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J L Martindale

Publications and source records attributed to J L Martindale.

11 recordsLinked to original sources
PubMed2001

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state.

gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.

Animals↗
PubMed2000

Requirement for ERK activation in cisplatin-induced apoptosis.

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Complexes containing activating transcription factor (ATF)/cAMP-responsive-element-binding protein (CREB) interact with the CCAAT/enhancer-binding protein (C/EBP)-ATF composite site to regulate Gadd153 expression during the stress response.

Gadd153, also known as chop, encodes a member of the CCAAT/enhancer-binding protein (C/EBP) transcription factor family and is transcriptionally activated by cellular stress signals. We recently demonstrated that arsenite treatment of rat pheochromocytoma PC12 cells results in the biphasic induction of Gadd153 mRNA expression, controlled in part through binding of C/EBPbeta and two uncharacterized protein complexes to the C/EBP-ATF (activating transcription factor) composite site in the Gadd153 promoter. In this report, we identified components of these additional complexes as two ATF/CREB (cAMP-responsive-element-binding protein) transcription factors having differential binding activities dependent upon the time of arsenite exposure. During arsenite treatment of PC12 cells, we observed enhanced binding of ATF4 to the C/EBP-ATF site at 2 h as Gadd153 mRNA levels increased, and enhanced binding of ATF3 complexes at 6 h as Gadd153 expression declined. We further demonstrated that ATF4 activates, while ATF3 represses, Gadd153 promoter activity through the C/EBP-ATF site. ATF3 also repressed ATF4-mediated transactivation and arsenite-induced activation of the Gadd153 promoter. Our results suggest that numerous members of the ATF/CREB family are involved in the cellular stress response, and that regulation of stress-induced biphasic Gadd153 expression in PC12 cells involves the ordered, sequential binding of multiple transcription factor complexes to the C/EBP-ATF composite site.

Animals↗
PubMed1999

Modulation of CCAAT/enhancer-binding protein-alpha gene expression by metabolic signals in rodent adipocytes.

The transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) is a positive modulator of transcription for several adipocyte-specific genes that play a role in energy metabolism. However, there is little information available regarding the regulation of its expression by metabolic signals. Exposure to insulin for 5-24 h attenuated C/EBPalpha expression when 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in 5.7 mM glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBPalpha gene, but not that of C/EBPbeta. Glucosamine, a product of the hexosamine pathway, in the presence of low glucose mimicked high glucose's ability to reduce C/EBPalpha messenger RNA expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites (e.g. UDP-N-acetylhexosamines) and/or changes in intracellular protein glycosylation with the ability of high glucose, glucosamine, or xylitol to down-regulate C/EBPalpha gene expression. None of these treatments caused a reduction in intracellular ATP levels. Stable transfection of 3T3-L1 cells with the 5'-flanking 468-bp sequence of the mouse C/EBPalpha gene fused to luciferase demonstrated that promoter activity was also reduced by these nutrients. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBPalpha messenger RNA levels in epididymal, but not omental, fat. Taken together, these results suggest that metabolic signals serve to down-regulate C/EBPalpha expression both in vitro and in vivo.

3T3 Cells↗
PubMed1998

The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival.

The mammalian response to stress is complex, often involving multiple signalling pathways that act in concert to influence cell fate. To examine potential interactions between the signalling cascades, we have focused on the effects of a model oxidant stress in a single cell type through an examination of the relative influences of mitogen-activated protein kinases (MAPKs) as well as two proposed apoptosis regulators, nuclear factor kappaB (NF-kappaB) and Bcl-2, in determining cell survival. Treatment of HeLa cells with H2O2 resulted in a time- and dose-dependent induction of apoptosis accompanied by sustained activation of all three MAPK subfamilies: extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38. This H2O2-induced apoptosis was markedly enhanced when ERK2 activation was selectively inhibited by PD098059. Apoptosis decreased when JNK/SAPK activation was inhibited by expression of a dominant negative mutant form of SAPK/ERK kinase 1. Inhibition of the p38 kinase activity with p38-specific inhibitors SB202190 and SB203580 had no effect on cell survival. Because NF-kappaB activation by H2O2 is potentially related to both the ERK and JNK/SAPK signalling pathways, we examined the effects of inhibiting the activation of NF-kappaB; this interference had no effect on the cellular response to H2O2. Overexpression of the anti-apoptotic protein Bcl-2 significantly decreased the apoptosis seen after treatment with H2O2 without altering ERK or JNK/SAPK activities. Our results suggest that ERK and JNK/SAPK act in opposition to influence cell survival in response to oxidative stress, whereas neither p38 nor NF-kappaB affects the outcome. Bcl-2 acts independently and downstream of ERK and JNK/SAPK to enhance the survival of H2O2-treated cells.

3T3 Cells↗
PubMed1998

Tumor promoter arsenite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc.

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

gadd153/Chop10, a potential target gene of the transcriptional repressor ATF3.

Recently, we demonstrated that the function of ATF3, a stress-inducible transcriptional repressor, is negatively regulated by a bZip protein, gadd153/Chop10. In this report, we present evidence that ATF3 can repress the expression of its own inhibitor, gadd153/Chop10. First, ATF3 represses a chloramphenicol acetyltransferase reporter gene driven by the gadd153/Chop10 promoter when assayed by a transfection assay in vivo and a transcription assay in vitro. Second, the gadd153/Chop10 promoter contains two functionally important binding sites for ATF3: an AP-1 site and a C/EBP-ATF composite site, a previously unidentified binding site for ATF3. The absence of either site reduces the ability of ATF3 to repress the promoter. Third, overexpression of ATF3 by transient transfection results in a reduction of the endogenous gadd153/Chop10 mRNA level. Fourth, as described previously, ATF3 is induced in the liver upon CCl4 treatment. Intriguingly, we show in this report that gadd153/Chop10 mRNA is not present in areas where ATF3 is induced. Taken together, these results strongly suggest that ATF3 represses the expression of gadd153/Chop10. The mutual negative regulation between ATF3 and gadd153/Chop10 is discussed.

Activating Transcription Factor 3↗
PubMed1996

Physical and functional association between GADD153 and CCAAT/enhancer-binding protein beta during cellular stress.

GADD153, a ubiquitously expressed member of the CCAAT/enhancer-binding protein (C/EBP) family is induced by a wide variety of growth-arresting and DNA-damaging agents. Functionally, GADD153 has been postulated to act as a dominant-negative regulator of C/EBPs. Therefore we sought to gain evidence for interactions between GADD153 and other C/EBPs during cellular responses to stress. In this report we have demonstrated that treatment of rat pheochromocytoma PC12 cells with sodium arsenite leads to enhanced expression of C/EBP-beta and GADD153 (growth arrest and DNA damage inducible gene 153) but not other C/EBPs. Coimmunoprecipitation experiments provided evidence for the formation of endogenous GADD153-C/EBP-beta complexes in arsenite-treated cells. Additional experiments were performed to determine the role of such complexes in regulating GADD153 expression. Previous studies in our laboratory demonstrated that the GADD153 promoter contains a C/EBP binding site through which other C/EBPs interact to transactivate GADD153 expression in liver hepatoma cells. Here, we demonstrate that extracts prepared from arsenite-treated PC12 cells likewise show increased amounts of factors capable of binding to the GADD153-C/EBP site and that these complexes are comprised at least in part of C/EBP-beta. Forced expression of C/EBP-beta was found to be capable of transactivating the GADD153 promoter in PC12 cells cotransfected with plasmids expressing a GADD153 reporter gene and C/EBP-beta protein. However, overexpression of GADD153 inhibited the transactivation of the GADD153 promoter by C/EBP-beta. These findings provide evidence for an autoregulatory loop in which stress-induced GADD153 feeds back to attenuate GADD153 expression during the cellular response to stress.

Adrenal Gland Neoplasms↗
PubMed1996

Regulation of p21WAF1/CIP1 expression through mitogen-activated protein kinase signaling pathway.

p21WAF1/CIP1 is a cyclin-dependent kinase inhibitor whose expression in mammalian tissues is highly induced in response to stress as well as during normal development and differentiation. Induction of p21WAF1/CIP1 in response to DNA damage occurs through a transcriptional mechanism that is dependent on the activation of the tumor suppressor protein p53. Recent evidence indicates that p21WAF1/CIP1 can also be induced independently of p53, but the signal transduction mechanisms involved in regulating p21WAF1/CIP1 expression in these situations have not been elucidated. In this study, we have addressed the role of the mitogen-activated protein kinase signaling pathway in the induction of p21WAF1/CIP1 in response to growth factor treatment. Using an experimental approach involving cotransfection of a p21WAF1/CIP1 promoter-luciferase construct with a variety of plasmids expressing dominant positive or dominant negative mutant proteins involved in this signaling pathway, we provide evidence to support a role for mitogen-activated protein kinase in the transcriptional activation of p21WAF1/CIP1 by growth factor stimulation.

Animals↗
PubMed1984

Subtle enablers.

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Alcoholics Anonymous↗