Human Insulin Receptor Substrate-2 (IRS-2) Is a Primary Progesterone Response Gene

Lothar Vaßen, Wojciech Wegrzyn and Ludger Klein-Hitpass

Institut für Zellbiologie (Tumorforschung) Universitätsklinikum Essen D-45122 Essen, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Elevated cAMP has been shown to unmask agonist activity of antiprogestin/antiglucocorticoid RU486. In our search for cellular target genes induced through this cross-talk mechanism, we identified human insulin receptor substrate-2 (IRS-2), a cytoplasmic signaling molecule that mediates effects of insulin, insulin-like growth factor-1 (IGF-I), and other cytokines by acting as a molecular adaptor between diverse receptor tyrosine kinases and downstream effectors. Our analysis of the regulation of IRS-2 in HeLa cell models shows that synergistic induction of IRS-2 by cAMP and RU486 can be mediated by progesterone receptors (PR) and glucocorticoid receptors (GR) and occurs through a relative slow mechanism that requires ongoing protein synthesis. Importantly, we demonstrate that IRS-2 mRNA is also inducible by progesterone, while glucocorticoid effects are only observed in the presence of cAMP. Up-regulation of IRS-2 by progesterone depends strictly on the presence of PR and occurs through a rapid mechanism, suggesting that it represents a primary transcriptional response. Furthermore, we show that expression of IRS-1, which also binds to receptors of insulin, IGF-I, and cytokines, is unaffected by progesterone. Thus, our results demonstrate that progesterone alters the ratio of IRS-1 and IRS-2 in PR-positive cells and implicate a mechanism through which progesterone can modulate the effects of insulin, IGF-I, and cytokines on cell proliferation, differentiation, and homeostasis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Progesterone and glucocorticoids regulate complex events in development, growth, differentiation, and cellular homeostasis. The underlying mechanisms are thought to be triggered through rapid transcriptional regulation of a set of primary response genes, some of which might have the potential to induce secondary responses of gene networks that contribute to the observed complex changes in gene expression. However, particularly in the case of progesterone, searches for primary cellular targets have resulted in only a very limited number of genes, most of which appear not to represent key factors in signaling cascades (1). The primary transcriptional responses to progesterone or glucocorticoids are mediated by the progesterone and glucocorticoid receptors (PR and GR, respectively), which belong to the nuclear hormone receptor superfamily (2). Upon activation by binding of the cognate agonist, these receptors can both bind to the same regulatory element, termed GRE/PRE (glucocorticoid response element/progesterone response element) (3, 4), located in target gene promoters or enhancers to stimulate transcription by RNA polymerase II.

In many cases the hormone-induced activation of target genes by agonist-loaded PR and GR can be completely antagonized by excess of RU486, a synthetic steroid that displays both antiprogestin and antiglucocorticoid activities (5). RU486 is used as an abortive drug and may also be useful to antagonize the growth-promoting effects of progesterone on breast tumors (6, 7). However, recent studies showed that the antagonist RU486 can be rendered into a pure agonist, if the protein kinase A (PKA) pathway is simultaneously activated by elevated cAMP levels (8, 9, 10, 11, 12). This cAMP-induced switching of RU486 antagonist/agonist activities is regarded as a potential mechanism through which antihormone-resistant cell populations might be selected during long-term therapy (13). The induction of GRE/PRE-containing reporter genes through cAMP and RU486 occurs in a clearly synergistic fashion and depends on the presence of GRE/PREs and on PR or GR, suggesting that the mechanism involves specific binding of RU486-loaded receptors to GRE/PREs (9, 11). Thus, gene induction by progesterone and glucocorticoids or by the combined action of RU486 and PKA activators share common regulatory elements and trans-acting factors. Yet the underlying mechanisms appear to be different, since gene induction by progesterone or cAMP/RU486 exhibit different kinetics and differential sensitivity to partial inhibition of protein synthesis (9).

To identify cellular genes synergistically induced by activated PKA and progesterone antagonist RU486, we have performed differential display using cDNA from appropriately treated HeLa3B2 cells, which stably express the PR isoform B (9). Since all cAMP/RU486-inducible reporter genes analyzed so far contain GRE/PREs and therefore are also progesterone and glucocorticoid responsive, we reasoned that a search for cAMP/RU486-inducible cellular genes could also lead us to potential progesterone and glucocorticoid target genes. By cloning of the corresponding full-length cDNA, we show here that a cDNA probe, which proved to be inducible by cAMP and RU486 in a synergistic manner, is derived from the human insulin receptor substrate-2 gene (hIRS-2), a key molecule in insulin-, insulin-like growth factor I (IGF-I)-, and cytokine-signaling pathways (14, 15, 16). In the current study we analyzed the regulation of IRS-2 by cAMP, RU486, progesterone, and dexamethasone. We demonstrate that IRS-2 is inducible by progesterone through a direct PR-mediated mechanism, while the related IRS-1 is unaffected. The progesterone-induced alteration of the relative abundance of IRS-1 and IRS-2 proteins provides a new cross-talk mechanism through which progesterone may modulate gene networks regulated by many growth factors, including insulin and IGF-1.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of hIRS-2 cDNA
To identify genes potentially responding to cAMP and RU486 in a synergistic manner, we employed the differential display RT-PCR (DDRT-PCR) method by comparing RNA from PR-positive HeLa cells (HeLa3B2) treated with either 8-Br-cAMP alone or 8-Br-cAMP plus RU486. By ribonuclease (RNase) protection analysis, we could confirm that a 273-bp DDRT-PCR probe indeed detected a transcript that was regulated by cAMP and RU486 in a synergistic fashion (see below). Sequencing of the 373-bp fragment revealed no extended open reading frame (ORF) and no homology to any known gene. To identify the corresponding gene, we used the isolated cDNA fragment to screen cDNA libraries from human fetal brain and R5020-induced HeLa3B2 cells. Overlapping cDNA clones isolated during multiple rounds of screening were used to assemble the 6996-bp cDNA sequence, which contains an ORF of 4014 bp and 5'- and 3'-untranslated regions of 516 bp and 2466 bp, respectively (Fig. 1Go, A and B). The 3'-untranslated region contains four consensus polyadenylation signals (AATAAA). Analysis of various cDNA clones and a number of expressed sequence tags (EST database, NCBI) showed that the two signals at positions 6891 and 6965 are functional and used at about equal frequency.



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Figure 1. A cAMP/RU486-Regulated DDRT-PCR Probe Is Derived from the Human IRS-2 Gene

A, Molecular cloning of hIRS-2 cDNA. Top, a schematic representation of the hIRS-2 cDNA is given. The location of the ORF as well as a number of restriction sites are indicated. Bottom, the partial cDNA probe (1.6) obtained by DDRT-PCR and overlapping cDNAs used to assemble the complete cDNA sequence are aligned. X, XhoI; K, KpnI; Bg, BglII; Ks, Ksp632I; S, SalI; RV, EcoRV. B, cDNA sequence and deduced protein sequence of hIRS-2. The putative Kozac start site at position 517 is shown in bold and the in-frame stop codon 5' of the translation start site is underlined. IH-1PH and IH-2PTB homology domains are shaded in gray and potential tyrosine phosphorylation motifs are highlighted as white characters on black background. Consensus polyadenylation signals within the 3'-untranslated region are in bold, and the part of the cDNA as isolated by DDRT-PCR is underlined. C, Schematic diagram of hIRS-2, showing regions of homology with mouse IRS-2 (mIRS-2) and human IRS-1 (hIRS-1). Numbers within boxes represent percentages of amino acid identity between hIRS-2 and the corresponding domains of mIRS-2 (18 ) and hIRS-1 (31 ) after pairwise alignment using Multalin version 5.3.3 (32 ). IH-1PH and IH-2PTB homology domains of IRS proteins are indicated as black boxes. Small numbers refer to amino acids bordering the homology regions.

 
The putative initiator methionine at nucleotide position 517 is preceded by an in-frame stop codon at position 397. The ORF encodes a protein of 1338 amino acids and a molecular mass of 138 kDa. A homology search revealed that this ORF showed similarities to various members of the insulin receptor substrate (IRS) gene family. IRS are cytoplasmic signal transduction proteins, which after binding to activated insulin and IGF-I receptors become phoshorylated at multiple tyrosine residues (14, 15, 16). Tyrosine-phosphorylated IRS proteins then serve as docking sites for a number of enzymes and effector proteins, which are linked to pathways regulating metabolism, differentiation, and cell growth. As indicated in Fig. 1CGo, the greatest homology was found with IRS-2 of the mouse (mIRS-2, Refs. 17, 18). The IRS-2 pleckstrin-homology [IH-1PH, amino acids (aa) 32–143] and the phosphotyrosine-binding (IH-2PTB, aa 195–353) domains, which are both important for binding and coupling to the phosphotyrosine kinase domains of insulin and IGF-I receptors (19), showed high amino acid sequence identities of 94% and 99%, respectively. A high level of amino acid sequence identity (83%) with mIRS-2 was also found within the C-terminal part of the protein (aa 355-1338), whereas the N-terminal domain (74% identity) and the region between IH-1PH and IH-2PTB (63% identity) were less conserved. Moreover, all potential tyrosine phosphorylation motifs identified previously in the mIRS-2 protein are well conserved in the predicted protein sequence (Fig. 1BGo). Within all domains, similarities with human IRS-1 (20) were clearly lower than those observed with mIRS-2, and homology with rat IRS-3 (21) and human IRS-4 (22) was only detected within the IH-1PH and IH-2PTB domains (Fig. 1CGo and data not shown). Together, these data indicate that the cloned cDNA represents the human homolog of mIRS-2.

Alignment of our sequence with a partial hIRS-2 cDNA sequence published during preparation of this manuscript (GeneBank accession no. AB000732) revealed a number of point mutations, which might represent polymorphisms or sequencing errors. However, compared with our hIRS-2 cDNA sequence, the nucleotide sequence published by Ogihara et al. (23) contains four 1-bp insertions within the last 80 bp of the coding region, which result in a C-terminal end of the protein that shows no extended homology to mIRS-2 any more (data not shown). We are positive that our nucleotide sequence and translation of the C-terminal end of the ORF represents the correct sequence, as our cDNA sequence is identical with human EST clone AA375248 in the NCBI database. Moreover, sequencing of the corresponding part of a genomic clone isolated from a phage library confirmed our cDNA sequence (data not shown).

Expression of hIRS-2 in Human Tissues
To determine the size and the expression profile of hIRS-2 mRNA, a Northern analysis of multiple tissue blots was performed. On a 2-day exposure, a single mRNA species of approximately 7.5 kb in size was detected in prostate, testis, ovary, small intestine, colon, peripheral blood, heart, brain, placenta, and skeletal muscle (Fig. 2Go). Longer exposure revealed also expression in spleen, thymus, kidney, liver, and pancreas (data not shown). Thus, in accordance with a Northern analysis of IRS-2 expression in mice (18), hIRS-2 appears to be expressed at low levels in all tissues analyzed. Assuming a poly(A) tail of 150–200 nucleotides in length, we conclude that the 6996-bp long hIRS-2 cDNA sequence represents full-length or nearly full-length transcripts.



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Figure 2. Expression of IRS-2 in Human Tissues

Premade Northern blots of poly A+ RNA (2 µg each) from human tissues were hybridized at high stringency with an IRS-2 cDNA probe derived from the 3'-untranslated region. The position of the 7.5-kb mol wt marker is indicated on the right.

 
Regulation of hIRS-2 mRNA by cAMP and Steroids
To study the regulation of hIRS-2 in detail, we analyzed RNA from PR-positive HeLa cells (HeLa3B2) treated with cAMP and various steroids by RNAse protection (Fig. 3AGo). The RNA antisense probe used was synthesized from a pBluescript plasmid containing the 273-bp IRS-2 cDNA fragment isolated by DDRT-PCR, which is derived from the most 3'-end of the hIRS-2 mRNA (see Fig. 1BGo). This probe generates two distinct protected bands since it covers the two functional polyadenylation sites at positions 6891 and 6965, which are used at about equal frequency. PR and {gamma}-actin mRNA, which did not respond to hormone treatment, were measured as internal controls. hIRS-2 mRNA was almost unaffected by RU486 alone (compare lane 1 with lane 3), while cAMP treatment (lane 4) resulted in about 2-fold induction. In contrast, combined treatment with 8-Br-cAMP and RU486 induced hIRS-2 mRNA levels 7.5-fold (lane 5). Thus, hIRS-2 responds to cAMP and RU486 in a synergistic fashion. As the synergistic induction of genes by cAMP and RU486 is a characteristic property of PR target genes, it was of interest to determine whether hIRS-2 mRNA could also be induced by PR agonists. Indeed, treatment with the synthetic progestin R5020 led to a 7-fold increase of hIRS-2 mRNA (Fig. 3AGo, lane 2). Thus, our study identifies hIRS-2 as a novel progesterone target gene, which can also be induced by RU486 upon costimulation of the PKA-signaling pathway.



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Figure 3. Regulation of hIRS-2 mRNA by cAMP and Steroids

A, Analysis of IRS-2 mRNA expression in HeLa3B2 (PR-positive) by RNase protection. Cells were treated for 20 h with R5020 (R; 10 nM), RU486 (RU; 10 nM), 8-Br-cAMP (100 µM), and cycloheximide (Chx, 40 µM) as indicated above the lanes. Protected bands resulting from hybridization of total RNA (20 µg each) with the IRS-2, PR, and {gamma}-actin probes after digestion with RNases T1 and A are indicated on the left. The IRS-2 probe covers two alternative polyadenylation sites that are used at about equal frequency, resulting in two protected bands. Left and right panels are derived from the same experiment and represent identical film exposure times. In the bar diagram below, absolute IRS-2 mRNA levels as determined by laser densitometer analysis of x-ray films are given in arbitrary units for each lane. Numbers above the bars represent induction factors normalized to {gamma}-actin mRNA. Induction factors of untreated controls in the absence (lane 1) or presence of cycloheximide (lane 6) were set to be 1. Similar results were obtained in three independent experiments. B, Hormonal regulation of IRS-2 mRNA in PR-negative HeLa cells. Total RNA (20 µg each) from HeLa3B2 cells treated for 20 h with hormones as indicated was analyzed by RNase protection analysis. Dexamethasone (D) was used at a concentration of 10 nM. Note the complete absence of PR transcripts. Lane 7 shows a control hybridization with 20 µg of yeast tRNA. Lane 8 contains pBR322 HpaII mol wt marker. C, Graphic representation of a representative RNase protection experiment analyzing induction of IRS-2 mRNA in HeLa3B2 cells after 4 (open bars) or 24 h (filled bars) of hormone treatment. Treatment of cells and RNA analysis was done as described in panel A. IRS-2 signals were quantified by laser densitometry of appropriate autoradiographs, normalized to {gamma}-actin signals, and are expressed relative to untreated controls. Qualitatively similar results were obtained in three independent experiments.

 
To confirm the role of PR in induction of hIRS-2 by progestins and cAMP/RU486, we investigated the regulation of IRS-2 in the parental HeLa cells, which do not express PR (Fig. 3BGo). In these cells, both in the absence and presence of 8-Br-cAMP, hIRS-2 mRNA levels could not be stimulated by R5020 (compare lane 1 with lanes 2 and 6) proving that progestin induction of hIRS-2 indeed requires the PR. RU486 (lane 3) or cAMP (lane 5) alone had no effect, but when applied together (lane 9), an increase of hIRS-2 mRNA became evident. Most likely, this induction is mediated by the GR, which is known to bind RU486 with high affinity and can bind to the same regulatory DNA element as PR.

Because high concentrations of dexamethasone can also activate the PR (L. Klein-Hitpass, unpublished observation), potential regulation of hIRS-2 mRNA levels by the synthetic glucocorticoid was also investigated in HeLa cells to rule out a possible interference of PR. Dexamethasone treatment alone did not significantly increase hIRS-2 mRNA (Fig. 3BGo, lane 4). However, upon costimulation with cAMP, dexamethasone increased hIRS-2 mRNA about 3-fold (lane 10). This result suggests that, at least in the presence of elevated cAMP, the hIRS-2 gene is also a target for GR.

Induction of hIRS-2 mRNA by Progesterone or cAMP/RU486 Occurs through Different Mechanisms
To investigate whether R5020 and 8-Br-cAMP/RU486 induction of hIRS-2 mRNA are primary responses, we analyzed the effect of cycloheximide in HeLa3B2 cells by RNase protection. As shown in Fig. 3AGo, cycloheximide treatment reproducibly elevated basal expression levels of IRS-2 about 3-fold (compare lane 1 with 6). However, this effect of the inhibitor on basal IRS-2 mRNA levels appears to be nonspecific, since PR mRNA levels were also about 3-fold stimulated by cycloheximide. Partial inhibition of protein synthesis by cycloheximide did not inhibit induction of hIRS-2 mRNA by progestin R5020 (compare lane 6 with lane 7), suggesting that R5020 induction of hIRS-2 occurs through a direct PR-mediated mechanism. In contrast, induction of hIRS-2 mRNA by cAMP/RU486 was largely prevented in the presence of the inhibitor (compare lane 6 with lane 10), indicating that cAMP/RU486 induction occurs through a distinct mechanism that requires ongoing protein synthesis.

To compare the kinetics of progesterone and cAMP/RU486 induction of hIRS-2 in the presence and absence of cAMP, we performed RNAse protection analysis with RNA from HeLa3B2 cells that were incubated with hormones for 4 or 24 h. As summarized in Fig. 3CGo, induction of hIRS-2 mRNA by progestin R5020 was already 12-fold after 4 h of treatment and decreased to about 8-fold at 24 h. In contrast, cotreatment with cAMP and RU486 had very little effect after 4 h, but resulted in about 20-fold induction after 24 h. cAMP or RU486 alone caused at most 2-fold induction of hIRS-2 mRNA both at 4- and 24-h time points. Taken together, our results show that progesterone induction of hIRS-2 mRNA is a rapid and direct transcriptional response, while induction by cAMP/RU486 occurs through a relatively slow mechanism that requires ongoing protein synthesis.

Differential Regulation of IRS-1 and IRS-2 by Steroids
To verify the induction of hIRS-2 by R5020 or cAMP/RU486 and their differential kinetics at the protein level, we performed Western blot analysis on whole cell extracts prepared from HeLa3B2 cells treated for 2–48 h. As shown in Fig. 4Go, A and B, by immunoblotting with an {alpha}hIRS-2 antibody, R5020 treatment resulted in a detectable induction of IRS-2 after as early as 4 h. Longer treatment led up to a 17-fold enhancement of IRS-2 expression at the 48-h time point. In agreement with the RNase protection data (Fig. 3Go), 8-Br-cAMP or RU486 alone had only minor effects on IRS-2 levels, while combined treatment with 8-Br-cAMP and RU486 resulted in about 8-fold induction after 48 h. Thus, both progesterone and cAMP/RU486 induction of IRS-2 are also clearly detectable at the protein level. Again, consistent with the RNA data, induction of hIRS-2 by cAMP/RU486 occurred much more slowly than by R5020 (Fig. 4BGo), supporting our conclusion that regulation of IRS-2 by R5020 and cAMP/RU486 occurs through different mechanisms. In the absence of cAMP, the induction of IRS-2 by R5020 observed in HeLa3B2 cells could be antagonized by a 100-fold molar excess of RU486, confirming the involvement of PR (Fig. 5Go). It should be noted that the level of IRS-2 protein induced by cAMP/RU486 remains lower than the R5020-induced IRS-2 level at all time points investigated (Fig. 4BGo), although cAMP/RU486-induced IRS-2 mRNA levels are at least as high as the R5020-induced mRNA at the 20 and 24 h time points (Fig. 3Go, A and C). We assume that this apparent discrepancy is due to a presumably long half-life time of the IRS-2 protein and the faster onset of the R5020 induction, which would allow a greater accumulation of the IRS-2 protein in the presence of R5020.



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Figure 4. Time Course of hIRS-2 Induction by Steroids and 8-Br-cAMP

A, Western blot analysis. HeLa3B2 cells were treated with steroids as indicated on the left (R5020, 10 nM; RU486, 10 nM) or 8-Br-cAMP (cAMP, 0.2 mM). Whole-cell extracts were harvested at the indicated time points after addition of hormones and analyzed by Western blot using antibody directed against IRS-2. Similar results were obtained in three to four independent experiments. B, Quantitative representation of the effects of steroids and cAMP on hIRS-2 expression after hormone addition. ECL signals were quantified using a CCD video camera (Raytest).

 


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Figure 5. Differential Regulation of IRS-1 and IRS-2 Proteins by Progestin R5020

Whole-cell extracts (20 µg/lane) were isolated from HeLa3B2 (left panel) and HeLa cells (right panel), which were incubated with the indicated hormones (cAMP, 0.2 mM; RU486, 10 nM; R5020, 10 nM; R5020/100xRU; 10 nM R5020 plus 1 µM RU486; Dex, 10 nM) for 10 h and analyzed by Western blot using antibodies directed against IRS-2 or IRS-1. The positions of IRS-2 and IRS-1 are indicated on the left. Similar results were obtained in two independent experiments.

 
Next we compared the expression of IRS-2 and IRS-1 in HeLa3B2 and HeLa cells, which were treated with cAMP, R5020, RU486, and dexamethasone for 10 h (Fig. 5Go). Confirming the result shown in Fig. 4Go, expression of IRS-2 was clearly induced by progestin R5020 in HeLa3B2 cells, whereas no effect was seen in PR-negative HeLa cells. Dexamethasone treatment for 10 h did not result in a comparable induction of IRS-2 either in HeLa 3B2 or in HeLa cells, although both cell lines contain functional GR. Similarly, cAMP/dexamethasone cotreatment for 10 h had very little or no effect on the level of IRS-2 protein in HeLa3B2 and HeLa cells (Fig. 5Go and data not shown), whereas about 3-fold induction of IRS-2 mRNA levels was observed in a 20-h experiment (Fig. 3BGo). This result suggests that induction of IRS-2 mRNA levels by cAMP/dexamethasone treatment follows a slow kinetic, which is similar to the kinetic of the cAMP/RU486 induction. Expression of the IRS-1 gene, which is homologous to IRS-2, was completely unaffected by treatment with cAMP, RU486, dexamethasone, and cotreatment with cAMP/dexamethasone or cAMP/RU486 both in HeLa3B2 and HeLa cells (Fig. 5Go and data not shown). In striking contrast to IRS-2, IRS-1 could not be induced by progestin treatment in the PR-positive cell line (Fig. 5Go). Together, these results demonstrate that progesterone, through a PR-mediated mechanism, specifically increases IRS-2 expression without altering IRS-1 levels, resulting in an increased ratio of IRS-2 to IRS-1 signaling proteins.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Induction of hIRS-2 by cAMP/RU486 Represents a Delayed PR-Mediated Response
Using a differential display technique and cDNA cloning, we found that the endogenous IRS-2 gene is synergistically induced by elevated cAMP and the antiprogestin RU486 in a HeLa-derived cell line that expresses human PRB. Consistent with a previous study in which we analyzed well defined transfected reporter genes (9), we show here that cAMP/RU486 induction of IRS-2 is prevented through partial inhibition of protein synthesis (Fig. 3AGo), suggesting that intermediate synthesis of a yet unknown cofactor is required. Moreover, we showed, both at the mRNA and protein level, that cAMP/RU486 induction of hIRS-2 occurs much slower than progesterone induction (Figs. 3CGo and 4Go), underlining our previous conclusion that induction of progesterone-responsive genes by cAMP/RU486 is not a direct transcriptional response mediated by PR (9). Whether cAMP/RU486-mediated induction of IRS-2 could represent a step of a mechanism through which antihormone-resistant tumor cell populations might be selected during therapy remains to be elucidated.

IRS-2 Is a Primary PR Target Gene but Responds Poorly to Glucocorticoid
Like the well defined cAMP/RU486-responsive reporter genes used in previous transfection studies (8, 9, 10, 11, 12), the endogenous IRS-2 gene proved to be strongly inducible by progesterone in the PR-expressing HeLa3B2 cell line. We showed that progesterone induction of hIRS-2 is dependent on the presence of PR, is largely prevented by excess of RU486 (in the absence of cAMP), is detectable as early as after 4 h, and is insensitive to cycloheximide ( Figs. 3–5GoGoGo). Together, these findings clearly suggest that progesterone induction of hIRS-2 occurs at the transcriptional level through a direct PR-mediated mechanism that most likely involves the binding of PR to one or more GRE/PREs located in the promoter or enhancer(s) of the hIRS-2 gene. In contrast to IRS-2, the endogenous IRS-1 gene proved to be unresponsive to progesterone in our experimental system, suggesting that the regulatory element(s) conferring inducibility have not been conserved throughout evolution of the IRS gene family.

Surprisingly, although HeLa cell lines contain functional GR that can bind to consensus GRE/PREs, treatment with dexamethasone alone did not result in a rapid induction of hIRS-2 mRNA and protein (Figs. 3BGo and 5Go). Thus, it seems possible that the hIRS-2 gene contains a special PRE that is not recognized by GR. However, this possibility seems unlikely, since both GR-mediated dexamethasone and RU486 effects were detectable upon cotreatment with cAMP for 20 h (Fig. 3BGo). Alternatively, in the absence of the potentiating action of cAMP, the level of GR present in our cell lines may be insufficient to ensure sufficient occupancy of the responsible elements of the IRS-2 gene. Clearly, identification of the elements that confer progesterone regulation to the IRS-2 gene in HeLa3B2 cells and an analysis of their interactions with PR and GR are required to gain further insight into the regulatory mechanism responsible for the differential inducibility of IRS-2 in response to progesterone and glucocorticoid treatment.

Possible Implications
IRS-1 knockout mice display a mild form of insulin resistance of peripheral tissues that can be overcome by increased insulin secretion, while IRS-2 knockout mice exhibit all characteristics of a type II diabetes disorder, including reduced ß-cell mass that prevents compensation through increased insulin secretion (24, 25, 26). Moreover, analysis of IRS-dependent signaling pathways in cells derived from IRS-1 knockout mice showed that IRS-2 overexpression cannot completely restore the impaired effect of IGF-I on cell cycle progression and that IRS-2 may not be necessary for activation of ERK1 and ERK2 (27). Together, these results suggest that IRS-1 and IRS-2, despite their many functional and structural similarities, are not completely interchangable. The molecular basis for the different signaling capabilities of IRS-1 and IRS-2, which seem to be coexpressed in many cell types (17, 28) and bind both to activated insulin and IGF-I receptors (19, 29), is not fully understood, but these differences could be due in part to different affinities for downstream SH2 domain interaction partners (17). We show here that progesterone treatment results in a up-regulation of IRS-2, whereas there is no effect of progesterone on IRS-1 expression (Fig. 5Go). This specific effect of progesterone on IRS-2 might affect the ability of cells to respond to signals, e.g. insulin and IGF-I, which are transduced via the various IRS proteins. We envision that the induction of IRS-2, which would occur only in PR-positive cells, could have both positive and negative effects on insulin and IGF-I signaling, depending on the progesterone level, the precise equipment with the various IRS proteins, and their relative affinities and concentrations. For example, in cells expressing limiting amounts of IRS proteins, modest induction of IRS-2 could simply enhance IRS-2-dependent signaling pathways without interfering with IRS-1-dependent signaling. In contrast, in cells expressing relatively high amounts of IRS-1, progesterone- induced overexpression of IRS-2 might interfere with IRS-1-specific signaling pathways, as an excess of IRS-2 might compete with IRS-1 for binding to activated phosphotyrosine receptors and downstream effector molecules. Further studies will be required to confirm whether progesterone-mediated induction of IRS-2 elicits secondary progesterone responses through IRS-2-dependent gene networks and to determine the importance of this potential cross-talk mechanism between progesterone and insulin, IGF-1, or cytokine pathways in the regulation of growth, differentiation, reproduction, and homeostasis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
The generation of HeLa3B2 cells stably expressing PR, cell culture, and hormone treatment of HeLa and HeLa3B2 cells have been described by Kahmann et al. (9).

Northern Analysis
Premade human multiple-tissue Northern blots (CLONTECH, Palo Alto, CA) containing approximately 2 µg of polyA+ mRNA per lane were hybridized with an hIRS-2 cDNA probe (nucleotides 5412–5873) labeled with [{alpha}-32P]dCTP using the RediPrime system (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. The blots were washed at high stringency (0.1x saline sodium citrate, 0.1% SDS at 64 C) and exposed to x-ray film.

SDS-PAGE and Western Blotting
Preparation of high-salt whole-cell extracts (0.5 M NaCl) of HeLa and HeLa3B2 cells, separation by SDS-PAGE, and transfer to nitrocellulose membranes were done as described by Kahmann et al. (9). After blocking, blots were probed with rabbit {alpha}IRS-2 or {alpha}IRS-1 antibodies. Blots were then incubated with a horseradish peroxidase-linked second antibody followed by chemiluminescence detection using SuperSignal (Pierce, Rockford, IL) as substrate. {alpha}IRS-1 antibodies directed against the 14 C-terminal residues of rat IRS-1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). {alpha}IRS-2 antibodies were prepared in rabbits (Eurogentec, Belgium) against a glutathione-S-transferase (GST) fusion protein containing the 76 C-terminal amino acids of human IRS-2, and specific antibodies were enriched by immunopurification on GST-IRS-2 cross-linked to tresyl-agarose. Signals were quantified by using a charge-coupled device camera system (Raytest, Straubenhardt, Germany).

DDRT-PCR
DDRT-PCR, gel electrophoresis on denaturing gels, and reamplification of bands were performed essentially as described by Bauer et al. (30). Reamplified cDNA probes were phosphorylated with T4 polynucleotide kinase and ligated into pBluescriptIISK+ (Stratagene, La Jolla, CA), which had been restricted with EcoRV and dephosphorylated with calf intestinal phosphatase. Plasmid sequencing of cloned fragments was done using an automated laser fluorescence sequencer (Pharmacia, Piscataway, NJ).

RNA Extraction and RNase Protection
Total RNA for DDRT-PCR was extracted from HeLa3B2 cells by using the Optiprep1 RNA isolation kit (Biometra, Göttingen, Germany). RNA for RNase protections was prepared using Trizol reagent (GIBCO-BRL, Eggenstein, Germany). 32P-labeled hIRS-2 antisense probe was synthesized with T7 RNA polymerase from a linearized pBluescript plasmid containing nucleotides 6751- 6987 of hIRS-2 cDNA (see Fig. 1BGo). An antisense PR probe was prepared by T7 RNA polymerase from a linearized pBluescript plasmid containing a 249-bp long cDNA fragment corresponding to aa 556–638 of human PR (isoform B), which was generated by PCR. The {gamma}-actin probe was as described by Kahmann et al. (9). Hybridization of cellular RNA and antisense probes, RNase digestion, and denaturing gel electrophoresis were done as described previously (9).

Isolation of cDNA Clones
A human fetal brain cDNA library in {lambda}gt10 from CLONTECH (HL3003a) and two different {lambda}gt11 libraries prepared from R5020 induced (6 h) HeLa3B2 cells by oligo dT priming or by priming with an hIRS-2 specific primer (Superscript {lambda} system for cDNA synthesis and cloning, GIBCO-BRL) were used during multiple rounds of screening to isolate an overlapping series of partial hIRS-2 cDNAs by plaque filter screening with 32P-labeled cDNA probes. Phage inserts were subcloned into pBluescriptIISK+ (Stratagene) or pZErO-1 (Invitrogen, San Diego, CA) and sequenced using T3, T7, and SP6 as well as gene- specific primers and an automated sequencer (Pharmacia).

Nucleotide Sequence Accession Numbers
The nucleotide sequence described here has been submitted to the NCBI nucleotide sequence database under accession number AF073310.


    ACKNOWLEDGMENTS
 
We are grateful to H. Gronemeyer (Strasbourg, France) and Ian Kerr (London, U.K.) for providing plasmids, and Roussel-Uclaf (France) for providing RU486. We thank G. U. Ryffel and S. Kahmann for a critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Ludger Klein-Hitpass, Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, Virchowstrasse 173, D-45122 Essen, Germany. E-mail: ludger.klein-hitpass{at}uni-essen.de

This work was supported by BMBF Grant 0310780 to L.K.-H. L.V. is a postdoctoral fellow of the Schering Research Foundation (Berlin).

Received for publication September 11, 1998. Revision received November 13, 1998. Accepted for publication December 8, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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