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Endocrinology Vol. 142, No. 12 5149-5157
Copyright © 2001 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

Regulation of Id2 Gene Expression by the Type 1 IGF Receptor and the Insulin Receptor Substrate-1

Magali Navarro, Barbara Valentinis1, Barbara Belletti, Gaetano Romano, Krysztof Reiss2 and Renato Baserga

Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Address all correspondence and requests for reprints to: Renato Baserga, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107. E-mail: r_baserga{at}lac.jci.tju.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70S6K, a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70S6K. Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FAMILY OF Id helix-loop-helix proteins is known to form heterodimers with a large family of proteins, mostly transcriptional activators, composed of a basic region and a helix-loop-helix region (bHLH) (1). Because the Id proteins lack a DNA binding region, their interaction with bHLH usually, but not always, results in a negative regulation of transcription (1, 2). MyoD and c-myc are the best known transcription factors that interact with the Id proteins, but the expression of other genes involved in the differentiation and proliferation of cells can also be regulated by Id proteins (reviewed in Ref. 3). Id gene expression is usually elevated in undifferentiated, cycling cells and tumor cell lines (4, 5). High levels of Id gene expression inhibit the differentiation of several types of cells (4, 6, 7, 8). Indeed, a number of investigators have suggested that down-regulation of Id gene expression may be a sine qua non for the differentiation of certain types of cells. Id gene expression can be cell cycle-regulated and has been reported to play a role in the G1-to-S transition (2, 3, 9). A connection between N-Myc, the retinoblastoma protein, and Id2 has been recently established in neuroblastoma cells (9).

In previous papers, we have reported that the type 1 IGF receptor (IGF-IR) up-regulates Id2 gene expression in 32D murine hemopoietic cells (10, 11). This function of the IGF-IR is strongly influenced by the presence of the insulin receptor substrate-1 (IRS-1, Ref. 10). 32D IGF-IR cells express a human IGF-IR; but, like parental 32D cells, they do not express IRS-1 or IRS-2 (12, 13). IGF-I induces a modest and short-lived increase in Id2 RNA levels in 32D IGF-IR cells. Ectopic expression of IRS-1 in 32D IGF-IR cells (32D IGF-IR/IRS-1 cells) elicits, in response to IGF-I, a dramatic increase in Id2 gene expression, which is also more prolonged than in the parental 32D IGF-IR cells (10). The effect of IRS-1 on Id2 gene expression depends largely on the activation of the PI3K pathway. This was demonstrated in several ways: 1) an inhibitor of PI3K activity, LY294002, markedly inhibited Id2 gene expression; 2) a constitutively active PI3K increased Id2 gene expression in 293 cells; and 3) ectopic expression of PTEN (an inhibitor of PI3K) in LNCaP prostate cancer cells reduced Id2 protein levels. The findings in 32D cells are especially interesting because Id2 gene expression was studied in the first 24 h after shifting 32D IGF-IR cells from IL-3 to IGF-I. Parental 32D cells rapidly undergo apoptosis after IL-3 withdrawal (14, 15). Both 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells survive and grow exponentially, doubling in number each 24 h, during the first 48 h after removal of IL-3 and supplementation with IGF-I. However, 32D IGF-IR cells stop growing 48 h after changing the medium to IGF-I and begin to differentiate along the granulocytic pathway (13). 32D IGF-IR/IRS-1 cells keep growing in the absence of IL-3 and can form tumors when injected into nude mice (16). Thus, the difference in Id2 gene expression between these two cell lines in the first 24 h did not reflect the actual proliferative status of the cells but their eventual fate. We have observed a similar situation when 32D IGF-IR cells are stably transfected with a plasmid expressing a dominant negative mutant of Stat3 (DNStat3). 32D IGF-IR/DNStat3 cells no longer differentiate after IGF-I stimulation, they express high levels of Id2 RNA and proteins (11), and the increase is still dependent on the addition of IGF-I.

Because IGF-I up-regulates Id2 gene expression in 32D cells, it is reasonable to ask which domains of the IGF-IR are necessary and sufficient for the activation of the Id2 genes. For this purpose, one can introduce into 32D cells mutant IGF-I receptors, a procedure we have already used to study IGF-IR signaling in these cells (13, 16, 17, 18). In addition, because IRS-1 plays such a preponderant role in the activation of Id2 gene expression by IGF-I, it seemed reasonable to investigate the IRS-1 domains required for such activation. IRS-1 is known to be a potent activator of the PI3K pathway (19), which, in turn, activates p70S6K (20, 21). In 32D IGF-IR cells, ectopic expression of IRS-1 results in a strong and sustained activation of p70S6K, which is a requirement for the inhibition of IGF-I-mediated differentiation of 32D IGF-IR cells (16). We therefore used several mutants of IRS-1 in an attempt to identify the IRS-1 domains required for the up-regulation of Id2 gene expression. At the same time, we tried to correlate Id2 gene expression with IGF-IR and IRS-1 signaling.

Although there are four Id proteins (3), our studies are limited to Id2 gene expression because, in 32D cells, the IGF-IR has little effect on the regulation of Id1 (10, 11), and 32D cells do not express Id3 and Id4 (22). At the same time, because p70S6K plays an important role in the inhibition of IGF-I-mediated differentiation (16), we have asked whether Id2 gene expression correlates with this signaling pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids
The plasmids expressing the human wild-type (WT) or mutant IGF-I receptors used in these experiments have been described and characterized in previous papers from this laboratory (23, 24). All plasmids for IRS-1 and its mutants described in this paper are based on a self-inactivating form of the MSCV retroviral vector system (25) and contain an internal CMV promoter derived from plasmid pcDNA3.1(+) (Invitrogen, Carlsbad, CA). The self-inactivating form of MSCV retroviral vector was generated as described elsewhere (26). The various mutants of IRS-1 were generated by site-directed mutagenesis, as previously described (24, 27). The mutants thus generated are described in Results. Briefly, all IRS-1 inserts are under the control of a CMV promoter. The WT mouse IRS-1 lacks the 3' untranslated region. The {delta}PTB mutant contains a mouse IRS-1 lacking the phosphotyrosine binding (PTB) domain (from amino acids 155–309), whereas the {delta}PH mutant contains a mouse IRS-1 gene lacking the Pleckstrin homology domain, (PH region, first 107 amino acids from the start codon).

The following point mutations on the IRS-1 sequence were also carried out: mut Grb2, Y891F (Grb2 binding site), mut p85 (with mutations at Y608 and Y935, binding sites for the p85 subunit of PI3K), and mut p85/Grb2 (combining the mutations of the last two mutants). The point mutations were generated with a site-directed mutagenesis kit (Stratagene, La Jolla, CA). All mutations were from tyrosine to phenylalanine. The primers used for site-directed mutagenesis are available on request. The sequences of all the mutations were monitored for the presence of the specific mutations and for possible misincorporations that could have been accidentally introduced.

Cells and cell culture
The 32D cells expressing either a WT or mutant IGF-IR have been described in detail in previous papers and are listed in Table 1Go. 32D IGF-IR cells expressing either a WT or mutant IRS-1 were generated by transducing cells with the appropriate retroviral expression vectors (24, 28). Cells were grown in RPMI 1640 medium supplemented with10% heat-inactivated FBS (Life Technologies, Inc., Rockville, MD), 10% WEHI cell-conditioned medium as a source of IL-3 and the required antibiotics to maintain the selective pressure (250 µg/ml G418; Life Technologies, Inc.), and 1 µg/ml Puromycin (Sigma, St. Louis, MO). Resulting clones were collected and used as mixed populations.


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  		Table 1. 32D-Derived cell populations expressing WT or mutant IGF-I receptors

 
Northern blots
Cells were seeded under the same conditions used for growth analysis (see below). At the indicated time points, cells were collected, and total RNA was extracted, using the RNeasy mini kit (QIAGEN, Valencia, CA). Northern blots were carried out by standard techniques. The radio-labeled probe used for hybridization was the full-length Id2 cDNA (10, 11).

Immunoblots
For the detection of the protein levels of WT and mutants IGF-IR and IRS-1, exponentially growing cells were lysed in ice for 30 min. Cell lysates were clarified by centrifugation at 13,000 rpm for 15 min, and equal amount of proteins were resolved by SDS-PAGE and transferred to a nitrocellulose filter. For the detection of phosphorylated proteins, exponentially growing cells were washed three times and incubated in serum-free medium (RPMI 1640 medium supplemented with 0.1% BSA) for 4 h before stimulation with 50 ng/ml IGF-I (Life Technologies, Inc.). At the desired time points, cells were collected and washed with cold phosphate buffer saline, and proteins were extracted as described above. For the detection of Id2 protein, exponentially growing cells were washed three times and incubated for the indicated times in IL3-free medium (RPMI 1640, containing 10% heat-inactivated FBS) supplemented with 50 ng/ml IGF-1. The proteins were then extracted and treated as above. For immunoblotting, membranes were blocked with 5% nonfat dry milk in TBST buffer [10 mM Tris (pH 8.0), 150 mM Na Cl, 0.1% Tween 20] and probed with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated antirabbit or antimouse Ig (Oncogene Science, Inc., Uniondale, NY). Blots were developed with the enhanced chemiluminescence system, according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ).

For the detection of IRS1 phosphorylation, exponentially growing cells were washed three times and incubated in serum-free medium for 4 h before stimulation with 50 ng/ml IGF-I. One milligram of whole cell lysate was then immunoprecipitated using a polyclonal antibody against the IRS1 C-terminus (Upstate Biotechnology, Inc., Lake Placid, NY). After resolution on SDS-PAGE and transfer onto nitrocellulose filter, a phosphotyrosine blot was performed with an antiphosphotyrosine peroxidase-conjugated antibody (Transduction Laboratories, Inc., Lexington, KY).

Antibodies
IGF-IR, IRS-1, and Id2 were detected with an antibody against the ß-subunit of the IGF-IR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an antibody against the carboxyterminal of IRS-1 (Upstate Biotechnology, Inc.), or an antibody against the Id2 C-terminus (Santa Cruz Biotechnology, Inc.), respectively. The phosphorylation of Thr389 in p70S6K was detected with an antibody against the phosphorylated amino acid, purchased from New England Biolabs, Inc., Beverly, MA. The total amount of p70S6K loaded was monitored after stripping the filters by immunoblotting with an anti p70S6K antibody (Santa Cruz Biotechnology, Inc.). The active form of Akt was detected by immunoblotting with the antiphospho-Akt Ser 473 (New England Biolabs, Inc.). Total amount of Akt was detected with anti-Akt polyclonal antibody (New England Biolabs, Inc.). Grb2 was immunoblotted with a monoclonal anti-Grb2 antibody (Transduction Laboratories, Inc.).

Growth and differentiation analysis
Cells exponentially growing were collected, washed three times, and seeded at a density of 5 x 104 cells/ml in IL-3-free medium (RPMI 1640 medium containing only 10% heat inactivated FBS) supplemented with 50 ng/ml IGF-1. After 2 and 4 d, viable cells were counted by trypan blue exclusion (Life Technologies, Inc.), and cytospins were performed for morphological analysis. To evaluate the degree of granulocytic differentiation, cytospins were Wright-Giemsa stained, and the cells in the different stages of differentiation were counted at the microscope (13). Differentiation was expressed as a percentage of bands and polymorphonuclear cells in the total number of cells scored.

Inhibitors
In some experiments, inhibitors were added to the cells, 30 min before IGF-I stimulation. The mTOR (target of Rapamycin) inhibitor Rapamycin (Sigma) was used at a concentration of 10 ng/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characteristics of 32D-derived cell lines
The mixed populations of 32D cells expressing either the WT or the mutant IGF-I receptors have been described in previous papers from this laboratory (13, 16, 17, 18, 28, 29). The mutations and the characteristics of the 32D-derived cell lines are summarized in Table 1Go, where we also list the abbreviations used. Briefly, the results from previous experiments indicate that: 1) Y950 is crucial for tyrosyl phosphorylation of Shc (13); 2) although there is a modest, but reproducible, activation of PI3K/Akt, even in 32D IGF-IR cells, expression of IRS-1 always markedly increases the activation of this pathway (10, 30); 3) MAPK activity is high in most cell lines, except in those carrying double mutations of the receptor (Y950/4S and Y950/{delta}1245). Ectopic expression of IRS-1 has little effect on MAPK activity in cells expressing the double mutants (18); 4) the WT receptor and the receptor truncated at residue 1245 ({delta}1245) protect 32D cells from apoptosis caused by IL-3 withdrawal (17, 29); 5) mutations at Y950 or at serines 1280–1283 impair survival (13, 18); 6) double mutants are indistinguishable from parental 32 cells, most of the cells dying in the first 24 h (18); and 7) ectopic expression of IRS-1 protects from apoptosis all cell lines with mutant receptors, except those expressing receptors with double mutations (18).

Induction of Id2 mRNA in 32D cells expressing WT or mutant IGF-I receptors
We have investigated Id2 gene expression in these various cell lines in the first 24 h after shifting the cells from IL-3 to IGF-I. We have determined the levels of Id2 mRNA, but we have already reported that the IGF-I-mediated increase in Id2 mRNA is accompanied by an increase in Id2 protein levels (10 , see also below). The results of a representative experiment are given in Fig. 1Go. As already reported (10, 11), IGF-I induces a modest, but reproducible, increase in Id2 mRNA levels in 32D IGF-IR cells (A). None of the mutant receptors examined were as effective as the WT receptor in up-regulating Id2 gene expression (A). The double-mutant receptors (Y950/{delta}1245 and Y950/4S) completely fail to elicit an increase in Id2 mRNA levels after the cells are shifted to IGF-I. Ectopic expression of IRS-1 in 32D IGF-IR cells (A) dramatically increases the levels of Id2 mRNA, as already reported by Belletti et al. (10). These experiments were repeated several times. It seems, therefore, that, in the absence of IRS-1, the IGF-IR up-regulates the expression of Id2 gene through at least two other domains, the Y950 and a second, less powerful domain in the C-terminus (tentatively, the 4S domain).



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  		Figure 1. Regulation of Id2 gene expression in 32D-derived cells by WT or mutant IGF-I receptors. In these experiments, 32D-derived cells were shifted from IL-3 to IGF-I (50 ng/ml) at time zero. The abbreviations and the mutations are explained in Table 1Go. RNAs were prepared from cells at the times indicated (in hours), and Northern blots for Id2 RNA were carried out as described in Materials and Methods. The amount of RNA in each lane was monitored by the amount of rRNA transferred. A, Id2 RNA levels in 32D cells expressing either the WT or mutant human IGF-I receptors. The last set shows the effect of ectopic expression of IRS-1 in 32D IGF-IR cells. B, Effect of ectopic expression of IRS-1 on Id2 RNA levels in 32D cells expressing either the WT or the mutant IGF-I receptors. Cells with the receptors are compared only with cells expressing both the receptors and the WT mouse IRS-1.

 
The results of Fig. 1BGo confirm that IRS-1 is a powerful inducer of Id2 gene expression. Ectopic expression of IRS-1 increases the levels of Id2 mRNA in all cell lines, even in the cell lines expressing the double-mutant receptors. Though the increase is not as dramatic as in cells expressing the WT IGF-IR, IRS-1 caused a modest (but reproducible) increase in Id2 gene expression, even in the Y950/{delta}1245 mutant. These results confirm that the IGF-I-mediated increase in Id2 gene expression is predominantly regulated by the activation of the IRS-1/PI3K pathway (10). On the other hand, Id2 gene expression is also up-regulated by other signals originating from the IGF-IR, from Y950 and a domain in the C-terminus (serines 1280–83). This is compatible with previous data indicating that both inhibitors of MAPK and of PI3K reduce the up-regulation of Id2 gene expression by the IGF-IR (11). These results are also in partial agreement with the biological behavior of these mixed populations. As summarized in Table 1Go, IRS-1 expression protects, from apoptosis, the cell lines expressing receptors with a single mutation but not the cells expressing double-mutant receptors (18). IRS-1 increases Id2 gene expression in all cell lines, including those expressing the double-mutant receptors (especially the Y-4S mutant), but cannot rescue these double-mutant cells from apoptosis (18). Because of the preponderant role of IRS-1 in IGF-I-mediated induction of Id2 gene expression, we have investigated how mutations in IRS-1 may affect Id2 gene regulation.

Generation of mixed populations of 32D IGF-IR cells expressing WT or mutant IRS-1
The IRS-1 mutants we have used are diagrammed in Fig. 2AGo. The levels of IRS-1 expression are given in Fig. 2BGo. All cell lines were mixed populations obtained by transduction of 32D IGF-IR cells with a retroviral vector expressing the appropriate IRS-1 construct (28). The mutants, generated by PCR mutagenesis (see Materials and Methods) included a deletion of the PTB domain ({delta}PTB); a deletion of the Pleckstrin homology domain ({delta}PH); and point mutants at Y608, Y891, and Y935, single or multiple. Y608 and Y935 are binding sites for the p85 subunit of PI3K, whereas Y891 is a binding site for Grb2 (31, 32). All IRS-1 mutants were well expressed. The {delta}PTB and the {delta}PH mutants are smaller in molecular size than the WT IRS-1. Several of these mutant IRS-1s have already been studied and, in 32D cells, by Yenush et al. (33). However, Yenush et al. (33) used these mutants in 32D cells overexpressing the insulin receptor, which, by itself, cannot protect 32D cells from apoptosis (29), even for the first 24 h. In Fig. 2BGo, we show the levels of IGF-IR in cells transduced with the mutant IRS-1 retroviral vectors. Occasionally, IGF-IR levels change in cells further transfected or transduced with other plasmids. Our blot shows that the IGF-IR levels are comparable in all IRS-1 cell lines, except for the cells transduced with the double IRS-1 mutant (PI3K and Grb2).



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  		Figure 2. IRS-1 mutants used in these experiments. A, Diagram of IRS-1 and the relevant domains and residues that were mutated (see text). B, Western blot of lysates from mixed populations of cells transduced with WT or mutant IRS-1 expressing plasmids. Below is a Western blot showing the levels of expression of the IGF-IR in the same cell lines. The upper band is the proreceptor.

 
Effect of IRS-1 mutations on the up-regulation of Id2 gene expression in 32D IGF-IR cells
The effects of IRS-1 mutations on IGF-I-mediated Id2 gene expression are shown in Fig. 3Go. We limited ourselves to IRS-1 (WT or mutants) expressed in 32D IGF-IR cells, because IRS-1, by itself, cannot protect parental 32D cells from apoptosis (13, 29, 34, 35). Id2 mRNA expression is increased in 32D IGF-IR cells transduced with all mutant IRS-1, except in those cells expressing the {delta}PTB mutant. These experiments were repeated several times, with reproducible results. It seems, therefore, that the PTB domain is necessary for the IRS-1-mediated increase in Id2 gene expression.



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  		Figure 3. Effect of IRS-1 mutations on the regulation of Id2 gene expression. 32D IGF-IR cells (WT IGF-IR) expressing the WT or mutant IRS-1 were shifted from IL-3 (time zero) to IGF-I (50 ng/ml), and lysates were prepared at the times indicated above the lanes (in hours). RNAs were isolated, and Northern blots for Id2 RNA were carried out as in Fig. 1Go. RNA amounts in each lane were monitored by the amount of rRNA transferred.

 
IRS-1 and Akt phosphorylation in 32D-derived cells
Tyrosylphosphorylation of IRS-1 (WT or mutants) after IGF-I stimulation is shown in Fig. 4AGo. WT and most mutants are tyrosyl phosphorylated by IGF-I, although to a variable degree. The {delta}PTB mutant gives a very weak band, which, significantly, is not increased by IGF-I (see Discussion). The effect of IRS-1 on PI3K activation in 32D cells has already been documented in previous papers (10, 28, 30). We therefore proceeded to determine Akt activation in the various 32D IGF-IR cells expressing either the WT or the mutant IRS-1 proteins (Fig. 4BGo). Akt is modestly, but reproducibly, activated by IGF-I, even in the parental 32D IGF-IR cells, an observation we have repeatedly made (13, 18), and that could find an explanation in the findings of Gu et al. (36). Akt activation by IGF-I is markedly decreased in the {delta}PTB and {delta}PH mutants, but is apparently normal in the other IRS-1 mutants. Interestingly, in 32D IGF-IR cells expressing the {delta}PH mutant of IRS-1, there is tyrosylphosphorylation of IRS-1 (Fig. 4AGo). However, Akt activation is very low in these cells, in fact, as low as the parental 32D IGF-IR cells or the 32D IGF-IR cells expressing the {delta}PTB mutant (Fig. 4BGo). These experiments have also been repeated several times, with reproducible results.



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  		Figure 4. IRS-1 and Akt phosphorylation in selected 32D-derived cells. A, Cell lines used were 32D IGF-IR cells expressing WT IRS-1 (first 2 lanes), pr the mutants of IRS-1. The cells were unstimulated (-) or stimulated with IGF-I (+) for 60 min. The lysates were immunoprecipitated with an antibody to IRS-1 and blotted with antiphosphotyrosine antibody. The amounts of IRS-1 were monitored after stripping and reprobing (not shown). B, Phosphorylation of Akt. Upper row, Western blot using an antibody against phospho-Akt (see Materials and Methods). Cell lines and time after IGF-I-stimulation are indicated above the lanes. Lower row, Total Akt in each lane.

 
Effect of IRS-1 mutations on the activation of p70S6K
Activation of the PI3K/Akt pathway usually results in the activation of p70S6K (37). Specifically, ectopic expression of IRS-1 markedly increases p70S6K activation in 32D IGF-IR cells and inhibits their differentiation (13, 16, 33). Because Id2 proteins are known to inhibit differentiation (see Introduction), it was logical to ask whether this was correlated to the activation of p70S6K. We have already reported our results on p70S6K activation in 32D IGF-IR cells expressing either the WT or mutant IRS-1 (28). Only in cells expressing the {delta}PTB mutant was there a decrease in p70S6K activation. With all the other IRS-1 mutants, p70S6K activation was as strong as with the WT IRS-1, and this included the {delta}PH mutant (28), despite the fact that the {delta}PH mutant activated Akt very weakly (see above).

The results for WT IRS-1 and the {delta}PH mutant are given in Fig. 5Go, A and B. Though it is clear that p70S6K is activated by the {delta}PH mutant, Fig. 5Go shows another interesting observation. In some cells, even when actively phosphorylated on Thr 389, p70S6K is rapidly degraded (Ref. 20 , and this paper), as evidenced by the appearance of smaller, specific bands (see Discussion). The bands are detectable using an antibody to phospho-Thr 389 (the upper band of these gels is a nonspecific protein that regularly interacts with this antibody). These degradation bands are clearly visible in lysates from 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells (Fig. 5AGo). When 32D IGF-IR cells are expressing the {delta}PH IRS-1 mutant, only one band is visible, of the correct size for p70S6K.



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  		Figure 5. Activation and expression levels of p70S6K in selected cell lines. A and B, The cell lines used (indicated above the lanes) were 32D IGF-IR, 32D IGF-IR/IRS-1 cells, and two mixed populations of 32D IGF-IR cells expressing the {delta}PH mutant of IRS.1. The cells were unstimulated or were stimulated with IGF-I (50 ng/ml) at the times (in minutes) indicated. Lysates were prepared, and Western blots were obtained using an antibody to phosphothreonine 389 (p-thr.389) of p70S6K (upper rows of both panels). The lower rows of both panels give the total amounts of p70S6K. C, Western blot of p70S6K in selected cell lines (upper row). The upper band is the p85 isoform of S6K1. The antibodies and the methodology used are given in Materials and Methods. Grb2 antibodies were used to monitor the protein amounts in each lane (last row).

 
Because degradation seems to be decreased, we asked whether the amounts of p70S6K could also be different. An increased amount of p70S6K was already suggested by using an antibody that detects total p70S6K (lower row of Fig. 5Go, A and B). We then focused on two separate mixed populations of 32D IGF-IR cells expressing the {delta}PH mutant (Fig. 5CGo). Interestingly, the levels of p70S6K were considerably increased in the two mixed populations expressing the {delta}PH mutant (the lower row gives Grb2 levels as controls for the amount of protein in each lane). In fact, not only are the levels of p70S6K increased, but now even the p85 isoform of S6K (38) is detectable in lysates from these cells. It suggests a possible negative effect of the PH domain on the levels of p70S6K.

Effect of Rapamycin on the regulation of Id2 gene expression
Valentinis et al. (16) have shown that Rapamycin effectively inhibits transformation and induces differentiation of 32D IGF-IR/IRS-1 cells. Rapamycin is a specific inhibitor of mTOR (39), which is required for the activation of p70S6K (37). When treated with Rapamycin, 32D IGF-IR/IRS-1 cells lose their transformed phenotype, and they undergo IGF-I- mediated differentiation, like the parental 32D IGF-IR cells (13, 16). We tested the effect of Rapamycin on the regulation of Id2 gene expression in 32D IGF-IR/IRS-1 cells. Fig. 6AGo shows the Id2 mRNA levels in parental 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells, either untreated or treated with Rapamycin (10 ng/ml). We confirm that expression of IRS-1 markedly increases the expression of Id2 mRNA (Ref. 10 , and Fig. 1Go in this paper). Rapamycin decreases (but does not completely abolish) the up-regulation of Id2 gene expression (Fig. 6AGo). The experiment was repeated several times, always with the same results. Id2 mRNA was somewhat decreased in Rapamycin-treated 32D IGF-IR/IRS-1 cells, but it remained above the levels of 32D IGF-IR cells. The results obtained with mRNA levels were confirmed by determining the levels of Id2 protein under the same conditions (Fig. 6BGo). Id2 protein levels are increased by IGF-I in 32D IGF-IR/IRS-1 cells. Rapamycin caused a decrease of approximately 50% in Id2 protein levels, but these levels were still higher than those in 32D IGF-IR cells.



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  		Figure 6. Effect of Rapamycin on Id2 gene expression. The cells used were 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells. The latter ones were left untreated or were treated with Rapamycin (10 ng/ml), 30 min before IGF-I stimulation (50 ng/ml), for the indicated times (in hours). A, Id2 RNA levels at various times after shifting from IL-3 (time zero) to IGF-I. RNAs were prepared, and Northern blots were carried out as in Fig. 1Go. RNA in each lane was monitored by the amount of rRNA transferred. B, Id2 protein levels in the same cells as in A. Grb2 levels were used to monitor protein amounts in each lane. C, Effect of Rapamycin on the growth and survival of 32D IGF-I/IRS-1 cells. Cell lines and treatment are indicated on the abscissa. The cells were shifted from IL-3 to IGF-I at d 0, and the number of cells was counted at 48 and 96 h. The results are expressed as percent increase over cells plated. D, Percent of differentiated cells in the same experiment described in C.

 
To rule out the possibility of a defective Rapamycin or a decreased uptake of the drug, we looked at the growth and differentiation of 32D IGF-IR cells and 32D IGF-IR/IRS-1 cells (Fig. 6Go, C and D). Rapamycin still inhibited the growth and induced differentiation of 32D IGF-IR/IRS-1 cells, as previously reported by Valentinis et al. (16). Thus, Rapamycin is still active in inhibiting 32D IGF-IR/IRS-1 cells, but it has only a partial inhibitory effect on the expression of Id2 mRNA and proteins.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Id proteins are known to play an important role in the proliferation and differentiation of cells (reviewed in Ref. 3). The Id proteins are involved in development (40, 41), differentiation (42, 43, 44, 45), growth (5, 9, 46, 47, 48, 49), angiogenesis (50), and cellular senescence (51, 52). Id2 gene expression is up-regulated by the IGF-IR in 32D cells, and this up-regulation is dramatically increased by IRS-1 (10, 11). The present communication confirms the preponderant role of IRS-1 in Id2 gene expression in 32D cells. Id2 mRNA is up-regulated, but Id2 proteins are also induced (Ref. 10 , and this paper). In the absence of IRS-1, Y950 and the C-terminus also send positive signals, given that a mutation at Y950 or the truncation of the receptor at residue 1245 markedly decreased Id2 gene expression, when compared with the WT receptor (Fig. 1AGo). A combination of a Y950 mutation and truncation at 1245 results in a complete abrogation of the IGF-IR signaling on Id2 gene expression, confirming that both domains send activating signals. These signals are weak, in comparison with the IRS-1 signal (Fig. 1Go, A and B). Ectopic expression of WT IRS-1 markedly increases Id2 gene expression in all cell lines, whether expressing WT or IGF-IR mutants, except the double-mutant Y-{delta} (Y950F plus truncation at 1245). Interestingly, IRS-1 will not rescue this double mutant from apoptosis induced by IL-3 withdrawal (18). 32D cells expressing the single mutants die when the cells are shifted from IL-3 to IGF-I (17, 18). However, all the cell lines expressing single mutations survive and grow, when IRS-1 is expressed, the only exception being the Y-{delta} mutant (18). In previous papers (18, 29), we formulated the hypothesis that the IGF-IR has three signaling pathways for survival, and that two of them (regardless of combination) are necessary and sufficient. However, Id2 gene expression does not seem to be sufficient for survival, because IRS-1 can increase Id2 gene expression (but not survival) in the Y-{delta} mutant. This is in agreement with previous observations (11, 22) that overexpression of Id2 proteins in 32D or 32D IGF-IR cells inhibits differentiation but does not abrogate IL-3 dependence.

As already mentioned, we limited ourselves to Id2 up-regulation, because, in 32D cells, Id1 expression is not affected by IGF-I (10, 11), and Id3 and Id4 are not expressed (22). Given the role of IRS-1 in the up-regulation of Id2 gene expression, it seemed reasonable to examine the effect of mutations in the IRS-1 sequence on the expression of Id2 in 32D cells with the WT IGF-IR. The results were somewhat disappointing, because only the {delta}PTB mutant was informative. This mutant is essentially an inactive mutant (33), defective in tyrosylphosphorylation (this paper) and signaling (53). The failure of the IRS-1 mutants ({delta}PTB excepted) to inhibit Id2 gene expression is compatible with our observation that the same mutants, like the WT IRS-1, inhibit IGF-I-mediated differentiation of 32D IGF-IR cells (28). The likely explanation for the failure of these mutants to show a phenotype is that there are other binding sites for PI3K and Grb2, as already pointed out by White (32) and Esposito et al. (54). We should add that, in a previous paper (10), we examined another IRS-1 mutant, the PH/PTB, which is a truncated IRS-1, comprising only the PH and PTB domains. This mutant is partially active in signaling (33), but it cannot activate PI3K, and it fails to induce Id2 gene expression (10).

More interesting were the results with the mTOR inhibitor, Rapamycin. Rapamycin is known to effectively and almost specifically inhibit p70S6K (37), and it also inhibits the growth of 32D IGF-IR/IRS-1 cells, which are induced to differentiate by Rapamycin (16). We expected Rapamycin to abrogate or markedly decrease Id2 gene expression, but this was not the case. Rapamycin, at a concentration that induced differentiation of 32D IGF-IR/IRS-1 cells and complete inhibition of p70S6K activation, only partially decreased Id2 gene expression. This experiment was repeated several times, but Id2 gene expression still remained above the level of the parental 32D IGF-IR cells. This was true for both mRNA and protein levels. One possible explanation could be found in the observation that Rapamycin inhibits phosphorylation of serine 727 of Stat3 (55), and inhibition of Stat3 does cause a dramatic increase in IGF-I-mediated Id2 gene expression (11). Alternatively, these results could be explained by the presence of more than one pathway by which the IGF-IR increases Id2 gene expression (this paper). Indeed, when we used inhibitors of either the PI3K or the MAPK pathways, either of them caused only a partial inhibition of Id2 gene expression (11). This explanation is supported by the finding that Y950 is one of the important domains for the activation of Id2 gene expression. Y950 is known to bind the Shc proteins (56, 57), which initiates the Ras/Raf/MAPK pathway (58). The role of MAPK (reviewed in Refs. 59 and 60) in Id2 gene expression is not clear.

It still remains puzzling that Rapamycin induces differentiation of 32D IGF-IR/IRS1 cells, yet causing only a modest increase in Id2 gene expression. Additional p7056K-pathways have been postulated by Brennan et al. (61), suggesting an explanation for our results. We have not dealt here with the mechanism of PI3K up-regulation of Id2 gene expression, because this was already discussed in a previous paper (10). Similarly, we omit here a detailed discussion of how p70S6K is activated in the absence of (or markedly decreased) IRS-1/PI3K activity. Some of the possibilities were discussed in previous papers (13, 16, 28).

Signaling in cells expressing the IRS-1 mutants follows their ability to affect differentiation (28) or Id2 gene expression (this paper). However, there is one exception that deserves a comment. The {delta}PH mutant behaves in an anomalous way. First of all, like the {delta}PTB mutant, it fails to phosphorylate Akt (Fig. 5Go), which is usually strongly activated by IRS-1/PI3K (62, 63). However, unlike {delta}PTB, it activates p70S6K (this paper). More interesting is the fact that {delta}PH seems to increase the levels of p70S6K protein. With the IGF-IR and/or with the other mutants of IRS-1, we have always observed several degradation bands of p70S6K. These bands can be detected only when using the antibody against the phosphothreonine 389 and are specific for this region, and for this phosphothreonine. The identity of the bands recognized by the phosphothreonine 389 antibody is supported by several observations: 1) they are absent in unstimulated cells; 2) they are undetectable when the cells are lysed directly in the buffer used for PAGE (unpublished data from our laboratory); and 3) they are also undetectable when the cells are treated with Rapamycin. It should be mentioned that the degradation products of p70S6K observed in most gels are not p70S6K2 (64), because they are not recognized by a p70S6K2 antibody (courtesy of Dr. George Thomas, Friedrich Miescher Institut, Basel, Switzerland). These bands also disappear under ordinary conditions when 32D cells express the {delta}PH mutant of IRS-1. Under these conditions, the amounts of p70S6K markedly increase (Figs. 5Go), and the p85 isoform becomes easily detectable. This increase in p70/p85 amounts was observed in two different mixed populations of 32D cells expressing the {delta}PH mutant, ruling out clonal variations. We have no explanation for the effect of the Pleckstrin homology domain of IRS-1 on the levels of p70S6K, but an attractive possibility is that PH domains, both functionally and by crystal structure, preferentially bind phosphoinositides (65, 66).

An important corollary of our results is that they suggest that up-regulation of Id2 gene expression may be necessary, but not sufficient, for transformation. Thus, ectopic expression of IRS-1 increases Id2 gene expression in the Y-4S mutant cell lines, but these cells are not IL-3-independent and die after shifting to IGF-I (18). Indeed, overexpression of Id2 in 32D or 32D IGF-IR cells does not transform cells (11, 22), although it does inhibit the differentiation program (11).

In conclusion, we have identified the domains of the IGF-IR that up-regulate the expression of Id2 RNA and proteins in 32D cells. There are at least three signals: IRS-1 (when expressed), Y950, and the C-terminus domain. IRS-1, when present, sends the preponderant signal, which can, to a certain extent, even compensate for the mutations in the IGF-IR that decrease Id2 up-regulation. Because Id2 proteins inhibit differentiation, and because Rapamycin induces differentiation of 32D IGF-IR/IRS-1 cells, it was logical to investigate the role of p70S6K in the regulation of Id2 gene expression. In this model, the role of p70S6K is, at best, ambiguous. Finally, the induction of Id2 gene expression in this model correlates with differentiation but not with transformation (IL-3 independence). Id2 gene expression in 32D cells may be important for inhibition of differentiation, but it is not sufficient for transformation.


   Footnotes
 
This work is supported by Grants CA-56309 and CA-78890 from the NIH.

1 Present address: GenEra S.p. A, Via Olgettina, 58, 20132, Milano, Italy. Back

2 Present address: Temple University, College of Science and Technology, Center for Neurovirology and Cancer Biology, 205 Biology Life Science Building, Room 238, 1900 North 12th Street, Philadelphia, Pennsylvania 19122. Back

Abbreviations: bHLH, Basic region and a helix-loop-helix region; DNStat3, dominant negative mutant of Stat3; IGF-IR, type 1 IGF receptor; IRS-1, insulin receptor substrate-1; PTB, phosphotyrosine binding; TOR, target of Rapamycin; WT, wild-type.

Received June 6, 2001.

Accepted for publication August 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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