Insulin Response Sequence-Dependent and -Independent Mechanisms Mediate Effects of Insulin on Glucocorticoid-Stimulated Insulin-Like Growth
http://www.100md.com
《内分泌学杂志》
Departments of Medicine and Physiology and Biophysics, University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
Abstract
IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor- coactivator-1, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in which insulin regulates IGFBP-1 expression through both IRS-dependent and IRS-independent mechanisms.
Introduction
IGF BINDING PROTEINS (IGFBPs) are a family of proteins that bind IGFs with high affinity and exert both IGF-dependent and -independent effects (1). IGFBP-1 is an approximately 30-kDa protein that is produced largely in the liver in which its expression is rapidly regulated at the level of gene transcription (2, 3). Circulating levels of IGFBP-1 are rapidly regulated in response to fasting and feeding, and circulating IGFBP-1 is thought to be the major short-term modulator of IGF bioavailability (4). IGFBP-1 also exerts local effects in the liver, in which it is thought to contribute to the capacity for regeneration in response to injury (5), and in the decidualized endometrium, in which it is a major secretory product and a marker of differentiation (5, 6).
Early studies regarding the regulation of IGFBP-1 revealed that its expression is stimulated by glucocorticoids and that this effect is suppressed by insulin in liver-derived cells (7, 8). Glucocorticoids regulate the abundance of IGFBP-1 mRNA in the liver through effects on both gene transcription (9) and mRNA stability (10, 11). Whereas the mechanisms mediating effects of glucocorticoids on IGFBP-1 mRNA stability are poorly defined, the effects of glucocorticoids on IGFBP-1 gene transcription are better understood. We previously identified a glucocorticoid response element (GRE) in the proximal IGFBP-1 promoter located approximately 100 bp upstream from the transcription start site based on DNase footprinting and reporter gene studies (12). This GRE is contiguous to two insulin response sequences [insulin response sequence (IRS)A and IRSB], which are essential for the ability of insulin to suppress basal IGFBP-1 promoter activity (13). Interestingly, mutation of the IRSA also reduces the ability of glucocorticoids to stimulate promoter activity (14, 15), suggesting that factors that interact with this IRS function cooperatively with glucocorticoids to stimulate promoter activity. This observation also suggested that disrupting the function of these factors might contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter activity (14). In the present study, we used reporter gene constructs to further define the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression.
Studies with the IGFBP-1 promoter have been important in defining the mechanisms by which insulin can suppress gene expression through an IRS. We reported that members of the Forkhead transcription factor family binding to this insulin response region (14) and that signaling through the phosphatidylinositol-3' kinase (PI3K)/protein kinase B (PKB) pathway is necessary and sufficient to mediate effects of insulin on basal promoter activity through an IRS (13). These results suggested that signaling by PI3K/PKB to Forkhead proteins may be important in mediating effects of insulin on gene expression. Subsequently, studies in Caenorhabditis elegans provided genetic evidence that members of the FoxO subfamily of Forkhead proteins are major targets of insulin-like signaling downstream from PKB (16, 17), and studies in this and other laboratories confirmed that FoxO proteins associate with IRSs in the IGFBP-1 promoter (18, 19, 20) and that phosphorylation by PKB promotes nuclear exclusion and suppresses transactivation by FoxO proteins (20, 21). The phosphorylation of FoxO proteins, and possibly other Forkhead proteins (22), is now thought to contribute to the effects of insulin on a number of genes involved in gluconeogenesis (23, 24, 25) and effects of growth factors on cell growth and survival (26, 27). At the same time, it is important to note that other factors interacting with IRS-like sequences also may contribute to the effects of insulin and growth factors on gene expression (28, 29, 30).
FoxO proteins also function cooperatively with glucocorticoids to stimulate the expression of IGFBP-1 (31) and several other genes, including pyruvate dehydrogenase kinase 4 (32) and atrogin (33), possibly by helping to recruit cAMP response element binding protein/p300 and/or other coactivator proteins (31). Disrupting FoxO functions, or possibly other factors that interact with the IRS, might help to mediate the effects of insulin on both basal and glucocorticoid-stimulated IGFBP-1 promoter activity.
To better understand the mechanisms mediating effects of insulin on gene expression, we examined the role of IRS-associated factors in mediating effects of insulin on the glucocorticoid-stimulated promoter. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal but not glucocorticoid-stimulated IGFBP-1 promoter activity, indicating that IRS-independent effects also contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter. Cotransfection studies indicate that this effect of insulin is not due to suppression of peroxisomal proliferator-activated receptor- coactivator (PGC)-1, an insulin-regulated coactivator that contributes to transcriptional activity of the glucocorticoid receptor (25, 34). Studies with kinase inhibitors indicate that multiple signaling pathways contribute to the effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. Together these results indicate that the regulation of IGFBP-1 gene expression by insulin and glucocorticoids reflects an integrated response that is mediated through both IRS-dependent and -independent mechanisms.
Materials and Methods
Reporter gene constructs and expression vectors
The SauI-HgaI fragment of the IGFBP-1 promoter was inserted into the pGL2 polylinker (Promega, Madison, WI) (BP1.luc) and the mutation of the IRSs (IRSmut) reporter gene construct containing mutations of IRSA and IRSB was created by site-directed mutagenesis as reported previously (12). The IRS-GRE construct was created by mutating both halves of the GRE in the IRSmut reporter gene construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The sequence of the mutated promoter was confirmed by dideoxy sequencing in the University of Illinois at Chicago Sequence Center and is provided in Fig. 1.
The expression vector for the human glucocorticoid receptor was provided by Muller et al. (35). The pTAT3.luc construct containing three copies of the GRE from the tyrosine aminotransferase gene immediately upstream of a minimal promoter was kindly provided by A. Kralli (University of Basel, Basel, Switzerland). The expression vector for the dominant-negative form of the p85 regulatory subunit of PI3K ( p85) was provided by Kasuga and colleagues (36), and the PGC-1 expression vector (pcDNA.flag.PGC-1-797) was provided by Kelly and colleagues (37). Plasmids expressing the Gal4 DNA binding domain in-frame with either full-length human PGC-1 (pA4.7-hPGC-1) or the truncated PGC-1 (pA4.7-hPGC-1.N482), in which the C-terminal region of PGC-1 has been deleted, were provided by Kralli and colleagues (38). The pG5e1b.luc reporter gene, which contains five copies of the Gal4 binding motif upstream of a minimal elb promoter, was provided by Dr. Peter Johnson (National Cancer Institute; Frederick, MD) as before (13).
Cell culture and transient transfection studies
HepG2 cells were plated in 65-mm dishes in Opti-MEM (Invitrogen, Carlsbad, CA) with 5% bovine calf serum (Invitrogen) and refed with DMEM with 10% bovine calf serum before transfection with calcium phosphate precipitates of plasmid DNAs, as previously reported (14). Transfected cells were rinsed twice with Hanks’ balanced salt solution (Sigma, St. Louis, MO) and then refed with DMEM with 1 mg/ml BSA with or without 10 μM dexamethasone and/or 100 nm recombinant human insulin for 18 h before lysis and analysis of luciferase activity.
Results
Effect of dexamethasone on IGFBP-1 promoter activity
To assess the role that IRSs play in glucocorticoid regulation of IGFBP-1 promoter activity, we performed reporter gene studies in HepG2 cells using luciferase reporter gene constructs containing the IGFBP-1 promoter with or without mutation of both the IRSs and the GRE (IRS/GRE). As shown in Fig. 1A, treatment with dexamethasone increases the activity of the intact IGFBP-1 promoter by 3-fold, whereas mutation of the IRSs reduces this effect by approximately 50%, supporting the concept that factors that interact with this region enhance the ability of glucocorticoids to stimulate promoter activity. Mutation of the GRE completely disrupts the remaining effect of glucocorticoids, indicating that the IRS-independent effect of glucocorticoids requires the presence of this GRE.
Because the expression of endogenous glucocorticoid receptors is thought to be low in HepG2 cells, we asked whether IRS-associated factors also contribute to the ability of glucocorticoids to stimulate promoter activity when the availability of glucocorticoid receptors is not limiting. As shown in Fig. 1B, we performed cotransfection studies with an expression vector for the glucocorticoid receptor (GR). Cotransfection with 30, 100, or 300 ng of the GR expression vector increases the ability of dexamethasone to stimulate the activity of both the wild-type and IRSmut IGFBP-1 promoter in a dose-dependent fashion. At each level of GR expression, the effect of dexamethasone on promoter activity is diminished by mutation of the IRSs, indicating that the effect of the IRSs on glucocorticoid function is independent of the level of GR expression.
Dexamethasone treatment enhances the effect of insulin
We also examined the effect of glucocorticoid treatment on the regulation of promoter activity by insulin using reporter gene constructs with/without mutation of the IRSs and/or GRE. As shown on the left in Fig. 2, insulin inhibits basal IGFBP-1 promoter activity by approximately 45% in the absence of dexamethasone. In contrast, no effect of insulin on basal promoter activity is seen in cells transfected with reporter gene constructs in which the IRSs have been mutated, consistent with previous reports (13). As shown by the solid bar on the right in Fig. 2, the ability of insulin to suppress IGFBP-1 promoter activity is enhanced in cells that have been treated with dexamethasone (65% inhibition by insulin, P < 0.01 vs. no dexamethasone). Mutation of the GRE disrupts the ability of dexamethasone to enhance the effect of insulin on promoter activity (data not shown), indicating that this effect is mediated through the GRE. To our knowledge, this result provides the first evidence that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that this effect is mediated through the GRE.
We next asked whether glucocorticoid treatment enhances the ability of insulin to suppress promoter activity through a mechanism that is IRS dependent or independent. As shown in the white bar on the right in Fig. 2, mutation of the IRSs reduces but does not completely disrupt the ability of insulin to suppress the dexamethasone-stimulated IGFBP-1 promoter. Mutation of the GRE together with the IRSs (hatched bar) completely disrupts the ability of insulin to suppress promoter activity in glucocorticoid-treated cells through an IRS-independent mechanism, indicating that this effect of insulin is mediated through the GRE. These results indicate that dexamethasone confers the ability of insulin to suppress IGFBP-1 promoter activity through an IRS-independent mechanism that is mediated through the GRE.
IRS-independent effect of insulin is context-dependent
We next asked whether the ability of insulin to suppress dexamethasone-stimulated promoter activity through an IRS-independent mechanism is context independent or whether other elements within the IGFBP-1 promoter also might be required. Here we performed transient transfection studies with a luciferase reporter gene construct containing three copies of the GRE from the tyrosine aminotransferase gene immediately upstream of a minimal promoter (pTAT3.luc) or the reporter gene construct containing the IGFBP-1 promoter in which the IRSs have been mutated (BP1.IRSmut). As shown in Fig. 3, insulin inhibits the ability of dexamethasone to stimulate the activity of the BP1.IRSmut promoter by approximately 40% in HepG2 cells (Fig. 3A) but does not inhibit the ability of dexamethasone to stimulate the activity of the pTAT3 promoter (Fig. 3B). This result indicates that the ability of insulin to inhibit the effect of dexamethasone on promoter activity is context dependent and is not due solely to the ability of insulin to suppress transactivation by the glucocorticoid receptor.
Effects of kinase inhibitors
We next explored the mechanism mediating IRS-independent effects of insulin on IGFBP-1 promoter activity. Initial studies indicated that treatment with LY294002, an inhibitor of PI3K, increases basal IGFBP-1 promoter activity approximately 3-fold, whereas treatment with PD98509, which inhibits the activation of the MAPK kinase (MAPKK), reduces basal promoter activity approximately 50%, and real-time PCR confirmed that LY294002 and PD98509 exert similar effects on the abundance of IGFBP-1 mRNA levels (data not shown), indicating that input from these pathways contribute to the regulation of basal IGFBP-1 promoter function and endogenous gene expression in HepG2 cells. As shown in Fig. 4 (solid bars), treatment with LY294002, which inhibits PI3 kinase activity, and the expression of the dominant-negative form of the regulatory subunit of PI3 kinase (p85) completely disrupts the ability of insulin to suppress basal IGFBP-1 promoter activity, consistent with previous studies indicating that the activation of PI3 kinase is required for this effect of insulin (10). Treatment with PD98059 also partially reduces the effect of insulin on basal activity of the IGFBP-1 promoter, indicating that input from MAPKK to the IGFBP-1 promoter also is important for the effect of insulin on basal promoter activity.
We next examined the effects of these compounds on the ability of insulin to inhibit IGFBP-1 promoter activity through an IRS-independent mechanism. Initial studies showed that these inhibitors had little effect on the ability of dexamethasone to stimulate IGFBP-1 promoter activity using reporter gene constructs containing the intact IGFBP-1 promoter or when both IRSs have been mutated (data not shown). As shown by Fig. 4 (hatched bars), the ability of insulin to inhibit the effect of dexamethasone on IGFBP-1 promoter activity when both IRSs are mutated is fully blocked by treatment with LY294002 and partially blocked by overexpression of a dominant-negative form of the regulatory subunit of PI3K (p85). These results indicate that activation of PI3K is required for the full IRS-independent effect of insulin on glucocorticoid-stimulated promoter activity. Treatment with PD98059 partially reduces this effect of insulin, indicating that input from MAPKK also is required for the full ability of insulin to inhibit glucocorticoid stimulation of the IGFBP-1 promoter through an IRS-independent mechanism.
Suppression of PGC-1 is not required for IRS-independent effect of insulin
PGC-1 is a coactivating protein that interacts with and contributes to the ability of the glucocorticoid receptor to stimulate gene expression (21, 27), and recent studies have suggested that insulin can suppress hepatic expression of PGC-1 (25). To determine whether insulin may suppress glucocorticoid-stimulated promoter activity by limiting the availability of PGC-1 in HepG2 cells, we performed cotransfection studies with a PGC-1 expression vector. As shown in Fig. 5A, the expression of PGC-1 enhances the ability of dexamethasone to stimulate the IRSmut promoter in a dose-dependent fashion (Fig. 5A, solid bars), consistent with the concept that PGC-1 can interact with and enhance the function of the GR. However, overexpression of PGC-1 does not limit the ability of insulin to suppress promoter activity (Fig. 5A, hatched bars). This result indicates that the ability of insulin to inhibit dexamethasone-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism in HepG2 cells is not due to limited availability of PGC-1 .
We also considered the possibility that insulin may inhibit the function of PGC-1 without suppressing its expression. The C-terminal region of PGC-1 contains an arginine/serine-rich region that contains several sites that might be phosphorylated by protein kinase B, based on a consensus sequence for protein kinase B phosphorylation sites (Arg-X-Arg-X-X-Ser/Thr), suggesting that signaling to this region might contribute to the ability of insulin to suppress transcription. Here we transfected HepG2 cells with vectors expressing the Gal4 DNA binding domain in-frame with either full-length PGC-1 (amino acids 1–797) or the truncated PGC-1 (amino acids 1–482), in which the C-terminal region has been deleted, together with the pG5e1b luciferase reporter gene construct, which contains five Gal4 binding sites immediately upstream of the e1b minimal promoter. As shown in Fig. 5B, full-length and truncated Gal4-PGC-1 fusion proteins potently stimulate pG5e1b promoter activity. Treatment with insulin does not suppress but rather enhances transactivation by full-length PGC-1 by approximately 50% in HepG2 cells, and this effect of insulin is not disrupted by deletion of the C-terminal region of PGC-1, indicating that this region is not required, and may interfere with, this effect of insulin. These results indicate that insulin does not suppress (and can enhance) transactivation by PGC-1 in HepG2 cells and that other mechanisms are required to mediate IRS-independent effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity.
Discussion
In the present study, we sought to better understand the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression. Because glucocorticoids can exert effects on IGFBP-1 expression through effects on both IGFBP-1 gene transcription and IGFBP-1 mRNA stability, we used reporter gene constructs to directly assess the effects of glucocorticoids and insulin on IGFBP-1 promoter activity. Previous studies have shown that glucocorticoids and insulin regulate IGFBP-1 promoter activity through IRSs and a GRE located in the proximal IGFBP-1 promoter (12, 39). Mutating the IRSs has been shown to reduce the ability of glucocorticoids to stimulate IGFBP-1 promoter activity (14), and this has suggested that insulin may suppress IGFBP-1 glucocorticoid-stimulated promoter activity by disrupting the function of trans-acting factors that interact with these IRSs. Here we used IGFBP-1 promoter constructs with/without mutations of the IRSs or GRE to better define the mechanisms mediating the effects of insulin and glucocorticoids on IGFBP-1 gene expression.
Initial studies revealed that glucocorticoid treatment increases the ability of insulin to suppress the function of the intact IGFBP-1 promoter and that mutation of the GRE disrupts this effect, indicating that interaction between the GR and the GRE is required for this effect. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal IGFBP-1 promoter activity in HepG2 cells, consistent with previous studies (13). In contrast, mutation of the IRSs impairs but does not completely disrupt the ability of insulin to suppress glucocorticoid-stimulated promoter activity. Mutation of the GRE also disrupts this IRS-independent effect of insulin, indicating that this effect of glucocorticoids also is mediated through the GRE. Together these results indicate that insulin suppresses glucocorticoid-stimulated IGFBP-1 promoter activity through both IRS-dependent and -independent mechanisms. To our knowledge, these studies provide the first report that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that it does so by conferring the ability of insulin to suppress promoter activity through an IRS-independent mechanism that is mediated through the GRE.
The IGFBP-1 promoter has proved to be an important model for defining mechanisms by which insulin regulates basal promoter activity through IRS-mediated mechanisms. Studies in other promoters in which insulin regulates gene expression through both IRS-dependent and -independent mechanisms, including the catalytic subunit of glucose-6 phosphatase (23) and phosphoenolpyruvate carboxykinase (40, 41, 42), have largely been conducted in the presence of cAMP agonists. Because cAMP stimulates the expression of PGC-1, an important coactivator for the glucocorticoid receptor (34) and FoxO proteins (25), some of these effects may be mediated through changes in the expression of PGC-1. Here we performed our experiments in the absence of cAMP agonists. Also, we found that overexpression of PGC-1 does not disrupt IRS-independent effects of insulin on glucocorticoid-stimulated promoter activity and that insulin does not suppress transactivation of PGC-1 in reporter gene studies. These results indicate that the IGFBP-1 promoter may prove to be a useful model for defining IRS-independent effects of insulin on glucocorticoid-stimulated gene expression under conditions in which PGC-1 expression and/or function is not a limiting factor.
We performed additional studies to characterize this effect of insulin. Whereas insulin suppressed the effect of glucocorticoids on the BP1.IRSmut construct, it failed to suppress the effects of glucocorticoids on an artificial promoter driven by an array of three GREs from the tyrosine aminotransferase gene (pTAT3.luc), indicating that this effect of insulin on IGFBP-1 promoter activity is context dependent in HepG2 cells and that other elements in the IGFBP-1 promoter may be required for insulin to suppress the effects of glucocorticoids on promoter activity through an IRS-independent mechanism in this cell culture model. However, it is important to note that Lemaigre and coworkers (43) have reported that insulin can exert direct effects on transactivation by the glucocorticoid receptor in other cell lines. This suggests that insulin may modulate the effects of glucocorticoids on gene expression through multiple mechanisms and that different mechanisms may be important in different settings. In the context of the IGFBP-1 promoter, Suh et al. (44) found that two elements outside the IRS/GRE region also contribute to the ability of glucocorticoids to stimulate promoter activity. This includes a site for hepatocyte nuclear factor-1 located downstream from the GRE and a site located approximately 150 bp upstream from the GRE. The protein factor(s) that binds to this upstream site remains to be identified. Additional studies are needed to identify cis-acting elements and trans-acting factors required for the ability of insulin to suppress glucocorticoid-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism.
Previous studies have shown that signaling through the PI3K pathway is critical for the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (13). In the present study, we found that PI3K also is important for the ability of insulin to suppress glucocorticoid-stimulated promoter activity through an IRS-independent mechanism. Whereas the effects of insulin on basal IGFBP-1 promoter activity in HepG2 cells appear to be mediated primarily through protein kinase B (13), other PI3K-dependent kinases may contribute to the regulation of IGFBP-1 expression in liver-derived cells that have been treated with glucocorticoids and cAMP (45, 46). Additional studies will be required to identify the role of specific effectors downstream from PI3K that are involved in mediating IRS-independent effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. We also found that inhibition of MAPKK activity reduces basal promoter activity and limits the ability of insulin to exert its effects on the IGFBP-1 promoter in HepG2 cells. These results indicate that multiple signaling pathways contribute to the regulation of the IGFBP-1 promoter and are required for the full effect of insulin on promoter activity.
Together, these findings suggest that insulin regulates IGFBP-1 promoter activity through multiple mechanisms. Whereas the effects of insulin on basal promoter activity is mediated through an IRS-dependent mechanism, stimulation of the promoter with glucocorticoids creates a more complex environment in which insulin exerts effects on promoter activity through both IRS-dependent and -independent mechanisms. Defining specific mechanisms mediating IRS-independent effects of insulin will be important for a more complete understanding of how insulin acts to regulate gene expression in the liver. The IGFBP-1 promoter promises to continue to provide a valuable model in this regard.
Footnotes
Abbreviations: GR, Glucocorticoid receptor; GRE, glucocorticoid response element; IGFBP-1, IGF binding protein-1; IRS, insulin response sequence; IRS/GRE, mutation of both the IRSs and the GRE; IRSmut, mutation of the IRSs; MAPKK, MAPK kinase; PGC, peroxisomal proliferator-activated receptor- coactivator; PI3K, phosphatidylinositol-3' kinase; PKB, protein kinase B.
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Abstract
IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor- coactivator-1, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in which insulin regulates IGFBP-1 expression through both IRS-dependent and IRS-independent mechanisms.
Introduction
IGF BINDING PROTEINS (IGFBPs) are a family of proteins that bind IGFs with high affinity and exert both IGF-dependent and -independent effects (1). IGFBP-1 is an approximately 30-kDa protein that is produced largely in the liver in which its expression is rapidly regulated at the level of gene transcription (2, 3). Circulating levels of IGFBP-1 are rapidly regulated in response to fasting and feeding, and circulating IGFBP-1 is thought to be the major short-term modulator of IGF bioavailability (4). IGFBP-1 also exerts local effects in the liver, in which it is thought to contribute to the capacity for regeneration in response to injury (5), and in the decidualized endometrium, in which it is a major secretory product and a marker of differentiation (5, 6).
Early studies regarding the regulation of IGFBP-1 revealed that its expression is stimulated by glucocorticoids and that this effect is suppressed by insulin in liver-derived cells (7, 8). Glucocorticoids regulate the abundance of IGFBP-1 mRNA in the liver through effects on both gene transcription (9) and mRNA stability (10, 11). Whereas the mechanisms mediating effects of glucocorticoids on IGFBP-1 mRNA stability are poorly defined, the effects of glucocorticoids on IGFBP-1 gene transcription are better understood. We previously identified a glucocorticoid response element (GRE) in the proximal IGFBP-1 promoter located approximately 100 bp upstream from the transcription start site based on DNase footprinting and reporter gene studies (12). This GRE is contiguous to two insulin response sequences [insulin response sequence (IRS)A and IRSB], which are essential for the ability of insulin to suppress basal IGFBP-1 promoter activity (13). Interestingly, mutation of the IRSA also reduces the ability of glucocorticoids to stimulate promoter activity (14, 15), suggesting that factors that interact with this IRS function cooperatively with glucocorticoids to stimulate promoter activity. This observation also suggested that disrupting the function of these factors might contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter activity (14). In the present study, we used reporter gene constructs to further define the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression.
Studies with the IGFBP-1 promoter have been important in defining the mechanisms by which insulin can suppress gene expression through an IRS. We reported that members of the Forkhead transcription factor family binding to this insulin response region (14) and that signaling through the phosphatidylinositol-3' kinase (PI3K)/protein kinase B (PKB) pathway is necessary and sufficient to mediate effects of insulin on basal promoter activity through an IRS (13). These results suggested that signaling by PI3K/PKB to Forkhead proteins may be important in mediating effects of insulin on gene expression. Subsequently, studies in Caenorhabditis elegans provided genetic evidence that members of the FoxO subfamily of Forkhead proteins are major targets of insulin-like signaling downstream from PKB (16, 17), and studies in this and other laboratories confirmed that FoxO proteins associate with IRSs in the IGFBP-1 promoter (18, 19, 20) and that phosphorylation by PKB promotes nuclear exclusion and suppresses transactivation by FoxO proteins (20, 21). The phosphorylation of FoxO proteins, and possibly other Forkhead proteins (22), is now thought to contribute to the effects of insulin on a number of genes involved in gluconeogenesis (23, 24, 25) and effects of growth factors on cell growth and survival (26, 27). At the same time, it is important to note that other factors interacting with IRS-like sequences also may contribute to the effects of insulin and growth factors on gene expression (28, 29, 30).
FoxO proteins also function cooperatively with glucocorticoids to stimulate the expression of IGFBP-1 (31) and several other genes, including pyruvate dehydrogenase kinase 4 (32) and atrogin (33), possibly by helping to recruit cAMP response element binding protein/p300 and/or other coactivator proteins (31). Disrupting FoxO functions, or possibly other factors that interact with the IRS, might help to mediate the effects of insulin on both basal and glucocorticoid-stimulated IGFBP-1 promoter activity.
To better understand the mechanisms mediating effects of insulin on gene expression, we examined the role of IRS-associated factors in mediating effects of insulin on the glucocorticoid-stimulated promoter. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal but not glucocorticoid-stimulated IGFBP-1 promoter activity, indicating that IRS-independent effects also contribute to the ability of insulin to suppress glucocorticoid-stimulated promoter. Cotransfection studies indicate that this effect of insulin is not due to suppression of peroxisomal proliferator-activated receptor- coactivator (PGC)-1, an insulin-regulated coactivator that contributes to transcriptional activity of the glucocorticoid receptor (25, 34). Studies with kinase inhibitors indicate that multiple signaling pathways contribute to the effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. Together these results indicate that the regulation of IGFBP-1 gene expression by insulin and glucocorticoids reflects an integrated response that is mediated through both IRS-dependent and -independent mechanisms.
Materials and Methods
Reporter gene constructs and expression vectors
The SauI-HgaI fragment of the IGFBP-1 promoter was inserted into the pGL2 polylinker (Promega, Madison, WI) (BP1.luc) and the mutation of the IRSs (IRSmut) reporter gene construct containing mutations of IRSA and IRSB was created by site-directed mutagenesis as reported previously (12). The IRS-GRE construct was created by mutating both halves of the GRE in the IRSmut reporter gene construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. The sequence of the mutated promoter was confirmed by dideoxy sequencing in the University of Illinois at Chicago Sequence Center and is provided in Fig. 1.
The expression vector for the human glucocorticoid receptor was provided by Muller et al. (35). The pTAT3.luc construct containing three copies of the GRE from the tyrosine aminotransferase gene immediately upstream of a minimal promoter was kindly provided by A. Kralli (University of Basel, Basel, Switzerland). The expression vector for the dominant-negative form of the p85 regulatory subunit of PI3K ( p85) was provided by Kasuga and colleagues (36), and the PGC-1 expression vector (pcDNA.flag.PGC-1-797) was provided by Kelly and colleagues (37). Plasmids expressing the Gal4 DNA binding domain in-frame with either full-length human PGC-1 (pA4.7-hPGC-1) or the truncated PGC-1 (pA4.7-hPGC-1.N482), in which the C-terminal region of PGC-1 has been deleted, were provided by Kralli and colleagues (38). The pG5e1b.luc reporter gene, which contains five copies of the Gal4 binding motif upstream of a minimal elb promoter, was provided by Dr. Peter Johnson (National Cancer Institute; Frederick, MD) as before (13).
Cell culture and transient transfection studies
HepG2 cells were plated in 65-mm dishes in Opti-MEM (Invitrogen, Carlsbad, CA) with 5% bovine calf serum (Invitrogen) and refed with DMEM with 10% bovine calf serum before transfection with calcium phosphate precipitates of plasmid DNAs, as previously reported (14). Transfected cells were rinsed twice with Hanks’ balanced salt solution (Sigma, St. Louis, MO) and then refed with DMEM with 1 mg/ml BSA with or without 10 μM dexamethasone and/or 100 nm recombinant human insulin for 18 h before lysis and analysis of luciferase activity.
Results
Effect of dexamethasone on IGFBP-1 promoter activity
To assess the role that IRSs play in glucocorticoid regulation of IGFBP-1 promoter activity, we performed reporter gene studies in HepG2 cells using luciferase reporter gene constructs containing the IGFBP-1 promoter with or without mutation of both the IRSs and the GRE (IRS/GRE). As shown in Fig. 1A, treatment with dexamethasone increases the activity of the intact IGFBP-1 promoter by 3-fold, whereas mutation of the IRSs reduces this effect by approximately 50%, supporting the concept that factors that interact with this region enhance the ability of glucocorticoids to stimulate promoter activity. Mutation of the GRE completely disrupts the remaining effect of glucocorticoids, indicating that the IRS-independent effect of glucocorticoids requires the presence of this GRE.
Because the expression of endogenous glucocorticoid receptors is thought to be low in HepG2 cells, we asked whether IRS-associated factors also contribute to the ability of glucocorticoids to stimulate promoter activity when the availability of glucocorticoid receptors is not limiting. As shown in Fig. 1B, we performed cotransfection studies with an expression vector for the glucocorticoid receptor (GR). Cotransfection with 30, 100, or 300 ng of the GR expression vector increases the ability of dexamethasone to stimulate the activity of both the wild-type and IRSmut IGFBP-1 promoter in a dose-dependent fashion. At each level of GR expression, the effect of dexamethasone on promoter activity is diminished by mutation of the IRSs, indicating that the effect of the IRSs on glucocorticoid function is independent of the level of GR expression.
Dexamethasone treatment enhances the effect of insulin
We also examined the effect of glucocorticoid treatment on the regulation of promoter activity by insulin using reporter gene constructs with/without mutation of the IRSs and/or GRE. As shown on the left in Fig. 2, insulin inhibits basal IGFBP-1 promoter activity by approximately 45% in the absence of dexamethasone. In contrast, no effect of insulin on basal promoter activity is seen in cells transfected with reporter gene constructs in which the IRSs have been mutated, consistent with previous reports (13). As shown by the solid bar on the right in Fig. 2, the ability of insulin to suppress IGFBP-1 promoter activity is enhanced in cells that have been treated with dexamethasone (65% inhibition by insulin, P < 0.01 vs. no dexamethasone). Mutation of the GRE disrupts the ability of dexamethasone to enhance the effect of insulin on promoter activity (data not shown), indicating that this effect is mediated through the GRE. To our knowledge, this result provides the first evidence that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that this effect is mediated through the GRE.
We next asked whether glucocorticoid treatment enhances the ability of insulin to suppress promoter activity through a mechanism that is IRS dependent or independent. As shown in the white bar on the right in Fig. 2, mutation of the IRSs reduces but does not completely disrupt the ability of insulin to suppress the dexamethasone-stimulated IGFBP-1 promoter. Mutation of the GRE together with the IRSs (hatched bar) completely disrupts the ability of insulin to suppress promoter activity in glucocorticoid-treated cells through an IRS-independent mechanism, indicating that this effect of insulin is mediated through the GRE. These results indicate that dexamethasone confers the ability of insulin to suppress IGFBP-1 promoter activity through an IRS-independent mechanism that is mediated through the GRE.
IRS-independent effect of insulin is context-dependent
We next asked whether the ability of insulin to suppress dexamethasone-stimulated promoter activity through an IRS-independent mechanism is context independent or whether other elements within the IGFBP-1 promoter also might be required. Here we performed transient transfection studies with a luciferase reporter gene construct containing three copies of the GRE from the tyrosine aminotransferase gene immediately upstream of a minimal promoter (pTAT3.luc) or the reporter gene construct containing the IGFBP-1 promoter in which the IRSs have been mutated (BP1.IRSmut). As shown in Fig. 3, insulin inhibits the ability of dexamethasone to stimulate the activity of the BP1.IRSmut promoter by approximately 40% in HepG2 cells (Fig. 3A) but does not inhibit the ability of dexamethasone to stimulate the activity of the pTAT3 promoter (Fig. 3B). This result indicates that the ability of insulin to inhibit the effect of dexamethasone on promoter activity is context dependent and is not due solely to the ability of insulin to suppress transactivation by the glucocorticoid receptor.
Effects of kinase inhibitors
We next explored the mechanism mediating IRS-independent effects of insulin on IGFBP-1 promoter activity. Initial studies indicated that treatment with LY294002, an inhibitor of PI3K, increases basal IGFBP-1 promoter activity approximately 3-fold, whereas treatment with PD98509, which inhibits the activation of the MAPK kinase (MAPKK), reduces basal promoter activity approximately 50%, and real-time PCR confirmed that LY294002 and PD98509 exert similar effects on the abundance of IGFBP-1 mRNA levels (data not shown), indicating that input from these pathways contribute to the regulation of basal IGFBP-1 promoter function and endogenous gene expression in HepG2 cells. As shown in Fig. 4 (solid bars), treatment with LY294002, which inhibits PI3 kinase activity, and the expression of the dominant-negative form of the regulatory subunit of PI3 kinase (p85) completely disrupts the ability of insulin to suppress basal IGFBP-1 promoter activity, consistent with previous studies indicating that the activation of PI3 kinase is required for this effect of insulin (10). Treatment with PD98059 also partially reduces the effect of insulin on basal activity of the IGFBP-1 promoter, indicating that input from MAPKK to the IGFBP-1 promoter also is important for the effect of insulin on basal promoter activity.
We next examined the effects of these compounds on the ability of insulin to inhibit IGFBP-1 promoter activity through an IRS-independent mechanism. Initial studies showed that these inhibitors had little effect on the ability of dexamethasone to stimulate IGFBP-1 promoter activity using reporter gene constructs containing the intact IGFBP-1 promoter or when both IRSs have been mutated (data not shown). As shown by Fig. 4 (hatched bars), the ability of insulin to inhibit the effect of dexamethasone on IGFBP-1 promoter activity when both IRSs are mutated is fully blocked by treatment with LY294002 and partially blocked by overexpression of a dominant-negative form of the regulatory subunit of PI3K (p85). These results indicate that activation of PI3K is required for the full IRS-independent effect of insulin on glucocorticoid-stimulated promoter activity. Treatment with PD98059 partially reduces this effect of insulin, indicating that input from MAPKK also is required for the full ability of insulin to inhibit glucocorticoid stimulation of the IGFBP-1 promoter through an IRS-independent mechanism.
Suppression of PGC-1 is not required for IRS-independent effect of insulin
PGC-1 is a coactivating protein that interacts with and contributes to the ability of the glucocorticoid receptor to stimulate gene expression (21, 27), and recent studies have suggested that insulin can suppress hepatic expression of PGC-1 (25). To determine whether insulin may suppress glucocorticoid-stimulated promoter activity by limiting the availability of PGC-1 in HepG2 cells, we performed cotransfection studies with a PGC-1 expression vector. As shown in Fig. 5A, the expression of PGC-1 enhances the ability of dexamethasone to stimulate the IRSmut promoter in a dose-dependent fashion (Fig. 5A, solid bars), consistent with the concept that PGC-1 can interact with and enhance the function of the GR. However, overexpression of PGC-1 does not limit the ability of insulin to suppress promoter activity (Fig. 5A, hatched bars). This result indicates that the ability of insulin to inhibit dexamethasone-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism in HepG2 cells is not due to limited availability of PGC-1 .
We also considered the possibility that insulin may inhibit the function of PGC-1 without suppressing its expression. The C-terminal region of PGC-1 contains an arginine/serine-rich region that contains several sites that might be phosphorylated by protein kinase B, based on a consensus sequence for protein kinase B phosphorylation sites (Arg-X-Arg-X-X-Ser/Thr), suggesting that signaling to this region might contribute to the ability of insulin to suppress transcription. Here we transfected HepG2 cells with vectors expressing the Gal4 DNA binding domain in-frame with either full-length PGC-1 (amino acids 1–797) or the truncated PGC-1 (amino acids 1–482), in which the C-terminal region has been deleted, together with the pG5e1b luciferase reporter gene construct, which contains five Gal4 binding sites immediately upstream of the e1b minimal promoter. As shown in Fig. 5B, full-length and truncated Gal4-PGC-1 fusion proteins potently stimulate pG5e1b promoter activity. Treatment with insulin does not suppress but rather enhances transactivation by full-length PGC-1 by approximately 50% in HepG2 cells, and this effect of insulin is not disrupted by deletion of the C-terminal region of PGC-1, indicating that this region is not required, and may interfere with, this effect of insulin. These results indicate that insulin does not suppress (and can enhance) transactivation by PGC-1 in HepG2 cells and that other mechanisms are required to mediate IRS-independent effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity.
Discussion
In the present study, we sought to better understand the mechanisms mediating effects of insulin and glucocorticoids on IGFBP-1 gene expression. Because glucocorticoids can exert effects on IGFBP-1 expression through effects on both IGFBP-1 gene transcription and IGFBP-1 mRNA stability, we used reporter gene constructs to directly assess the effects of glucocorticoids and insulin on IGFBP-1 promoter activity. Previous studies have shown that glucocorticoids and insulin regulate IGFBP-1 promoter activity through IRSs and a GRE located in the proximal IGFBP-1 promoter (12, 39). Mutating the IRSs has been shown to reduce the ability of glucocorticoids to stimulate IGFBP-1 promoter activity (14), and this has suggested that insulin may suppress IGFBP-1 glucocorticoid-stimulated promoter activity by disrupting the function of trans-acting factors that interact with these IRSs. Here we used IGFBP-1 promoter constructs with/without mutations of the IRSs or GRE to better define the mechanisms mediating the effects of insulin and glucocorticoids on IGFBP-1 gene expression.
Initial studies revealed that glucocorticoid treatment increases the ability of insulin to suppress the function of the intact IGFBP-1 promoter and that mutation of the GRE disrupts this effect, indicating that interaction between the GR and the GRE is required for this effect. Mutation of the IRSs completely disrupts the ability of insulin to suppress basal IGFBP-1 promoter activity in HepG2 cells, consistent with previous studies (13). In contrast, mutation of the IRSs impairs but does not completely disrupt the ability of insulin to suppress glucocorticoid-stimulated promoter activity. Mutation of the GRE also disrupts this IRS-independent effect of insulin, indicating that this effect of glucocorticoids also is mediated through the GRE. Together these results indicate that insulin suppresses glucocorticoid-stimulated IGFBP-1 promoter activity through both IRS-dependent and -independent mechanisms. To our knowledge, these studies provide the first report that glucocorticoid treatment enhances the ability of insulin to suppress IGFBP-1 promoter activity and that it does so by conferring the ability of insulin to suppress promoter activity through an IRS-independent mechanism that is mediated through the GRE.
The IGFBP-1 promoter has proved to be an important model for defining mechanisms by which insulin regulates basal promoter activity through IRS-mediated mechanisms. Studies in other promoters in which insulin regulates gene expression through both IRS-dependent and -independent mechanisms, including the catalytic subunit of glucose-6 phosphatase (23) and phosphoenolpyruvate carboxykinase (40, 41, 42), have largely been conducted in the presence of cAMP agonists. Because cAMP stimulates the expression of PGC-1, an important coactivator for the glucocorticoid receptor (34) and FoxO proteins (25), some of these effects may be mediated through changes in the expression of PGC-1. Here we performed our experiments in the absence of cAMP agonists. Also, we found that overexpression of PGC-1 does not disrupt IRS-independent effects of insulin on glucocorticoid-stimulated promoter activity and that insulin does not suppress transactivation of PGC-1 in reporter gene studies. These results indicate that the IGFBP-1 promoter may prove to be a useful model for defining IRS-independent effects of insulin on glucocorticoid-stimulated gene expression under conditions in which PGC-1 expression and/or function is not a limiting factor.
We performed additional studies to characterize this effect of insulin. Whereas insulin suppressed the effect of glucocorticoids on the BP1.IRSmut construct, it failed to suppress the effects of glucocorticoids on an artificial promoter driven by an array of three GREs from the tyrosine aminotransferase gene (pTAT3.luc), indicating that this effect of insulin on IGFBP-1 promoter activity is context dependent in HepG2 cells and that other elements in the IGFBP-1 promoter may be required for insulin to suppress the effects of glucocorticoids on promoter activity through an IRS-independent mechanism in this cell culture model. However, it is important to note that Lemaigre and coworkers (43) have reported that insulin can exert direct effects on transactivation by the glucocorticoid receptor in other cell lines. This suggests that insulin may modulate the effects of glucocorticoids on gene expression through multiple mechanisms and that different mechanisms may be important in different settings. In the context of the IGFBP-1 promoter, Suh et al. (44) found that two elements outside the IRS/GRE region also contribute to the ability of glucocorticoids to stimulate promoter activity. This includes a site for hepatocyte nuclear factor-1 located downstream from the GRE and a site located approximately 150 bp upstream from the GRE. The protein factor(s) that binds to this upstream site remains to be identified. Additional studies are needed to identify cis-acting elements and trans-acting factors required for the ability of insulin to suppress glucocorticoid-stimulated IGFBP-1 promoter activity through an IRS-independent mechanism.
Previous studies have shown that signaling through the PI3K pathway is critical for the ability of insulin to suppress basal IGFBP-1 promoter activity through an IRS (13). In the present study, we found that PI3K also is important for the ability of insulin to suppress glucocorticoid-stimulated promoter activity through an IRS-independent mechanism. Whereas the effects of insulin on basal IGFBP-1 promoter activity in HepG2 cells appear to be mediated primarily through protein kinase B (13), other PI3K-dependent kinases may contribute to the regulation of IGFBP-1 expression in liver-derived cells that have been treated with glucocorticoids and cAMP (45, 46). Additional studies will be required to identify the role of specific effectors downstream from PI3K that are involved in mediating IRS-independent effects of insulin on glucocorticoid-stimulated IGFBP-1 promoter activity. We also found that inhibition of MAPKK activity reduces basal promoter activity and limits the ability of insulin to exert its effects on the IGFBP-1 promoter in HepG2 cells. These results indicate that multiple signaling pathways contribute to the regulation of the IGFBP-1 promoter and are required for the full effect of insulin on promoter activity.
Together, these findings suggest that insulin regulates IGFBP-1 promoter activity through multiple mechanisms. Whereas the effects of insulin on basal promoter activity is mediated through an IRS-dependent mechanism, stimulation of the promoter with glucocorticoids creates a more complex environment in which insulin exerts effects on promoter activity through both IRS-dependent and -independent mechanisms. Defining specific mechanisms mediating IRS-independent effects of insulin will be important for a more complete understanding of how insulin acts to regulate gene expression in the liver. The IGFBP-1 promoter promises to continue to provide a valuable model in this regard.
Footnotes
Abbreviations: GR, Glucocorticoid receptor; GRE, glucocorticoid response element; IGFBP-1, IGF binding protein-1; IRS, insulin response sequence; IRS/GRE, mutation of both the IRSs and the GRE; IRSmut, mutation of the IRSs; MAPKK, MAPK kinase; PGC, peroxisomal proliferator-activated receptor- coactivator; PI3K, phosphatidylinositol-3' kinase; PKB, protein kinase B.
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