scholarly journals Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol.

1987 ◽  
Vol 7 (8) ◽  
pp. 2974-2976 ◽  
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Ionophore A23187 ◽  
Grp78 Promoter ◽  
Steady State Levels ◽  
Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol

1987 ◽  
Vol 7 (8) ◽  
pp. 2974-2976
Author(s):  
Y K Kim ◽  
A S Lee
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Protein Glycosylation ◽  
Temperature Sensitive ◽  
Ionophore A23187 ◽  
Grp78 Promoter ◽  
Steady State Levels ◽  
Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

scholarly journals Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1362-1370 ◽  
Author(s):  
JO Hensold ◽  
G Dubyak ◽  
DE Housman
Keyword(s):  
Transcriptional Activation ◽  
Calcium Ionophore ◽  
Erythroid Differentiation ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cellular Processes ◽  
Cellular Events ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Abstract Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

scholarly journals Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1362-1370 ◽  
Author(s):  
JO Hensold ◽  
G Dubyak ◽  
DE Housman
Keyword(s):  
Transcriptional Activation ◽  
Calcium Ionophore ◽  
Erythroid Differentiation ◽  
Cytosolic Calcium ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cellular Processes ◽  
Cellular Events ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.

Complex regulation of heat shock- and glucose-responsive genes in human cells

1988 ◽  
Vol 8 (1) ◽  
pp. 393-405
Author(s):  
S S Watowich ◽  
R I Morimoto
Keyword(s):  
Transcriptional Activation ◽  
Posttranscriptional Regulation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Hsp70 Expression ◽  
Gene Probe ◽  
Ionophore A23187 ◽  
Glycoprotein Processing ◽  
Cloned Gene ◽  
Hsp70 Synthesis

We have isolated a human genomic clone that encodes the glucose-responsive protein GRP78 and have used this cloned gene probe, together with a cloned HSP70 gene, to study the expression of both stress-induced genes in response to inhibitors of cellular metabolism. On the basis of the effects of this group of chemicals on GRP78 and HSP70 expression, we have identified three classes of stress gene inducers. The first class induces GRP78 expression and includes inhibitors of glycoprotein processing. The second class results in coordinate activation of both GRP78 and HSP70 synthesis and includes amino acid analogs and heavy metals. Chemicals in the third class coordinately induce GRP78 and repress HSP70 expression; this class includes the calcium ionophore A23187 and the glucose analog 2-deoxyglucose. Whereas induction of GRP78 or HSP70 expression is primarily due to transcriptional activation, chemicals that repress HSP70 expression act through posttranscriptional regulation. These results reveal that the regulation of GRP78 and HSP70 expression is complex and may be dependent on the specificity and magnitude of physiological damage.

scholarly journals Calcium ionophore A23187 as a regulator of gene expression in mammalian cells.

1986 ◽  
Vol 103 (6) ◽  
pp. 2145-2152 ◽  
Author(s):  
E Resendez ◽  
J Ting ◽  
K S Kim ◽  
S K Wooden ◽  
A S Lee
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Similar Response ◽  
Calmodulin Antagonist ◽  
Induction Process ◽  
Continuous Presence ◽  
Lower Magnitude

The calcium ionophore A23187 can reversibly induce the expression of two glucose-regulated genes, p3C5 and p4A3. This induction requires a continuous presence of the ionophore for over 2 h. Although extracellular Ca2+ is important for the optimal effect of A23187, it is not necessary for the induction, since a similar response with a lower magnitude can be triggered in cells cultured in low Ca2+ medium buffered with EGTA. Both the basal and induced levels of p3C5 and p4A3 transcripts can be modulated by the calmodulin antagonist W-7, indicating the involvement of Ca2+/calmodulin-associated pathways. In addition, the sensitivity of the A23187 induction to cycloheximide suggests that the induction process is dependent on de novo protein synthesis.

scholarly journals Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I.

1991 ◽  
Vol 11 (11) ◽  
pp. 5612-5623 ◽  
Author(s):  
S K Wooden ◽  
L J Li ◽  
D Navarro ◽  
I Qadri ◽  
L Pereira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Promoter Activity ◽  
Calcium Ionophore ◽  
Regulatory Elements ◽  
Proximal Region ◽  
Calcium Ionophore A23187 ◽  
Viral Envelope ◽  
Transcriptional Level ◽  
Ionophore A23187 ◽  
Grp78 Promoter

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.

Transactivation of the grp78 promoter by malfolded proteins, glycosylation block, and calcium ionophore is mediated through a proximal region containing a CCAAT motif which interacts with CTF/NF-I

1991 ◽  
Vol 11 (11) ◽  
pp. 5612-5623
Author(s):  
S K Wooden ◽  
L J Li ◽  
D Navarro ◽  
I Qadri ◽  
L Pereira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Promoter Activity ◽  
Calcium Ionophore ◽  
Regulatory Elements ◽  
Proximal Region ◽  
Calcium Ionophore A23187 ◽  
Viral Envelope ◽  
Transcriptional Level ◽  
Ionophore A23187 ◽  
Grp78 Promoter

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.

scholarly journals Complex regulation of heat shock- and glucose-responsive genes in human cells.

1988 ◽  
Vol 8 (1) ◽  
pp. 393-405 ◽  
Author(s):  
S S Watowich ◽  
R I Morimoto
Keyword(s):  
Transcriptional Activation ◽  
Posttranscriptional Regulation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Hsp70 Expression ◽  
Gene Probe ◽  
Ionophore A23187 ◽  
Glycoprotein Processing ◽  
Cloned Gene ◽  
Hsp70 Synthesis

We have isolated a human genomic clone that encodes the glucose-responsive protein GRP78 and have used this cloned gene probe, together with a cloned HSP70 gene, to study the expression of both stress-induced genes in response to inhibitors of cellular metabolism. On the basis of the effects of this group of chemicals on GRP78 and HSP70 expression, we have identified three classes of stress gene inducers. The first class induces GRP78 expression and includes inhibitors of glycoprotein processing. The second class results in coordinate activation of both GRP78 and HSP70 synthesis and includes amino acid analogs and heavy metals. Chemicals in the third class coordinately induce GRP78 and repress HSP70 expression; this class includes the calcium ionophore A23187 and the glucose analog 2-deoxyglucose. Whereas induction of GRP78 or HSP70 expression is primarily due to transcriptional activation, chemicals that repress HSP70 expression act through posttranscriptional regulation. These results reveal that the regulation of GRP78 and HSP70 expression is complex and may be dependent on the specificity and magnitude of physiological damage.

scholarly journals 12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

2020 ◽  
Vol 295 (16) ◽  
pp. 5307-5320 ◽  
Author(s):  
Xinping Liu ◽  
Harold F. Sims ◽  
Christopher M. Jenkins ◽  
Shaoping Guan ◽  
Beverly G. Dilthey ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Oxidation Products ◽  
Therapeutic Interventions ◽  
Enzymatic Oxidation ◽  
Ionophore A23187 ◽  
Genetic Ablation ◽  
Age Related ◽  
12 Lipoxygenase

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

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