Antagonistic effects of phorbol esters on insulin regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) but not glucose-6-phosphatase gene expression

2001 ◽  
Vol 359 (3) ◽  
pp. 611-619 ◽  
Author(s):  
Satish PATEL ◽  
Pamela A. LOCHHEAD ◽  
Graham RENA ◽  
Calum SUTHERLAND
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Map Kinases ◽  
Phorbol Esters ◽  
Binding Protein ◽  
Gene Promoter ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Factor Binding

Glucose-6-phosphatase (G6Pase) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes contain a homologous promoter sequence that is required for gene repression by insulin. Interestingly, this element interacts with members of the forkhead family of transcription factors [e.g. HNF3 (hepatic nuclear factor 3), FKHR (forkhead in rhabdomyosarcoma)] in vitro, while insulin promotes the phosphorylation and inactivation of FKHR in a phosphatidylinositol 3-kinase- and protein kinase B (PKB)-dependent manner. This mechanism has been proposed to underlie insulin action on G6Pase and IGFBP-1 gene transcription. However, we find that treatment of cells with phorbol esters mimics the effect of insulin on G6Pase, but not IGFBP-1, gene expression. Indeed, phorbol ester treatment actually blocks the ability of insulin to repress IGFBP-1 gene expression. In addition, the action of phorbol esters is significantly reduced by inhibition of the p42/p44 mitogen-activated protein (MAP) kinase pathway. However insulin-induced phosphorylation of PKB or FKHR is not affected by the presence of phorbol esters. Therefore we suggest that activation of p42/p44 MAP kinases will reduce the sensitivity of the IGFBP-1 gene promoter, but not the G6Pase gene promoter, to insulin. Importantly, the activation of PKB and phosphorylation of FKHR is not, in itself, sufficient to reduce IGFBP-1 gene expression in the presence of phorbol esters.

scholarly journals Transcriptional Activation of Insulin-Like Growth Factor-Binding Protein-4 by 17β-Estradiol in MCF-7 Cells: Role of Estrogen Receptor-Sp1 Complexes*

Endocrinology ◽  
1999 ◽  
Vol 140 (6) ◽  
pp. 2501-2508 ◽  
Author(s):  
Chunhua Qin ◽  
Pomila Singh ◽  
Stephen Safe
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Growth Factor ◽  
Binding Protein ◽  
Gene Promoter ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Human Estrogen Receptor ◽  
17Β Estradiol ◽  
Mcf 7

Abstract Insulin-like growth factor-binding protein-4 (IGFBP-4) is expressed in MCF-7 human breast cancer cells, and treatment of these cells with 17β-estradiol (E2) resulted in induction of IGFBP-4 gene expression (>3-fold) and protein secretion (>6-fold). To identify genomic sequences associated with E2 responsiveness, the 5′-promoter region (−1214 to +18) of the IGFBP-4 gene was cloned into a vector upstream from the firefly luciferase reporter gene, and E2 induced a 10-fold increase in luciferase activity in MCF-7 cells transiently transfected with this construct. Deletion analysis of this region of the IGFBP-4 gene promoter identified two GC-rich sequences at −559 to −553 and −72 to −64 that were important for E2-induced trans-activation. Gel mobility shift assays using 32P-labeled −569 to −540 and −83 to −54 oligonucleotides from the IGFBP-4 gene promoter showed that Sp1 protein bound these oligonucleotides to form a retarded band, and the intensity of the band was competitively decreased after coincubation with unlabeled IGFBP-4-derived and consensus Sp1 oligonucleotides. Mutation of the GC-rich sites within these sequences resulted in loss of the retarded band formation. Wild-type human estrogen receptor did not bind directly to the IGFBP-4 oligonucleotides; however, human estrogen receptor enhanced Sp1-DNA binding in a concentration-dependent manner. The results of this study demonstrate that at least two GC-rich sequences at −559 to −553 and− 72 to −64 are required for induction of IGFBP-4 gene expression by E2 in MCF-7 cells.

scholarly journals Tri-iodothyronine increases insulin-like growth factor binding protein-2 expression in cultured hepatocytes from hypothyroid rats

1999 ◽  
Vol 161 (3) ◽  
pp. 465-474 ◽  
Author(s):  
I Demori ◽  
C Bottazzi ◽  
E Fugassa
Keyword(s):  
Growth Factor ◽  
Thyroid Hormone ◽  
Binding Protein ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Insulin Like Growth Factor ◽  
Cultured Hepatocytes ◽  
Factor Binding

Previous evidence suggests the existence of a thyroid hormone-IGF axis in the liver and changes in hepatic insulin-like growth factor binding protein (IGFBP) expression in rats with altered thyroid status have been previously reported. The aim of this study was to check if the higher IGFBP-2 mRNA levels observed in liver of hypothyroid rats could be due to a direct effect of thyroid hormone on the IGFBP-2 gene. In our experiments, cultured hepatocytes isolated from normal and hypothyroid adult rats were used. Northern blot analysis revealed barely detectable IGFBP-2 mRNA in normal rat hepatocytes, but easily detectable signal in hypothyroid rat cells. Therefore, the effect of tri-iodothyronine (T3) was investigated using cultured hepatocytes from hypothyroid rats as an in vitro model. The IGFBP-2 message was increased in a dose-dependent manner in hepatocytes cultured for 12-24 h in the presence of T3. A similar increase occurred in accumulation of IGFBP-2 in the culture medium, as measured by RIA. The effect of T3 on IGFBP-2 transcript levels appeared to consist of enhanced gene transcription and was independent of ongoing protein synthesis, but it was completely abolished by the incubation of hepatocytes with insulin. The latter result confirmed the dominant role of insulin in regulating IGFBP-2 expression by cultured hepatocytes. In vivo experiments confirmed an increase in hepatic IGFBP-2 mRNA and serum IGFBP-2 levels in hypothyroid rats and demonstrated, in addition, a significant increase in these measures in T3-treated rats. Taken together, our in vitro and in vivo results support a role for a thyroid hormone-IGF axis in the liver and suggest that other factors, such as insulin, interact in vivo with thryoid hormone in regulating hepatic IGFBP-2 expression.

INSULIN AND VARIATION IN GLUCOSE LEVELS MODIFY THE SECRETION RATES OF THE GROWTH HORMONE-INDEPENDENT INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 IN THE HUMAN HEPATOBLASTOMA CELL LINE HEP G2

1989 ◽  
Vol 123 (3) ◽  
pp. R17-R20 ◽  
Author(s):  
A.M. Cotterill ◽  
C.T. Cowell ◽  
M. Silink
Keyword(s):  
Growth Factor ◽  
Cell Line ◽  
Glucose Concentration ◽  
Binding Protein ◽  
Suitable Model ◽  
Insulin Like Growth Factor ◽  
Hep G2 ◽  
Factor Binding ◽  
Glucose Levels

ABSTRACT The plasma level of the GH-independent insulin-like growth factor binding-protein-1 (IGFBP-1) is regulated inversely by insulin. In this study the effect of insulin and changes in the glucose concentration on in-vitro IGFBP-1 secretion by the Hep G2 cell line was studied. Media from confluent cells in 12 replicates were collected for consecutive periods: initial control (20 h), study(6 h) and recovery (20 h). Insulin suppressed IGFBP-1 secretion maximally at 100 mU/1 (−32%) within 6 h. The secretion of IGFBP-1 was stimulated by a decrease in the glucose concentration in the medium, maximally (+25%) with a decrease from 24 to 6 mmol/l. Stimulation by varying glucose levels and suppression by insulin of IGFBP-1 secretion persisted on return to control conditions after the removal of physiological concentrations of glucose (4 - 12 mmol/l) and insulin (50 - 500 mU/1). The findings in the Hep G2 cell line that a variation in the physiological concentrations of glucose and insulin each independently regulate IGFBP-1 secretion suggest that this cell line may by a suitable model for further in-vitro studies of the regulation of secretion of IGFBP-1.

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