The Experts below are selected from a list of 3135 Experts worldwide ranked by ideXlab platform
Gary L Firestone - One of the best experts on this subject based on the ideXlab platform.
-
indole 3 carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor alpha and Sp1 Transcription Factor interactions within the htert promoter and mediates the g1 cell cycle arrest of human breast cancer cells
Carcinogenesis, 2011Co-Authors: Crystal N Marconett, Leonard F Bjeldanes, Shyam N Sundar, Min Tseng, Kalvin Q Tran, Kelly Mahuron, Gary L FirestoneAbstract:Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G1 cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 Transcription Factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element–Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1–DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven Transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.
-
indole 3 carbinol and 3 3 diindolylmethane antiproliferative signaling pathways control cell cycle gene Transcription in human breast cancer cells by regulating promoter Sp1 Transcription Factor interactions
Journal of Nutrition, 2003Co-Authors: Gary L Firestone, Leonard F BjeldanesAbstract:Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1) gene expression. Construction and transfection of a series of promoter-reporter plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1) levels are due to specific effects on their corresponding promoters. Mutagenic analysis reveals that I3C signaling targets a composite Transcriptional element in the Cdk6 promoter that requires both Sp1 and Ets Transcription Factors for transactivation function. Analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1 DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein complex that contains the Sp1 Transcription Factor. In indole-treated cells, a fraction of [(3)H]I3C was converted into its natural diindole product (3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus; this suggests that DIM may have a role in the Transcriptional activities of I3C. Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) Transcription through an indole-responsive region of the promoter that contains multiple Sp1 consensus sequences. Furthermore, DIM treatment regulates the presence of a nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and p21(Waf1/Cip1) promoters are newly defined downstream targets of the indole-signaling pathway, and that the observed Transcriptional effects are due to a combination of the cellular activities of I3C and DIM.
-
Indole-3-Carbinol and 3-3′-Diindolylmethane Antiproliferative Signaling Pathways Control Cell-Cycle Gene Transcription in Human Breast Cancer Cells by Regulating Promoter–Sp1 Transcription Factor Interactions
Journal of Nutrition, 2003Co-Authors: Gary L Firestone, Leonard F BjeldanesAbstract:Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1) gene expression. Construction and transfection of a series of promoter-reporter plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1) levels are due to specific effects on their corresponding promoters. Mutagenic analysis reveals that I3C signaling targets a composite Transcriptional element in the Cdk6 promoter that requires both Sp1 and Ets Transcription Factors for transactivation function. Analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1 DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein complex that contains the Sp1 Transcription Factor. In indole-treated cells, a fraction of [(3)H]I3C was converted into its natural diindole product (3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus; this suggests that DIM may have a role in the Transcriptional activities of I3C. Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) Transcription through an indole-responsive region of the promoter that contains multiple Sp1 consensus sequences. Furthermore, DIM treatment regulates the presence of a nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and p21(Waf1/Cip1) promoters are newly defined downstream targets of the indole-signaling pathway, and that the observed Transcriptional effects are due to a combination of the cellular activities of I3C and DIM.
-
indole 3 carbinol inhibits cdk6 expression in human mcf 7 breast cancer cells by disrupting Sp1 Transcription Factor interactions with a composite element in the cdk6 gene promoter
Journal of Biological Chemistry, 2001Co-Authors: Erin J Cram, Leonard F Bjeldanes, Gary L FirestoneAbstract:Abstract Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G1 cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of theCDK6 promoter in order to determine whether I3C inhibits CDK6 Transcription. In MCF-7 cells stably transfected withCDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between −805 and −638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 Transcription Factor. Taken together, our results suggest that I3C down-regulates CDK6 Transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.
-
hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum and glucocorticoid inducible protein kinase sgk by a p38 mapk dependent pathway
Journal of Biological Chemistry, 2000Co-Authors: Lisa M Bell, Meredith L Leong, Eddie Wang, Jongsun Park, Brian A. Hemmings, Gary L FirestoneAbstract:Abstract We have established that the serum- and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5′ deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation ofsgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 Transcription Factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction ofsgk promoter activity or protein production. Using anin vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.
Masahiko Kurabayashi - One of the best experts on this subject based on the ideXlab platform.
-
dimethylarginine dimethylaminohydrolase 2 increases vascular endothelial growth Factor expression through Sp1 Transcription Factor in endothelial cells
Arteriosclerosis Thrombosis and Vascular Biology, 2006Co-Authors: Kazuhiro Hasegawa, Shu Wakino, Toru Tanaka, Masumi Kimoto, Satoru Tatematsu, Takeshi Kanda, Kyoko Yoshioka, Koichiro Homma, Naoki Sugano, Masahiko KurabayashiAbstract:Objectives— Dimethylarginie dimethylaminohydrolase (DDAH) is a degrading enzyme for asymmetrical dimethylarginine, an endogenous NO synthase inhibitor. The molecular mechanism for DDAH-induced vascular endothelial growth Factor (VEGF) expression was examined. Methods and Results— Although the transfection of expression vectors for 2 isoforms of DDAH, DDAH1, or DDAH2 increased DDAH activity in bovine aortic endothelial cells and human umbilical vein endothelial cells, expression and secretion of VEGF were increased only in DDAH2-transfected cells. Knocking down the DDAH2 gene reduced VEGF production, and DDAH2 overexpression enhanced both proliferation and migration of endothelial cells. The VEGF promoter activity was increased by DDAH2 transfection, which was not blocked by an NO synthase (NOS) inhibitor but required the Sp1 sites. DDAH2 overexpression increased nuclear protein levels bound to Sp1 oligonucleotides in endothelial cells. Sp1 small interfering RNA blocked DDAH2-induced upregulation of VEGF. DDAH2 transfection increased nuclear and threonine-phosphorylation levels of Sp1 in a protein kinase A (PKA)–dependent manner. Protein–protein interaction between DDAH2 and PKA was enhanced in DDAH2-transfected cells. Conclusions— DDAH2 upregulated the expression of VEGF through Sp1-dependent and NO/NOS system-independent promoter activation. DDAH2-increased Sp1 DNA binding activity was PKA dependent. These mechanisms may provide a novel therapeutic strategy for VEGF-related vasculopathies such as atherosclerosis.
Robert H Horvitz - One of the best experts on this subject based on the ideXlab platform.
-
an Sp1 Transcription Factor coordinates caspase dependent and independent apoptotic pathways
Nature, 2013Co-Authors: Takashi Hirose, Robert H HorvitzAbstract:Removal of cells during development in Caenorhabditis elegans requires the precise execution of cell-death programs, which can include both caspase-dependent and -independent pathways; here it is shown that a single upstream Transcription Factor can drive both, in parallel, to destroy a single cell. The removal of cells during development requires the precise execution of cell-death programs, which can include both caspase-dependent and caspase-independent pathways. Takashi Hirose and H. Robert Horvitz report that a single upstream Factor can drive both, in parallel, to destroy a single cell. They show in C. elegans that the Transcription Factor SPTF-3 not only drives the Transcription of the egl-1 gene, which promotes apoptosis through the activation of caspases, but also the Transcription of pig-1, which codes for a protein kinase and kills cells in a caspase-independent manner. Thus different cell-killing pathways can be coordinated by a single Transcription Factor. The authors suggest that such 'cell-death nodes' might be important therapeutic targets for diseases caused by excessive cell death. During animal development, the proper regulation of apoptosis requires the precise spatial and temporal execution of cell-death programs, which can include both caspase-dependent and caspase-independent pathways1,2. Although the mechanisms of caspase-dependent and -independent cell killing have been examined extensively, how these pathways are coordinated within a single cell that is fated to die is unknown. Here we show that the Caenorhabditis elegans Sp1 Transcription Factor SPTF-3 specifies the programmed cell deaths of at least two cells—the sisters of the pharyngeal M4 motor neuron and the AQR sensory neuron—by Transcriptionally activating both caspase-dependent and -independent apoptotic pathways. SPTF-3 directly drives the Transcription of the gene egl-1, which encodes a BH3-only protein that promotes apoptosis through the activation of the CED-3 caspase3. In addition, SPTF-3 directly drives the Transcription of the AMP-activated protein kinase-related gene pig-1, which encodes a protein kinase and functions in apoptosis of the M4 sister and AQR sister independently of the pathway that activates CED-3 (refs 4, 5). Thus, a single Transcription Factor controls two distinct cell-killing programs that act in parallel to drive apoptosis. Our findings reveal a bivalent regulatory node for caspase-dependent and -independent pathways in the regulation of cell-type-specific apoptosis. We propose that such nodes might act as features of a general mechanism for regulating cell-type-specific apoptosis and could be therapeutic targets for diseases involving the dysregulation of apoptosis through multiple cell-killing mechanisms.
Muhammad Zafarullah - One of the best experts on this subject based on the ideXlab platform.
-
Role of Sp1 Transcription Factor in Interleukin-1-induced ADAMTS-4 (aggrecanase-1) gene expression in human articular chondrocytes
Rheumatology International, 2011Co-Authors: Judith Sylvester, Rasheed Ahmad, Muhammad ZafarullahAbstract:Proinflammatory cytokines such as interleukin-1 beta (IL-1β) stimulate cartilage extracellular matrix aggrecan degradation by aggrecanases or ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) during the pathogenesis of arthritis. Human aggrecanase-1 (ADAMTS-4) gene promoter contains at least one specificity protein-1 (Sp1)-Transcription Factor–binding site. We investigated the previously unknown role of Sp1 in the regulation of ADAMTS-4 gene expression in human articular chondrocytes. Mithramycin and WP631, the specific inhibitors of guanine cytosine (GC)-rich Sp1 DNA binding, partially suppressed IL-1-induced ADAMTS-4 expression and activity. Genetic inhibition of Sp1 by antisense oligonucleotide or by small interfering RNA (siRNA)-mediated Sp1 knockdown partially inhibited ADAMTS-4 induction by IL-1. Sense oligonucleotide and negative control siRNA had no effect. In contrast, cytomegalovirus promoter–driven Sp1 overexpression further enhanced IL-1-induced ADAMTS-4 expression and activity. Constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not affected by any of the agents. These results provide pharmacological and genetic evidence for the importance of Sp1 in ADAMTS-4 gene regulation by IL-1. Thus, Sp1 could be potentially targeted to reduce arthritis-associated cartilage aggrecan loss.
-
Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in human chondrocytes
Cellular Signalling, 2007Co-Authors: Hamid Yaqoob Qureshi, Judith Sylvester, Rasheed Ahmad, Muhammad ZafarullahAbstract:Abstract Transforming growth Factor beta (TGF-β1) induces cartilage extracellular matrix synthesis and tissue inhibitor of metalloproteinases-3 (TIMP-3), an important natural inhibitor of matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme, which are implicated in cartilage degradation and joint inflammation. This study tested the hypothesis that Akt/protein kinase B signaling pathway could mediate TGF-β1 induction of TIMP-3 in human articular chondrocytes. TGF-β activated phosphorylation of Akt in a delayed and sustained fashion that correlated with TIMP-3 mRNA induction. Phosphatidylinositol kinase (PI3K) inhibitors, Wortmannin and LY294002 and Akt inhibitor (NL-71-101) significantly inhibited TGF-β-induced Akt phosphorylation, TIMP-3 expression, TIMP-3 promoter (− 940 to + 376)-driven luciferase activity and Sp1 Transcription Factor binding. PI3K p85, Akt and Sp1 small interfering RNA (siRNA)-driven knockdown of the respective gene products significantly suppressed TGF-β-induced TIMP-3 gene expression. TGF-β-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-β induces TIMP-3 gene expression in human chondrocytes partly through PI3K/Akt pathway and Sp1 Transcription Factor and by translational mechanisms via mammalian target of rapamycin (mTOR) signaling. TGF-β induction of pro-survival Akt cascade and TIMP-3 may be related to strengthening of cartilage extracellular matrix, increased chondrocyte viability and maintenance of joint tissue integrity.
-
tgf β induced expression of tissue inhibitor of metalloproteinases 3 gene in chondrocytes is mediated by extracellular signal regulated kinase pathway and Sp1 Transcription Factor
Journal of Cellular Physiology, 2005Co-Authors: Hamid Yaqoob Qureshi, Judith Sylvester, Mohammed El Mabrouk, Muhammad ZafarullahAbstract:Transforming growth Factor (TGF-β1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 Transcription Factor in TGF-β-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-β time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF-β-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Sp1-binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF-β-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-β-stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (−940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-β-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Sp1 Transcription Factor play a pivotal role in the induction of TIMP-3 by TGF-β in chondrocytes. © 2004 Wiley-Liss, Inc.
Kazuhiro Hasegawa - One of the best experts on this subject based on the ideXlab platform.
-
dimethylarginine dimethylaminohydrolase 2 increases vascular endothelial growth Factor expression through Sp1 Transcription Factor in endothelial cells
Arteriosclerosis Thrombosis and Vascular Biology, 2006Co-Authors: Kazuhiro Hasegawa, Shu Wakino, Toru Tanaka, Masumi Kimoto, Satoru Tatematsu, Takeshi Kanda, Kyoko Yoshioka, Koichiro Homma, Naoki Sugano, Masahiko KurabayashiAbstract:Objectives— Dimethylarginie dimethylaminohydrolase (DDAH) is a degrading enzyme for asymmetrical dimethylarginine, an endogenous NO synthase inhibitor. The molecular mechanism for DDAH-induced vascular endothelial growth Factor (VEGF) expression was examined. Methods and Results— Although the transfection of expression vectors for 2 isoforms of DDAH, DDAH1, or DDAH2 increased DDAH activity in bovine aortic endothelial cells and human umbilical vein endothelial cells, expression and secretion of VEGF were increased only in DDAH2-transfected cells. Knocking down the DDAH2 gene reduced VEGF production, and DDAH2 overexpression enhanced both proliferation and migration of endothelial cells. The VEGF promoter activity was increased by DDAH2 transfection, which was not blocked by an NO synthase (NOS) inhibitor but required the Sp1 sites. DDAH2 overexpression increased nuclear protein levels bound to Sp1 oligonucleotides in endothelial cells. Sp1 small interfering RNA blocked DDAH2-induced upregulation of VEGF. DDAH2 transfection increased nuclear and threonine-phosphorylation levels of Sp1 in a protein kinase A (PKA)–dependent manner. Protein–protein interaction between DDAH2 and PKA was enhanced in DDAH2-transfected cells. Conclusions— DDAH2 upregulated the expression of VEGF through Sp1-dependent and NO/NOS system-independent promoter activation. DDAH2-increased Sp1 DNA binding activity was PKA dependent. These mechanisms may provide a novel therapeutic strategy for VEGF-related vasculopathies such as atherosclerosis.