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Endocrinology Vol. 141, No. 2 544-550
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor I Activates c-Jun N-Terminal Kinase in MCF-7 Breast Cancer Cells1

Satoshi Monno, Marsha V. Newman, Michelle Cook and William L. Lowe, Jr.

Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Veterans Administration Chicago Healthcare System-Lakeside Division and Northwestern University Medical School, Chicago, Illinois 60611

Address all correspondence and requests for reprints to: William L. Lowe, Jr., M.D., Center for Endocrinology, Metabolism, and Molecular Medicine, Tarry 15–703, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: wlowe{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin-like growth factor I (IGF-I) is an important mediator of breast cancer cell growth, although the signaling pathways important for IGF-I-mediated effects in breast cancer cells are still being elucidated. We had demonstrated previously that increased intracellular cAMP in MCF-7 breast cancer cells inhibited cell growth and IGF-I-induced gene expression, as determined using a reporter gene assay. This effect of cAMP on IGF-I signaling was independent of IGF-I-induced activation of the mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (ERK1 and -2). To determine whether this effect of cAMP may be mediated via another mitogen-activated protein kinase, the ability of IGF-I to activate the c-Jun N-terminal kinases (JNKs) was investigated. Treatment of MCF-7 cells with 100 ng/ml IGF-I increased the level of phosphorylated JNK, as determined by Western blot analysis. JNK phosphorylation was not evident until 15 min after treatment with IGF-I, and peak levels of phosphorylation were present at 30–60 min. This was in contrast to ERK phosphorylation, which was present within 7.5 min of IGF-I treatment. Determination of JNK activity using an immune complex assay demonstrated a 3.3- and 3.5-fold increase in JNK1 and -2 activity, respectively, 30 min after treatment with 100 ng/ml IGF-I. The use of PD98059, which inhibits activation of ERK1 and -2, and LY 294002, an inhibitor of phosphatidylinositol 3-kinase, demonstrated that IGF-I-induced activation of JNK1 is independent of ERK and phosphatidylinositol 3-kinase activation. In contrast, increasing intracellular cAMP with forskolin resulted in abrogation of IGF-I-induced JNK activity. In summary, these data demonstrate that IGF-I activates the JNKs in MCF-7 breast cancer cells and, taken together with the results of our previous study, suggest that JNK may contribute to IGF-I-mediated gene expression and, possibly, cell growth in MCF-7 breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) and its receptor mediate a variety of growth-promoting and metabolic effects in different cell types (1, 2). The cellular events that follow IGF-I binding to its receptor and account for its many biological effects are still being elucidated, but a number of the signaling pathways that are activated upon IGF-I binding have been defined. Ligand binding to the IGF-I receptor results in receptor autophosphorylation, increased receptor tyrosine kinase activity, and tyrosine phosphorylation of a number of substrate molecules, including members of a family of insulin receptor substrates, Shc, Crk, and Grb2 (3, 4). Subsequent to these early signaling events, two signaling pathways, the phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK) pathways, that mediate many of the biological effects of the IGFs are activated (3, 4).

The MAPKs are a family of serine-threonine kinases that are activated in response to a variety of extracellular stimuli (5, 6). Activation of the MAPKs occurs via a protein kinase cascade in which MAPK kinase kinases phosphorylate MAPK kinases (MEKs), which, in turn, phosphorylate and activate MAPKs (5, 6). There are several different MAPKs that have different substrate specificities, are activated by different MEKs, and are, in general, linked to different signals at the plasma membrane (5, 6). The first family of MAPKs to be identified was the extracellular signal-regulated kinases (ERKs), which are referred to as ERK1 and -2. These kinases are activated primarily in response to proliferative stimuli (7). Additional MAPKs that respond primarily to cell stresses have also been identified. These include the c-Jun N-terminal kinases (JNKs) and the p38 kinases. Multiple JNK isoforms that originate from three homologous genes, JNK1, JNK2, and JNK3, have been identified (8). JNK1 and -2 are expressed in a relatively ubiquitous fashion, whereas JNK3 is expressed in brain. The JNKs are activated primarily in response to cytokines, UV radiation, and environmental stresses (8, 9). The third family of MAPKs is the p38 MAPKs. Four p38 genes, {alpha}, ß, {gamma}, and {delta}, have been described to date (8, 10, 11). Like the JNKs, these kinases are activated primarily in response to environmental stresses (8).

In a previous study we examined the effect of increased intracellular cAMP on proliferation of MCF-7 breast cancer cells and IGF-I signaling (12). cAMP inhibited both MCF-7 cell growth and IGF-I-induced signaling, as measured in a reporter gene assay. These same studies demonstrated that IGF-I activated the ERKs in MCF-7 cells, but that cAMP had no effect on IGF-I-induced ERK activity, suggesting that the effect of cAMP on IGF-I-induced signaling was independent of ERK activation. As other growth factors, e.g. epidermal growth factor, have been shown to activate the JNKs (13), the present study was designed to determine whether IGF-I was able to activate the JNKs in MCF-7 cells with the ultimate goal of determining the role of the JNKs in IGF-I-induced effects in breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
MCF-7 cells were provided by Dr. Craig Jordan (Lurie Cancer Center, Northwestern University Medical School, Chicago, IL). The cells were maintained in 75-cm2 flasks in MEM supplemented with 5% calf serum, 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50 U/ml) at 37 C in 5% CO2. Upon reaching confluence, the cells were replated at a dilution of 1:4. In all experiments the cells were preincubated for the indicated period of time in phenol red-free MEM with 0.1% charcoal-stripped calf serum.

JNK immune complex assay
Antibodies directed against JNK1 and -2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The substrate protein for the JNK immune complex assay was a glutathione-S-transferase (GST)-c-Jun fusion protein. To generate a complementary DNA for the synthesis of this fusion protein, a fragment of DNA encoding the amino-terminal 81 amino acids of c-Jun was amplified using PCR with a rat c-jun complementary DNA as template DNA. The amplified fragment, the identity of which was confirmed by DNA sequencing, was purified and cloned into pGEX4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) in-frame with the GST gene. The resulting fusion protein (GST-c-Jun81) was purified from bacterial lysates using glutathione-agarose beads according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

This immune complex assay was performed using a previously described method (14). Briefly, before treatment with IGF-I, cells were placed into MEM and 0.25% BSA for 48 h. The cells were then treated with 100 ng/ml IGF-I for the indicated period of time and solubilized in lysis buffer [20 mM Tris (pH 7.4), 1% Triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM ß-glycerophosphate, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, 10 µg/ml leupeptin, and 1 mM phenylmethanesulfonylfluoride]. The protein concentration of the cell lysate was determined using the Coomassie blue protein assay (Bio-Rad Laboratories, Inc., Richmond, CA). After clarification, 100–150 µg cell lysate protein were incubated for 2 h at 4 C with anti-JNK1 or anti-JNK2 antibodies that had been prebound to protein A-agarose beads. The beads were collected, washed, and resuspended in kinase buffer [25 mM HEPES (pH 7.4), 25 mM ß-glycerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM sodium orthovanadate]. The kinase reaction was then initiated by adding 12.5 µCi [{gamma}-32P]ATP and 1 µg GST-c-Jun81 and was allowed to proceed for 30 min at 30 C. The reaction was terminated by the addition of Laemmli sample buffer. The proteins were eluted from the beads by heating at 95 C for 5 min and were separated by SDS-PAGE on a 15% polyacrylamide gel. The resulting gel was dried and exposed to x-ray film or used in a STORM 860 PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) to calculate 32P incorporation into GST-c-Jun81. All assays were performed in duplicate.

Western blot analysis
Western blots were probed with a monoclonal antibody directed against phospho-ERK1/2 (New England Biolabs, Inc., Beverly, MA) or with a polyclonal antibody directed against phospho-SAPK/JNK (New England Biolabs, Inc.), phospho-Akt (New England Biolabs, Inc.), ERK2 (Santa Cruz Biotechnology, Inc.), Akt (New England Biolabs, Inc.), or JNK1 (Santa Cruz Biotechnology, Inc.). All phospho-specific antibodies were used at a dilution of 1:1000. Anti-JNK1, -JNK2, and -ERK2 antibodies were used at a dilution of 1:7500, whereas the anti-Akt antibody was used at a dilution of 1:1000.

For Western blot analyses, cell lysates were prepared in the cell lysis buffer used for the JNK immune complex assays as described above, and the protein content of the lysate was determined using the Coomassie blue protein assay. Fifty micrograms of protein were then diluted 1:4 in sample buffer [62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 1% bromophenol blue], boiled for 5 min, and size-separated using SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane using a semidry apparatus in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). For Western blot analysis, membranes were blocked in TBST containing 5% nonfat dry milk for 90 min at room temperature. Membranes were incubated for 90 min at 22 C in TBST containing nonfat milk and primary antibody, washed three times for 15 min each time at 22 C in 20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween-20 (TBST), and incubated for 90 min at room temperature in TBST containing nonfat dry milk and secondary antibody (1:7500 dilution). After three washes in TBST, immunoreactive bands were detected using the enhanced chemiluminescence detection system from Amersham Pharmacia Biotech (Arlington Heights, IL), according to the manufacturer’s instructions.

Statistical analysis
Values are reported as the mean ± SEM P values were calculated using the Mann-Whitney rank sum test or Kruskal-Wallis one-way ANOVA on ranks with the Dunnett’s pairwise multiple comparison procedure, as appropriate, using SigmaStat 2.0 software (Jandel Corp., San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
JNK activation is dependent upon tyrosine and threonine phosphorylation by specific MEKs (9). To determine whether the JNKs were activated by IGF-I in MCF-7 breast cancer cells, MCF-7 cells were treated for varying periods of time with 100 ng/ml IGF-I, and Western blot analyses were performed using an antibody specific for the phosphorylated form of JNK (Fig. 1AGo). To compare the effect of IGF-I on JNK phosphorylation with its effect on another member of the MAPKs, Western blot analyses also were performed using an antibody specific for phosphorylated ERK1 and 2 (Fig. 1BGo). In a previous study we had demonstrated that IGF-I activates the ERKs in MCF-7 cells (12). As can be seen, IGF-I was able to stimulate phosphorylation of both the JNKs and ERKs in MCF-7 cells, although the time course of the effect was quite different. Similar to what has been found in multiple cell types, phosphorylation of the ERKs was already evident 7.5 min after IGF-I treatment, and consistent with the results of our previous study, the effect of IGF-I was diminished 60 min after treatment (12). In contrast, phosphorylation of the JNKs was not apparent until 15 min after treatment with IGF-I, and peak phosphorylation appeared to occur 30–60 min after treatment with IGF-I.



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  		Figure 1. A, Western blot analysis of phospho-JNK and JNK1 levels in MCF-7 cells. Cells were treated for the indicated periods of time with MEM and 0.25% BSA without or with 100 ng/ml IGF-I. Proteins in the cell lysates were size-separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blot analysis was performed as described in Materials and Methods using a 1:1000 dilution of antibody directed against phospho-JNK. After detection of phospho-JNK, the blot was stripped and reprobed with a 1:7500 dilution of antibody directed against JNK1. The findings are representative of the results of two independent experiments performed using different lysates. B, Western blot analysis of phospho-ERK and ERK2 levels in MCF-7 cells. Cells were treated, and Western blot analysis was performed using a 1:1000 dilution of antibody directed against phospho-ERK. After detection of phospho-ERK, the blot was stripped and reprobed with a 1:7500 dilution of antibody directed against ERK2. The findings are representative of the results of two independent experiments performed using different lysates.

 
To determine whether JNK phosphorylation was accompanied by an increase in JNK activity, an immune complex assay was used to determine the time course of the effect of IGF-I on JNK1 activity. As shown in Fig. 2AGo, treatment with 100 ng/ml IGF-I increased JNK1 activity, as reflected by IGF-I-induced phosphorylation of a c-Jun substrate protein. Quantification of the effect of IGF-I on JNK1 activity demonstrated that IGF-I induced a 3.3-fold increase in JNK1 activity, and consistent with the studies examining IGF-I-induced JNK phosphorylation, the peak activity of JNK1 occurred 30–60 min after treatment with IGF-I (Fig. 2BGo). Given the protracted time course of the effect of IGF-I on JNK activity, Western blot analyses of cell lysates were performed to demonstrate that the effect of IGF-I on JNK activity was not secondary to an increase in the level of JNK1 protein (Fig. 2CGo). IGF-I had a similar effect on JNK2 activity, stimulating 3.5 ± 0.9- and 3.1 ± 1.1-fold (mean ± SEM; n = 3) increases in activity at 30 and 60 min, respectively.



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  		Figure 2. The time course of IGF-I-induced JNK1 activity in MCF-7 cells. A, Autoradiogram of phosphorylated GST-c-Jun81 substrate protein. Cells were treated with MEM and 0.25% BSA without or with 100 ng/ml IGF-I for the indicated periods of time. JNK1 activity was measured using an immune complex assay as described in Materials and Methods. A representative autoradiogram is shown. B, Time course of the effect of IGF-I on JNK1 activity. JNK1 activity was measured using an immune complex assay, and kinase activity was quantified by measuring 32P incorporation into the substrate protein using a PhosphorImager. Values represent the relative 32P incorporation into GST-c-Jun81 using lysates from cells treated for the indicated period of time with 100 ng/ml IGF-I compared with 32P incorporation using lysates from cells maintained for the same period of time in the absence of IGF-I, which was defined as 1.0, and are the mean ± SEM of three independent experiments. *, P < 0.05 compared with the activity in cells maintained for the same period of time in the absence of IGF-I. C, Western blot analysis of JNK1 levels in cells treated with MEM and 0.25% BSA without or with 100 ng/ml IGF-I for the indicated period of time. Western blot analysis was performed using a 1:7500 dilution of antibody directed against JNK1 as described in Materials and Methods.

 
Studies were performed next to define the mechanism for IGF-I-induced JNK1 activity. As described above, one of the major pathways that mediates the cellular effects of IGF-I is the ERK pathway (3, 4). Given the apparent delay in the activation of JNK1 after treatment with IGF-I, studies were performed to address whether ERK activation is required for IGF-I-induced activation of JNK1. Cells were pretreated for 30 min with 10 µM PD98059, an inhibitor of MEK1 and -2, the MAPK kinases that are responsible for activation of ERK1 and -2. Treatment with 100 ng/ml IGF-I for 30 min increased the activity of JNK1 2.5- to 3-fold in both the absence and presence of PD98059 (Fig. 3AGo). To confirm that PD98059 was effective, Western blot analyses using antibodies specific for phospho-ERK were performed using lysates from IGF-I-treated and control cells incubated in the absence and presence of PD98059 (Fig. 3BGo). PD98059 was responsible for a marked decrease in both basal and IGF-I-stimulated phospho-ERK levels, although residual IGF-I-stimulated ERK phosphorylation was evident. To determine whether higher doses of PD98059 would abrogate this residual phosphorylation, studies were performed using 100 µM PD98059. Even with this high dose of inhibitor, a similar amount of residual IGF-I-induced ERK phosphorylation was present (data not shown). Moreover, interpretation of the kinase assay data using this concentration of PD98059 was complicated, as 100 µM PD98059 alone stimulated a 2.6 ± 0.6-fold (mean ± SEM; n = 7) increase in JNK1 activity compared with the activity in cells not treated with PD98059. Despite that, JNK1 activity increased 3.0 ± 0.7-fold (mean ± SEM; n = 7) in cells treated with IGF-I and PD98059 compared with that in cells treated with PD98059 alone. This was similar to the 3.4 ± 0.9-fold (mean ± SEM; n = 7) increase in JNK1 activity that was observed in IGF-I-treated compared with untreated cells in the absence of PD98059. Together, these data are consistent with IGF-I-induced ERK activity not being required for IGF-I-induced JNK1 activity.



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  		Figure 3. Effect of PD98059 on IGF-I-induced JNK activity and ERK phosphorylation. A, Effect of PD98059 on IGF-I-induced JNK1 activity. Cells were pretreated for 30 min without or with 10 µM PD98059 and then treated for 30 min without or with 100 ng/ml IGF-I. JNK1 activity was determined using an immune complex assay as described in Materials and Methods. Values represent the relative level of JNK1 activity compared with the level in cells maintained in MEM and 0.25% BSA in the absence of PD98059 and IGF-I, which was defined as 1.0, and are the mean ± SEM of five independent experiments. *, P < 0.01 compared with the activity in cells maintained in the absence of PD98059 and IGF-I; +, P < 0.05 compared with the activity in cells treated with PD98059 alone. B, Western blot analysis of effect of PD98059 on IGF-I-induced ERK phosphorylation. Cells were treated as described above, and Western blot analysis was performed using a 1:1000 dilution of antiphospho-ERK antibody as described in Materials and Methods. The blot was then stripped and reprobed using a 1:7500 dilution of antibody directed against ERK2.

 
A second major signaling pathway activated by IGF-I is phosphatidylinositol (PI) 3-kinase and its downstream effectors. In a previous study PI 3-kinase activity was shown to be required for JNK activation by epidermal growth factor in HeLa cells (13). To determine whether PI 3-kinase also was required for IGF-I-induced JNK1 activity, cells were pretreated for 30 min in the absence and presence of 50 µM LY 294002, an inhibitor of PI 3-kinase. In the absence of LY 294002, treatment for 30 min with 100 ng/ml IGF-I stimulated a 3.8-fold increase in JNK1 activity compared with activity in cells maintained in MEM and 0.25% BSA (Fig. 4AGo). When cells were pretreated with LY 294002, IGF-I still stimulated a 3.2-fold increase in JNK1 activity compared with that in cells treated with LY 294002 alone (Fig. 4AGo). These data suggest that activation of PI 3-kinase is not required for IGF-I-induced activation of JNK1. To confirm that LY 294002 was efficacious, its effect on IGF-I-induced phosphorylation of Akt or protein kinase B, a kinase that is activated by IGF-I in a PI 3-kinase-dependent fashion, was examined (Fig. 4BGo). Treatment with 100 ng/ml IGF-I for 30 min stimulated a marked increase in Akt activity, which was completely abrogated by 50 µM LY 294002.



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  		Figure 4. Effect of LY 294002 on IGF-I-induced JNK1 activity and Akt phosphorylation. A, Effect of LY 294002 on IGF-I-induced JNK1 activity. Cells were pretreated for 30 min without or with 50 µM LY 294002 and then treated for 30 min without or with 100 ng/ml IGF-I. JNK1 activity was determined using an immune complex assay as described in Materials and Methods. Values represent the relative level of JNK1 activity compared with the level in cells maintained in MEM and 0.25% BSA in the absence of LY 294002 and IGF-I, which was defined as 1.0, and are the mean ± SEM of four independent experiments. *, P < 0.05 compared with the activity in cells maintained in the absence of LY 294002 and IGF-I. The increase in JNK1 activity in cells treated with LY 294002 and IGF-I compared with the activity in cells treated with LY 294002 alone was of borderline statistical significance (P = 0.057). B, Western blot analysis of effect of LY 294002 on IGF-I-induced Akt phosphorylation. Cells were treated as described above, and Western blot analysis was performed using a 1:1000 dilution of antiphospho-Akt antibody as described in Materials and Methods. The blot was stripped and reprobed using a 1:1000 dilution of antibody directed against Akt.

 
Consistent with the above findings, rapamycin had no effect on IGF-I-induced activation of JNK1. Rapamycin is thought to work primarily by inhibiting activation of p70S6 kinase, which, in the case of IGF-I and insulin, is thought to be activated downstream of PI 3-kinase (15), although activation of p70S6 kinase via pathways that are independent of PI-3 kinase has been described (16). To examine the effect of rapamycin on IGF-I-induced JNK1 activity, cells were pretreated for 30 min in the absence or presence of 100 nM rapamycin. In the absence of rapamycin, treatment for 30 min with 100 ng/ml IGF-I stimulated a 2.2 ± 0.3-fold increase in JNK1 activity compared with basal activity in cells maintained in MEM and 0.25% BSA (mean ± SEM; n = 4; P < 0.05). Compared with basal activity, JNK1 activity increased 3.1 ± 0.4-fold in cells treated with 100 ng/ml IGF-I in the presence of rapamycin, whereas JNK1 activity increased only 1.3 ± 0.2-fold in cells treated with rapamycin alone (mean ± SEM; n = 4; P = 0.01 for difference between groups). These data suggest that the effect of IGF-I on JNK1 activity was not mediated via a rapamycin-sensitive pathway.

Previously, we used a reporter gene assay to demonstrate that pretreating cells for 30 min with 10 µM forskolin, an activator of adenylate cyclase that increases intracellular cAMP, inhibited IGF-I-induced activity of the serum response element (SRE) (12). This effect of cAMP occurred independent of IGF-I-induced ERK activation. To determine whether IGF-I-induced JNK1 activity was sensitive to increased intracellular cAMP, cells were pretreated for 30 min with 10 µM forskolin before treatment with 100 ng/ml IGF-I for either 30 or 60 min (Fig. 5Go). Pretreatment with forskolin resulted in complete abrogation of IGF-I-induced JNK1 activity regardless of the period of treatment with IGF-I. A similar effect of forskolin on IGF-I-induced JNK2 activity was observed (data not shown).



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  		Figure 5. Effect of forskolin on IGF-I-induced JNK1 activity. Cells were pretreated for 30 min without or with 10 µM forskolin and then treated for 30 min (left panel) or 60 min (right panel) without or with 100 ng/ml IGF-I. JNK1 activity was determined using an immune complex assay as described in Materials and Methods. Values represent the relative level of JNK1 activity compared with the level in cells maintained in MEM and 0.25% BSA in the absence of forskolin and IGF-I, which was defined as 1.0, and are the mean ± SEM of three independent experiments. *, P < 0.05 compared with the activity in cells maintained in the absence of forskolin and IGF-I.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have demonstrated growth-promoting effects of IGF-I in MCF-7 cells (17, 18), although the signaling pathways that are required for these effects of IGF-I have not been completely elucidated. Recent studies have suggested that activation of PI 3-kinase and/or the ERKs is required for the growth-promoting effects of IGF-I in MCF-7 cells (19, 20). Whether other signaling systems contribute to IGF-I-mediated effects in MCF-7 cells has not been specifically addressed. In the present study we have demonstrated that IGF-I also increases JNK1 and -2 activity in MCF-7 cells. Interestingly, the time course of JNK activation by IGF-I was distinct from that of ERK activation, with peak activity occurring 30–60 min after IGF-I treatment. Additional studies demonstrated that IGF-I-induced JNK activity was not dependent upon activation of either the ERKs or PI 3-kinase. In fact, inhibition of ERK activity using the MEK1 inhibitor PD98059 or PI 3-kinase activity using LY 294002 increased basal JNK activity. Whether these inhibitors activate a cell stress response and, subsequently, the stress-activated protein kinases, or inhibition of these pathways relieves the JNK pathway of tonic inhibition is unclear.

The contribution of IGF-I-induced JNK activation to IGF-I-mediated effects in MCF-7 cells remains unclear. In a previous study we used a reporter gene assay to demonstrate that increased intracellular cAMP attenuated the ability of IGF-I to stimulate the activity of the SRE (12), a promoter element that is growth factor responsive and activated in response to a variety of growth-promoting events (21). Interestingly, IGF-I-induced ERK activity was unaffected by cAMP in these studies, suggesting that the effect of cAMP on SRE activity was independent of IGF-I-induced ERK activation. The activity of the SRE is modulated by activation of a ternary complex factor that binds to the SRE. Ternary complex factor is one of several Ets domain DNA-binding proteins that include Elk-1 (21, 22). Modulation of tran-scriptional activity by Elk-1 is dependent upon Elk-1 phosphorylation (21, 22). Interestingly, among the kinases capable of phosphorylating Elk-1 are not only the ERKs, but the JNKs as well (23, 24). In the present study increased intracellular cAMP completely abrogated the effect of IGF-I on JNK activation. Thus, taken together with the observation that increased intracellular cAMP inhibits IGF-I-induced SRE activity, these data suggest that JNK activation may mediate the effect of IGF-I on SRE activity.

In addition to the effect of cAMP on SRE activity, we and others have demonstrated inhibition of breast cancer cell growth by cAMP in MCF-7 and other breast cancer cells (12, 25, 26). A second possibility is that JNK participates in the stimulation of MCF-7 cell growth by IGF-I. Increased activity of the JNKs typically has been associated with inhibition of cell growth and/or apoptosis (8, 9), but there are data to suggest that JNK may have growth-promoting effects under certain conditions. In vascular smooth muscle cells, inhibition of thrombin-induced DNA synthesis by cAMP is accompanied by inhibition of JNK activity, whereas cAMP has no effect on ERK activity in these cells (14). In quiescent fibroblasts, microinjection of the small G proteins, Rho, Rac, and Cdc42, stimulated progression through the cell cycle and DNA synthesis (27). Interestingly, microinjection of these proteins did not activate the ERKs; rather, it resulted in JNK activation. Finally, JNK is activated in thyroid cells after treatment with TSH, a growth-promoting factor in thyroid cells (28). The mechanism for the inhibitory effect of cAMP on MCF-7 cell growth has not been defined, but, as described, ERK activity is not affected by cAMP in MCF-7 cells (12). It is now clear, however, that increased cAMP is accompanied by an inhibition of IGF-I-induced JNK activity, suggesting that the JNKs may participate in IGF-I-mediated growth of breast cancer cells. Given the previous data that activation of PI 3-kinase and the ERKs is required for the growth-promoting effects of IGF-I in MCF-7 cells (19, 20) and our finding that activation of these pathways is not required for JNK activation by IGF-I, the JNKs are unlikely to be the sole mediator of the effects of IGF-I on cell growth, but, together with the findings of our previous study (12), the data in the present study suggest that the JNKs may contribute to IGF-I-induced growth of MCF-7 cells. To further address this important issue will require the use of specific inhibitors of the JNKs, which are not currently available.

The ability of IGF-I to increase JNK activity stands in contrast to recently reported findings in other cell types. In SH-SY5Y cells, a human neuroblastoma cell line, and 293 cells, an embryonic kidney cell line, IGF-I treatment alone had little or no effect on JNK activity, but in both cell lines, IGF-I was able to inhibit stress-induced JNK activity (29, 30). In SH-SY5Y cells, IGF-I inhibited hyperglycemia-induced JNK activity, whereas in 293 cells, IGF-I inhibited anisomycin- and tumor necrosis factor-{alpha}-induced JNK activity. Interestingly, different signaling pathways were used by IGF-I in the two cell types to effect these changes in JNK activity. In 293 cells, IGF-I-induced PI 3-kinase activity was required, whereas ERK activation was required in SH-SY5Y cells. In contrast to IGF-I, the related peptide, insulin, has been shown to increase JNK activity in some cell types. In vivo, insulin stimulated a rapid and transient increase in JNK activity in skeletal muscle (31). JNK activity increased within 30 sec in response to insulin treatment and returned nearly to baseline by 4 min. Similarly, rapid activation of JNK by insulin has been demonstrated in rat adipocytes in primary culture and in L6 myotubes (32). In contrast, insulin has no effect on JNK activity in either Chinese hamster ovary cells overexpressing the insulin receptor or 3T3L1 adipocytes (33). Thus, the effect of IGF-I and insulin on JNK activity is dependent upon both the specific cell type being examined and, in the case of IGF-I, the context of IGF-I treatment.

The signal transduction pathway responsible for IGF-I-induced JNK activation in MCF-7 cells is unknown at present. Our studies demonstrate that neither PI 3-kinase activation, ERK activation, nor a rapamycin-sensitive pathway is required. As described, a family of small G proteins, including Rac, Rho, and Cdc42, is able to mediate JNK activation (27, 34). Whether IGF-I is able to activate this family of proteins has not been examined directly, but this family of guanosine triphosphatases regulates actin-based cytoskeletal reorganization, a process that IGF-I is known to affect in different cell types, including MCF-7 cells (35, 36, 37). An alternative, but not mutually exclusive, possibility is that IGF-I activates a JNK-specific MEK via a pathway that is sensitive to inhibition by cAMP. Future studies will be needed to determine the specific pathway that mediates the effect of IGF-I on JNK activation.


   Acknowledgments
 
The authors thank Dr. Eva Feldman for helpful discussions.


   Footnotes
 
1 This work was supported by a grant from the Butz Foundation and by the Northwestern Memorial Foundation. Back

Received June 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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