c-Jun and phosphorylated c-Jun immunoreactivity in cells treated with PDGF and cells that were transfected with wild-type c-jun plasmid using immunocytochemistry and Western blot analyses, and our results indicate that pentoxifylline inhibited phosphorylation of c-Jun on serine 73.

Animals↗