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Endocrinology Vol. 141, No. 9 3156-3164
Copyright © 2000 by The Endocrine Society

ARTICLES

Stimulation of Insulin-Like Growth Factor Binding Protein-1 Synthesis by Interleukin-1ß: Requirement of the Mitogen-Activated Protein Kinase Pathway1

Robert A. Frost, Gerald J. Nystrom and Charles H. Lang

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Address correspondence and reprint requests to: Robert A. Frost, Ph.D., Department of Cellular and Molecular Physiology, Penn State University College of Medicine, Hershey Medical Center: H166, Hershey, Pennsylvania 17033. E-mail: rfrost{at}psu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is a 28-kDa plasma protein that binds to IGF-I and IGF-II with high affinity. IGFBP-1 is elevated in the blood as a result of sepsis, AIDS, excessive alcohol consumption, and diabetes and may, in part, be responsible for the wasting observed during these pathophysiological conditions. The liver is the principal site of IGFBP-1 synthesis, and we have previously shown that proinflammatory cytokines can directly stimulate IGFBP-1 secretion in a human hepatoma cell line (HepG2). The purpose of the present study was to investigate the role of the MAP kinase pathway in regulating IGFBP-1 synthesis by IL-1ß. We show that IL-1ß stimulates the phosphorylation of ERK-1 and -2 in a time- and dose-dependent manner. In addition, the MAP kinase-kinase MEK-1 and the ribosomal S6-kinase RSK-1 are also phosphorylated in response to IL-1ß. The transcription factor CREB, a potential substrate of both protein kinase A (PKA) and RSK-1, is phosphorylated in response to IL-1ß and cAMP in HepG2 cells. The ability of IL-1ß to stimulate the expression of IGFBP-1 and the phosphorylation of the above kinases was specifically inhibited by PD98059, a MEK-1 inhibitor. cAMP also stimulated IGFBP-1 synthesis, but PD98059 failed to block the cAMP effect. Conversely, a PKA inhibitor (H-89) inhibited the ability of cAMP, but not IL-1ß to stimulate IGFBP-1 synthesis. The effect of IL-1ß and cAMP on IGFBP-1 messenger RNA (mRNA) accumulation was additive. IL-1ß, cAMP, PD98059, and H-89 had similar effects on the accumulation of IGFBP-1 protein and mRNA. IL-1ß and cAMP did not change the half-life of IGFBP-1 mRNA, but PD98059 and SB202190, a p38 MAP kinase inhibitor, destabilized IGFBP-1 mRNA and blocked the phosphorylation of RSK-1 in response to IL-1ß. Our data demonstrate that the MAP kinase signal transduction pathway plays an important role in the regulation of IGFBP-1 synthesis by IL-1ß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor (IGF) binding protein-1 (IGFBP-1) is a 28-kDa plasma protein that binds IGF-I and IGF-II with high affinity (1). Consequently, IGFBP-1 inhibits many IGF-dependent processes including glucose and amino acid uptake and protein synthesis (2). Expression of IGFBP-1 in transgenic mice produces IGF-I resistance and results in an elevation in the plasma concentration of glucose and amino acids (3). IGFBP-1 is elevated in the blood in response to sepsis, AIDS, alcohol consumption, and diabetes and may, in part, be responsible for the wasting observed during these pathophysiological conditions (4, 5, 6, 7). An understanding of the regulation of IGFBP-1 synthesis is therefore important because overexpression of IGFBP-1 can reduce free IGF-I and inhibit IGF-dependent processes (8).

The plasma concentration of IGFBP-1 is increased during sepsis and inflammation primarily due to immune activation. IGFBP-1 is rapidly elevated in humans and rats injected with gram-negative endotoxin (9, 10). In addition, the plasma concentration of IGFBP-1 is rapidly increased after injection of cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF{alpha}) (10, 11). Prophylactic infusion of an IL-1ß receptor antagonist completely prevents the sepsis-induced increase in IGFBP-1. Administration of the glucocorticoid receptor antagonist RU486, on the other hand, fails to attenuate the increase in IGFBP-1 (4, 12). Endogenously produced IL-1ß therefore appears to be a major regulator of IGFBP-1 synthesis during inflammatory conditions.

The liver is the principal site of IGFBP-1 synthesis and IL-1ß, TNF{alpha}, and IL-6 can directly stimulate IGFBP-1 secretion in the human hepatoma HepG2 cell line (13). IL-1ß also markedly increases IGFBP-1 messenger RNA (mRNA) in HepG2 cells, but little is known about the signal transduction pathway this cytokine uses to enhance IGFBP-1 synthesis (14). The IL-1ß receptor has no inherent enzymatic activity but interacts with two interleukin receptor associated kinases. IL-1ß treatment also activates kinases that phosphorylate a number of downstream targets, including an inhibitor of the transcription factor nuclear factor {kappa}B (NF{kappa}B) called I {kappa}B (15). Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF {kappa}B activation, only partially attenuates the IL-1ß induced increase in IGFBP-1 in HepG2 cells (14). This response suggests that IL-1ß may stimulate IGFBP-1 synthesis by additional signal transduction pathways, which may include activation of protein kinase A (PKA) as well as phosphorylation of downstream targets such as the cAMP response element binding protein (CREB). Alternatively, IL-1ß may activate other kinases that affect IGFBP-1 transcription, mRNA stability, or translation.

Many growth factors and cytokines elicit their physiological response via mitogen-activated protein kinases (MAPK). Three independent MAP kinase pathways have been identified including pathways that lead to the activation of extracellular signal regulated kinases (ERK-1 and -2), p38 MAP kinase, and jun N-terminal kinase (JNK). These kinases are part of kinase cascades consisting of upstream MAP kinase-kinases and downstream targets such as the 90-kDa ribosomal S6 kinases (RSK). IL-1ß transiently activates all three of the MAP kinase pathways in HepG2 cells (16, 17). Therefore, the purpose of the present study was to investigate the role of the MAP kinase and PKA pathways in the regulation of IGFBP-1 synthesis in response to IL-1ß.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
A human hepatoma cell line (Hep G2) was used for all studies. These cells were originally shown to synthesize and secrete IGFBP-1 by Julkunen et al. (18) and have subsequently been used for the majority of studies that have examined the regulation of IGFBP-1 synthesis by hormones and cytokines (18). Cells were grown in six-well cluster dishes (Greiner, Lake Mary, FL). This allowed for the simultaneous measurement of IGFBP-1 protein and mRNA. Cells were cultured in minimal essential media (MEM, Sigma St. Louis, MO) containing 5% new born calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml) and Amphotericin (25 µg/ml). Cells were grown to 70–80% confluence and switched to serum-free MEM for all experiments. Experiments were routinely performed with IL-1ß (Biological Response Modifiers Program, NCI, 50 ng/ml), dibutryl cyclic AMP (dbcAMP; BIOMOL, Plymouth Meeting, PA, 2 mM), theophylline (Sigma, 2 mM), PD98059 (Calbiochem, La Jolla, CA, 20 µM), H-89 (BIOMOL, 30 µM), or SB202190 (Calbiochem, 10 µM). In some experiments, transcription was inhibited with 5,6-dichloro-B-D-ribofuranosyl-benzimidazole (DRB, 75 µM; Calbiochem) for the subsequent measurement of mRNA decay over a 9-h period.

IGFBP-1 immunoradiometric assay
Conditioned media was collected after 16 h incubation and frozen at -70 C. Thawed samples were subsequently diluted in a serum-based diluent and assayed with an IGFBP-1 immunoradiometric assay (IRMA), as described by the manufacturer (Diagnostics Systems Laboratories, Inc., Webster TX).

Western blot analysis
Conditioned media or cell extracts were electrophoresed on denaturing polyacrylamide gels and electrophoretically transferred to nitrocellulose with a semidry blotter (Bio-Rad Laboratories, Inc., Melville, NY). The resulting blots were blocked with 5% nonfat dry milk for 1.5 h and incubated with antibodies against either human IGFBP-1 (Upstate Biotechnology, Inc., Lake Placid, NY), phosphorylated or total CREB, MEK, RSK1, ATF-2, (New England Biolabs, Inc., Beverly, MA), phosphorylated or total ERK or Elk-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Unbound primary antibody was removed by washing with Tris-buffered saline containing 0.05% Tween-20, and blots were incubated with antirabbit immunoglobulin conjugated with horseradish peroxidase. Blots were briefly incubated with the components of an enhanced chemiluminescent detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Dried blots were used to expose x-ray film for 1–3 min.

RNA isolation and Northern blotting
Total RNA, DNA, and protein were extracted from HepG2 cells in a mixture of phenol and guanidine thiocyanate (TRI Reagent LS, Molecular Research Center, Inc., Cincinnati, OH). RNA was separated from protein and DNA by the addition of bromocholoropropane and precipitation in isopropanol. RNA was subsequently washed with ethanol and quantitated using a spectrophotometer (Beckman Coulter, Inc. DU640B, Fullerton, CA). Total RNA (10 µg) was electrophoresed under denaturing conditions on a 1% agarose gel containing 6% formaldehyde. RNA was transferred to a Zeta-Probe GT membrane using the Turbo Blotting System (Ambion, Inc. Austin, TX). The membrane was probed with a human IGFBP-1 oligonucleotide probe (Oncogene Research Products, Cambridge, MA) that was end-labeled with [{gamma}32P] ATP. For normalization of RNA loading, a human ß-actin oligonucleotide was radioactively labeled using terminal deoxynucleotidyl transferase and used to reprobe all blots. Membranes were baked, hybridized, and washed according to the formamide protocol provided with the {zeta}-Probe GT. Membranes were exposed to a phosphoimager screen (Molecular Dynamics, Inc., Sunnyvale, CA) and the resultant data were quantitated using ImageQuant software.

Statistics
Values are means ± SEM. Unless otherwise noted, each experimental condition was tested in sets of six, and each experiment was repeated three times. Data were analyzed by ANOVA followed by Student’s-Newman-Keuls test. IGFBP-1 mRNA half-life was calculated from the slope of the regression line using the formula t1/2= 0.5/m, where m is the slope of the line in arbitrary units (AU) per hour. Half-lives were compared by t test where t=((m1-m2)/({surd}(SE12 + SE22)) (19). Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERK and p38 MAP kinase inhibitors block IL-1ß but not cAMP induced IGFBP-1 synthesis
Because IL-1ß is known to activate multiple MAP kinase pathways in HepG2 cells, we tested whether PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, could prevent IL-1ß induced IGFBP-1 synthesis. The two inhibitors displayed different effects on the basal expression of IGFBP-1. PD98059 decreased the basal synthesis of IGFBP-1 mRNA dose dependently (Fig. 1AGo). Maximal inhibition occurred between 10 and 40 µM PD98059. In contrast, SB202190 exhibited a biphasic effect on IGFBP-1 mRNA expression (Fig. 1BGo). Low doses of SB202190 (2–5 µM) stimulated the accumulation of IGFBP-1 mRNA. A dose of 10 µM or greater had no effect on the basal expression of IGFBP-1. IL-1ß stimulated IGFBP-1 synthesis and secretion, and this response was inhibited by both PD98059 (20 µM) and SB202190 (10 µM) (Fig. 1CGo). IL-1ß increased the concentration of IGFBP-1 by 50% in the conditioned media of HepG2 cells, and this was inhibited dose dependently by PD98059 (Fig. 1DGo). Complete inhibition occurred at a concentration of 40 µM PD98059.



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  		Figure 1. Effect of IL-1ß, PD98059, and SB202190 on IGFBP-1 synthesis in HepG2 cells. HepG2 cells were grown to 80% confluence in six-well cluster dishes. Cells were switched to serum-free media and treated overnight with an increasing dose of either PD98059 (a MEK inhibitor, panel A) or SB202190 (a p38 inhibitor, panel B) for 16 h. Total RNA was isolated from the cells and IGFBP-1 mRNA detected by Northern blotting. RNA from triplicate wells is shown (A and B). Cells were also treated overnight with IL-1ß (50 ng/ml) and PD98059 (20 µM) or SB202190 (10 µM). Conditioned media from the cells was run on an SDS-PAGE gel and probed for IGFBP-1 by Western blotting. Media from triplicate wells are shown (C). HepG2 cells were also incubated with IL-1ß (50 ng/ml) and an increasing concentration of PD98059. After a 16 h incubation, media was collected, and frozen for subsequent measurement of IGFBP-1 by IRMA (D), as described in Materials and Methods. Values are the mean ± SE (n = 6 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different.

 
The expression of IGFBP-1 in HepG2 cells is positively regulated not only by cytokines, such as IL-1ß, but also by agents that elevate cAMP (20). Figure 2Go illustrates that IL-1ß and cAMP increased the synthesis of IGFBP-1 protein up to 3-fold and IGFBP-1 mRNA by 6-fold (Fig. 2Go, top and bottom panels, respectively). To determine whether the MAP kinase pathway was necessary for IGFBP-1 stimulation by both agents, HepG2 cells were coincubated with either cAMP and PD98059 or IL-1ß and PD98059. Figure 2Go showed that PD98059 effectively inhibited IL-1ß- induced IGFBP-1 synthesis. In contrast, PD98059 failed to inhibit the ability of cAMP to stimulate IGFBP-1 synthesis. IGFBP-1 mRNA and protein were coordinately regulated under these conditions.



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  		Figure 2. Effect of IL-1ß, cAMP, and PD98059 on IGFBP-1 protein and mRNA. HepG2 cells were grown as described in Fig. 1Go and treated with IL-1ß (50 ng/ml) or cAMP and theophylline (2 mM each), either alone or in the presence of PD98059 (20 µM). Conditioned media was collected after 16 h and cellular RNA isolated from each well in TRI-Reagent LS, as described in Materials and Methods. RNA was quantitated by analyzing the signal on Northern blots with a phosphoimager and ImageQuant software as described. Values are the mean ± SE (n = 6 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different.

 
IL-1ß and cAMP stimulate the phosphorylation of CREB through a PKA-mediated pathway
Because IL-1ß has previously been shown to stimulate cAMP synthesis in some cell types (21), we examined whether IL-1ß could stimulate the phosphorylation of the transcription factor CREB, a downstream target of PKA. Western blot analysis of HepG2 cell extracts revealed that cells treated with either cAMP or IL-1ß showed a rapid 10- to 12-fold increase in the phosphorylation of CREB (Fig. 3Go). PD98059 inhibited the ability IL-1ß to stimulate CREB phosphorylation by 60% without altering the stimulatory effect of cAMP. None of the treatments altered the total amount of CREB in the cell extracts. Similar results were obtained for ATF-1, a transcription factor that also binds to cAMP- responsive elements (CRE) and shares carboxy-terminal homology with CREB (Fig. 3Go) (22).



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  		Figure 3. Effect of IL-1ß, cAMP, and PD98059 on the phosphorylation of CREB. HepG2 cells were grown as described in Fig. 1Go and switched to serum-free media for 2 h. Cells were pretreated with PD98059 (or vehicle) for 30 min and subsequently challenged with either IL-1ß (50 ng/ml) or cAMP and theophylline (2 mM each). Cell extracts from triplicate wells were isolated in SDS-PAGE sample buffer and run on a 10% SDS-PAGE gel. CREB phosphorylation was analyzed by Western blotting for total CREB and CREB phosphorylated on Ser 133 (pCREB). A representative blot is shown (top panel). Phosphorylated ATF-1 (pATF-1) shares a phospho-peptide sequence with pCREB and is also recognized by this antibody. A quantitative analysis of this and additional blots is shown (bottom panel). Values are the mean ± SE (n = 6 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different.

 
Elevated levels of cAMP are known to activate PKA, which subsequently phosphorylates the transcription factor CREB (23). CREB has also been shown to be a substrate of other kinases (23, 24, 25, 26). We examined whether CREB phosphorylation was PKA dependent because IL-1ß might stimulate CREB through multiple pathways. IL-1ß and cAMP stimulated CREB phosphorylation, and the response to both agents was completely blocked by pre-incubation of cells with a PKA inhibitor (H-89) (Fig. 4Go). Similar results were found for the phosphorylation of ATF-1. This suggests that both IL-1ß and cAMP stimulate the phosphorylation of CREB through a PKA-mediated pathway in HepG2 cells.



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  		Figure 4. Effect of IL-1ß, cAMP, and H-89 on the phosphorylation of CREB. Cells were pretreated with either H-89 (30 µM, a PKA inhibitor), or vehicle for 30 min and then challenged with either IL-1ß (50 ng/ml) or cAMP and theophylline (2 mM each). Cell extracts from triplicate wells were isolated in SDS-PAGE sample buffer and run on a 10% SDS-PAGE gel. After transfer of proteins to nitrocellulose, CREB phosphorylation was analyzed by Western blotting for pCREB. A representative blot is shown (top panel). A quantitative analysis of this and additional blots is shown (bottom panel). Values are the mean ± SE (n = 6 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different.

 
Given that IL-1ß stimulated CREB phosphorylation through a PKA-dependent pathway, we examined whether IGFBP-1 synthesis was also PKA dependent. IL-1ß and cAMP both stimulated IGFBP-1 synthesis, but the PKA inhibitor H-89 only inhibited the ability of cAMP to stimulate IGFBP-1 synthesis (Fig. 5Go). H-89 alone had no effect on IGFBP-1 synthesis (data not shown).



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  		Figure 5. Effect of H-89 on the accumulation of IGFBP-1 protein in the media of IL-1ß and cAMP-treated cells. Cells were grown as described in Fig. 1Go and switched to serum-free media. Cells were treated with either cAMP and theophylline (2 mM each) or IL-1ß (50 ng/ml). Some cells also received the PKA inhibitor H-89 (30 µM). After 16 h incubation, media were collected and run on a 12% SDS-PAGE gel. After transfer to nitrocellulose, the blots were probed for human IGFBP-1. Two representative Western blots are shown.

 
IL-1ß and cAMP have an additive effect on IGFBP-1 synthesis
The above data suggested that cAMP and IL-1ß stimulate IGFBP-1 synthesis by PKA-dependent and -independent pathways, respectively. Therefore, we predicted that IL-1ß and cAMP would have an additive effect on IGFBP-1 synthesis. Addition of IL-1ß, cAMP, and theophylline individually approximately doubled IGFBP-1 mRNA accumulation in HepG2 cells (Fig. 6Go). A combination of each of the individual agents was additive. Cells treated with a combination of IL-1ß, theophylline, and cAMP demonstrated the greatest expression of IGFBP-1 mRNA. Theophylline and cAMP have previously been shown to have an additive effect on IGFBP-1 synthesis by Suwanichkul et al. (20).



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  		Figure 6. Additive effect of IL-1ß and cAMP on IGFBP-1 mRNA expression. Cells were grown as described in Fig. 1Go and switched to serum-free media. Cells were treated with cAMP (2 mM), theophylline (2 mM), IL-1ß (50 ng/ml) or a combination of these agents for 16 h. Cellular RNA was isolated in TRI-Reagent LS and the resulting Northern blot quantitated as in Materials and Methods. Values are the mean ± SE (n = 3 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different.

 
IL-1ß stimulates a time- and dose-dependent increase in the phosphorylation of ERK and potential ERK substrates
The selective inhibition of IGFBP-1 synthesis by PD98059, demonstrated in Figs. 1Go and 2Go, suggested that IL-1ß might stimulate the phosphorylation of ERK-1 and -2 in HepG2 cells. HepG2 cells exhibited a constitutive expression and phosphorylation of ERK (Fig 7Go, top panel). Maximal stimulation of ERK-1 and -2 by IL-1ß occurred after 20 min and returned to a basal phosphorylation state by 180 min. Additionally, IL-1ß increased ERK phosphorylation in a dose-dependent manner (Fig. 7Go, bottom panel). Maximal stimulation occurred at an IL-1ß concentration of 15–40 ng/ml (Fig. 7Go, bottom panel).



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  		Figure 7. Stimulation of ERK phosphorylation by IL-1ß. Cells were grown as described in Fig. 1Go and switched to serum-free media for 2 h. Cells were treated with IL-1ß for either various periods of time (top panel) or with an increasing concentration of IL-1ß for 20 min (bottom panel). Cell extracts from triplicate wells were isolated in SDS-PAGE sample buffer and run on a 10% SDS-PAGE gel. After transfer of proteins to nitrocellulose, ERK phosphorylation was analyzed by Western blotting and normalized for the total amount of ERK in each extract. Values are the mean ± SE (n = 6 per condition). Groups with different letters are significantly different from each other (P < 0.05). Groups with the same letter are not significantly different. Note that the top panel demonstrates a time-dependent increase in the phosphorylation of ERK, whereas the bottom panel demonstrates a dose-dependent stimulation of ERK in response to IL-1ß.

 
The activation of ERK by IL-1ß suggested that additional downstream substrates might be involved in the IL-1ß induced increase in IGFBP-1 synthesis. We examined the phosphorylation of MEK-1, ERK, and RSK-1 because the RSK kinases dock with ERK, and they have been shown to be ERK substrates in other cell types (27). IL-1ß stimulated the phosphorylation of MEK-1, ERK-1 and -2, and RSK-1 by 225%, 90%, and 290% respectively. The phosphorylation of all these proteins was inhibited by PD98059 (Fig. 8Go). The p38 MAP kinase also phosphorylates RSK isoforms. A p38 kinase inhibitor (SB202190) inhibited the ability of IL-1ß to stimulate the phosphorylation of MEK-3 and RSK-1. A combination of the PD and SB compounds was more effective than either compound alone (Fig. 8Go). This suggests that part of the IL-1ß signal may be transmitted through both ERK and p38. None of the compounds altered the total amount of ERK-1, ERK-2 or RSK-1 in the cell extracts.



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  		Figure 8. Composite effect of IL-1ß, PD98059, and SB202190 on elements of the MAP kinase pathway. Cells were grown as described in Fig. 1Go and switched to serum-free media for 2 h. Cells were pretreated with either PD98059 (20 µM) or SB202190 (10 µM) for 30 min followed by IL-1ß (50 ng/ml) for 20 min. Cell extracts from triplicate wells were isolated in SDS-PAGE sample buffer and run on a 10% SDS-PAGE gel. After transfer of proteins to nitrocellulose, MEK-1 and -3, ERK-1 and -2, and RSK-1 phosphorylation were analyzed by Western blotting with phospho-specific antibodies. A composite figure showing the phosphorylation of each of these kinases, and the effect of each kinase inhibitor is shown. The total amount of ERK and RSK-1 in each extract is also shown.

 
PD98059 and SB202190 alter IGFBP-1 mRNA stability
The IL-1ß induced increase in IGFBP-1 mRNA could be due to an increase in IGFBP-1 gene transcription, RNA stability, or both. We examined the ability of IL-1ß and cAMP to alter IGFBP-1 mRNA levels in the presence of a transcription inhibitor (DRB). Both IL-1ß and cAMP increased IGFBP-1 protein and mRNA after an overnight incubation (Fig. 9Go, A and B, respectively). IGFBP-1 mRNA from IL-1ß and cAMP pretreated cells had similar half-lives, and neither stimulus resulted in a significant change in the half-life of IGFBP-1 mRNA compared with control values (Fig. 9BGo). Cells grown in the absence of DRB maintained a constant amount of IGFBP-1 mRNA over the course of the experiment, whereas DRB-treated cells showed progressively less IGFBP-1 mRNA over time, confirming the ability of DRB to inhibit transcription in these experiments (Fig. 9CGo). PD98059 shortened the half-life of IGFBP-1 mRNA from 2.5 h to 1.8 h in IL-1ß pretreated cells (Fig. 9DGo; P < 0.05). SB202190 also destabilized IGFBP-1 mRNA in IL-1ß pretreated cells.



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  		Figure 9. Effect of IL-1ß and cAMP on IGFBP-1 mRNA half-life. HepG2 cells were grown in triplicate wells of six-well cluster dishes as described in Fig 2Go. Cells were cultured either in serum-free media alone (control) or in the presence of either cAMP and theophylline (2 mM each) or IL-1ß (50 ng/ml) for 16 h. Conditioned media were collected, run on an SDS-PAGE gel, and probed for IGFBP-1. A representative Western blot is shown (A). After 16 h, cells were treated with DRB to inhibit transcription and RNA was isolated from triplicate wells at 3, 6, and 9 h. IGFBP-1 mRNA was detected by Northern blotting and normalized to the amount of ß-actin in the same sample. Arbitrary units (AU) determined by phosphoimager analysis are shown (B). IGFBP-1 mRNA in cells treated with IL-1ß followed by incubation with or without DRB to inhibit transcription is shown (C). The effects of PD98059 and SB202190 on IGFBP-1 mRNA half-life were determined by growing cells in the presence of IL-1ß for 16 h followed by either DRB alone or DRB and PD98059 or SB202190 (D). Note the ability of the PD and SB compounds to decrease IGFBP-1 mRNA half-life.

 
To determine if the PD and SB compounds affect the half-life of IGFBP-1 mRNA in unstimulated cells, we treated cells with DRB for 4h in the presence of either PD98059 or SB202190. After 4 h, cells had 28% (DRB alone), 17% (DRB + PD), or 11% (DRB + SB) of control IGFBP-1 mRNA levels remaining. Thus, both inhibitors decreased mRNA stability by between 40–60% (Fig. 10AGo).



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  		Figure 10. Effect of PD98059, SB202190, and cAMP on the half-life of IGFBP-1 mRNA. HepG2 cells were grown in presence of DRB for 4 h to inhibit transcription. Some cells also received PD98059 or SB202190. RNA was isolated after 4 h and IGBP-1 mRNA quantitated by Northern blotting (top panel). After 4 h approximately 28% (DRB alone), 17% (DRB + PD) and 11% (DRB +SB) of the control levels (t = 0h) of IGFBP-1 mRNA remained. Cells were also grown in the presence of cAMP for 16 h and then treated with DRB in the presence or absence of additional cAMP and PD98059. RNA was isolated from triplicate wells at 3, 6, and 9 h. IGFBP-1 mRNA was detected by Northern blotting and normalized to the amount of ß-actin in the same sample. The decay of IGFBP-1 mRNA in cells treated with cAMP for 16 h followed by PD98059 in the presence or absence of additional cAMP over the ensuing 9 h is shown (bottom panel). Note the effect of additional cAMP in blunting the ability of PD98059 to shorten the half-life of IGFBP-1 mRNA.

 
Because the efficacy of cAMP and IL-1ß may be diminished after a 16 h incubation, we pretreated cells with cAMP and then added additional cAMP to determine if this prevented PD98059 from altering the half-life of IGFBP-1 mRNA. IGFBP-1 mRNA had a similar half-life when cells were pretreated with cAMP overnight, and then treated with or without additional cAMP during the half-life experiment (data not shown). Additional cAMP added to the media during the half-life experiment prevented PD98059 from altering IGFBP-1 mRNA half-life (Fig. 10BGo). In contrast, PD98059 shortened the half-life of IGFBP-1 mRNA regardless of whether additional IL-1ß was present during the half-life experiment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that IL-1ß and cAMP stimulate the synthesis and secretion of IGFBP-1 by HepG2 cells. The effects of both agents are additive, suggesting that they use different signal transduction pathways to stimulate IGFBP-1 synthesis. The response to IL-1ß can be prevented by PD98059 a specific inhibitor of the MAP kinase-kinase, MEK-1. In contrast, cAMP-stimulated IGFBP-1 synthesis was unaffected by PD98059. IL-1ß also activated multiple protein kinase cascades in HepG2 cells, and this led to the phosphorylation of transcription factors such as CREB, ATF-1, and Elk-1.

Little is known about how IL-1ß increases IGFBP-1 synthesis. Our data suggest that the increase in IGFBP-1 mRNA is not due to an altered mRNA half-life. Cells pretreated with IL-1ß or cAMP show an increase in IGFBP-1 mRNA, but in the presence of DRB, all treatments exhibited essentially identical decay kinetics. This would suggest that IL-1ß increases IGFBP-1 transcription. Benbassat et al. (28) have reported that IL-1{alpha} increases IGFBP-1 mRNA in HepG2 cells. Yet, these authors found that IL-1{alpha} decreased IGFBP-1 promoter activity (28). It is possible that an additional IL-1 responsive element might lie outside of the region of the gene that these authors examined.

The effect of IL-1ß on IGFBP-1 synthesis may be tissue specific. Frank et al. (29) have found that IL-1ß inhibits IGFBP-1 synthesis in endometrial stromal cells. These investigators did not examine the signal transduction pathway responsible for this effect but did report that IL-1ß also impaired the synthesis of other endometrial proteins and suggested that IL-1ß may influence the differentiation of these cells. Gao et al. (30, 31) have demonstrated the presence of an enhancer element in the IGFBP-1 gene, and it is possible that this element responds differently to signals generated by IL-1ß in a hepatic vs. a uterine environment. Nuutila et al. (32) have also recently shown that a prostaglandin E2 (PGE2) gel can increase the cervical concentration of IGFBP-1. PGE2 is an additional possible mediator of IL-1ß action, but we were unable to demonstrate an increase in IGFBP-1 synthesis in response to PGE2 in HepG2 cells (RAF unpublished observation).

The ERK and p38 kinases appear to be important for both basal and IL-1ß stimulated synthesis of IGFBP-1. IL-1ß activates upstream MEK kinase kinases as well as the downstream kinase RSK-1. This signal may ultimately result in the phosphorylation of a transcription factor that enhances the expression of the IGFBP-1 gene. This would be consistent with a transient increase in ERK phosphorylation resulting in more sustained changes in gene transcription (33). Potential transcription factors would have to display the same positive regulation by IL-1ß and negative regulation by PD98059, as do ERK, RSK1, and IGFBP-1. ATF-1, ATF-2, and CREB display the same positive regulation in response to IL-1ß, but the ATF and CREB responses were not blocked completely by PD98059. In addition, IGFBP-1 synthesis remained robust when CREB phosphorylation was inhibited with a PKA inhibitor. Our data suggest that CREB mediates the stimulatory effect of cAMP, but not that of IL-1ß, on IGFBP-1 synthesis. In addition, cAMP can prevent the destabilizing effect on IGFBP-1 mRNA induced by inhibition of the MAP kinase pathway.

SB202190 displayed a paradoxical ability to stimulate IGFBP-1 synthesis at low doses but this effect was absent at doses of 10 µM or greater. Another pyridinyl imidazole inhibitor (SB203580) has recently been shown to inhibit the protein kinase B kinase, phosphoinositide-dependent protein kinase-1 (34). Thus, this class of inhibitors potentially has dual effects on basal and IL-1ß stimulated IGFBP-1 synthesis. Inhibition of the PI 3-kinae pathway with wortmannin has previously been shown to increase the transcription of IGFBP-1 (35). Interestingly, we find that PD98059 also inhibits the ability of wortmannin to increase IGFBP-1 mRNA, suggesting that the ERK pathway is critical for the maintenance of IGFBP-1 mRNA.

A combination of MEK and p38 inhibitors was more potent than either inhibitor alone at blocking the phosphorylation of RSK-1 in response to IL-1ß. This response suggests that both the p38 and ERK pathways contribute to phosphorylation of this substrate. We find that a combination of the two inhibitors is also more potent at inhibiting IGFBP-1 synthesis. Miwa et al. (33) have recently shown that IL-1ß activates both ERK and p38 kinases in osteoblastic MC3T3-E1 cells. In these cells, IL-6 synthesis is inhibited by PD98059 and SB203580, suggesting a critical role of these kinase pathways in cytokine production. Because IL-6 also stimulates BP-1 synthesis in hepatocytes (13), it is possible that paracrine production of IL-6 by Kupffer cells may contribute to a positive feedback loop that continuously promotes IGFBP-1 synthesis during inflammatory conditions.

Initially, the MAP kinase pathway would appear to be a poor candidate for the positive regulation of IGFBP-1 synthesis. It is well known that insulin and IGF-I stimulate ERK phosphorylation, and both peptides are negative regulators of IGFBP-1 synthesis. Yet, Cichy et al. (35) have shown that insulin does not inhibit IGFBP-1 synthesis through the MAP kinase pathway. Instead, insulin and IGF-I activate protein kinase B to phosphorylate forkhead transcription factors (35, 36). Under most circumstances, therefore, insulin and IGF-I are dominant negative regulators of IGFBP-1 synthesis. Akt has recently been shown to phosphorylate and inactivate the MEK kinase Raf by creating a binding site for 14–3-3 proteins (37). Inhibition of the MAP kinase pathway by Akt may be an additional mechanism by which insulin inhibits IGFBP-1 synthesis.

The importance of the MAP kinase pathway in stimulating IGFBP-1 synthesis is consistent with a previous study in which epidermal growth factor (EGF) stimulated IGFBP-1 synthesis (38). EGF, like IL-1ß, is a well-known activator of ERK-1 and -2 (39). ERK and p38 MAP kinase are also activated by protein synthesis inhibitors. Addition of anisomycin or cycloheximide to cells results in a prolonged activation of the p38 MAP kinase pathway and phosphorylation of ELK-1 (40). Ooi et al. (41), have also shown that cycloheximide dramatically increases IGFBP-1 mRNA stability in H4-II-E hepatoma cells.

IL-1ß neither altered the half-life of IGFBP-1 mRNA nor prevented the drop in mRNA stability that we observed in the presence of PD98059. The PD compound diminished IGFBP-1 mRNA stability by 30–40% in the basal state as well as in IL-1ß pretreated cells. This suggests that flux through the MAP kinase pathway is necessary to maintain the stability of IGFBP-1 mRNA. PD98059 may alter the phosphorylation of factors that bind to IGFBP-1 mRNA. The destabilizing effect of PD98059 can be overcome by cAMP, but given its labile nature, this agent must be present throughout the experiment. Flux through the MAP kinase pathway is also required for maintaining the stability of mRNAs for the muscarinic acetylcholine receptor (42) and macrophage inflammatory protein-2 (43). In addition, transient transfection of MEK kinase-1 into HeLa cells has recently been shown to increase the stability of IL-6 and IL-8 mRNA (44). Both mRNAs contain AU-rich sequences similar to those in the 3' untranslated region of the IGFBP-1 message. Progressive deletion of AU rich sequences in the IGFBP-1 mRNA have recently been shown to dramatically stabilize the message (45).

In summary, the results of the present study indicate that IL-1ß positively regulates IGFBP-1 synthesis via a MAP kinase signal transduction pathway. This effect is specific because it can be inhibited by PD98059, whereas this compound has no effect on the ability of cAMP to stimulate IGFBP-1 synthesis. Transient phosphorylation of a downstream kinase, such as RSK-1, presumably activates transcription factor(s) that subsequently stimulate IGFBP-1 gene transcription and result in long-term changes in IGFBP-1 mRNA and protein levels. Although IL-1ß does not alter the half-life of IGFBP-1 mRNA, flux through the MAP kinase pathway appears to be necessary for maintaining the stability of IGFBP-1 mRNA. Hence, IL-1ß regulates IGFBP-1 synthesis in a manner that is unique from previously studied activators, such as cAMP.


   Acknowledgments
 
We thank the Biological Resources Branch of the Biological Response Modifiers Program Division of Cancer Treatment, National Cancer Institute, for providing recombinant human IL-1ß.


   Footnotes
 
1 This work was supported in part by the National Institutes of Health Grants GM-38032 and AA-11290. Back

Received January 12, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lee PD, Giudice LC, Conover CA, Powell DR 1997 Insulin-like growth factor binding protein-1: recent findings and new directions. Proc Soc Exp Biol Med 216:319–357[CrossRef][Medline]
  2. Frost RA, Lang CH 1999 Differential effects of insulin-like growth factor I (IGF-I) and IGF- binding protein-1 on protein metabolism in human skeletal muscle cells. Endocrinology 140:3962–3970[Abstract/Free Full Text]
  3. Rajkumar K, Krsek M, Dheen ST, Murphy LJ 1996 Impaired glucose homeostasis in insulin-like growth factor binding protein-1 transgenic mice. J Clin Invest 98:1818–1825[Medline]
  4. Lang CH, Fan J, Cooney R, Vary TC 1996 IL-1 receptor antagonist attenuates sepsis-induced alterations in the IGF system and protein synthesis. Am J Physiol 270:E430–E437
  5. Frost RA, Fuhrer J, Steigbigel R, Mariuz P, Lang CH, Gelato MC 1996 Wasting in the acquired immune deficiency syndrome is associated with multiple defects in the serum insulin-like growth factor system. Clin Endocrinol (Oxf) 44:501–514[CrossRef][Medline]
  6. Mynarcik DC, Frost RA, Lang CH, DeCristofaro K, McNurlan MA, Garlick PJ, Steigbigel RT, Fuhrer J, Ahnn S, Gelato MC 1999 Insulin-like growth factor system in patients with HIV infection: effect of exogenous growth hormone administration. J Acquir Immune Defic Syndr 22:49–55
  7. Bereket A, Lang CH, Wilson TA 1999 Alterations in the growth hormone-insulin-like growth factor axis in insulin dependent diabetes mellitus. Horm Metab Res 31:172–181[Medline]
  8. Zapf J 1997 Total and free IGF serum levels. Eur J Endocrinol 136:146–147[Abstract/Free Full Text]
  9. Lang CH, Pollard V, Fan J, Traber LD, Traber DL, Frost RA, Gelato MC, Prough DS 1997 Acute alterations in growth hormone-insulin-like growth factor axis in humans injected with endotoxin. Am J Physiol 273:R371–R378
  10. Fan J, Molina PE, Gelato MC, Lang CH 1994 Differential tissue regulation of insulin-like growth factor-I content and binding proteins after endotoxin. Endocrinology 134:1685–1692[Abstract/Free Full Text]
  11. Fan J, Wojnar MM, Theodorakis M, Lang CH 1996 Regulation of insulin-like growth factor (IGF)-I mRNA and peptide and IGF-binding proteins by interleukin-1. Am J Physiol 270:R621–R629
  12. Li YH, Fan J, Lang CH 1997 Differential role of glucocorticoids in mediating endotoxin-induced changes in IGF-I and IGFBP-1. Am J Physiol 272:R1990–R1997
  13. Samstein B, Hoimes ML, Fan J, Frost RA, Gelato MC, Lang CH 1996 IL-6 stimulation of insulin-like growth factor binding protein (IGFBP)-1 production. Biochem Biophys Res Commun 228:611–615[CrossRef][Medline]
  14. Lang CH, Nystrom GJ, Frost RA 1999 Regulation of IGF binding protein-1 in Hep G2 cells by cytokines and reactive oxygen species. Am J Physiol 276:G719–G727
  15. Saklatvala J, Dean J, Finch A 1999 Protein kinase cascades in intracellular signalling by interleukin-I and tumour necrosis factor. Biochem Soc Symp 64:63–77[Medline]
  16. Kumar A, Middleton A, Chambers TC, Mehta KD 1998 Differential roles of extracellular signal-regulated kinase-1/2 and p38(MAPK) in interleukin-1ß- and tumor necrosis factor-{alpha}-induced low density lipoprotein receptor expression in HepG2 cells. J Biol Chem 273:15742–15748[Abstract/Free Full Text]
  17. Stylianou E, Saklatvala J 1998 Interleukin-1. Int J Biochem Cell Biol 30:1075–1079[CrossRef][Medline]
  18. Julkunen M, Koistinen R, Aalto-Setala K, Seppala M, Janne OA, Kontula K 1988 Primary structure of human insulin-like growth factor-binding protein/placental protein 12 and tissue-specific expression of its mRNA. FEBS Lett 236:295–302[CrossRef][Medline]
  19. Glantz 1992 Primer of Biostatistics. McGraw-Hill, New York
  20. Suwanichkul A, DePaolis LA, Lee PD, Powell DR 1993 Identification of a promoter element which participates in cAMP-stimulated expression of human insulin-like growth factor-binding protein-1. J Biol Chem 268:9730–9736[Abstract/Free Full Text]
  21. Beasley D 1999 COX-2 and cytosolic PLA2 mediate IL-1ß-induced cAMP production in human vascular smooth muscle cells. Am J Physiol 276:H1369–H1378
  22. Rehfuss RP, Walton KM, Loriaux MM, Goodman RH 1991 The cAMP-regulated enhancer-binding protein ATF-1 activates transcription in response to cAMP-dependent protein kinase A. J Biol Chem 266:18431–18434[Abstract/Free Full Text]
  23. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[CrossRef][Medline]
  24. Pugazhenthi S, Boras T, O’Connor D, Meintzer MK, Heidenreich KA, Reusch JE 1999 Insulin-like growth factor I-mediated activation of the transcription factor cAMP response element-binding protein in PC12 cells. Involvement of p38 mitogen-activated protein kinase-mediated pathway. J Biol Chem 274:2829–2837[Abstract/Free Full Text]
  25. Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959–963[Abstract]
  26. Hansen TV, Rehfeld JF, Nielsen FC 1999 Mitogen-activated protein kinase and protein kinase A signaling pathways stimulate cholecystokinin transcription via activation of cyclic adenosine 3',5'-monophosphate response element-binding protein. Mol Endocrinol 13:466–475[Abstract/Free Full Text]
  27. Smith JA, Poteet-Smith CE, Malarkey K, Sturgill TW 1999 Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo. J Biol Chem 274:2893–2898[Abstract/Free Full Text]
  28. Benbassat CA, Lazarus DD, Cichy SB, Evans TM, Moldawer LL, Lowry SF, Unterman TG 1999 Interleukin-1 {alpha} (IL-1{alpha}) and tumor necrosis factor alpha (TNF alpha) regulate insulin-like growth factor binding protein-1 (IGFBP-1) levels and mRNA abundance in vivo and in vitro. Horm Metab Res 31:209–215[Medline]
  29. Frank GR, Brar AK, Jikihara H, Cedars MI, Handwerger S 1995 Interleukin-1 beta and the endometrium: an inhibitor of stromal cell differentiation and possible autoregulator of decidualization in humans. Biol Reprod 52:184–191[Abstract]
  30. Tseng L, Gao J, Mazella J, Zhu HH, Lane B 1997 Differentiation-dependent and cell-specific regulation of the hIGFBP-1 gene in human endometrium. Ann NY Acad Sci 828:27–37[CrossRef][Medline]
  31. Gao J, Tseng L 1996 Distal Sp3 binding sites in the hIGBP-1 gene promoter suppress transcriptional repression in decidualized human endometrial stromal cells: identification of a novel Sp3 form in decidual cells. Mol Endocrinol 10:613–621[Abstract/Free Full Text]
  32. Nuutila M, Hiilesmaa V, Karkkainen T, Ylikorkala O, Rutanen EM 1999 Phosphorylated isoforms of insulin-like growth factor binding protein-1 in the cervix as a predictor of cervical ripeness. Obstet Gynecol 94:243–249[CrossRef][Medline]
  33. Miwa M, Kozawa O, Tokuda H, Uematsu T 1999 Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C. Endocrinology 140:5120–5125[Abstract/Free Full Text]
  34. Lali FV, Hunt AE, Turner SJ, Foxwell BM 2000 The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem 275:7395–7402[Abstract/Free Full Text]
  35. Cichy SB, Uddin S, Danilkovich A, Guo S, Klippel A, Unterman TG 1998 Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence. J Biol Chem 273:6482–6487[Abstract/Free Full Text]
  36. Guo S, Rena G, Cichy S, He X, Cohen P, Unterman T 1999 Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274:17184–17192[Abstract/Free Full Text]
  37. Zimmermann S, Moelling K 1999 Phosphorylation and regulation of Raf by Akt (protein kinase B). Science 286:1741–1744[Abstract/Free Full Text]
  38. Angervo M 1992 Epidermal growth factor enhances insulin-like growth factor binding protein-1 synthesis in human hepatoma cells. Biochem Biophys Res Commun 189:1177–1183[CrossRef][Medline]
  39. Putz T, Culig Z, Eder IE, Nessler-Menardi C, Bartsch G, Grunicke H, Uberall F, Klocker H 1999 Epidermal growth factor (EGF) receptor blockade inhibits the action of EGF, insulin-like growth factor I, and a protein kinase A activator on the mitogen-activated protein kinase pathway in prostate cancer cell lines. Cancer Res 59:227–233[Abstract/Free Full Text]
  40. Zinck R, Cahill MA, Kracht M, Sachsenmaier C, Hipskind RA, Nordheim A 1995 Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1. Mol Cell Biol 15:4930–4938[Abstract]
  41. Ooi GT, Brown DR, Suh DS, Tseng LY, Rechler MM 1993 Cycloheximide stabilizes insulin-like growth factor-binding protein-1 (IGFBP-1) mRNA and inhibits IGFBP-1 transcription in H4-II-E rat hepatoma cells. J Biol Chem 268:16664–16672[Abstract/Free Full Text]
  42. Lee NH, Malek RL 1998 Nerve growth factor regulation of m4 muscarinic receptor mRNA stability but not gene transcription requires mitogen-activated protein kinase activity. J Biol Chem 273:22317–22325[Abstract/Free Full Text]
  43. Xiao YQ, Someya K, Morita H, Takahashi K, Ohuchi K 1999 Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils. Biochim Biophys Acta 1450:155–163[Medline]
  44. Winzen R, Kracht M, Ritter B, Wilhelm A, Chen CY, Shyu AB, Muller M, Gaestel M, Resch K, Holtmann H 1999 The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J 18:4969–4980[CrossRef][Medline]
  45. Gay E, Babajko S 2000 AUUUA sequences compromise human insulin-like growth factor binding protein-1 mRNA stability. Biochem Biophys Res Commun 267:509–515[CrossRef][Medline]



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