Alpha 1-acid Glycoprotein Gene Expression Research Articles

Peroxisome Proliferator-Activated Receptor α Physically Interacts with CCAAT/Enhancer Binding Protein (C/EBPβ) to Inhibit C/EBPβ-Responsive α1-Acid Glycoprotein Gene Expression

Recently, the role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in the hepatic inflammatory response has been associated to the decrease of acute phase protein transcription, although the molecular mechanisms are still to be elucidated. Here, we were interested in the regulation by Wy-14643 (PPARalpha agonist) of alpha1-acid glycoprotein (AGP), a positive acute phase protein, after stimulation by Dexamethasone (Dex), a major modulator of the inflammatory response. In cultured rat hepatocytes, we demonstrate that PPARalpha inhibits at the transcriptional level the Dex-induced AGP gene expression. PPARalpha exerts this inhibitory effect by antagonizing the CCAAT/enhancer binding protein (C/EBPbeta) transcription factor that is involved in Dex-dependent up-regulation of AGP gene expression. Overexpression of C/EBPbeta alleviates the repressive effect of PPARalpha, thus restoring the Dex-stimulated AGP promoter activity. Furthermore, glutathione-S-transferase GST pull-down and coimmunoprecipitation experiments evidenced, for the first time, a physical interaction between PPARalpha and the C-terminal DNA binding region of C/EBPbeta, thus preventing it from binding to specific sequence elements of the AGP promoter. Altogether, these results provide an additional molecular mechanism of negative regulation of acute phase protein gene expression by sequestration of the C/EBPbeta transcription factor by PPARalpha and reveal the high potency of the latter in controlling inflammation.

Growth hormone down-regulation of Interleukin-1beta and Interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3.

Severe burn induces the hepatic acute phase response. We previously showed that recombinant human growth hormone (GH) treatment after burn down-regulated acute phase protein (APP) production and gene expression in vivo. In this study, we hypothesized that the inhibitory effect of GH on the hepatic acute phase response was due to increased suppressor of cytokine signaling (SOCS) gene expression. HepG2 cells were treated with Interleukin-1beta (IL-1beta; 2 ng/mL) and interleukin 6 (IL-6; 20 ng/mL) alone or combined with GH (2 microg/mL) for 15 and 30 min, and 1, 2, and 4 h. The levels of gene expression for alpha1-acid glycoprotein (AGP), alpha1-antitrypsin (ATT), and SOCS (CIS, SOCS-1, 2, and 3) were measured by reverse transcript-polymerase chain reaction (RT-PCR). APP levels in the supernatant were determined by enzyme-linked immunosorbent sandwich assay (ELISA). The gene expression of AGP and ATT were also measured in HepG2 cells transfected with pEF-Flag-l/mSOCS-3 plasmid after IL-1beta or IL-6 treatment. Data are expressed as means +/- SEM, and statistical analysis was performed by one- or two-way analysis of variance. IL-1beta and IL-6 induced AGP and ATT gene expression and protein production, respectively, which was down-regulated by GH treatment. SOCS-3 but not CIS, SOCS-1, or SOCS-2 gene expression was significantly increased by GH treatment. APP gene expression was significantly decreased in cells transfected with plasmid over expressing SOCS-3 after IL-6 and IL-1beta treatment. GH attenuates IL-1beta or IL-6 induced APP gene expression, which is associated with increased expression of SOCS-3. This study suggests that SOCS-3 plays an important role in the suppression of cytokine signaling by GH in down-regulating the acute phase response after injury.

Growth hormone inhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.

The gene encoding alpha-1-acid glycoprotein (AGP), one of the major acute-phase proteins, is positively controlled at the transcriptional level by cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) and glucocorticoids. Here, we show that growth hormone (GH) treatment of isolated rat hepatocytes in vitro reduces AGP messenger RNA (mRNA) expression. AGP gene expression remained inducible by IL-1, IL-6, and phenobarbital (PB) in GH-treated hepatocytes. Interestingly, the repressive effect of GH on AGP gene expression was also observed in vivo: liver AGP mRNA content was strongly increased in hypophysectomized rats, and GH treatment of these animals led to a decrease in mRNA to levels lower than those in untreated control animals. Moreover, the inhibitory effect of GH mainly occurs at the transcriptional level and can be observed as little as 0.5 hours after GH adding in vitro to isolated hepatocytes. These results show negative regulation of AGP gene expression and strongly suggest that GH is a major endogenous regulator of constitutive AGP gene expression. Moreover, transfection assays showed that the region of the AGP promoter located at position -147 to -123 is involved in AGP gene regulation by GH. Furthermore, GH deeply modifies the pattern of nuclear protein binding to this region. GH treatment of hypophysectomized rats led to the release of proteins of 42 to 45 and 80 kd and to the binding of proteins of 48 to 50 and 90 kd.

CCAAT/enhancer binding proteins beta and delta regulate alpha1-acid glycoprotein gene expression in rat intestinal epithelial cells.

Isoforms of CCAAT/enhancer binding protein (C/EBP) are expressed in rodent intestine as well as in the rat intestinal epithelial cell line IEC-6. However, no specific roles have been attributed to these isoforms in intestinal epithelial cells. To determine whether C/EBP family members could be implicated in the regulation of acute-phase response gene expression in intestinal epithelial cells, we have studied the effect of glucocorticoids on expression of the alpha1-acid glycoprotein gene and C/EBP isoforms in IEC-6 cells. Glucocorticoids induced alpha1-acid glycoprotein gene expression in these cells. This induction coincided with an increase of DNA-binding capacity of both C/EBPbeta and C/EBPdelta to the B1 and B2 C/EBP-interacting sites localized in the rat AGP promoter. Transforming growth factor beta, (TGFbeta), a cytokine involved in the transcriptional regulation of several acute-phase plasma proteins, antagonized the glucocorticoid-dependent induction of alpha1-acid glycoprotein gene expression. In parallel, TGFbeta downregulated the DNA-binding capacities of both the C/EBPbeta and C/EBPdelta isoforms. Mutations of the B1 or the B2 C/EBP-interacting site strongly reduced the responsiveness of the alpha1-acid glycoprotein promoter to glucocorticoids and TGFbeta. These results demonstrate a functional role for C/EBPbeta and C/EBPdelta in rat intestinal epithelial cells and suggest that these isoforms represent important modulators of the acute-phase response and of glucocorticoid, as well as TGFbeta, responsiveness.

Thyroid hormone, all-trans retinoic acid, and 9-cis retinoic acid functioned as negative modulators of the effect of glucocorticoid on induction of alpha 1-acid glycoprotein mRNA in RLN-10 cells.

The expression of acute-phase protein genes is controlled by many factors, such as IL-1, IL-6, glucocorticoids, thyroid hormone (T3), and retinoic acids. We studied the interaction of T3, glucocorticoids, all-trans retinoic acid (RA), and 9-cis retinoic acid (9cRA) on the expression of the rat alpha 1-acid glycoprotein (AGP) gene in vitro. Dexamethasone (Dex) activated AGP gene expression in a rat liver derived cell line, RLN-10. Although T3, RA, and 9cRA by themselves had no effect on AGP production, they reduced the response to Dex of the AGP gene.

Effects of insulin, dexamethasone and cytokines on ?1-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

While the effects of insulin, dexamethasone and cytokines on alpha 1-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of alpha 1-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of alpha 1-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify alpha 1-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of alpha 1-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines.

Regulation of rabbit alpha 1-acid glycoprotein gene expression in acute-phase liver. Identification of inducible and constitutive proteins like CCAAT-enhancer binding protein that interact with the 5'-proximal promoter elements.

To identify the cis-acting DNA sequences responsible for inducible transcription of rabbit alpha 1-acid glycoprotein gene, 5'-flanking region containing 529 bp of this gene and its various 5'-deletions were linked to the reporter gene coding for the bacterial chloramphenicol acetyltransferase (CAT) and analyzed for their ability to confer cytokine-mediated inducibility to the reporter CAT gene in liver cells. Deletion analysis has identified a 151-bp region from the sequence -186 to -35, that contains the regulatory promoter element(s) responsible for stimulation mediated by cytokines present in the conditioned-medium. Using mobility shift assays, we have identified highly inducible nuclear factors in acute liver nuclear extract that interact with this regulatory promoter region. DNase I footprint analysis has revealed two adjacent nuclear factor binding sites and competition of DNA-binding activity has indicated that the distal element of these two sites has higher affinity for nuclear factors than the proximal one. Both of these two regions have been found to be capable of directing conditioned-medium-induced transcription. Studies on the characterization of nuclear factors binding to these elements have shown that they belong to a class of transcription factors called CCAAT-enhancer binding protein (C/EBP). Our results indicate that binding of C/EBP-like factors to the inducible promoter elements of rabbit alpha 1-acid glycoprotein gene is highly specific and the induction of this gene under acute-phase conditions may involve their participation.

Induction of Liver Alpha-1 Acid Glycoprotein Gene Expression Involves Both Positive and Negative Transcription Factors

Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene.

Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene.

Molecular cloning and nucleotide sequence of complementary DNA encoding rabbit α1-acid glycoprotein

Alpha 1-acid glycoprotein (AGP) complementary DNA clone has been isolated from an acute phase rabbit liver library. Complete and full-length nucleotide sequence of this cDNA has been determined. The cDNA contains 35 nucleotides 5′ untranslated region followed by 606 nucleotides of coding region and a 112 nucleotides 3′ untranslated region. Comparison of this sequence with that of human, rat and mouse reveals a high degree of homology with human α 1-AGP. Northern blot analysis of messenger RNA for α 1-AGP in normal and acute livers demonstrates remarkable induction of transcription of this gene in rabbit in response to acute inflammation by the administration of turpentine. Such a response of AGP gene expression makes it a major member of the acute phase responsive genes in rabbit liver.

Transcriptional control of the rat alpha 1-acid glycoprotein gene.

Dexamethasone strongly stimulates the production of alpha 1-acid glycoprotein (AGP) in rat liver. The regulated expression of AGP in cultured liver cells has been variably ascribed to the activation of AGP gene transcription, to change in the nuclear processing of AGP gene transcripts, or to both. Treatment of HTC cells with dexamethasone proportionally enhanced the rate of AGP gene transcription and the concentration of AGP mRNA. To assess whether hormone treatment is indeed capable of affecting posttranscriptionally the level of rat AGP mRNA, a hormone-independent expression vector containing the rat AGP gene was transiently introduced into HepG2 cells. None of the hormone combinations known to modulate expression of the endogenous AGP influenced the steady-state concentration of mature mRNA that was derived from the transgene. Hence, the existence of a hormone-sensitive posttranscriptional regulatory pathway for rat AGP gene expression could not be substantiated.

Localization of DNA sequences involved in dexamethasone-dependent expression of the rat alpha 1-acid glycoprotein gene.

Synthesis of rat alpha 1-acid glycoprotein (AGP), one of the major inflammation-induced plasma proteins, is positively regulated by dexamethasone. To define the dexamethasone-responsive genetic element, we isolated and tested AGP gene sequences for the ability to confer glucocorticoid induction to the bacterial chloramphenicol acetyltransferase (CAT) gene in L cells. A 141-base-pair region of the AGP gene, including 120 base pairs of DNA upstream from the start site of transcription and 21 base pairs of the 5' untranslated region, was sufficient for maximal CAT gene induction by dexamethasone. To localize more precisely the AGP glucocorticoid-responsive element, parts of this 141-base-pair region were inserted 5' to either an AGP promoter-CAT gene or a human triosephosphate isomerase promoter-CAT gene, both of which lacked a response to the steroid. The AGP gene region between 120 and 42 base pairs upstream from the start site of transcription was found to mediate maximal dexamethasone induction of CAT enzyme levels. This result was unexpected because this region does not contain sequence homologies to known glucocorticoid receptor-binding sites and those AGP gene regions that lay further upstream and were homologous to other glucocorticoid receptor-binding sites were inactive in the CAT assay. The fact that the AGP gene region mediating dexamethasone regulation was distinct from the transcribed region indicates that glucocorticoids increase AGP gene expression primarily at the transcriptional rather than the posttranscriptional level.

Localization of DNA sequences involved in dexamethasone-dependent expression of the rat alpha 1-acid glycoprotein gene.

Synthesis of rat alpha 1-acid glycoprotein (AGP), one of the major inflammation-induced plasma proteins, is positively regulated by dexamethasone. To define the dexamethasone-responsive genetic element, we isolated and tested AGP gene sequences for the ability to confer glucocorticoid induction to the bacterial chloramphenicol acetyltransferase (CAT) gene in L cells. A 141-base-pair region of the AGP gene, including 120 base pairs of DNA upstream from the start site of transcription and 21 base pairs of the 5' untranslated region, was sufficient for maximal CAT gene induction by dexamethasone. To localize more precisely the AGP glucocorticoid-responsive element, parts of this 141-base-pair region were inserted 5' to either an AGP promoter-CAT gene or a human triosephosphate isomerase promoter-CAT gene, both of which lacked a response to the steroid. The AGP gene region between 120 and 42 base pairs upstream from the start site of transcription was found to mediate maximal dexamethasone induction of CAT enzyme levels. This result was unexpected because this region does not contain sequence homologies to known glucocorticoid receptor-binding sites and those AGP gene regions that lay further upstream and were homologous to other glucocorticoid receptor-binding sites were inactive in the CAT assay. The fact that the AGP gene region mediating dexamethasone regulation was distinct from the transcribed region indicates that glucocorticoids increase AGP gene expression primarily at the transcriptional rather than the posttranscriptional level.