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Endocrinology, doi:10.1210/en.2003-0592
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Endocrinology Vol. 144, No. 12 5402-5410
Copyright © 2003 by The Endocrine Society

Insulin-Regulated Expression of Egr-1 and Krox20: Dependence on ERK1/2 and Interaction with p38 and PI3-Kinase Pathways

Adam B. Keeton, Katherine D. Bortoff, William L. Bennett, J. Lee Franklin, Derwei Y. Venable and Joseph L. Messina

Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019

Address all correspondence and requests for reprints to: Joseph L. Messina, Ph.D., Department of Pathology, Division of Molecular and Cellular Pathology, Volker Hall, G019, 1670 University Boulevard, University of Alabama at Birmingham, Birmingham, Alabama 35294-0019. E-mail: messina{at}path.uab.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In addition to its ability to rapidly alter metabolism, insulin is also able to regulate the expression of numerous genes via activation of the PI3-kinase (PI3-K), MAPK kinase (MEK)-ERK, or p38 pathways. Using differential screening of H4IIE cells, we have identified two members of the Egr zinc-finger transcription factor family of early response genes, Egr-1 and Krox20, whose transcription is induced by insulin treatment. Egr-1 may be involved in insulin’s regulation of hepatic gene expression. Krox20 regulation and expression have been primarily studied in neural cells and tissues, but little has been previously reported on the presence of Krox20 in cells of hepatic origin or its regulation by insulin. In the present studies, insulin treatment rapidly increased transcription of both Egr-1 and Krox20. In cells pretreated with a PI3-K inhibitor, there was no reduction in the effect of insulin on Egr-1 and Krox20, but an increase in Egr-1 transcription. The rapid induction of ERK1/2 phosphorylation was completely blocked by pretreatment with a MEK1 inhibitor and was associated with a nearly complete inhibition of insulin-stimulated induction of both Egr-1and Krox20, indicating this pathway is necessary for insulin’s effect on these genes. Finally, inhibition of the p38 pathway, followed by insulin addition, caused an additive induction of both Egr-1and Krox20. In conclusion, these genes are induced by insulin via coordinated regulation of the MEK-ERK and p38 pathways and, in the case of Egr-1, the PI3-K pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN TARGET TISSUES, INSULIN is an important modulator of glucose homeostasis and energy reserves. In cells of hepatic origin, insulin has been shown to regulate the expression and activity of metabolic genes (1, 2, 3). Insulin also regulates the expression of a large number of genes not directly involved in metabolism, such as mitogen-responsive immediate-early genes (1, 4). Insulin’s signal is transduced from its heterotetrameric transmembrane receptor via tyrosine phosphorylation events and subsequent activation of at least two main pathways, the PI3-kinase (PI3-K) and ERK/MAPK cascades (5, 6, 7). Though the effect of insulin on the parallel p38 MAPK pathway has not been as extensively studied, recent reports indicate it is necessary for insulin-mediated glucose transporter activation (8, 9). Our lab and others have recently described an apparent cross talk between the p38 and ERK1/2 pathways (10, 11, 12).

Egr-1 (also known as NGF-1A, Krox24, zif268, and TIS8) and Krox20 (also known as Egr-2) are members of a family of zinc-finger transcription factors (Egr-1–4) with an amino-terminal activation domain, a central domain that interacts with the co-repressor proteins NAB1 and NAB2, and a DNA binding domain consisting of three Cys2-His2 zinc fingers near the carboxy-terminal end of the protein sequence (13, 14, 15, 16, 17). Egr-1 and Krox20 (Egr-2), as well as Egr-3 and Egr-4, bind the consensus nucleotide sequence GCGGGGGCG to regulate expression of target genes (18).

Regulation of the expression of Egr-1 has been studied in a variety of tissues and cell types. Egr-1 is expressed in liver, in liver-derived cell lines (as in the present work), and in neoplasms from human, mouse, and rat (19, 20, 21, 22, 23, 24). Outside the liver, Egr-1 mRNA has been shown to be expressed in heart, brain, spleen, skeletal muscle, and renal cells of mice in response to growth factor treatment (21, 25). Alterations of Egr-1 by mitogenic stimuli have been reported in vivo, especially in normal and pathophysiological states associated with rapid cellular proliferation (19, 21, 26). Human, mouse, and rat fibroblasts express Egr-1 in response to either mitogenic or stress stimulation (19, 27, 28), and Egr-1 is induced in both peripheral and liver resident immune cells by lipopolysaccharide treatment (20, 29, 30).

Insulin induces Egr-1 mRNA in hepatoma cells and in non-liver-derived cells transfected to overexpress insulin receptors (19, 28, 31, 32). Induction of Egr-1 may be vital to insulin’s ability to stimulate proliferation and wound healing in rat aortic endothelial cells (33). Egr-1 is a vital transcription factor for many genes, and recent studies implicate Egr-1 in the control of the hepatic malic enzyme, the phosphatase of regenerating liver (PRL-1), and the apolipoprotein AI genes (22, 23, 34, 35, 36, 37). Egr-1 may also play a role in the expression of cytokines and growth factors such as TNF-{alpha}, PDGF-A, and IGF-II (29, 38). Yoo et al. (39) have described a mechanism whereby Egr-1 may be involved in the induction of primary liver tumors associated with Hepatitis B viral infection.

Normal expression of Krox20 is required for appropriate development of the primordial hindbrain and associated cranial sensory ganglia during embryogenesis (40, 41, 42). Krox20 is also important in myelination of peripheral nerves and maturation of Schwann cells in animal models as well as human disease (40, 43, 44). In addition to its expression in the developing hindbrain, Krox20 expression can be up-regulated by stimuli that induce long-term potentiation of dentate gyrus granule cells (45).

Expression of Krox20 outside of neural or neuroendocrine tissue has been less well studied. A low basal expression of Krox20 is measurable in rat liver and hepatoma cells (46, 19). Murine fibroblasts and preadipocytes, as well as Chinese hamster ovary cells, which overexpress the human insulin receptor, also express the Krox20 gene (19, 32, 47). Endometrial and ovarian tumors express low levels of Krox20, and overexpression of Krox20 reduces colony formation, whereas antisense Krox20 enhances proliferation (48). Peripheral blood monocytes and promyelocytic and T-cell leukemia cells also express Krox20 where it seems to be important in maturation and regulation of Fas ligand expression (49, 50, 51, 52). Finally, like Egr-1, Krox20 is induced in murine fibroblasts by serum or adipocyte differentiation medium (15, 19, 47).

In the present work, we describe the regulation of Egr-1 and Krox 20 by insulin in the rat H4IIE hepatoma cell line. Induction of both genes seems to require engagement of the ERK1/2 pathway. Whereas this effect of insulin does not require activation of the PI3-K pathway, there is cross talk of the PI3-K pathway which modulates Egr-1 expression. Finally, we report that regulation of both Egr-1 and Krox20 genes seems to be subject to cross talk between the p38 and ERK1/2 pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
H4IIE cells were maintained in Swim’s 77 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 2% fetal bovine serum, 3% calf serum, and 5% horse serum. Serum was withdrawn from subconfluent cultures, 24–48 h before experimental treatments. Insulin was obtained from Sigma; SB202190, SB203580, and SB202474 were obtained from Calbiochem (San Diego, CA); LY294002 was obtained from Biomol (Plymouth Meeting, PA); and U0126 was obtained from Promega, Inc. (Madison, WI). PD98059 was obtained from Cell Signaling Technology (Beverly, MA), as were Phospho-(active)-ERK1/2, Total-ERK, Phospho-Akt, and secondary rabbit antisera.

Differential screening of cDNA libraries
Libraries were constructed from H4IIE cells treated with insulin and anisomycin, as described by Bortoff et al. (53). Briefly, the cDNA library colonies were plated, transferred in duplicate to nylon membranes, and denatured. They were then probed with radiolabeled cDNAs synthesized from mRNA isolated from H4IIE cells treated with insulin and anisomycin for 2 h, or cDNAs synthesized from mRNA from untreated cells. Colonies that indicated differential expression were selected for further screening, and the desired colonies were selected for DNA sequence analysis. Two genes induced by insulin identified in this manner were Egr-1 and Krox20. Their identity was established by sequence analysis and homology searching the NCBI GenBank database.

Transcription
Transcription rates were assayed by the nuclear run-on method as previously described (10, 53, 54). Transcriptionally active nuclei were labeled with 32P-UTP and incubated to allow extension of nascent mRNA. Transcripts were isolated and hybridized with cDNAs spotted on nitrocellulose, followed by autoradiography. Densitometric data were analyzed using ZeroD Scan from Scanalytics (Fairfax, VA); and values from experimental treatments, compared with untreated controls, after background subtraction, were expressed as fold-change.

Western blot analysis
SDS whole-cell lysates were resolved by PAGE and transferred to Protran BA85 membranes (Schleicher & Schuell, Keene, NH), developed with ECL Plus (Amersham Biosciences, Buckinghamshire, UK), and visualized by autoradiography or direct digital imaging of chemiluminescent blots using the Fluorchem FC imager system (Alpha Innotech, San Leandro, CA) (10, 55, 56). Antisera to activated p38 was obtained from Biosource International, Inc. (Camarillo, CA), and Egr-1 and Krox20 antisera were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For the comparison of band intensity of Western blot data within each experiment, the value of 100% was arbitrarily assigned to samples treated with insulin for 5 min, and others were expressed in relation to this value. Relative band intensity quantitation and background subtraction were carried out using the onboard software of the Fluorchem FC digital imager (10, 57, 58).

In vitro kinase assays
The nonradioactive kinase assay kit was obtained from Cell Signaling Technology and used according to the supplied protocols. Briefly, p38 was purified by immunoprecipitation from 0.3 mg of soluble cell lysate, by standard methods. The immune complex was washed four times, then incubated at 30 C for 30 min with 200 mM ATP and 2 mg of the purified recombinant substrate, glutathione-S-transferase (GST)-ATF-2 (activating transcription factor 2) (59, 60). The kinase reaction was terminated by the addition of 1% SDS loading buffer and boiling for 5 min. Phosphorylated substrate was detected by Western blotting with antisera specific for the phosphorylated form of ATF-2.

Northern blot analysis
As previously described, total RNA was obtained from cultured H4IIE cells using the Ultraspec reagent (Biotecx, Inc., Houston, TX) (61). Briefly, cultures were washed with PBS, then Ultraspec reagent added. Cell lysate was collected by scraping and the RNA isolated by organic extraction, followed by precipitation in ethanol. Equal amounts of RNA (10 µg) were electrophoresed in 1.2% agarose formaldehyde denaturing gels, then transferred to Bright Star-Plus membranes (Ambion, Austin, TX) using the Turbo-blotter apparatus (Schleicher & Schuell, Inc.). Membranes were probed with radiolabeled cDNA probes prepared using the Prime-It II Kit (Stratagene, La Jolla, CA), followed by autoradiography.

Statistical analysis
Student’s t test and ANOVA were performed using the Instat version 3.0 software (GraphPad, Inc., San Diego, CA). Fold increases in transcription were compared with untreated or vehicle-treated control cultures. In Western blot experiments, values obtained at 5 min insulin treatment were assigned the maximum value of 100% for comparison with other treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of steady-state mRNA and protein products
To confirm preliminary observations obtained in the cDNA library screening, mRNA levels of both Egr-1 and Krox20 were assayed by Northern blot analysis. A time course of insulin treatment was performed in H4IIE cells and compared with the addition of the translational inhibitor, anisomycin, which had also been present during the preparation of the cDNA library. As shown in Fig. 1AGo, Egr-1 mRNA was nearly undetectable in untreated cells. It was rapidly induced by insulin treatment, reaching a maximum level by 30 min, rapidly decreasing by 60 min, and returning to nearly undetectable levels by 120 min. The Krox20 mRNA level was also induced by insulin treatment and followed approximately the same time course as Egr-1 (Fig. 1BGo). Treatment with anisomycin resulted in large increases in both Egr-1 and Krox20 mRNA.



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  		FIG. 1. Induction of Egr-1 and Krox20 mRNA and protein by insulin and anisomycin. Serum-deprived H4IIE cells were treated with 10 nM insulin or 30 µM anisomycin as indicated. Total RNA was analyzed by Northern blot with (A) Egr-1 and (B) Krox20 radiolabeled cDNA followed by autoradiography as described in Materials and Methods. Representative Northern blots are shown. Whole-cell lysates were analyzed by Western blot for expression of (C) Egr-1 and (D) Krox20 as described in Materials and Methods.

 
To determine whether the mRNA of these transcription factors was translated into their respective protein products, Western blot analysis was performed on whole-cell lysates of H4IIE cells. The levels of both proteins were near the limit of detection in serum-starved cells. Following a time course of insulin treatment, expression of both Egr-1 and Krox20 were induced, both reaching peak levels at approximately 4 h, and declining thereafter (Fig. 1Go, C and D).

Transcriptional regulation of Egr-1 and Krox20
To determine whether these genes were induced by insulin at the level of transcription, nuclear run-on assays were performed as described in the experimental procedures. Serum-starved H4IIE cells were treated with 10 nM insulin over a time course from 7.5–180 min. After subtraction of background hybridization, insulin treatment caused a peak in Egr-1 transcription of approximately 40-fold by 15 min, compared with untreated cells (Fig. 2Go). After 60 min, Egr-1 transcription was only 8-fold above control and returned to the basal level by 180 min. Krox20 transcription also peaked at 15 min of insulin treatment but at a lower maximum of 11-fold above control and returning to the basal level by 120 min (Fig. 2Go). This induction of transcription is specific, because transcription of ß-tubulin was not regulated by insulin treatment (data not shown).



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  		FIG. 2. Kinetics of insulin-induced transcription of Egr-1 and Krox20. Serum-deprived H4IIE cells were treated with 10 nM insulin for the indicated times and transcription measured by nuclear run-on assay as described in Materials and Methods. A representative autoradiogram is shown along with mean data collected from three or more separate experiments. Fold induction is compared with vehicle-treated cells after background subtraction. Statistical significance of differences from control: §, P < 0.05 vs. serum-free control (SF Cont); #, P < 0.001 vs. SF Cont.

 
Identification of required signal transduction pathway
The PI3-K-Akt pathway and the MAPK kinase (MEK)-ERK pathways are activated by insulin treatment in cells of hepatic origin. To determine whether either or perhaps both of these pathways were required for insulin’s effect on Egr-1 and Krox20, pathway-specific inhibitors were used. LY294002 is an inhibitor of the PI3-K catalytic subunit, preventing activation of components downstream in this cascade. The serine kinase Akt lies downstream of PI3-K, and its phosphorylation at serine 473 was used as a marker of insulin-activated PI3-K signaling. A maximal activation of phosphorylated (P-)Akt occurred within 5 min and remained at approximately 90% of peak levels for 60 min (Fig. 3Go, A and B). Treatment of H4IIE cells with 50 µM LY294002 completely blocked the insulin-mediated increase in P-Akt.



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  		FIG. 3. Kinetics of activation of MAPK and PI3-kinase pathways by insulin. Serum-deprived H4IIE cells were treated with 10 nM insulin alone or in the presence of kinase inhibitors. Whole-cell lysates were analyzed by Western blot with phospho-specific antisera. A and B) Cells were pretreated for 30 min with 50 µM LY294002 or vehicle, followed by insulin treatment. Akt phosphorylation was determined by Western blot. C, D, and E) Cells were pretreated for 30 min with 50 µM PD98059, 10 µM U0126, or vehicle, followed by insulin treatment. A representative autoradiogram is shown (A, C, and E) along with mean data collected from three or more separate experiments (B and D). {theta}, P < 0.01 vs. SF Cont; {lambda}, P < 0.01 vs. 5-min insulin.

 
The ERK1/2 kinase cascade is also strongly activated by insulin treatment. Insulin treatment of H4IIE cells caused a rapid increase in ERK1/2 phosphorylation/activation, peaking by 5 min and returning to a low (but slightly increased) level, even after 2 h (Fig. 3Go, C and D). Activation of MEK1, which lies immediately upstream of the ERK1/2 MAPKs in this signaling cascade, is specifically inhibited by PD98059 or U0126. MEK1 kinase activity is required for activation of ERK1/2 by insulin in H4IIE cells, because pretreatment with PD98059 or U0126 completely blocked insulin-induced ERK1/2 phosphorylation (Fig. 3Go, C–E) (10).

Having established that the insulin induction of the PI3-K and MEK/ERK signaling pathways can be specifically blocked with these inhibitors in H4IIE cells, the respective effects of these inhibitors on transcription of Egr-1 and Krox20 were examined. The PI3-K inhibitor by itself had no effect, and inhibition of PI3-K did not block insulin-induced transcription of either gene (Fig. 4Go, A and B). In fact, a significant enhancement of the effect of insulin on Egr-1 transcription resulted when cells were pretreated with LY294002 and then treated with insulin, compared with cells treated with insulin alone. Inhibition of insulin-induced MEK1/ERK1/2 signaling by PD98059 pretreatment completely blocked the insulin induction of both Egr-1 and Krox20 transcription, indicating the requirement of this signaling pathway for insulin’s regulation of these genes. These effects on transcription were specific, because ß-tubulin transcription was not affected by any of these treatments (data not shown).



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  		FIG. 4. Insulin-induced transcription of Egr-1 and Krox20 requires MEK-ERK but not PI3-kinase activation. Serum-deprived H4IIE cells were pretreated for 30 min with 50 µM LY294002 (LY) or 50 µM PD98059 (PD) or vehicle, then stimulated with 10 nM insulin for 30 min (Ins 30). Transcription of (A) Egr-1 and (B) Krox20 was measured by nuclear run-on assay and compared with vehicle controls (note different scales). {pi}, not significant vs. SF Cont; {sigma}, P < 0.01 vs. Ins 30; {ddagger}, P < 0.05 vs. Ins 30.

 
Investigation of tonic suppression by p38 MAPK
Recent work suggests cross talk between the p38 and ERK1/2 pathways (10, 11). We have proposed that, in H4IIE cells, p38 may inhibit the ERK1/2 pathway and subsequent gene transcription (10). Inhibition of p38 with the specific inhibitor SB202190 results in activation of ERK1/2 comparable with the peak level caused by insulin (Fig. 5AGo, lane 4 vs. lane 2). When H4IIE cells are pretreated with the p38 inhibitor, followed by stimulation with insulin, ERK1/2 is activated to an even greater extent than with either treatment alone. Furthermore, this activation is prolonged, compared with the transient activation after insulin alone (Fig. 5AGo, lanes 5 and 6, compared with Fig. 3Go, C and D). The distinct p38 inhibitor SB203580 elicited a similar effect on ERK phosphorylation alone and in combination with insulin, but the inactive analog SB202474 did not alter ERK1/2 activation (Fig. 5Go, B and C). Measurement of both phosphorylation and kinase activity revealed that a readily detectable level of p38 activity is present in untreated cells and that this is further enhanced by insulin treatment (Fig. 5Go, D and E).



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  		FIG. 5. Insulin-induced phosphorylation of ERK1/2 is enhanced by inhibition of basal and insulin-induced p38. Serum-deprived H4IIE cells were treated with 10 nM insulin for the indicated times with or without 30 min pretreatment with 10 µM (A) SB202190, (B) SB203580, or (C) SB202474. Presented are representative Western blots of whole-cell lysates probed with anti-Phospho-ERK1/2 antiserum. D, Phosphorylation of p38 was determined using activation-state-specific antisera in Western blots of whole-cell lysates. E, Activation of p38 kinase activity was measured by in vitro kinase assay followed by Western blot analysis of the phosphorylated substrate, GST-ATF-2, as described in Materials and Methods.

 
Because both Egr-1 and Krox20 require ERK1/2 activation for their induction by insulin, we investigated whether alterations in ERK1/2 activity by inhibition of p38 could affect their expression. Cells were treated with either the p38 inhibitor alone or for 60 min before a further 30-min insulin stimulation. SB202190 treatment alone induced Egr-1 transcription to a greater extent than did insulin alone (Fig. 6Go). Inhibition of p38 activity followed by insulin addition resulted in an additive induction of Egr-1 transcription of 64-fold. In contrast, the effect of SB202190 alone on Krox20 was not significantly different from that of insulin added alone (Fig. 7Go). Inhibition of p38 before insulin treatment augmented the effect of insulin, resulting in an at least additive increase in Krox20 transcription of 18-fold (Fig. 7Go). As observed for activation of ERK1/2, the effects of SB203580 on gene transcription were similar to those of SB202190 (data not shown). The transcription rate of the ß-tubulin gene was assayed in parallel (Figs. 6Go and 7Go) and was not altered by any of the treatments described, indicating that the effects on Egr-1 and Krox20 transcription are specific actions of these treatments and do not reflect global changes in transcription.



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  		FIG. 6. Inhibition of p38 induces Egr-1 transcription more than insulin and augments insulin-induced transcription of Egr-1. Serum-deprived H4IIE cells were pretreated with 50 µM PD98059, 10 µM SB202190, or vehicle, then stimulated with insulin for 30 min or given no further treatment. Transcription of Egr-1 (and ß-tubulin as a nonregulated control) was measured by nuclear run-on assay and compared with vehicle controls. The means of three or more experiments are presented. {theta}, P < 0.01 vs. SF Cont; {pi}, not significant vs. SF Cont; §, P < 0.05 vs. SF Cont; {psi}, P < 0.001 vs. Ins 30; {ddagger}, P < 0.05 vs. Ins 30.

 


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  		FIG. 7. Inhibition of p38 induces transcription and augments insulin-induced transcription of Krox20 but not ß-tubulin. Serum-deprived H4IIE cells were pretreated with 50 µM PD98059, 10 µM SB202190, or vehicle, then stimulated with insulin for 30 min or given no further treatment. Transcription of Krox20 and ß-tubulin were measured by nuclear run-on assay and compared with vehicle controls. The means of three or more experiments are presented. {pi}, Not significant vs. SF Cont; §, P < 0.05 vs. SF Cont; {theta}, P < 0.01 vs. SF Cont; {psi}, P < 0.001 vs. Ins 30; {Omega}, not significant vs. Ins 30.

 
As with insulin alone (Fig. 4Go), the transcriptional effect of SB202190 was blocked by inhibition of the MEK1/ERK1/2 pathway. When p38 was inhibited by SB202190, the induction of both Egr-1 (Fig. 6Go) and Krox20 (Fig. 7Go) transcription was inhibited by addition of PD98059. The additive effects of p38 inhibition in combination with insulin treatment also required activation of the MEK-ERK pathway, because addition of PD98059 resulted in a nearly complete blockage of the large induction of transcription by SB202190 plus insulin of both the Egr-1 and Krox20 genes (90.7% and 89.2%, respectively) (Figs. 6Go and 7Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcription of both Egr-1 and Krox20 were rapidly and transiently induced by insulin treatment, both reaching maximum levels by 15 min. These increases in transcription were followed by concomitant increases in steady-state levels of mRNA, reaching maximal levels within only 30 min.

Previous studies have implicated both the PI3-K and MEK-ERK signaling pathways in insulin regulation of hepatic genes. In the present work, peak activation of each of these pathways occurs within 5 min, precedes maximum activation of insulin-induced transcription of either gene studied, and results in accumulation of both Egr-1 and Krox20 mRNA. Further, this is the first report of induction of Krox20 transcription by insulin. Our results with the inhibitor of MEK1 activation, PD98059, also indicate that the MEK-ERK signaling pathway is required for insulin’s induction of the Krox20 gene, because the MEK1 inhibitor PD98059 was able to block insulin’s effect on transcription. Our results also show that ERK1/2 activation is blocked when cells are pretreated with the MEK1/2 inhibitor, U0126. It has been reported that U0126 may also partially inhibit the activity of p38 kinase in vitro and, of direct importance to the present work, in intact H4IIE cells. Therefore, this distinct inhibitor of ERK1/2 activation was not used in transcriptional studies because of this possible confounding factor (62, 63).

The present studies demonstrate a requirement for MEK-ERK activation for induction of Egr-1 transcription by insulin. This supports findings that, in other cell lines, activation of ERK1/2 is necessary for an increase in the level of Egr-1 mRNA in response to insulin (19, 28, 31, 34). However, this is not a uniform finding, because treatment of peripheral blood monocytes with insulin was found to reduce intranuclear Egr-1 (64). Thus, the effects of insulin on Egr-1 are tissue-type specific. Little work has been accomplished, before the present study, indicating a transcriptional vs. a posttranscriptional effect of insulin. Clearly, in liver-derived cells, insulin leads to a rapid induction of Egr-1 transcription, as well as a rapid induction of Krox20 transcription, a related zinc-finger transcription factor.

Previous studies present conflicting data. For example, in rat fibroblasts overexpressing the human insulin receptor, inhibition of PI3-K with wortmannin, or by expressing a mutant GAB-1 gene, partially blocks insulin induction of Egr-1 mRNA (31). However, overexpression of an inducible form of Akt reduced Egr-1 mRNA in murine fibroblasts (65). The present work is more in line with this latter publication. Inhibition of PI3-K did not affect transcription by itself or insulin’s effect on Krox20 transcription. However, when cells were pretreated with LY294002, there was a significant augmentation of insulin-induced Egr-1 transcription. Attempts to use wortmannin to further study this phenomenon could not be pursued, because it can have other effects on insulin signaling [(66, 67); unpublished observations].

This may indicate that there is an additional level of intracellular signaling involved in insulin’s regulation of Egr-1 and that insulin-induced PI3-K activity is involved in turning off the insulin induction of this gene via the MEK-ERK pathway. This modulation of Egr-1 transcription must occur downstream or independently of the activation of ERK1/2, because LY294002 does not cause a concomitant ERK1/2 activation in H4IIE cells (data not shown), and this effect was not seen on the Krox20 gene, which is also ERK1/2 dependent. Thus, a PI3-K-activated inhibitory pathway is proposed. A similar effect on Egr-1 expression was reported in monocytes and endothelial cells, in which ERK1/2-dependent induction of Egr-1 by LPS or thrombin is augmented by either LY294002 or a dominant negative Akt mutant (25, 30). In contrast to our data, induction of ERK1/2 activity was coordinately increased, suggesting the effect was through augmenting activation of the MEK-ERK pathway and subsequent gene transcription. Such findings may indicate an inhibitory action of Akt itself on Raf1, the kinase upstream of MEK1, and subsequent MEK/ERK activation as previously described in cells of renal origin (68). Different signal transduction cascades are expected to be engaged by these diverse stimuli, such as insulin vs. LPS or thrombin, which act through different receptors. Thus, it is not surprising that different activators of MEK-ERK may also activate additional or separate feedback pathways.

Another possible interpretation of our findings is that insulin may simultaneously have two actions on Egr-1 transcription, a strong induction and a weaker secondary inhibition. There are several previous findings of biphasic (both stimulatory and inhibitory) actions of insulin on a single gene (69, 70). In the present work, insulin may decrease Egr-1 transcription by increasing Akt activity via PI3-K. Thus, inhibition of insulin-induced PI3-K activity without inhibition of the parallel stimulatory MAPK pathway could result in the enhanced insulin effect observed here. This suggests that there is a mechanism for very fine control of Egr-1 transcription, in which stimuli such as insulin that activate both the ERK1/2 pathway and the PI3-K pathway have a qualitatively different effect than stimuli that may activate only one or the other of these pathways. Further studies to understand these complex regulatory patterns are warranted and may require detailed analysis of elements in the Egr-1 promoter that respond to insulin treatment and other stimuli in H4IIE cells. This is especially true because insulin’s ability to activate the PI3-K pathway can be dramatically altered in insulin-resistant cells and animal models (71).

Our laboratory and others have identified an apparent cross talk between the p38 and ERK1/2 MAPK pathways. In various cell types, including human and rat hepatoma cells, activity of p38 has been found to reciprocally affect ERK1/2 activity and MEK-ERK-dependent effects (11, 12, 72). We have previously reported that the induction of the immediate early genes c-Fos and Pip92 by insulin are affected by such cross talk in H4IIE cells (10). In the present work, the inhibition of p38 activity by the specific inhibitor SB202190 results in activation of the ERK1/2 pathway and the induction of both Egr-1 and Krox20 transcription. An additive induction of both genes was produced when cells were stimulated with the combination of the p38 inhibitor and insulin. The induction of Krox20 by the combination of SB202190 and insulin may be greater than additive, something we have found with both c-Fos and Pip92 (10). As in the present studies, in rat mesangial cells, another p38 inhibitor (SB203580) induced increases in Egr-1 mRNA. However, this p38 inhibitor did not cooperate with LPA in this induction (73).

The effects of the p38 inhibitor and its involvement in insulin-mediated transcription of these genes occur at or above the level of MEK1 in the ERK1/2 pathway. Treatment with SB202190 resulted in activation of ERK1/2 phosphorylation, which was further enhanced by subsequent insulin treatment. When cells were pretreated with the MEK1 inhibitor PD98059, induction of gene transcription by p38 inhibition alone or in combination with insulin was blocked. Taken together, these results suggest that p38 may play a role in the regulation of the ERK1/2 pathway and subsequent transcription of the Egr-1 and Krox 20 genes by insulin.

Other groups have identified similar cross talk between the p38 and ERK1/2 pathways that may effect gene transcription. Cyclin D1 expression is induced in lung fibroblasts by upstream activators of ERK1/2 and inhibited by suppression of the MEK-ERK1 pathway. Inhibition of p38 by SB203580 also resulted in induction of this ERK-dependent gene, whereas activation of the p38 pathway inhibits expression of cyclin D1 (74). These findings are similar to the present work, where inhibition of p38 activity induced expression of insulin-regulated, ERK-dependent genes.

Other studies have also found induction of P-ERK1/2 by inhibitors of the p38 pathway in rat pineal cells. This induction was sensitive to MEK1 inhibitors but insensitive to inhibitors of other kinases (cyclic GMP-dependent protein kinase, protein kinase C, and calmodulin-dependent protein kinase). Additionally, this effect seemed to be independent of phosphatases such as PP1, PP2A, PTP-1B, or SHPTP-1 (12). Together with our work, these studies suggest that p38 exerts an inhibitory effect on the MEK-ERK pathway independent of these kinases or phosphatases. Together, this evidence suggests that the effect of p38 inhibition to activate ERK1/2 may proceed via regulation of one or more unidentified kinases or signaling intermediates that act at MEK1 or upstream of MEK1. Future studies will seek to positively identify the component of the MEK-ERK pathway that is directly affected.

Egr-1 and Krox20 mRNA were also strongly induced in the present studies by anisomycin treatment. Anisomycin is also able to strongly induce the phosphorylation of all three MAPK pathways [ERK, c-jun N-terminal kinase (JNK), and p38, data not shown]. This is in contrast to the modest effect of insulin on p38 observed here. In PC12 and NIH3T3 cells, anisomycin’s ability to induce Egr-1 transcription is independent of ERK activation (75, 76). In fact, inhibition of p38 blocks this effect of anisomycin on Egr-1 expression in these cell types. This suggests that there are significant differences in p38’s role in regulating Egr-1 in different cell types.

In summary, we have presented direct evidence of the transcriptional regulation of the two zinc-finger transcription factors, Egr-1 and Krox20, by insulin in H4IIE rat hepatoma cells. This effect of insulin required activation of the ERK1/2 pathway, which could be further modulated by alterations in the activity of the parallel p38 MAPK pathway. Insulin’s effect on either gene did not require activation of the PI3-K pathway. However, inhibition of the PI3-K pathway augmented insulin’s effect on Egr-1, suggesting that some factor downstream of PI3-K may partially inhibit induction of Egr-1. Consistent with these findings is our proposed model (Fig. 8Go) of insulin’s regulation of Egr-1 and Krox20 expression illustrating the apparent feedback between the p38 and MEK-ERK pathways, the inhibitory effect of the PI3-K pathway on Egr-1 transcription alone, and how inhibition of these pathways may affect insulin’s activation of ERK1/2 and Egr-1 and Krox 20 transcription.



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  		FIG. 8. Proposed model of insulin regulation of Egr-1 and Krox20. In H4IIE cells, the interactions are represented by: ->, activation; {perp}, inhibition; IR, insulin receptor.

 


   Acknowledgments
 
We thank Drs. S. A. Abdulkadir and Y. Ma and L. T. Holland for their helpful and insightful discussions and suggestions in preparation of this manuscript.


   Footnotes
 
This work was supported by a grant from the American Diabetes Association and by National Institutes of Health Grant DK40456 (to J.L.M.). A Department of Education Fellowship provided support for W.L.B.

Abbreviations: ATF, Activating transcription factor; JNK, c-jun N-terminal kinase; MEK, MAPK kinase; P-, phosphorylated; PI3-K, PI3-kinase.

Received May 14, 2003.

Accepted for publication August 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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