scholarly journals Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2.

1989 ◽  
Vol 9 (2) ◽  
pp. 396-405 ◽  
Author(s):  
M Pech ◽  
C D Rao ◽  
K C Robbins ◽  
S A Aaronson
Keyword(s):  
Growth Factor ◽  
Reporter Gene ◽  
Promoter Region ◽  
Promoter Activity ◽  
K562 Cells ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Platelet Derived Growth Factor ◽  
Regulatory Sequence ◽  
Mrna Levels

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2

1989 ◽  
Vol 9 (2) ◽  
pp. 396-405
Author(s):  
M Pech ◽  
C D Rao ◽  
K C Robbins ◽  
S A Aaronson
Keyword(s):  
Growth Factor ◽  
Reporter Gene ◽  
Promoter Region ◽  
Promoter Activity ◽  
K562 Cells ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Platelet Derived Growth Factor ◽  
Regulatory Sequence ◽  
Mrna Levels

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

Shear stress increases endothelial platelet-derived growth factor mRNA levels

1991 ◽  
Vol 260 (2) ◽  
pp. H642-H646 ◽  
Author(s):  
H. J. Hsieh ◽  
N. Q. Li ◽  
J. A. Frangos
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Fold Increase ◽  
Platelet Derived Growth Factor ◽  
Mrna Levels ◽  
Cell Mitogen ◽  
Stress Dependent

We have investigated the effect of shear stress on platelet-derived growth factor (PDGF) A and B chain mRNA levels in cultured human umbilical vein endothelial cells (hUVEC). The levels of both PDGF A and B mRNA in hUVEC were increased by a physiological shear stress (16 dyn/cm2), reaching a maximum approximately 1.5-2 h after the onset of shear stress and returning almost to control values at 4 h. The peak levels showed a more than 10-fold enhancement for PDGF A mRNA and a 2- to 3-fold increase for PDGF B mRNA (P less than 0.05). PDGF A mRNA also showed a shear-dependent increase from 0 to 6 dyn/cm2 (P less than 0.05) and then plateaued from 6 to 51 dyn/cm2. PDGF B mRNA levels were elevated as shear stress increased from 0 to 6 dyn/cm2 then declined gradually to a minimum at 31 dyn/cm2 (P less than 0.05) and increased again when shear stress rose to 51 dyn/cm2 (P less than 0.05). PDGF, a potent smooth muscle cell mitogen and vasoconstrictor, released from the endothelium may regulate the blood flow in vivo. The shear stress-dependent elevation of PDGF A and B mRNA in endothelial cells may be involved in the adaptation of blood vessels to flow mediated by the endothelium.

Hoxa5 overexpression correlates with IGFBP1 upregulation and postnatal dwarfism: evidence for an interaction between Hoxa5 and Forkhead box transcription factors

Development ◽  
2002 ◽  
Vol 129 (17) ◽  
pp. 4065-4074
Author(s):  
Isabelle Foucher ◽  
Michel Volovitch ◽  
Monique Frain ◽  
J. Julie Kim ◽  
Jean-Claude Souberbielle ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Growth Factor ◽  
Promoter Activity ◽  
Homeobox Gene ◽  
Direct Interaction ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Insulin Like Growth Factor ◽  
Forkhead Box

Transgenic mice expressing the homeobox gene Hoxa5 under the control of Hoxb2 regulatory elements present a growth arrest during weeks two and three of postnatal development, resulting in proportionate dwarfism. These mice present a liver phenotype illustrated by a 12-fold increase in liver insulin-like growth factor binding protein 1 (IGFBP1) mRNA and a 50% decrease in liver insulin-like growth factor 1 (IGF1) mRNA correlated with a 50% decrease in circulating IGF1. We show that the Hoxa5 transgene is expressed in the liver of these mice, leading to an overexpression of total (endogenous plus transgene) Hoxa5 mRNA in this tissue. We have used several cell lines to investigate a possible physiological interaction of Hoxa5 with the main regulator of IGFBP1 promoter activity, the Forkhead box transcription factor FKHR. In HepG2 cells, Hoxa5 has little effect by itself but inhibits the FKHR-dependent activation of the IGFBP1 promoter. In HuF cells, Hoxa5 cooperates with FKHR to dramatically enhance IGFBP1 promoter activity. This context-dependent physiological interaction probably corresponds to the existence of a direct interaction between Hoxa5 and FKHR and FoxA2/HNF3β, as demonstrated by pull-down experiments achieved either in vitro or after cellular co-expression. In conclusion, we propose that the impaired growth observed in this transgenic line relates to a liver phenotype best explained by a direct interaction between Hoxa5 and liver-specific Forkhead box transcription factors, in particular FKHR but also Foxa2/HNF3β. Because Hoxa5 and homeogenes of the same paralog group are normally expressed in the liver, the present results raise the possibility that homeoproteins, in addition to their established role during early development, regulate systemic physiological functions.

Vascular Endothelial Growth Factor (VEGF) Increases KCC4 Expression In Erythroid Cells Via Signaling Pathways Involving HIF-1α

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2039-2039
Author(s):  
Caryn S Gonsalves ◽  
Clinton H. Joiner
Keyword(s):  
Transcription Factor ◽  
Growth Factor ◽  
Volume Regulation ◽  
Promoter Activity ◽  
Phenotypic Variability ◽  
K562 Cells ◽  
Vegf Receptor ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract Abstract 2039 The K-Cl co-transporters are key components of volume regulation in human reticulocytes. K-Cl transport activity is increased in sickle red blood cells (SS RBC) and is thought to contribute to SS RBC dehydration, a process that potentiates sickling. Four mammalian KCC isoforms have been cloned and characterized, of which three are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Studies in sickle and normal reticulocytes indicate substantial variation in the relative expression of these genes among individuals, suggesting a potential source of phenotypic variability in sickle cell disease (SCD). Relatively little is known about the regulation of KCC expression. Previous studies have shown an increase in mRNA levels of KCC3a in HUVEC cells treated with the angiogenic factor vascular endothelial growth factor (VEGF) (Hiki, et.al. (1999) J. Biol. Chem. 274, 10661–10667). Because VEGF and the related placenta growth factor (PlGF) levels are elevated in SCD, we evaluated the potential effects of the vascular growth factors on KCC expression in erythroid cells. RT-PCR revealed that erythroid K562 cells expressed the VEGF receptor-1 (VEGF-R1, or Flt-1) but not VEGF receptor-2, (VEGF-R2 or Flk-1). Treatment of K562 cells with VEGF (50 ng/ml) showed an increase in KCC4 mRNA levels at 8 hours, with no significant change in KCC1, KCC3a and KCC3b expression. An inhibitor specific for the VEGF receptor-1 (VEGF-R1), SU5416, ablated the effect of VEGF, suggesting a role for this receptor in the signaling pathway. Studies with pharmacological inhibitors for specific kinases indicated that VEGF-stimulated KCC4 expression involves PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), which have been implicated in other VEGF effects. Analysis of the KCC4 promoter revealed binding sites for transcription factor SP-1 at positions -35 to -44 and -56 to -64, relative to the transcriptional start site. In addition, seven putative binding sites for transcription factor HIF-1α were found in the KCC4 promoter, suggesting a role for HIF-1α in VEGF-stimulated KCC4 expression, which occurs under non-hypoxic conditions. We examined KCC4 promoter activity using KCC4 luciferase promoter constructs expressed in K562 cells. Luciferase activity was stimulated by VEGF treatment, with maximum activity from the promoter constructs spanning 1200 bp and 875 bp from the start site. Minimal promoter activity was seen in the -65bp construct. Additionally, a mutation in the HIF-1α binding site at -73 to -76bp significantly inhibited promoter activity. Site-directed mutagenesis of the SP-1 sites at position -35 to -44 and position -56 to -64 also attenuated promoter activities upon VEGF treatment. These results suggest that activation of VEGF-R1 by VEGF, and presumably its other ligand, PlGF, leads to non-hypoxic activation of HIF-1α and SP-1-mediated up-regulation of KCC4 expression in erythroid K562 cells via its canonical signaling pathways. Variation in KCC gene expression and its modulation by cytokines and growth factors may be a source of inter-individual variation in SS RBC volume regulation and thus of phenotypic variability of SCD. Identifying the factors that modulate transcriptional control of KCC4 expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC. Disclosures: No relevant conflicts of interest to declare.

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells

1989 ◽  
Vol 9 (7) ◽  
pp. 2934-2943
Author(s):  
M I Wahl ◽  
N E Olashaw ◽  
S Nishibe ◽  
S G Rhee ◽  
W J Pledger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Hormonal Regulation ◽  
Growth Factor Receptor ◽  
Fold Increase ◽  
Platelet Derived Growth Factor ◽  
3T3 Cells ◽  
Pdgf Stimulation

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.

Functional analysis of the long terminal repeats of intracisternal A-particle genes: sequences within the U3 region determine both the efficiency and direction of promoter activity

1988 ◽  
Vol 8 (3) ◽  
pp. 1093-1102
Author(s):  
R J Christy ◽  
R C Huang
Keyword(s):  
Reporter Gene ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Antisense Transcription ◽  
Regulatory Elements ◽  
Deletion Mapping ◽  
Long Terminal Repeats ◽  
Terminal Repeats ◽  
Nuclease Mapping ◽  
Intracisternal A Particle

The transcriptional activity of five intracisternal A-particle (IAP) long terminal repeats (LTRs) in mouse embryonal carcinoma PCC3-A/1 cells and in Ltk- cells was determined. We tested the promoter activity of the LTRs by coupling them to the reporter gene chloramphenicol acetyltransferase (CAT) or guanosine-xanthine phosphoribosyltransferase (gpt). Each LTR was tested for promoter function in both the sense (5' to 3') and antisense (3' to 5') orientation preceding the reporter gene. The transcriptional activity of individual IAP gene LTRs varied considerably, and the LTR from IAP81 possessed promoter activity in both directions. The bidirectional activity of the IAP81 LTR confirmed by monitoring Ecogpt expression in stably transfected Ltk- cells, with the initiation sites for sense and antisense transcription being localized to within the IAP81 LTR by S1 nuclease mapping. Deletions of LTR81 show that for normal 5'-to-3' gene transcription (sense direction), the 3'U3/R region determines the basal level of transcription, whereas sequences within the 5'U3 region enhance transcription four- to fivefold. Deletion mapping for antisense transcription indicates that a 64-base-pair region (nucleotides 47 to 110) within the U3 region is essential for activity. These data indicate that the U3 region contains all the regulatory elements for bidirectional transcription in IAP LTRs.

Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells

1993 ◽  
Vol 265 (1) ◽  
pp. H3-H8 ◽  
Author(s):  
M. Mitsumata ◽  
R. S. Fishel ◽  
R. M. Nerem ◽  
R. W. Alexander ◽  
B. C. Berk
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Growth Factor ◽  
Fluid Shear Stress ◽  
Critical Role ◽  
Fold Increase ◽  
Flow Chamber ◽  
Platelet Derived Growth Factor ◽  
Aortic Endothelial Cells ◽  
A Chain

Fluid flow and the associated shear stress play a critical role in vascular growth and remodeling. Recent data suggest that increased endothelial cell expression of platelet-derived growth factor (PDGF) A- and B-chain by flow may participate in these events. In the present study, we examined the mechanism for flow-induced PDGF expression, focusing on protein kinase C (PKC). Bovine aortic endothelial cells were exposed to flow (shear stress = 30 dyn/cm2) in a parallel-plate flow chamber. Increases in PDGF B-chain, but not PDGF A-chain, were observed within 3 h, maximal within 6 h (13-fold increase), and sustained for 24 h. PKC appeared to be involved because phorbol 12-myristate 13-acetate induced PDGF B-chain mRNA. Activation of PKC alone, however, was insufficient to induce PDGF mRNA because the selective PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, did not induce PDGF expression. A PKC-independent pathway was suggested by the fact that inhibition of PKC (downregulation with phorbol 12,13-dibutyrate or exposure to staurosporine) failed to block PMA or flow-induced PDGF B-chain expression. These results demonstrate flow-induced PDGF B-chain expression in endothelial cells that appears to be mediated, in part, by a PKC-independent pathway.

Cortisol inhibits hepatocyte growth factor/scatter factor expression and induces c-met transcripts in osteoblasts

2000 ◽  
Vol 278 (3) ◽  
pp. E509-E515 ◽  
Author(s):  
Frederic Blanquaert ◽  
Renata C. Pereira ◽  
Ernesto Canalis
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Fibroblast Growth Factor 2 ◽  
Mrna Levels ◽  
Fibroblast Growth ◽  
Scatter Factor ◽  
Fetal Rat ◽  
Factor Expression ◽  
Hepatocyte Growth

Hepatocyte growth factor/scatter factor (HGF/SF) is expressed by osteoblasts and has important effects on repair and bone remodeling. Because glucocorticoids regulate these two functions, we tested the effects of cortisol on the expression of HGF/SF and c-met, the protooncogene encoding the HGF/SF receptor, in cultures of osteoblast-enriched cells from 22-day fetal rat calvariae (Ob cells). Cortisol decreased HGF/SF mRNA levels and diminished the induction of HGF/SF transcripts by fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor BB (PDGF BB). Cortisol also decreased FGF-2 and PDGF BB-induced HGF/SF mRNA and polypeptide levels in MC3T3 cells. In contrast, cortisol enhanced the expression of c-met transcripts in Ob cells. Cortisol did not modify the half-life of HGF/SF or of c-met mRNA in transcriptionally arrested cells, and it increased the rate of transcription of c-met. In conclusion, cortisol decreases HGF/SF transcripts in Ob cells and enhances c-met expression transcriptionally. The effects of cortisol on HGF/SF could be relevant to its inhibitory actions on bone formation and repair.

Development of a novel reporter gene assay for platelet-derived growth factor-BB bioactivity

Biologicals ◽  
2020 ◽  
Vol 63 ◽  
pp. 68-73
Author(s):  
Anna Niu ◽  
Zhang Zhang ◽  
Xiaolin Wang ◽  
Jing Wang ◽  
Junjie Xu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Platelet Derived Growth Factor ◽  
Gene Assay
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