Distinct and Common Pathways in the Regulation of Insulin-like Growth Factor-1 Receptor Gene Expression by Angiotensin II and Basic Fibroblast Growth Factor*

Kathrin J. Scheidegger, Jie DuDagger , and Patrick Delafontaine§

From the Division of Cardiology, University Hospital of Geneva, Switzerland and the Dagger  Department of Medicine, Emory University, Atlanta, Georgia 30322

    ABSTRACT
Top
Abstract
Introduction
References

Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF) are important modulators of cell growth under physiological and pathophysiological conditions. We and others have previously shown that these growth factors increase insulin-like growth factor-1 receptor (IGF-1R) number and mRNA in vascular smooth muscle cells and that this effect is transcriptionally regulated. To study the mechanisms and the signaling pathways involved, IGF-1R promoter reporter constructs were transiently transfected in CHO-AT1 cells that overexpress angiotensin AT1 receptors. Our findings indicate that Ang II and bFGF significantly increased IGF-1R promoter activity up to 7- and 3-fold, respectively. The effect induced by Ang II was mediated via a tyrosine kinase-dependent mechanism, since tyrphostin A25 largely inhibited the Ang II-induced increase in promoter activity. In addition, co-transfection of dominant negative Ras, Raf, and MEK1 or pretreatment with the MEK inhibitor PD 98059 dose-dependently decreased both the Ang II- and bFGF-induced increase in IGF-1R transcription and protein expression, suggesting that the Ras-Raf-mitogen-activated protein kinase kinase pathway is required for both growth factors. Reactive oxygen species have been shown to act as second messengers in Ang II-induced signaling, and activation of the transcription factor NF-kappa B is redox-sensitive. While co-transfection of dominant negative Ikappa Balpha mutant completely inhibited the Ang II-induced increase in transcription, it had no effect on the bFGF signaling. In contrast, co-transfection studies indicated that the transcription factors STAT1, STAT3, and c-Jun and the Janus kinase 2 kinase are required in the signaling pathway of bFGF, whereas only dominant c-Jun inhibited the Ang II-induced effect. In summary, these data demonstrate that Ang II and bFGF increase IGF-1R gene transcription via distinct as well as shared pathways and have important implications for understanding growth-stimulatory effects of these growth factors on vascular cells.

    INTRODUCTION
Top
Abstract
Introduction
References

The vascular response to injury requires a coordinated interaction between hemostatic and inflammatory systems and is regulated by cytokines and growth factors that act locally to regulate cellular proliferation and tissue repair. Among the many growth factors that have been shown to be implicated in the response to vascular injury, angiotensin II (Ang II)1 is of particular interest. It stimulates a variety of physiological responses related to regulation of blood pressure, salt, and fluid homeostasis (1). However, Ang II has also been shown to function, either directly or indirectly, as a growth factor for vascular smooth muscle cells, cardiac fibroblasts, and cardiac myocytes (2-7). The array of genes induced by Ang II includes proto-oncogenes such as c-fos, c-jun, c-myc, and egr-1 (5, 7-9), genes encoding extracellular matrix proteins such as collagen, fibronectin, and tenascin (10-12), and genes for growth factors like transforming growth factor beta 1, platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and its receptor (5, 7, 12-14). Similarly, basic fibroblast growth factor (bFGF) has been implicated in the vascular injury response. In particular, bFGF increases endothelial cell migration and proliferation and also stimulates angiogenesis in vitro and in vivo (15, 16). The role of bFGF in vessel injury and repair is further supported by evidence that bFGF is released from vessel wall cells after injury (17) and that bFGF mRNA is up-regulated in atherosclerotic lesions (18).

Accumulating evidence has shown that the insulin-like growth factor-1 receptor (IGF-1R) is a convergence point of the control of cell growth. Thus, a functional IGF-1R autocrine loop is required for the mitogenic effects of various growth factors, such as PDGF (19, 20), epidermal growth factor (20-22), thrombin (23), bFGF (24-26), and Ang II (14). Furthermore, we and others have demonstrated that PDGF (24-26), thrombin (27), bFGF (25, 26, 28), and Ang II (25) increase IGF-1R density on vascular cells. Inhibition of this effect by IGF-1R antisense phosphorothioate oligonucleotides inhibits the Ang II-induced cellular growth (29).

Ang II exerts its effects through specific G-protein-coupled receptors, predominantly through the AT1 receptor subtype. These receptors induce intracellular calcium mobilization; activation of tyrosine kinases such as p125FAK, p46SHC, and p54SHC; induction of serine/threonine kinases, including protein kinase C and mitogen-activated protein kinases (MAPKs) (13, 30-35); and stimulation of the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway (36). The bFGF receptor, however, belongs to the receptor tyrosine kinase family (37-39) and couples to a variety of signaling pathways, including phospholipase C-gamma and phospholipase A2 activation (40, 41), activation of the MAPK pathway (42-44), and activation of the Jak/STAT cascade (45, 46). Similarly to Ang II, bFGF has been shown to induce the expression of the early response gene c-fos (47). However, the signaling pathways by which Ang II or bFGF increase transcriptional activity of the IGF-1R gene are unknown, with the exception that the bFGF, but not the Ang II effect, is protein kinase C-dependent (48).

The purpose of the present studies was to localize the Ang II- and the bFGF-responsive elements in the IGF-1R promoter and define the signaling cascades whereby these two growth factors increase IGF-1R gene transcription or protein expression. We show that Ang II and bFGF positively regulate transcriptional activity of the IGF-1R gene and that they increase IGF-1R gene expression via common as well as distinct signaling pathways.

    EXPERIMENTAL PROCEDURES

Materials-- Cell culture media and LipofectAMINE were purchased from Life Technologies (Basel, Switzerland), and human Ang II was from Sigma. Human recombinant bFGF, PD 98059, genistein, tyrphostin A25, eicosatetrayonic acid (ETYA), SB 203580, and BAPTA/AM were from Calbiochem. The Dual-LuciferaseTM Reporter Assay was from Promega (Madison, WI), and salmon sperm DNA was purchased from Stratagene (La Jolla, CA). The enhanced chemiluminescence reagents and the horseradish peroxidase-conjugated anti-rabbit immunoglobulin were from Amersham (Pharmacia Biotech). The polivinylidene fluoride blotting membranes were from Millipore Corp. (Bedford, MA), and the anti-MAPK and phosphospecific anti-MAPK (p42/p44) antibodies were purchased from New England Biolabs (Beverly, MA). The antibody against the beta -subunit of the IGF-1 receptor was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Culture-- CHO-AT1 cells (kindly provided by Dr. E. Clauser, INSERM, Paris) stably overexpressing the Ang II AT1A receptor (49) were grown in Ham's F-12 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.75 mg/ml G418 and incubated at 37 °C in a humidified 5% CO2 atmosphere. The parental cell line CHO-K1 (ATCC, Rockville, MD) was cultured under the same conditions but without the addition of G418.

Plasmids and Transfections-- The full length promoter of the IGF-1R (-2350/+640-Luc) and a shorter promoter construct (-476/+640-Luc) were a generous gift from Dr. H. Werner (National Institutes of Health, Bethesda, MD). Deletion fragments were made from the full-length promoter construct and subcloned upstream of the firefly luciferase cDNA, resulting in fragments extending from nucleotides -476 to +21, -416 to +21, -330 to +21, -270 to +21, and -135 to +21. The following constructs have previously been described: dominant negative mutant p21ras (N17) (50), dominant negative Raf (301) (51), dominant negative MAPKK 1 (MEK1) mutant A221 (52), dominant negative Jun kinase (stress-activated extracellular signal-regulated kinase (SEK1)) (53), dominant negative c-Jun (Tam67) (54), dominant negative STAT1 Tyr701 (55), dominant negative STAT3 Tyr705 (56), kinase-deficient Jak2 kinase (57), and dominant negative Ikappa Balpha K21/22R (58). To control for transfection efficiency and interwell variation, cells were co-transfected with the internal control vector pRL-TK according to the manufacturer (Herpes simplex virus thymidine kinase promoter region driving Renilla luciferase expression). Cells were plated in 24-well plates and transfected with 1 µg of reporter plasmid and 5 ng of pRL-TK/well with LipofectAMINE reagent. In co-transfection experiments with dominant negative Ras, Raf, MEK1, STAT1, STAT3, Jak2, c-Jun, SEK1, and the Ikappa Balpha mutant, cells were transfected as described above with the addition of increasing amounts of the above mentioned plasmids. The total amount of DNA transfected was kept constant using salmon sperm DNA. Twenty hours after transfection, the DNA-containing medium was changed to Ham's F-12, and the cells were treated with or without Ang II (100 nM) or bFGF (10 ng/ml). To determine the signaling pathways, transfected cells were incubated with inhibitors for 1 h prior to the addition of Ang II or bFGF. After 24 h, cells were washed and lysed according to the manufacturer's instruction. Firefly and Renilla luciferase activities were measured using an EG & G Berthold luminometer (Bad Wildbad, Germany). Firefly luciferase activity was normalized to the internal control Renilla luciferase (Luc/Ren).

Western Blot Analysis-- Cultured CHO-AT1 or CHO-K1 cells were serum-starved for 24 h prior to the addition of Ang II (100 nM) or bFGF (10 ng/ml) for the times indicated. In some experiments, cells were incubated with the MAPKK inhibitor PD 98059 (10 µM), the tyrosine kinase inhibitor tyrphostin (10 µM), the p38 MAPK inhibitor SB 203580 (10 µM), or ETYA (10 µM) 1 h prior to the addition of Ang II or bFGF. Cells were washed in ice-cold phosphate-buffered saline and lysed in lysis buffer containing 150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM epsilon -aminocaproic acid, 1 mM sodium orthovanadate, 0.1 M okadaic acid, 0.1 µM aprotinin, 10 µg/ml leupeptin, and 10 mM NaF. Lysates were subjected to SDS-PAGE on 7.5 or 12% gels, and separated proteins were transferred to polivinylidene fluoride membranes. Blots were blocked with 5% dry milk; incubated with polyclonal anti-MAPK, anti-phosphospecific MAPK (p44, p42), or anti-IGF-1Rbeta antibodies; and then incubated with peroxidase-conjugated donkey anti-rabbit antibody. Immunopositive bands were visualized by enhanced chemiluminescence. Purified MAPK protein was included as positive control; nonimmune rabbit IgG was used as negative control.

    RESULTS

Effect of Ang II on IGF-1R Promoter Activity and Localization of the Ang II-responsive Element-- To measure the effect of Ang II on IGF-1R gene expression and to localize the Ang II-responsive region, CHO-AT1 cells were transiently transfected with IGF-1R promoter constructs containing a luciferase reporter gene under the control of the proximal promoter region of the IGF-1R gene together with the Renilla luciformis thymidine kinase expression vector (pRL-TK) as internal control. Ang II (100 nM) significantly increased IGF-1R promoter activity between 2.3- and 7-fold depending on the promoter constructs (Fig. 1B). No effect was observed when the promoterless pOLUC was used (data not shown). The biggest effect induced by Ang II was seen with the promoter construct containing 476 base pairs of the 5'-flanking region and 640 base pairs of the 5'- untranslated region (UTR) (p(-476/+640-Luc)), and a lesser response occurred with the construct containing a shorter 5'-UTR. It appeared that there were some negative regulatory elements between nucleotides -2350 and -476, because the longest reporter construct responded less to Ang II stimulation, compared with (-476/+640-Luc). When the sequence between nucleotides -270 and -135 was deleted, the stimulatory effect of Ang II was greatly diminished, although not completely, suggesting that the major Ang II-responsive element may be located between nucleotides -270 and -135 of the 5'-flanking region.


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Fig. 1.   Regulation of IGF-1R promoter activity by Ang II and bFGF. A, IGF-1R promoter constructs used. CHO-AT1 cells were transiently transfected with 1 µg of the various reporter plasmids and 5 ng of pRL-TK and 20 h later incubated with or without Ang II (100 nM) (B) or bFGF (10 ng/ml) (C) for 24 h. The luciferase values are normalized to Renilla luciferase activity and presented as mean ± S.E. from five separate experiments. Hatched bars, control cells; filled bars, cells stimulated with Ang II or bFGF, respectively.

Effect of bFGF on IGF-1R Gene Transcription-- The stimulatory effect induced by bFGF in CHO-AT1 cells ranged between 1.7- and 3.2-fold (Fig. 1C), and the same was found in the parental cell line CHO-K1 (data not shown). Previous studies have suggested that the bFGF-response element may be located between nucleotides -476 and -188 of the 5'-flanking region (59). In our CHO-AT1 cells, the -476/+640 construct responded well to bFGF (~3-fold increase in luciferase activity). In contrast to Ang II, there was no evidence of a repressor sequence in the larger construct (-2350/+640). The removal of the majority of the 5'-UTR resulted similarly in a reduction in basal activity, but the bFGF response persisted. Progressive deletion of the cis-acting sequence up to -135/+21 still yielded a ~2-fold increase in luciferase activity after bFGF treatment (Fig. 1C).

Effect of Ang II and bFGF on IGF-1R Protein Levels-- To assess whether Ang II or bFGF increased IGF-1R protein levels, cell lysates of cells treated with or without the corresponding growth factor were assayed for IGF-1R protein levels by Western immunoblot. Fifty micrograms were fractionated on a 7.5% reducing SDS-PAGE. Ang II and bFGF increased IGF-1R protein expression, with a maximum seen after 24 h of incubation (Fig. 2). Incubation with Ang II or bFGF for 48 h did not further increase IGF-1R protein levels (not shown).


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Fig. 2.   Western blot analysis of the effect of Ang II and bFGF on IGF-1R levels in CHO-AT1 cells. After 24 h of serum starvation, cells were treated with 100 nM Ang II or 10 ng/ml bFGF for the times indicated. Total proteins from cell lysates were subjected to SDS-PAGE under reducing conditions on 7.5% gels and transferred to polyvinylidene fluoride membranes. Membranes were then probed with an antibody recognizing the beta -subunit of the IGF-1R. Lanes 1 and 5, control cells; lanes 2-4 and 6-8, cells were incubated with Ang II or with bFGF for 4, 8, and 24 h, respectively. Fifty µg of protein were loaded.

Common and Distinct Signaling Pathways of Ang II and bFGF on the Stimulation of IGF-1R Gene Expression and on Protein Level-- The Ang II AT1 receptor belongs to the family of G-protein-coupled seven-transmembrane domain receptors (60), whereas the bFGF receptor is characterized by a single transmembrane domain that has intrinsic tyrosine kinase activity (37-39). To define signal transduction pathways by which Ang II and bFGF stimulate transcription of the IGF-1R, cells were transfected with the indicated promoter reporter constructs and either pretreated with various inhibitors or co-transfected with increasing doses of dominant negative expression constructs prior to the addition of Ang II or bFGF. The protein-tyrosine kinase inhibitor tyrphostin A25 (10 µM) decreased the Ang II response by 60-70%, suggesting that protein tyrosine phosphorylation is involved in the pathway of Ang II (Fig. 3A). Similar results were obtained when genistein, another tyrosine kinase inhibitor was used (54 ± 1.1 to 82 ± 5.3% inhibition of the Ang II response, depending on the promoter construct used; mean ± S.E. of three experiments).


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Fig. 3.   Effect of protein tyrosine kinase inhibition and lipoxygenase inhibition on the Ang II-induced increase in IGF-1R transcription and on AngII-induced MAPK phosphorylation. A, CHO-AT1 cells were transfected with the p(-476/+640-Luc) and the p(-270/+21-Luc) construct and the pRL-TK plasmid to correct for interwell variation. Twenty hours after transfection, cells were incubated with or without tyrphostin A25 (10 µM) for 1 h prior to the addition of Ang II (100 nM). Twenty four hours later, cells were lysed, and luciferases were measured. Data are presented as Luc/Ren and are mean ± S.E. of three separate experiments. Tyrphostin A25 alone had no effect on basal luciferase activity. Tyrphostin A25 had the same inhibitory effect on all other IGF-1R promoter constructs (data not shown). B, cells were treated with or without genistein (60 nM) or ETYA (10 µM) prior to the addition of Ang II for 24 h. Ten micrograms of total protein were subjected to SDS-PAGE on a 12% reducing gel and probed with anti-phosphospecific p44/p42 antibody.

We have previously shown that Ang II signals through a lipoxygenase-dependent pathway to increase macrophage-mediated oxidative modification of low density lipoprotein (61). Therefore, we were interested to see if Ang II would also signal through that pathway to increase IGF-1R transcription. Indeed, ETYA almost completely inhibited the stimulatory effect of Ang II on IGF-1R transcription (representative experiments as follows: for -476/+640, control, 9.5; Ang II, 35.6; and ETYA/Ang II, 17.9 Luc/Ren, respectively; or for -416/+21, control, 5.4; Ang II, 16.5; and ETYA/Ang II, 7.2 Luc/Ren), whereas it had no effect on the increase induced by bFGF (-2350/+640: control, 10.1 ± 3.6; bFGF, 16.8 ± 1.4; ETYA, 14.7 ± 4.8; and ETYA/bFGF, 19.8 ± 5.9, Luc/Ren respectively (mean ± S.D. of two experiments). ETYA alone had no effect on basal luciferase activity (data not shown). In agreement with the above mentioned experiments using genistein or ETYA to block the Ang II stimulation of IGF-1R gene transcription, both blockers also inhibited Ang II-induced MAPK phosphorylation, suggesting that a tyrosine kinase- and a lipoxygenase-dependent step are upstream of MAPK activation (Fig. 3B).

It is documented that Ang II activates the MAPK pathway in vascular smooth muscle cells (62, 63) and that this activation is partially dependent on protein kinase C (62) and apparently requires prior activation of a Ca2+-dependent tyrosine kinase (64). Co-transfection experiments with dominant negative Ras, Raf, and MEK1 suggested that the Ras-Raf-MAPKK pathway is involved in the transcriptional activation of the IGF-1R by Ang II because all inhibited the luciferase response induced by Ang II (Fig. 4), whereas the empty vectors had no effect (data not shown). Similarly, the specific MAPKK inhibitor PD 98059 (100 µM) blocked the Ang II response to control levels in all promoter constructs without having any effect on basal luciferase activity (-2350/+640: control, 3.3 ± 0.8; Ang II, 13.9 ± 2.7; PD 98059/Ang II, 5.1 ± 1.2 Luc/Ren, respectively; -135/+21: control, 6.8 ± 1.1; Ang II, 20.7 ± 2.2; and PD 98059/Ang II, 8.6 ± 1.9 Luc/Ren, respectively (mean ± S.E. of five independent experiments)). In contrast, while the response induced by Ang II required the p44/p42 MAPK activation, the p38 MAPK inhibitor SB 203580 had no effect on the Ang II response, suggesting that p38 MAPK was not involved (-476/+21: control, 6.5 ± 0.6; SB 203580, 5.7 ± 0.6; Ang II, 28.8 ± 1.8; and SB 203580/Ang II, 24.2 ± 1.7 Luc/Ren, respectively (mean ± S.E. of four experiments)). To confirm the specificity of these findings, cells were treated with or without Ang II for various times, and total proteins were immunoblotted with phosphospecific antibodies against p44 and p42 (extracellular signal-regulated kinases 1 and 2). Ang II induced a rapid phosphorylation of p44/p42 already after 2 min, with a maximum at 5 min. PD 98059 completely inhibited the Ang II-induced phosphorylation of p44/p42 (data not shown). It is known that MAPKs in turn phosphorylate numerous cellular proteins, including c-Jun among many others (65). When dominant negative c-Jun (Tam67) was co-transfected with the p(-476/+21-Luc), it completely reduced the stimulatory effect of Ang II to control values, whereas the empty vector had no effect (Fig. 5). This inhibitory effect of dominant negative c-Jun could also be observed with the smaller IGF-1R promoter constructs p(-270/+21-Luc) and p(-135/+21-Luc) (data not shown).


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Fig. 4.   The Raf-Ras-MAPK kinase pathway is required in the transcriptional stimulation of the IGF-1R promoter and IGF-1R protein by Ang II. CHO-AT1 cells were transfected with the p(-476/+640-Luc) IGF-1R promoter construct and increasing doses of dominant negative Raf, Ras, and MEK1. After transfection, cells were then stimulated with or without Ang II (100 nM) for 24 h. The experiments were performed twice with essentially identical results.


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Fig. 5.   Effect of dominant negative c-Jun (Tam67) on the Ang II activation of IGF-1R promoter activity. Cells were transfected with the p(-476/+21-Luc) construct and increasing doses of Tam67. Twenty hours later, cells were stimulated with or without Ang II. Data are expressed as mean ± S.E. of three experiments.

We have seen that the Ang II-induced IGF-1R gene expression is calcium-dependent and is mediated via a redox-sensitive pathway.2 Indeed, intracellular Ca2+ chelation using BAPTA/AM (10 µM) decreased the stimulatory effect of Ang II to control levels without having any effect on basal luciferase activity, suggesting that intracellular Ca2+ is required (-476/+640: control, 10.8 ± 1.3; BAPTA/AM, 13.4 ± 1.9; Ang II, 31.5 ± 2.5; and BAPTA/AM/Ang II, 8.7 ± 7.0 Luc/Ren, respectively (mean ± S.D. from two experiments)). Also, reactive oxygen species have been shown to act as second messengers in Ang II-induced signaling (66) and activation of the transcription factor NF-kappa B is redox-sensitive (67). Co-transfection of the IGF-1R promoter construct p(-476/+640-Luc) with K21/22R, the Ikappa Balpha mutant that shows a defect in degradation and in ubiquitin conjugation and therefore inhibits translocation of NF-kappa B to the nucleus (58), completely inhibited the Ang II-induced increase in IGF-1R transcription (Fig. 6A). This was also true when the shorter construct p(-270/+21-Luc) was used (data not shown). Interestingly, the Ikappa Balpha mutant had no effect on the Ang II response when the short IGF-1R promoter construct p(-135/+21-Luc) was used, suggesting that a putative NF-kappa B site is located 5' of the nucleotide -135 (Fig. 6B). Quite in contrast to Ang II, however, co-transfection with Ikappa Balpha lysine mutant did not decrease the bFGF-induced activation of IGF-1R expression (data not shown).


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Fig. 6.   Effect of Ikappa B mutant on the Ang II-induced increase in IGF-1R transcriptional activity. CHO-AT1 cells were transfected with the p(-476/+640-Luc) (A) or the short p(-135/+21-Luc) (B) construct and increasing doses of dominant Ikappa B (K21/22R) mutant. After transfection, cells were stimulated with or without Ang II for 24 h. The Ikappa B mutant significantly and dose-dependently (p = 0.02, Student's t test) reduced the stimulatory effect of Ang II. Note that the inhibitory effect of K21/22R is lost when the short construct is used. Data are mean ± S.E. of three experiments.

While both Ang II and bFGF stimulated IGF-1R gene transcription (Fig. 1), the signal pathway by which these two growth factors mediate the increase in IGF-1R expression showed common but also distinct features. Thus, tyrphostin A25 reduced the stimulatory effect of bFGF on IGF-1R transcription by 44.4 ± 3.8% (-476/+21: control, 3.8 ± 0.10; tyrphostin A25, 3.1 ± 0.08; bFGF, 11.8 ± 0.46; and tyrphostin A25/bFGF, 6.5 ± 0.29, respectively (mean ± S.E. of four experiments)). The Ras-Raf-MAPK pathway seemed also to be required in the transcriptional activation of the IGF-1R by bFGF, as we found with Ang II. Dominant negative Ras, Raf, and MEK1 inhibited the bFGF-induced increase in luciferase activity to a similar degree as seen with Ang II (Fig. 7). Accordingly, the MAPKK inhibitor PD 98059 (100 µM) completely abrogated the bFGF-induced stimulation of IGF-1R transcription and inhibited the bFGF-induced phosphorylation of p44/p42 MAPK (data not shown). Furthermore, BAPTA/AM inhibited the bFGF-induced stimulation of IGF-1R expression to control values (representative experiment -476/+640: control, 9.9; bFGF, 33.3; and BAPTA/bFGF, 13.7 Luc/Ren, respectively); however, the p38 MAPK inhibitor SB 203580 did not inhibit the bFGF response similarly to Ang II-stimulated IGF-1R expression (data not shown). The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) has been shown to phosphorylate and regulate the activity of several transcription factors including c-Jun, ELK-1, and ATF-2 (68-72). The JNK/SAPK is phosphorylated, resulting in its activation by JNK kinase (JNK kinase/SEK1) (53, 73-75). Increasing doses of dominant negative SEK1 expression construct produced a dose-dependent decrease in the IGF-1R transcriptional activity induced by bFGF when the p(-476/+21-Luc) reporter construct was used, suggesting that the SEK1/JNK/SAPK pathway was involved (Fig. 8A). It is of interest that the same dominant negative SEK1 did not have any effect on IGF-1R transcriptional stimulation by Ang II (data not shown), despite the inhibitory effect of dominant negative c-Jun in the response to Ang II (Fig. 5). It is of note that dominant negative c-Jun also dose-dependently reduced the stimulatory effect seen with bFGF (Fig. 8B).


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Fig. 7.   Involvement of the Ras-Raf-MAPK kinase pathway in the stimulation of IGF-1R transcriptional activity by bFGF. CHO-AT1 cells were transfected with the p(-270/+21-Luc) reporter construct and increasing doses of dominant negative Ras, Raf, and MEK1. Cells were then treated with or without bFGF (10 ng/ml) for 24 h. Values are mean ± S.E. of three experiments.


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Fig. 8.   Effect of dominant negative SEK1 and c-Jun on the bFGF-induced stimulation of the IGF-1R promoter. A, increasing amounts of dominant negative SEK1 were transfected with the p(-476/+21-Luc) IGF-1R promoter construct, and 20 h later cells were stimulated with or without bFGF (10 ng/ml). B, CHO-AT1 cells were transfected with dominant negative c-Jun (Tam67) and with p(-476/+21-Luc). Transfected cells were then incubated with or without bFGF for 24 h. Data are mean ± S.E. of three experiments.

More recently, a novel nuclear signaling pathway has been described that regulates a large family of transcription factors called STATs (76). This pathway, initially described for the interferon receptors, has subsequently been shown to be involved in hormone and growth factor signaling, such as growth hormone (77), Ang II (36), or bFGF (45, 46). We have previously shown that Ang II directly stimulates the Jak/STAT pathway in rat aortic smooth muscle cells by phosphorylation of the intracellular Jak2 kinase and its substrates STAT1 and STAT2 (36). We therefore investigated whether kinase-deficient Jak2 or dominant negative STAT1 Tyr701 and STAT3 Tyr705 mutants could inhibit the increase in IGF-1R transcriptional activity induced by Ang II or bFGF. While all mutants had no effect on the Ang II response (data not shown), they greatly reduced the effect seen with bFGF (Fig. 9), suggesting that the Jak/STAT pathway is involved in the response to bFGF but not to Ang II in this cell model.


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Fig. 9.   Inhibitory effect of dominant negative STAT1, STAT3, and Jak2 on the bFGF-induced stimulation of the IGF-1R promoter. Increasing amount of dominant negative STAT1 Y701F or STAT3 Y705F and the p(-270/+21-Luc) as well as kinase-deficient Jak2 together with p(-476/-21-Luc) were transfected as described. Twenty hours later, cells were treated with or without bFGF (10 ng/ml). Data are mean ± S.E. of three experiments.

Similarly to the transcriptional assays, the MAPKK inhibitor PD 98059 and the protein tyrosine kinase inhibitor tyrphostin A25 decreased the stimulatory effect of Ang II or bFGF on IGF-1R protein expression, whereas the lipoxygenase inhibitor ETYA blocked only the Ang II response. Furthermore, the p38 MAPK inhibitor SB 203580 did not inhibit the Ang II or bFGF effect (Fig. 10), confirming the results observed in the reporter assays.


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Fig. 10.   Effect of various inhibitors on the Ang II- or bFGF-induced increase in IGF-1R protein expression. Total protein from cells treated with or without Ang II or bFGF were subjected to SDS-PAGE under reducing conditions on 7.5% gels, transferred to polyvinylidene fluoride membranes, and then blotted with anti-IGF-1Rbeta . Lane 1, control cells (Con); lane 2, Ang II or bFGF, respectively; lane 3, PD 98059/Ang II and PD 98059/bFGF, respectively; lane 4, tyrphostin A25/Ang II and tyrphostin A25/bFGF, respectively; lane 5, SB 203580/Ang II and SB 203580/bFGF, respectively; lane 6, ETYA/Ang II and ETYA/bFGF, respectively. In all lanes, 50 µg of protein were loaded.


    DISCUSSION

It has previously been demonstrated that growth factors such as PDGF, thrombin, Ang II, and bFGF increase IGF-1R on vascular smooth muscle cells and that this effect is transcriptionally regulated (23, 25, 28). Furthermore, the ability of Ang II to up-regulate IGF-1R is a critical determinant of its mitogenic activity on vascular cells, since the Ang II-induced increase in DNA synthesis was inhibited by IGF-1R-specific antisense oligonucleotides (29). The present studies show by which mechanisms and signaling pathways Ang II and bFGF increase IGF-1R gene transcription. By deletional analysis of the IGF-1R promoter region, we determined that the Ang II-responsive region is located in the proximal promoter, between nucleotides -270 and -135 upstream of the transcription start site, as is the bFGF-responsive element. In addition, stimulation of IGF-1R gene promoter activity by Ang II or bFGF in transient transfection experiments correlates well with its effect on endogenous IGF-1R protein levels. Both increased IGF-1R protein expression after 8-24 h. This is in good agreement with the previous reports of Du et al. (48) and Ververis et al. (25), which showed that Ang II and bFGF caused a significant increase in IGF-1R mRNA peaking at 3 h and 6-9 h, respectively. Of note, Hernandez-Sanchez et al. (59) reported that the bFGF-responsive element was located between nucleotides -476 and -188. These findings are somewhat different from ours; however, our studies were performed using different cells, and our deletion constructs contained less 5'-UTR sequence. Our data indicate loss of basal activity between nucleotides -476 and -135 but conservation of a bFGF-responsive element.

There has been significant interest generated by the observation that growth factors and cytokines, which possess structurally different receptors, with or without intrinsic tyrosine kinase activity, may signal through a common pathway to the nucleus. In order to define the mechanisms and the signaling cascade involved in the Ang II or bFGF regulation of IGF-1R gene expression, we transiently transfected various IGF-1R promoter constructs into CHO-AT1 or CHO-K1 cells and used different approaches to block the signaling pathways at different levels. Our findings clearly show that Ang II and bFGF share common but also quite distinct pathways. Thus, both Ang II and bFGF increase IGF-1R transcriptional activity via the Ras-Raf-MAPKK-MAPK pathway, since transfection of dominant negative expression constructs for Ras, Raf, or MEK1 dose-dependently reduced the stimulatory effects of these growth factors on IGF-1R promoter activity, whereas they had no effect on IGF-1R promoter activation in the absence of Ang II or bFGF. Further evidence for the involvement of this signaling pathway in the activation of the IGF-1R promoter by Ang II or bFGF was provided by experiments using PD 98059. This compound, which is a specific inhibitor of MAPKK phosphorylation and activation (78, 79), completely reversed the stimulatory effect on luciferase activity induced by Ang II or bFGF. Furthermore, analysis at the protein level clearly demonstrated that both Ang II and bFGF induced a rapid phosphorylation of MAPK, which was inhibited by upstream blockade of MAPKK by PD 98059, and inhibition of the MAPKK reduced the stimulatory effect of Ang II and bFGF on IGF-1R protein levels. Thus, the Ras-Raf-MAPK pathway is clearly required for Ang II and bFGF induction of IGF-1R gene and protein expression.

Although the Ang II AT1 receptor does not possess intrinsic tyrosine kinase activity, its activation leads to intracellular second messenger protein tyrosine phosphorylation by cytosolic tyrosine kinases (80). Thus, our finding that the protein-tyrosine kinase inhibitors genistein and tyrphostin A25 inhibited the Ang II-induced stimulation of IGF-1R gene expression and phosphorylation of MAPK demonstrates a requirement for protein-tyrosine kinase(s) in Ang II-stimulated IGF-1R expression. We have previously shown that lipoxygenases may be involved in the signaling pathway of Ang II (61). Our present study demonstrates that Ang II-induced activation of the IGF-1R promoter requires lipoxygenase activity, since this stimulation was blocked by ETYA, a lipoxygenase inhibitor (81). ETYA and genistein not only reduced the stimulatory effect of Ang II on IGF-1R promoter transcriptional activity to basal levels but also inhibited the Ang II-induced phosphorylation of MAPK, suggesting that protein tyrosine phosphorylation and lipoxygenase activation is upstream of MAPK activation. Consistent with the results observed in the transcriptional assays, tyrosine kinase and lipoxygenase inhibition abolished the increase in IGF-1R protein level induced by Ang II.

The regulation of IGF-1R transcription is not well understood. The IGF-1R gene promoter lacks TATA or CAAT motifs; thus, transcription starts from a unique initiator sequence (82). The present experiments are therefore of interest in characterizing the signaling pathway of Ang II or bFGF in stimulating the IGF-1R promoter and in determining the interaction between transcription factors and the IGF-1R promoter. The region of the IGF-1R promoter extending from nucleotide -2350 in the 5'-flanking region to nucleotide +640 in the 5'-UTR, contains putative consensus sequences for a number of well defined regulatory elements, including Egr-1 (83) and Sp1 (84), as well as a PDGF-responsive element (85) and potential AP-2 (86), AP-1, and NF-kappa B sites.3 To gain insights into the promoter region of the IGF-1R gene responsive to Ang II or bFGF, cells were transfected with expression vectors encoding dominant negative forms of Ikappa B (lysine mutant), JNK kinase, c-Jun, and the transcription factors STAT1 and STAT3. The results of these experiments suggest that one of the other common pathways by which Ang II or bFGF increases IGF-1R gene transcription was the involvement of c-Jun. Thus, our data indicated that dominant negative c-Jun dose-dependently inhibited the Ang II- as well as the bFGF-induced increase in IGF-1R promoter activity. c-Jun is one of the components of the transcription factor AP-1, its best known partner being c-Fos (87). However, the activity known as AP-1 can consist of heterodimers between any of the Jun proteins and any of the Fos proteins (87). The finding that dominant negative c-Jun inhibited the increase in IGF-1R promoter activity by Ang II or bFGF, whereas dominant negative JNK kinase (SEK1), which activates JNK/SAPK and ultimately activates c-Jun, dose-dependently blocked the effect induced by bFGF and not by Ang II can be explained by differential pathways whereby these two growth factors signal. It is possible that although Ang II and bFGF both activate MAP kinases, Ang II may predominantly induce c-Fos through the MAPK pathway, whereas bFGF induces c-Jun through activation of the JNK/SAPK pathway. Obviously, more data are needed to fully comprehend and define this difference.

Another important mitogenic cascade that is activated by cytokines and growth factors involves the Jak family of cytoplasmic tyrosine kinases (76, 88). Jak-mediated tyrosine phosphorylation of STATs promotes the translocation of these growth factors to the nucleus, where they bind to specific DNA motifs and induce c-fos gene transcription (76, 88-90). Marrero et al. (36) have previously demonstrated that Ang II stimulates tyrosine phosphorylation of Jak isoforms, tyrosine kinase activity of Jak2, and tyrosine phosphorylation of STATs in vascular smooth muscle cells. Using a kinase-deficient Jak2 or dominant negative STAT1 or STAT3, we were unable to inhibit the Ang II-induced stimulation of the IGF-1R promoter in transient transfection assays using CHO-AT1 cells, indicating that the Jak/STAT pathway is not involved in Ang II-induced IGF-1R gene expression. Quite in contrast to Ang II, kinase-deficient Jak2, dominant negative STAT1, and STAT3 completely inhibited the stimulatory effect of bFGF on IGF-1R transcription, whereas neither empty vectors nor both dominant negative expression constructs had any effect on the IGF-1R transcriptional activity in the absence of bFGF. These findings demonstrate that the Jak/STAT pathway and more precisely Jak2, STAT1, and STAT3 are required in the transcriptional activation of the IGF-1R promoter by bFGF but are not involved in the stimulation by Ang II.

The involvement of the transcription factor NF-kappa B seems to be restricted to the stimulatory effect induced by Ang II and not by bFGF, since the Ikappa Balpha mutant only inhibited the activation of luciferase activity by Ang II. Furthermore, it was found that by deleting nucleotides between -270 and -135, the inhibitory effect of the Ikappa Balpha mutant on the Ang II-induced effect was lost, implying that a putative NF-kappa b site is located between nucleotides -270 and -135. These results suggest that Ang II and bFGF utilize distinct and specific transcription factors to stimulate the IGF-1R gene promoter.

In conclusion, we have characterized the regulation of IGF-1R gene expression by Ang II and bFGF and the main signaling pathways by which these growth factors increase IGF-1R transcriptional activity and IGF-1R protein expression. Both growth factors increase IGF-1R promoter activity by acting on the proximal promoter region upstream of the transcription start site. Although Ang II and bFGF possess structurally different receptors, they transduce signaling through common pathways, notably the Ras-Raf-MAPKK-MAPK and c-Jun pathways. However, they also use unique signaling pathways, such as lipoxygenase-mediated MAPK activation or the involvement of the transcription factor NF-kappa B in the case of Ang II or the JNK/SAPK cascade and the Jun kinase and Jak/STAT pathway in the bFGF-induced regulation of IGF-1R gene expression. These studies have important implications for understanding growth-stimulatory effects of these growth factors.

    ACKNOWLEDGEMENTS

We thank Dr. B. Cenni for helpful discussions, Dr. H. Werner for the generous supply of the full-length IGF-1R promoter constructs, Dr. M. Birrer (National Institutes of Health, Bethesda, MD) for the gift of dominant negative c-Jun (Tam67), and Dr. D. M. Wojchowski (Pennsylvania State University) for the supply of kinase-deficient Jak2.

    FOOTNOTES

* These studies were supported by NHLBI, National Institutes of Health, Grants HL 47035 and HL 45317, the Swiss Cardiology Foundation, Swiss National Science Foundation Grant FNSR3100-050799.97, and the Gerbex-Bourget Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Div. of Cardiology, University Hospital of Geneva, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland. Tel.: 41 22 372 7192; Fax: 41 22 372 7229; E-mail: Patrice.Delafontaine{at}hcuge.ch.

The abbreviations used are: Ang II, angiotensin II; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; ETYA, eicosatetrayonic acid; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; STAT, signal transducer and activator of transcription; UTR, untranslated region; PAGE, polyacrylamide gel electrophoresis; SEK, stress-activated extracellular signal-regulated kinase; Jak, Janus kinase; BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester.

2 J. Du, T. Peng, K. Scheidegger, and P. Delafontaine, submitted for publication.

3 J. Du, unpublished results.

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