scholarly journals Cell-shape-dependent modulation of p52(PAI-1) gene expression involves a secondary response pathway

1995 ◽  
Vol 306 (2) ◽  
pp. 497-504 ◽  
Author(s):  
P J Higgins ◽  
L Staiano-Coico ◽  
M P Ryan
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Activation ◽  
Actinomycin D ◽  
De Novo ◽  
Rat Kidney ◽  
Secondary Response ◽  
Protein Levels ◽  
Run Off ◽  
Dependent Pathway ◽  
Pai 1

Expression of the rat p52(PAI-1) gene is positively regulated by agents that influence cellular microfilament organization and/or cell-to-substrate adhesion [e.g. cytochalasin D (CD) and sodium n-butyrate (NaB)] [Higgins, Chaudhari and Ryan (1991) Biochem. J. 273, 651-658; Higgins, Ryan and Providence (1994) J. Cell. Physiol. 159, 187-195]. As shape-responsive genes may be subject to inducer-specific controls, the biochemical mechanisms underlying the shape-dependent pathway of p52(PAI-1) gene regulation were examined in v-ras-transformed rat kidney (KNRK) cells. NaB and/or CD effectively stimulated p52(PAI-1) run-off transcription and augmented de novo p52(PAI-1) mRNA and protein synthesis in KNRK cells; induction at both the mRNA and protein levels was inhibited by actinomycin D. Pretreatment with cycloheximide (CX) markedly attenuated NaB- and/or CD-stimulated p52(PAI-1) expression. CX alone, however, induced low levels of p52(PAI-1) mRNA; increased p52(PAI-1) protein synthesis was evident after release of KNRK cells from CX blockade. Such CX-mediated induction was also sensitive to actinomycin D. Full stimulation of p52(PAI-1) expression in KNRK cells in response to the shape modulators NaB and/or CD involves transcriptional activation of the p52(PAI-1) gene, requires de novo RNA synthesis and occurs through a secondary-response (i.e. protein-synthesis-dependent) pathway.

scholarly journals Cell-shape-associated transcriptional activation of the p52(PAI-1) gene in rat kidney cells

1992 ◽  
Vol 288 (3) ◽  
pp. 1017-1024 ◽  
Author(s):  
P J Higgins ◽  
M P Ryan ◽  
A Ahmed
Keyword(s):  
Growth Factors ◽  
Transcriptional Activation ◽  
Cell Shape ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Rat Kidney ◽  
Regulatory Element ◽  
Mrna Abundance ◽  
Matrix Deposition ◽  
Pai 1

The microfilament-disrupting agent cytochalasin D (CD) increased (by 10-22-fold) the synthesis de novo and extracellular matrix deposition of plasminogen-activator inhibitor type-1 [p52(PAI-1)] in normal rat kidney (NRK) cells. Transition from a flat to a round phenotype occurred concomitantly with, and may actually precede, p52(PAI-1) induction; both the morphological and p52(PAI-1) responses were dose-dependent. Augmented synthesis became evident between 4 and 5 h of treatment of NRK cells with 100 microM-CD, correlating with a transition from 25 to more than 60% rounded cells. CD-associated increases in p52(PAI-1) mRNA abundance and protein biosynthesis were maximal between 6 and 8 h of continuous CD exposure, declined by 50% thereafter, but remained elevated (by at least 6-21-fold respectively over control values) for 24 h. Changes in p52(PAI-1) mRNA abundance at this 24 h point reflected an approx. 5-fold increase in p52(PAI-1)-gene transcription. These data confirm previous suggestions, based on actinomycin D-sensitivity of the inductive response [Higgins & Ryan (1992) Biochem. J. 284, 433-439], that CD-mediated increases in p52(PAI-1) expression are at least partly due to transcription-level events. Since CD also augments specific cellular responses to growth factors or cytokines, the potential effectiveness of this inducer was evaluated both in the presence and absence of serum growth factors using quiescent NRK cells [a growth state in which p52(PAI-1) is not expressed] as a model system. Induction of p52(PAI-1) synthesis and matrix deposition in CD-stimulated quiescent NRK cells was as efficient under growth-factor-deficient conditions as when CD was added simultaneously with serum. CD alone is thus a complete inducer of p52(PAI-1) expression in NRK cells, an observation that supports the contention that cell shape is an important regulatory element in p52(PAI-1)-gene control.

Lipopolysaccharide-induced sensitization of adenylyl cyclase activity in murine macrophages

2006 ◽  
Vol 290 (1) ◽  
pp. C143-C151 ◽  
Author(s):  
Y. Osawa ◽  
H. T. Lee ◽  
C. A. Hirshman ◽  
D. Xu ◽  
C. W. Emala
Keyword(s):  
Protein Synthesis ◽  
Adenylyl Cyclase ◽  
Actinomycin D ◽  
Inhibitory Effect ◽  
Cytokine Release ◽  
Adenylyl Cyclase Activity ◽  
Phosphorylation State ◽  
Murine Macrophages ◽  
Dependent Pathway ◽  
New Protein

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, Giproteins, and NF-κB translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5–10,000 ng/ml)- and time (1–8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-κB inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-κB-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-κB had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive.

Glucocorticoids inhibit IL-1β-induced GM-CSF expression at multiple levels: roles for the ERK pathway and repression by MKP-1

2010 ◽  
Vol 427 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Robert Newton ◽  
Elizabeth M. King ◽  
Wei Gong ◽  
Christopher F. Rider ◽  
Karl J. Staples ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Transcriptional Repression ◽  
Actinomycin D ◽  
De Novo ◽  
A549 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Erk Pathway ◽  
Gm Csf ◽  
Erk Phosphorylation

In the present study, IL (interleukin)-1β increased GM-CSF (granulocyte/macrophage colony-stimulating factor) expression from pulmonary A549 cells and primary HBE (human bronchial epithelial) cells. These responses were repressed by the glucocorticoid dexamethasone, allowing the use of A549 cells as a relevant model. IL-1β induced GM-CSF release into the culture medium by 6 h and in cell lysates (cytosolic) at 2 h. These effects were profoundly inhibited by dexamethasone, yet IL-1β-induced GM-CSF mRNA and unspliced nRNA (nuclear RNA; a surrogate of transcription rate) were modestly inhibited by dexamethasone at times up to 2 h. Although this indicates an effect on protein synthesis, actinomycin D chase experiments also indicated post-transcriptional repression by dexamethasone. Dexamethasone-dependent mRNA repression increased with time and was prevented by translational blockade. In addition, dexamethasone and the dissociated steroid RU24858 repressed GM-CSF release in an actinomycin D-sensitive manner, thereby implicating glucocorticoid-induced gene expression. At 2 h, IL-1β-induced expression of GM-CSF protein, but not mRNA, was sensitive to the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitors PD098059 and U0126. Although this indicates a role for the MEK/ERK pathway in GM-CSF translation, PD098059 subsequently destabilized GM-CSF mRNA. Dexamethasone and RU24858 both reduced IL-1β-induced ERK phosphorylation and increased MKP-1 (MAPK phosphatase-1) expression. Inhibition of ERK phosphorylation was reproduced by MKP-1 overexpression and prevented by MKP-1-targeting siRNA (small interfering RNA). Since MKP-1 prevented GM-CSF expression by transcriptional, post-transcriptional and translational processes, we propose that glucocorticoids induce MKP-1 expression to reduce both MEK/ERK activation and GM-CSF protein synthesis. Thus de novo gene expression, particularly of MKP-1, is involved in the repressive effects of glucocorticoids.

scholarly journals Activation of the inducible orphan receptor gene nur77 by serum growth factors: dissociation of immediate-early and delayed-early responses.

1993 ◽  
Vol 13 (10) ◽  
pp. 6124-6136 ◽  
Author(s):  
G T Williams ◽  
L F Lau
Keyword(s):  
Protein Synthesis ◽  
Growth Factors ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
De Novo ◽  
Early Gene ◽  
Immediate Early ◽  
Early Component ◽  
Early Expression ◽  
De Novo Protein Synthesis

We have characterized the genetic elements that mediate the transcriptional activation of nur77, a growth factor-inducible gene encoding a member of the steroid/thyroid hormone receptor superfamily. Although initially identified as a serum-inducible immediate-early gene with expression kinetics similar to those of c-fos, we found that transcriptional activation of nur77 by serum growth factors in fibroblasts is in fact composed of two components: an immediate-early component, which can occur in the absence of de novo protein synthesis, and a delayed-early component, which is dependent on de novo protein synthesis. The expression of nur77 following serum stimulation reflects the superimposition of immediate-early and delayed-early expression. Immediate-early and delayed-early expression can be dissociated from one another by deletion or base substitution mutations of the nur77 promoter. Immediate-early expression of nur77 is mediated primarily by sequences located between nucleotides -86 and -126 upstream of the transcription start site. This region includes a sequence that resembles but differs from the CArG element found in other serum-inducible promoters. Upstream of the CArG-like element is a potential binding site for a transcription factor of the Ets family; the presence of this site is required for significant transcriptional induction. Delayed-early expression of nur77 is mediated by multiple AP-1-like and GC-rich elements, which can interact with products of immediate-early genes such as Fos/Jun and Zif268, respectively. Furthermore, we show that Zif268 can activate transcription of the nur77 promoter, suggesting that it may play a role in the delayed-early expression of nur77.

Studies on the nature of the seromucoid and haptoglobin responses to experimental inflammation

1968 ◽  
Vol 46 (5) ◽  
pp. 477-481 ◽  
Author(s):  
M. Maung ◽  
D. G. Baker ◽  
R. K. Murray
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
De Novo ◽  
De Novo Synthesis ◽  
Experimental Inflammation ◽  
Haptoglobin Level

The effects of the administration of actinomycin D, ethionine, and puromycin on the elevations of the total seromucoid fraction and of one of its components (haptoglobin) occurring during experimental inflammation have been studied. All three inhibitors of protein synthesis abolished the elevation of haptoglobin level. Ethionine and puromycin also completely suppressed the elevation of total seromucoid level, whereas actinomycin D only partially suppressed it. The seromucoid and haptoglobin levels in control animals injected with only the inhibitors of protein synthesis were not in general significantly different from those of the animals injected with turpentine and these agents. The results are consistent with the concept that the elevation of various plasma glycoproteins occurring during inflammation is principally due to de novo synthesis of these proteins rather than release of preformed proteins from tissue pools.

scholarly journals Apoptosis Triggered by Myc-Induced Suppression of Bcl-XL or Bcl-2 Is Bypassed during Lymphomagenesis

2001 ◽  
Vol 21 (15) ◽  
pp. 5063-5070 ◽  
Author(s):  
Christine M. Eischen ◽  
David Woo ◽  
Martine F. Roussel ◽  
John L. Cleveland
Keyword(s):  
Protein Synthesis ◽  
Transgenic Mice ◽  
Tumor Cells ◽  
De Novo ◽  
Apoptotic Pathway ◽  
Antiapoptotic Protein ◽  
Protein Levels ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
De Novo Protein Synthesis

ABSTRACT Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-XL or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-XL RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-XL and Bcl-2 expression is inhibited by Myc in precancerous B cells from Eμ-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-XL by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-XLlevels are markedly elevated in over one-half of all lymphomas arising in Eμ-myc transgenic mice. Bcl-2 and/or Bcl-XL overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-XL or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.

Activation of the inducible orphan receptor gene nur77 by serum growth factors: dissociation of immediate-early and delayed-early responses

1993 ◽  
Vol 13 (10) ◽  
pp. 6124-6136
Author(s):  
G T Williams ◽  
L F Lau
Keyword(s):  
Protein Synthesis ◽  
Growth Factors ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
De Novo ◽  
Early Gene ◽  
Immediate Early ◽  
Early Component ◽  
Early Expression ◽  
De Novo Protein Synthesis

We have characterized the genetic elements that mediate the transcriptional activation of nur77, a growth factor-inducible gene encoding a member of the steroid/thyroid hormone receptor superfamily. Although initially identified as a serum-inducible immediate-early gene with expression kinetics similar to those of c-fos, we found that transcriptional activation of nur77 by serum growth factors in fibroblasts is in fact composed of two components: an immediate-early component, which can occur in the absence of de novo protein synthesis, and a delayed-early component, which is dependent on de novo protein synthesis. The expression of nur77 following serum stimulation reflects the superimposition of immediate-early and delayed-early expression. Immediate-early and delayed-early expression can be dissociated from one another by deletion or base substitution mutations of the nur77 promoter. Immediate-early expression of nur77 is mediated primarily by sequences located between nucleotides -86 and -126 upstream of the transcription start site. This region includes a sequence that resembles but differs from the CArG element found in other serum-inducible promoters. Upstream of the CArG-like element is a potential binding site for a transcription factor of the Ets family; the presence of this site is required for significant transcriptional induction. Delayed-early expression of nur77 is mediated by multiple AP-1-like and GC-rich elements, which can interact with products of immediate-early genes such as Fos/Jun and Zif268, respectively. Furthermore, we show that Zif268 can activate transcription of the nur77 promoter, suggesting that it may play a role in the delayed-early expression of nur77.

scholarly journals Glucocorticoids Increase VE-Cadherin Expression and Cause Cytoskeletal Rearrangements in Murine Brain Endothelial cEND Cells

2008 ◽  
Vol 28 (6) ◽  
pp. 1139-1149 ◽  
Author(s):  
Kinga G Blecharz ◽  
Detlev Drenckhahn ◽  
Carola Y Förster
Keyword(s):  
Protein Synthesis ◽  
Tight Junction ◽  
Transcriptional Activation ◽  
Adherens Junction ◽  
Barrier Properties ◽  
Positive Influence ◽  
Brain Capillary Endothelial Cell ◽  
Protein Levels ◽  
Junction Protein ◽  
Adherens Junction Proteins

Recent studies have shown the influence of glucocorticoids on the expression of the tight junction protein occludin in the brain capillary endothelial cell line cEND, contributing to improvement in endothelial barrier functions. In this study, we investigated glucocorticoid effects on the expression of the adherens junction proteins VE- (vascular-endothelial) cadherin, α-catenin and β-catenin as well as that of ZO-1, the plaque protein shared by both adherens and tight junctions on stimulation with dexamethasone. We were able to show a positive influence of dexamethasone administration on VE-cadherin protein levels as well as a rearrangement of VE-cadherin protein to the cytoskeleton after dexamethasone treatment. Investigation of transcriptional activation of the VE-cadherin promoter by dexamethasone, however, did not point to direct glucocorticoid-mediated VE-cadherin gene induction but rather suggested indirect steroid effects leading to increased VE-cadherin protein synthesis. Dexamethasone was further shown to induce cellular differentiation into a cobblestone cellular morphology and reinforcement of adherens junctions concomitant with the increased anchorage of VE-cadherin to the actin cytoskeleton. We thus propose that glucocorticoid effects on VE-cadherin protein synthesis and organization are important for the formation of both adherens and tight junction, and for improved barrier properties in microvascular brain endothelial cells.

scholarly journals Posttranslational, Reversible O-Glycosylation Is Stimulated by High Glucose and Mediates Plasminogen Activator Inhibitor-1 Gene Expression and Sp1 Transcriptional Activity in Glomerular Mesangial Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (1) ◽  
pp. 222-231 ◽  
Author(s):  
Howard J. Goldberg ◽  
Catharine I. Whiteside ◽  
Gerald W. Hart ◽  
I. George Fantus
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
High Glucose ◽  
Mesangial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Glomerular Mesangial Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
Protein Levels ◽  
Activator Inhibitor ◽  
Pai 1

Metabolic flux through the hexosamine biosynthetic pathway (HBP) is increased in the presence of high glucose (HG) and potentially stimulates the expression of genes associated with the development of diabetic nephropathy. A number of synthetic processes are coupled to the HBP, including enzymatic intracellular O-glycosylation (O-GlcNAcylation), the addition of single O-linked N-acetylglucosamine monosaccharides to serine or threonine residues. Despite much data linking flow through the HBP and gene expression, the exact contribution of O-GlcNAcylation to HG-stimulated gene expression remains unclear. In glomerular mesangial cells, HG-stimulated plasminogen activator inhibitor-1 (PAI-1) gene expression requires the HBP and the transcription factor, Sp1. In this study, the specific role of O-GlcNAcylation in HG-induced PAI-1 expression was tested by limiting this modification with a dominant-negative O-linked N-acetylglucosamine transferase, by overexpression of neutral β-N-acetylglucosaminidase, and by knockdown of O-linked β-N-acetylglucosamine transferase expression by RNA interference. Decreasing O-GlcNAcylation by these means inhibited the ability of HG to increase endogenous PAI-1 mRNA and protein levels, the activity of a PAI-1 promoter-luciferase reporter gene, and Sp1 transcriptional activation. Conversely, treatment with the β-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, in the presence of normal glucose increased Sp1 O-GlcNAcylation and PAI-1 mRNA and protein levels. These findings demonstrate for the first time that among the pathways served by the HBP, O-GlcNAcylation, is obligatory for HG-induced PAI-1 gene expression and Sp1 transcriptional activation in mesangial cells.

Sign in / Sign up

Export Citation Format

Share Document