Differential Glucocorticoid Enhancement of the Cytokine-Driven Transcriptional Activation of the Human Acute Phase , SAA1 and SAA2 This information is current as of September 28, 2021. Caroline F. Thorn and Alexander S. Whitehead J Immunol 2002; 169:399-406; ; doi: 10.4049/jimmunol.169.1.399 http://www.jimmunol.org/content/169/1/399 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Glucocorticoid Enhancement of the Cytokine-Driven Transcriptional Activation of the Human Acute Phase Serum Amyloid A Genes, SAA1 and SAA2

Caroline F. Thorn and Alexander S. Whitehead1

The human acute phase serum amyloid A (A-SAA) genes, SAA1 and SAA2, have a high degree of sequence identity that extends ϳ450 bp upstream of their transcription start sites. Each promoter contains analogously positioned functional binding sites for the transcription factors NF-␬B and NF-IL6. In human HepG2 hepatoma cells transfected with SAA promoter luciferase reporter constructs, administration of IL-1 and IL-6, singly or in combination, induced SAA1 and SAA2 transcriptional readouts that were qualitatively indistinguishable. However, under induced conditions, the SAA2 promoter had a significant quantitative transcrip-

tional advantage over the SAA1 promoter. The application of the synthetic glucocorticoid dexamethasone in the context of cytokine Downloaded from stimulation enhanced the transcriptional activity of the SAA1, but not the SAA2, promoter such that readout from the former became equivalent to that from the latter. A putative glucocorticoid response element (GRE) is present (between residues ؊208 and ؊194) only in the SAA1 ; a similar sequence in the corresponding region of the SAA2 gene is disrupted by a nine-residue insertion. The SAA1 GRE was shown to be functionally active and the SAA2 disrupted GRE was shown to be functionally inactive in experiments using reporter constructs carrying SAA1 and SAA2 promoters that had been modified by site-specific mutagenesis.

Quantitative analysis of transcript-specific RT-PCR products, derived from SAA1 and SAA2 mRNAs after treatment of HepG2 http://www.jimmunol.org/ cells with cytokines in the presence or absence of dexamethasone, confirmed that the endogenous SAA1 gene has a cytokine-driven transcriptional disadvantage that is superseded by a marginal transcriptional advantage when glucocorticoids are present. The Journal of Immunology, 2002, 169: 399Ð406.

he acute phase response (APR)2 occurs in the hours im- which encodes the constitutively expressed C-SAA (7, 8). In mediately following an inflammatory stimulus such as in- mouse, the Saa genes are arranged in a similar configuration on a T fection or trauma (1). It is initiated, and subsequently region of distal 7 that has synteny with human chro- maintained, modulated, and terminated, by the coordinated release mosome 11p15 (9). The sequences of A-SAAs from vertebrate of proinflammatory cytokines, anaphylatoxins, anti-inflammatory species that last shared a common ancestor hundreds of millions of by guest on September 28, 2021 cytokines, cytokine antagonists, and glucocorticoids (2). During years ago are remarkably conserved (10), indicating that these the APR, characteristic changes occur in the synthetic profile of the APPs have a fundamentally important protective function that has liver leading to altered regulation of a subset of products called been retained via positive selection of critical structural motifs. acute phase (APPs). These include positively regulated The sequence similarities between the A-SAA genes within a given APPs such as acute phase serum amyloid A (A-SAA), C-reactive species are striking and extend beyond the coding regions to the , complement C3 and C5, and negatively regulated APPs mRNA untranslated regions (UTRs), introns, and upstream pro- such as albumin (3). A-SAA is a major APP in that it is subject to moter elements, suggesting that they may have been subject to a quantitatively dramatic induction; at the peak of an APR it can regular homogenizing events by a mechanism such as gene con- reach concentrations of Ͼ1 mg/ml (i.e., levels exceeding those version (11). Based on the evolutionary considerations outlined observed under normal physiological conditions by Ͼ1000-fold) above, and on direct experimental observations in vivo and in (4). vitro, it has generally been assumed that the SAA1 and SAA2 genes Human A-SAA comprises two , SAA1 and are coordinately regulated and that their protein products play es- SAA2, the products of the SAA1 and SAA2 genes, which map to a sentially identical roles in the APR. However, the apparent para- cluster of related genes on chromosome 11p15.1 (5). The other dox of why higher mammals have at least two A-SAAs has thus far members of the cluster are SAA3, a pseudogene (6), and SAA4, not been resolved. During an APR the A-SAAs are expressed at high levels in liver and at more modest levels in many other tissues (12Ð15). A-SAAs Department of Pharmacology and Center for Pharmacogenetics, University of Penn- are the circulating precursors of the insoluble cleavage product sylvania School of Medicine, Philadelphia, PA 19104 amyloid A that is deposited in major organs in secondary amy- Received for publication February 12, 2002. Accepted for publication April 25, 2002. loidosis, a progressive and fatal disease that is an occasional con- The costs of publication of this article were defrayed in part by the payment of page sequence of chronic or episodic inflammatory conditions such as charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. rheumatoid arthritis and leprosy (16). In addition to this well-doc- 1 Address correspondence and reprint requests to Dr. Alexander S. Whitehead, De- umented role in the etiology of secondary amyloidosis, the A- partment of Pharmacology and Center for Pharmacogenetics, University of Pennsyl- SAAs may have other pathogenic actions: 1) they associate with vania School of Medicine, 159 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, HDL3 particles, replacing ApoA1 as the predominant apolipopro- PA 19104. E-mail address: [email protected] tein (17), and may therefore compromise reverse cholesterol trans- 2 Abbreviations used in this paper: APR, acute phase response; SAA, serum amyloid A; A-SAA, acute SAA; UTR, untranslated region; APP, acute phase protein; GR, port and indirectly contribute to atherogenesis in those with glucocorticoid receptor; GRE, glucocorticoid response element. chronic inflammatory conditions; 2) they are synthesized in the

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 400 GLUCOCORTICOID ENHANCEMENT OF SAA1 TRANSCRIPTION

foam cells, smooth muscle cells, and endothelial cells of athero- was performed in triplicate. IL-1 was purchased from PeproTech (Rocky sclerotic plaques (18) and are chemotactic for monocytes and T Hill, NJ). IL-6 was obtained from AstraZeneca (Wilmington, DE). Dexa- cells (19, 20), suggesting that they may directly participate in le- methasone and RU-486 were from Sigma-Aldrich (St. Louis, MO). sion formation; and 3) they are produced in synoviocytes and ap- Luciferase assays pear to function as autocrine inducers of collagenase (21), indicat- Cells were harvested at various times posttreatment, washed in PBS, and ing a possible role in the etiology of degenerative joint diseases. resuspended in passive lysis buffer (Promega, Madison, WI). Lysates were Given the known and presumptive clinical consequences of assayed for luciferase and Renilla activity using the LLR and Stop and Glo chronically high systemic concentrations of A-SAA and the sus- reagents (Promega) in a dual-injection luminometer (Turner Designs, tained local synthesis of A-SAA, it is important to fully charac- Sunnyvale, CA). The mean ratios of luciferase to Renilla activity for each terize the mechanisms whereby the synthesis of this archetypal treatment (performed in triplicate) were calculated. The fold induction for each treatment at each time point, together with the SDs, were calculated major APP is induced, and subsequently controlled, under a vari- relative to the ratios derived from triplicate controls treated with medium ety of pro- and anti-inflammatory conditions. Over the past decade only for the same length of time. Each experiment was performed at least studies of SAA1 and SAA2 promoter-reporter constructs (22Ð24) three times. Statistical significance was measured by a t test. have helped to define the parameters that govern the responsive- RT-PCR ness of each of these genes to IL-1, TNF, and IL-6, alone and in combination, and have identified NF-␬B and NF-IL6 transcription Total RNA was prepared by LiCl urea extraction (34) from HepG2 cells factor binding sites that facilitate responses of both genes to cy- treated for 24 h under various experimental conditions. RT-PCR was con- ducted in a two-step process. cDNA was reverse transcribed from 3 ␮gof tokine signaling that are qualitatively indistinguishable. However, total cellular RNA in a 25-␮l reaction containing oligo(dT) primer, RNasin no published studies to date have explored the mechanisms under- RNase inhibitor, and Moloney murine leukemia virus reverse transcriptase Downloaded from lying the striking enhancement of cytokine-driven A-SAA mRNA (Promega) at 42¡C for 1 h. PCR was performed using 2 ␮l of cDNA ␮ ␮ ␮ and protein synthesis that is conferred by glucocorticoids both in product in a 50- l reaction containing 125 M primers, 200 M dNTPs (Amersham Pharmacia Biotech, Piscataway, NJ), 1ϫ PCR buffer, 5 mM vivo and in vitro (13Ð15, 25Ð29). MgCl2, and AmpliTaq polymerase (PerkinElmer, Wellesley, MA). The forward primer was 5Ј-CAGACAAATACTTCCATGCT-3Ј; the reverse Materials and Methods primer was an equal mix of 5Ј-TTTTTTCCACCTCTTAAGTATTTAT Ј Ј Ј SAA promoter luciferase reporter constructs TAGA-3 and 5 -TTTTTTCCACCTCTTAAGCATTTATTAGA-3 . PCR http://www.jimmunol.org/ conditions were as follows: 95¡C for 5 min, followed by 25 cycles at 94¡C Generation of the pGL2-SAA2pt construct, which contains 1.2 kb of the for 20 s, 51¡C for 1 min, and 72¡C for 1 min, followed by 72¡C for 5 min. human SAA2 promoter upstream of a luciferase reporter gene, is described Products were separated on 8% polyacrylamide gels at 50 V for 24 h, elsewhere (24). The pGL2-SAA1pt construct was generated as follows. stained with ethidium bromide, and quantified by NIH Image. The proximal 3.1 kb of genomic sequence upstream of the human SAA1 transcription start site plus the full 37 bases of 5ЈUTR was amplified by PCR Computer analysis from human genomic DNA (Roche, Indianapolis, IN) using forward and re- verse primers, HSAA1PF (5Ј-GAATTCACGCGTTTGGGCAGGGAAT Transcription factor consensus binding site predictions were made using ATACTTATTTATGGAAG-3Ј) and HSAAPR (5Ј-GAATTCCCATGGT the Signalscan and TESS programs available from the Center for Bioin- GCTGATCTGTGCTGTAGCTGAGCTGCGGG-3Ј), that incorporate Mlu1 formatics (University of Pennsylvania, Philadelphia, PA) at http://www. and NcoI restriction sites, respectively. The product was digested with Mlu1 cbil.upenn.edu. Sequence alignments were conducted using the ClustalW by guest on September 28, 2021 program (35) available at http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl? and NcoI and directionally cloned into a pGL2 vector that had been modified ϭ to include an NcoI site at the start of the luciferase coding sequence and con- page /NPSA/npsa_clustalwan.html. tains the human SAA2 3ЈUTR (30). Constructs pGL2-SAA1[0.7] and pGL2- SAA1[0.25] containing 704 and 233 bases of promoter sequence, respectively, Results were generated from pGL2-SAA1pt by PCR using the forward primers Studies of A-SAA transcriptional activation in a range of species 5Ј-GAATTCACGCGTGCGTGATTATAGCTCACTGCAGCCTTGACC-3Ј have identified a number of A-SAA promoter elements that are and 5Ј-GAATTCACGCGTGGTCTCCTGCCTG-3Ј, respectively, and the re- ␬ verse primer HSAAPR. Constructs pGL2-SAA2[0.7] and pGL2-SAA2[0.25] targets for critical transactivating factors including NF- B, NF- containing 700 and 239 bases of promoter sequence, respectively, were IL6, SEF, YY-1, AP-2, SAF, and Sp-1 (36Ð41). Equivalent con- generated from pGL2-SAA2pt by PCR using the forward primers 5Ј- sensus binding sites for several of these factors are present in the TATAACGCGTCCTATTTAACGCACCACACTCT-3Ј and 5Ј-GAATT human SAA1 and SAA2 promoters (Fig. 1). Of these, the NF-␬B Ј CACGCGTGATCTAGCACCTG-3 , respectively, and the reverse primer site (from bases Ϫ91 to Ϫ82) and the NF-IL6 site (from bases HSAAPR. Ϫ Ϫ The GREI construct was generated by PCR mutagenesis of pGL2- 184 to 171) of SAA2 have been shown by functional studies to SAA1[0.7] using primers GREIF (5Ј-CAGCAAACCTCTCTTGTCCC-3Ј) be important for facilitating the response to IL-1 and IL-6, respec- and GREIR (5Ј-AGAGAGGTTTGCTGTGCCT-3Ј). The GRED construct tively (23). Glucocorticoids have been shown, both in vitro and in was generated by PCR mutagenesis of pGL2-SAA2[0.7] using primers vivo, to enhance the cytokine-driven induction of A-SAA mRNA GREDF (5Ј-CAAGGCACATCTTGTTCCCATAGGT-3Ј) and GREDR (5Ј-GGAACAAGATGTGCCTTGGCAATG-3Ј). and protein (25Ð29). However, no studies to date have character- The integrity of all constructs was verified by DNA sequencing. ized the intrinsic genetic elements that govern the glucocorticoid- mediated enhancement of A-SAA synthesis. Other plasmids The Renilla transfection control plasmid is described elsewhere (31). The Sequence alignment of the proximal promoters of constitutive human glucocorticoid receptor (GR)-␣ expression plasmid, SAA1 and SAA2 CMX-GR, was a gift of Dr. R. Evans (Salk Institute, La Jolla, CA) (32). To explore the basis of the glucocorticoid enhancement of A-SAA Cell culture and transient transfection synthesis we initially undertook a comprehensive computational analysis of the promoters of the SAA1 and SAA2 genes. Alignment Human HepG2 hepatoma cells (American Type Culture Collection, Man- assas, VA) were cultured in DMEM containing 10% FCS, gentamicin, of 0.7 kb of the SAA1 and SAA2 sequences immediately upstream sodium pyruvate, and nonessential amino acids (Life Technologies, Rock- of their respective transcription start sites, using the ClustalW pro- ville, MD). Cells were seeded into 24-well plates 24 h before transfection gram, revealed a very high degree of sequence identity (87%) in using FuGENE (Roche) as previously described (33). Cells transfected the proximal ϳ450 bp, upstream of which the sequences exhibit a with SAA promoter luciferase reporter constructs and Renilla control plas- mid were incubated for 16Ð20 h before replacement of culture medium markedly decreased level of identity and many regions of noncon- with fresh medium alone or fresh medium containing 10 ng/ml cytokines tiguity. The alignment of the highly conserved proximal promoter and/or dexamethasone and/or RU-486 (mifepristone). Each experiment regions is depicted in Fig. 1. Within this alignment there are two The Journal of Immunology 401 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Alignment of SAA1 and SAA2 promoters. The proximal 450 bases of the human SAA1 and SAA2 promoters were aligned using the ClustalW program. Putative transcription factor binding sites are underlined once (predicted by TESS program), with a dotted line (predicted by Signalscan program), or twice (predicted by visual inspection). Dashes represent gaps in one sequence relative to the other. short noncontiguous regions that each mandate the introduction of gistically induced by the simultaneous addition of both cytokines a gap of more than one residue into one of the promoter sequences. (Fig. 2A). IL-1-driven readout increased from 3 to 24 h, whereas These deletions are in SAA1 relative to positions Ϫ207 to Ϫ199 of IL-6-driven readout was highest at 3 h and decreased through 24 h. SAA2 and in SAA2 relative to positions Ϫ43 to Ϫ40 of SAA1. The synergistic response to dual treatment with IL-1 plus IL-6 Close visual inspection revealed a putative 15-bp glucocorticoid followed a kinetic profile similar to that observed for IL-6 alone. response element (GRE) consensus sequence (GGCACATCTT These results were all in accord with data previously reported by Ϫ Ϫ GTTCC) (42) in SAA1 (from 208 to 194) that encompasses the us (24). The SAA1 promoter exhibited transcription induction pro- first of these noncontiguous regions. These residues are also files in response to single and dual cytokine treatments that were Ϫ Ϫ present in SAA2 (within the sequence from 213 to 190) but are qualitatively and kinetically similar to those of the SAA2 promoter Ϫ Ϫ disrupted by nine residues from 207 to 199 that have no coun- (Fig. 2B). This is consistent with our a priori expectation that the terparts in the corresponding location in SAA1 (i.e., between SAA1 analogously positioned NF-␬B and NF-IL6 sites in the SAA1 and residues Ϫ203 and Ϫ202). SAA2 promoters would mandate similar cytokine responses for To establish the extent to which the SAA1 and SAA2 promoters each. However, SAA2 appears to have a considerable (ϳ2- to exhibit qualitatively and quantitatively similar responses to cyto- kines, and to determine whether the putative SAA1 GRE defined 3-fold) quantitative transcriptional advantage over SAA1 in re- above is functionally active, various reporter constructs containing sponse to all three cytokine treatments. native and modified SAA1 and SAA2 promoters were tested for Treatment of transfected cells with dexamethasone alone had no their responsiveness to cytokines and glucocorticoids in vitro. effect on the SAA1 promoter. However, cotreatment of transfected cells with 50 nM dexamethasone enhanced the cytokine-driven Transcriptional regulation of the SAA1 and SAA2 promoters in induction of the SAA1 promoter ϳ2-fold for all treatments at all vitro by cytokines and glucocorticoids time points (Fig. 2B). In contrast, dexamethasone had no measur- HepG2 cells transfected with either of the A-SAA promoter lucif- able effect on SAA2 promoter activity under any of the test con- erase reporter constructs, pGL2-SAA1pt or pGL2-SAA2pt, were ditions (Fig. 2A). These data establish that the SAA1 and SAA2 treated with cytokines for 3, 6, 9, and 24 h. The SAA2 promoter genes respond differentially to glucocorticoids in the context of an was moderately induced by IL-1 alone or IL-6 alone and syner- ongoing cytokine-dependent transcriptional induction. 402 GLUCOCORTICOID ENHANCEMENT OF SAA1 TRANSCRIPTION

moter to dexamethasone in the context of cytokine induction, was due to suboptimal dosing, a range of dexamethasone concentra- tions was tested. HepG2 cells transfected with either pGL2- SAA1pt or pGL2-SAA2pt were treated with 10, 50, 100, or 500 nM or 1 ␮M dexamethasone in the presence or absence of IL-1 plus IL-6 for 4 h. Neither promoter showed any response to dexa- methasone alone, even at the highest dose used (data not shown). In contrast to the cytokine-driven transcriptional readout from the SAA2 promoter, which could not be enhanced by dexamethasone at any concentration, the SAA1 promoter exhibited a clear dose-de- pendent enhancement of transcriptional activity (data not shown). This suggests that the dexamethasone enhancement of cytokine- driven SAA1 promoter activity involves specific receptor-mediated events, most likely via GRs.

The enhancement of cytokine-driven SAA1 promoter transcriptional activity by glucocorticoids is GR dependent To definitively establish that the enhancement of cytokine-depen- dent SAA1 promoter transcriptional activity by glucocorticoids is Downloaded from mediated by the GR, cytokine and dexamethasone treatments sim- ilar to those described above were conducted in the presence of the GR antagonist RU-486 (mifepristone). HepG2 cells transfected with pGL2-SAA1pt were treated with IL-1 plus IL-6, in the pres- ence or absence of 100 nM dexamethasone and/or 10 nM RU-486

for 4 h (Fig. 3). The presence of RU-486 alone had no measurable http://www.jimmunol.org/ effect on the level of cytokine-driven SAA1 promoter transcrip- tional activity. However, RU-486 completely blocked the capacity of dexamethasone to quantitatively enhance the induction of the SAA1 promoter by cytokines, limiting the transcriptional readout to that observed in transfected cells treated only with cytokines. This establishes that the GR is a requisite component in mediating the dexamethasone enhancement of cytokine-driven SAA1 transcrip- tional activity. by guest on September 28, 2021 The effect of GR overexpression on the capacity of dexamethasone to modify SAA1 and SAA2 transcriptional activity HepG2 cells have been reported to express only low levels of GR FIGURE 2. Time course of cytokine/dexamethasone induction of SAA1 (43). To determine whether the nonresponse of both promoters to and SAA2 promoter luciferase reporter constructs. HepG2 cells transfected with pGL2-SAA2pt (A) or pGL2-SAA1pt (B) luciferase reporter constructs were treated with medium only, dexamethasone (50 nM), IL-1 (10 ng/ml), IL-1 plus dexamethasone, IL-6 (10 ng/ml), IL-6 plus dexamethasone, IL-1 plus IL-6, or IL-1 plus IL-6 plus dexamethasone. Cells were harvested 3, 6, 9, and 24 h after treatment and relative luciferase values were calculated and compared with untreated controls. The difference between pGL2- SAA1pt luciferase reporter construct treated with IL-1 plus IL-6 and treated with IL-1 plus IL-6 plus dexamethasone was statistically significant at all time points (p Յ 0.02).

A construct containing only 235 bases of the SAA1 promoter (pGL2-SAA1[0.25]) retained all of the quantitative, qualitative, and kinetic aspects of cytokine responsiveness and glucocorticoid enhancement exhibited by constructs containing 3.1 kb of se- quence upstream of the SAA1 transcription start site, suggesting that all of the critical control elements engaged by the most important inflammatory mediators are located in this short region FIGURE 3. SAA1 glucocorticoid responsiveness is GR dependent. (data not shown). HepG2 cells were transfected with pGL2-SAA1pt and treated with 10 ng/ml IL-1 plus 10 ng/ml IL-6, alone and in the presence of 10 nM RU-486 Dose-dependent glucocorticoid enhancement of cytokine-driven and/or 100 nM dexamethasone. Cells were harvested 4 h after treatment SAA1 promoter activity and relative luciferase values were calculated and compared with untreated -The difference between treatment with cytokines plus dexa ,ء .controls To eliminate the trivial possibility that the nonresponse of both methasone and cytokines plus dexamethasone plus RU-486 was statisti- A-SAA promoters to dexamethasone alone, and of the SAA2 pro- cally significant (p ϭ 0.05). The Journal of Immunology 403 dexamethasone alone, and that of the SAA2 promoter to dexameth- SAA2 disrupted GRE, mandate the differential dexamethasone re- asone in the context of cytokine induction, is due to cellular GR sponsiveness of the genes. Two modified constructs were gener- levels that are below a functional threshold, SAA1 and SAA2 tran- ated: the GREI construct contains an SAA1 promoter with a nine- scriptional readout was measured in HepG2 cells cotransfected residue sequence (GCAAACCTC) inserted into the GRE to form with a constitutive GR expression construct after treatment with an SAA2-like disrupted GRE; the GRED construct contains an various combinations of cytokines and dexamethasone. The SAA1 SAA2 promoter in which the same nine residues have been deleted and SAA2 promoters were unresponsive to dexamethasone alone in to form an SAA1-like GRE (Fig. 4A). The GREI and GRED con- HepG2 cells cotransfected with a GR expression construct together structs each retained the basal and cytokine-driven levels of tran- with pGL2-SAA1pt or pGL2-SAA2pt (data not shown). However, scriptional activity that are characteristic of the unmodified paren- in the context of cytokine induction of the SAA1 promoter, the tal promoters from which they were derived. However, the GREI dexamethasone enhancement of cytokine-driven transcriptional construct had lost the capacity to respond to dexamethasone in the readout could be augmented by cotransfection with GR expression presence of cytokines, whereas the GRED construct had gained construct (data not shown), suggesting that the glucocorticoid sig- this property (Fig. 4B), thereby establishing that the SAA1 GRE is naling capacity of native HepG2 cells is not maximized with re- functional and is both necessary and sufficient to confer glucocor- spect to engagement of the SAA1 promoter GRE. In contrast, co- ticoid responsiveness in the context of cytokine stimulation. transfection of GR expression vector could not bring about a dexamethasone-dependent enhancement of cytokine-driven tran- Differential transcriptional regulation of the endogenous human scriptional readout from the SAA2 promoter (data not shown), es- SAA1 and SAA2 genes in response to glucocorticoids tablishing that the SAA2 promoter is truly unresponsive to To determine whether the results obtained using the SAA1 and Downloaded from glucocorticoids. SAA2 promoter luciferase reporter constructs accurately reflect the regulation of the endogenous genes with respect to cytokines and Confirmation that the putative GRE in SAA1 is functional glucocorticoids, we developed an RT-PCR method whereby the Site-directed mutagenesis experiments were performed to deter- relative proportions of the transcription products of each gene mine whether the putative SAA1 GRE is functional and to exclude could be directly compared. The SAA1 and SAA2 mRNAs, al- the possibility that subtle differences in genomic context, rather though highly similar (91% identical overall), differ significantly http://www.jimmunol.org/ than intrinsic sequence differences in the putative SAA1 GRE and in the central region of their 3ЈUTRs; the SAA1 mRNA 3ЈUTR relative to that of the SAA2 mRNA 3ЈUTR has four deletions totaling 26 residues (Fig. 5A). We designed forward and reverse PCR primers, each of which can bind cDNA derived from either A-SAA mRNA (Fig. 5A). RT-PCR using these primers generates bands of 335 and 361 bp corresponding to products generated from the SAA1 and SAA2 mRNAs, respectively. In addition, the prim- ers span intron 3 of each gene, thereby permitting products of amplification from contaminating genomic DNA to be identified. by guest on September 28, 2021 The ratio of 335- to 361-bp products, as determined by image analysis following resolution on 8% polyacrylamide gels, reflects the relative concentrations of cellular SAA1 and SAA2 mRNAs and serves as a surrogate measure of the relative transcriptional activation of the SAA1 and SAA2 promoters. The above RT-PCR method was applied to total RNA from untreated and treated HepG2 cells. Products derived from SAA1 or SAA2 mRNA were not detected in untreated cells or following treatment with dexamethasone alone (Fig. 5B, lanes 1 and 2). However, the ratio of SAA1:SAA2 PCR product was ϳ2:5 fol- lowing treatment with IL-1 and IL-6 (Fig. 5B, lane 3), indicating that the endogenous SAA2 gene has a significant transcriptional advantage (i.e., ϳ2.5-fold) when induced by cytokines in the ab- sence of glucocorticoids. In contrast, the ratio of SAA1:SAA2 PCR products from cells treated with IL-1 and IL-6 plus dexa- methasone was 5:4 (Fig. 5B, lane 4), indicating that the above cytokine-driven transcriptional advantage of the SAA2 promoter is superceded by a modest SAA1 transcriptional advantage in the presence of glucocorticoids. Thus, the responses of the endoge- nous genes to different combinations of proinflammatory media- tors parallel those observed in experiments using isolated promot- FIGURE 4. GRE mutant A-SAA promoters. A, Alignment of the region ers to drive a quantifiable reporter, thereby establishing that the of SAA1 encompassing the GRE, the corresponding regions of SAA2, the SAA1 GRE is functional when in its native genomic and cellular mutants GREI and GRED, and the consensus GRE sequence. B, HepG2 environment. cells were transfected with pGL2-SAA1, GREI, GRED, or pGL2-SAA2 constructs and treated with medium only, 50 nM dexamethasone, 10 ng/ml IL-1 plus 10 ng/ml IL-6, or IL-1 plus IL-6 plus dexamethasone. Cells were Discussion harvested 4 h after treatment and relative luciferase values were calculated The human SAA1 and SAA2 genes are highly conserved and con- -The difference between GRED sequently have been considered to be coordinately regulated dur ,ء .and compared with untreated controls treated with cytokines and GRED treated with cytokines plus dexametha- ing the APR. In this paper we present evidence that glucocorticoids sone was statistically significant (p ϭ 0.003). are able to enhance the transcriptional activity of the SAA1 gene, 404 GLUCOCORTICOID ENHANCEMENT OF SAA1 TRANSCRIPTION

FIGURE 5. Response of the endogenous human SAA1 and SAA2 genes to cytokines and dexamethasone. A, Alignment of the 335-bp SAA1 and 361-bp SAA2 RT-PCR product sequences. The numbering is in rela- tion to the full mRNA sequence. The intron exon boundary is marked with a vertical line; amplification from genomic DNA would generate a product that in- corporates sequence encompassing the 384-bp (SAA1) or 394-bp (SAA2) intron at this position. The 3ЈUTRs are underlined once and the primer sequences are un- derlined twice. Diagonal lines represent 121 bases of aligned exon 4 coding sequences that contain no gaps.

Dashes represent regions of the SAA2 3ЈUTR that have Downloaded from no counterpart in the SAA1 3ЈUTR. B, HepG2 cells were treated with medium only, 100 nM dexametha- sone, 10 ng/ml IL-1 plus 10 ng/ml IL-6, or IL-1 plus IL-6 plus dexamethasone for 24 h. RNA was extracted, reverse transcribed, and amplified as described. PCR products were separated by 8% PAGE. http://www.jimmunol.org/

but not the SAA2 gene, via a GR-dependent targeting of a GRE that During cytokine-driven induction the SAA1 and SAA2 promoters by guest on September 28, 2021 is functional only in the former, and that this capability is only are both engaged by the transcription factors NF-␬B and NF-IL6, realized in HepG2 hepatoma cells in the context of concomitant with SAA2 having a transcriptional advantage over SAA1. When signaling by proinflammatory cytokines. glucocorticoids are also present, the SAA1 promoter, but not the Our data from experiments using promoter reporter constructs SAA2 promoter, is additionally engaged by GR, the net result of strongly indicate that in HepG2 cells glucocorticoids cannot con- which is to enhance SAA1 transcriptional activity to a level similar tribute to enhanced SAA1 transcription in the absence of cytokines. to that of SAA2. We hypothesize the presence of a repressor that Indeed, even under experimental conditions in which augmenta- constitutively binds to the A-SAA promoters in untreated HepG2 tion of HepG2 intracellular levels of GR, a critical component of cells such that access to the SAA1 GRE is physically blocked. We the glucocorticoid signaling pathway, was combined with excess further hypothesize that the repressor can be displaced by cyto- glucocorticoid there was no induction of transcription in the ab- kine-driven NF-␬B activation and engagement of the NF-␬B sites sence of cytokines. These in vitro findings in a hepatic cell line are in the A-SAA promoters, thereby exposing the SAA1 GRE and per- in accord with the reported in vivo observation that patients who mitting GR binding and transcriptional enhancement. A similar were undergoing long-term glucocorticoid therapy but had no mechanism is evident in the rat SAA1 promoter in which YY-1- overt inflammation did not have elevated A-SAA protein concen- dependent transcriptional repression is overcome following its dis- trations (28). In contrast, patients who were receiving long-term placement by the binding of activated NF-␬B to an adjacent target glucocorticoid therapy and subsequently contracted an infectious site (37). However, the possible involvement of GR as a transcrip- disease had A-SAA protein concentrations that were markedly in- tional enhancer has not been explored in the SAA genes of the rat creased relative to those seen in patients with an infectious disease or any other species to date. who had not been receiving glucocorticoids (28). Furthermore, The data presented in this paper are derived from experiments Yamada et al. (29) observed significantly higher A-SAA:C-reac- using cultured hepatoma cells and reflect the regulatory criteria tive protein ratios in rheumatoid arthritis patients treated with glu- that govern transcription of the SAA1 and SAA2 genes in the liver. cocorticoids than in those treated with other disease-modifying Most A-SAA protein in the circulation is liver derived, and its anti-rheumatic drugs. The published studies outlined above sug- increased synthesis during an APR is achieved largely through gest that glucocorticoids alone have no effect on hepatic A-SAA increased transcription (4), with only a minor contribution from expression, but that under conditions in which there is an under- posttranscriptional mechanisms (30). Therefore, the transcriptional lying inflammation, and hence ongoing cytokine signaling, hepatic hypothesis proposed above may be directly relevant to circulating A-SAA expression is greatly enhanced by the presence of A-SAA protein concentrations. Thus, the ratios of SAA1 and glucocorticoids. SAA2 proteins may change over time with a bias strongly in favor Up-regulation of hepatic A-SAA protein synthesis during the of SAA2 in the early APR giving way to increasing relative APR involves both cytokine- and glucocorticoid-mediated effects. amounts of SAA1 after the systemic release of glucocorticoids. The Journal of Immunology 405

Furthermore, the absolute concentrations of each of the A-SAAs 14. Urieli-Shoval, S., R. L. Meek, R. H. Hanson, N. Eriksen, and E. P. Benditt. 1994. during chronic inflammation may depend on the nature of the un- Human serum amyloid A genes are expressed in monocyte/ cell lines. Am. J. Pathol. 145:650. derlying disease and thus the mix of pro- and anti-inflammatory 15. Urieli-Shoval, S., P. Cohen, S. Eisenberg, and Y. Matzner. 1998. Widespread mediators that are present. The introduction of anti-inflammatory expression of serum amyloid A in histologically normal human tissues: predom- steroid therapy may further modify the ratio of SAA1 and SAA2 inant localization to the epithelium. J. Histochem. Cytochem. 46:1377. 16. Husby, G., A. Husebekk, B. Skogen, K. Sletten, G. Marhaug, J. Magnus, and proteins depending on the type and therapeutic dose of synthetic V. Syversen. 1988. Serum amyloid A (SAA): the precursor of protein AA in glucocorticoids used. Such changes in the ratios of SAA1 and secondary amyloidosis. Adv. Exp. Med. Biol. 243:185. 17. Coetzee, G. A., A. F. Strachan, D. R. van der Westhuyzen, H. C. Hoppe, SAA2 may have important clinical as well as biological implica- M. S. Jeenah, and F. C. de Beer. 1986. Serum amyloid A-containing human high tions, especially given that SAA1 has been reported to be the pre- density lipoprotein 3: density, size, and composition. J. Biol. dominant isoform found in human secondary amyloid deposits Chem. 261:9644. 18. Meek, R. L., S. Urieli-Shoval, and E. P. Benditt. 1994. Expression of apolipopro- (44). tein serum amyloid A mRNA in human atherosclerotic lesions and cultured vas- Many nonhepatic cell types including monocytes, endothelial cular cells: implications for serum amyloid A function. Proc. Natl. Acad. Sci. cells, epithelial cells, and smooth muscle cells have been shown to USA 91:3186. 19. Badolato, R., J. M. Wang, W. J. Murphy, A. R. Lloyd, D. F. Michiel, express A-SAA when stimulated with cytokines and glucocorti- L. L. Bausserman, D. J. Kelvin, and J. J. Oppenheim. 1994. Serum amyloid A is coids. There have been reports that in some of these, namely a chemoattractant: induction of migration, adhesion, and tissue infiltration of THP-1 monocytes, KB oral epithelial cells, and primary aortic monocytes and polymorphonuclear leukocytes. J. Exp. Med. 180:203. 20. Badolato, R., J. A. Johnston, J. M. Wang, D. McVicar, L. L. Xu, J. J. Oppenheim, smooth muscle cells, A-SAA synthesis can be induced by dexa- and D. J. Kelvin. 1995. Serum amyloid A induces calcium mobilization and methasone alone (13, 45, 46). This suggests that glucocorticoids chemotaxis of human monocytes by activating a pertussis toxin-sensitive signal- may be the predominant inflammatory modulator with respect to ing pathway. J. Immunol. 155:4004. Downloaded from 21. Mitchell, T. I., C. I. Coon, and C. E. Brinckerhoff. 1991. Serum amyloid A A-SAA synthesis in some local tissues and further leads to the (SAA3) produced by rabbit synovial fibroblasts treated with phorbol esters or speculation that in such locations A-SAA synthesis may be re- interleukin 1 induces synthesis of collagenase and is neutralized with specific stricted to, or heavily biased toward, SAA1. As alluded to above, antiserum. J. Clin. Invest. 87:1177. 22. Edbrooke, M. R., J. Foldi, J. K. Cheshire, F. Li, D. J. Faulkes, and P. Woo. 1991. the differential effect of glucocorticoids on SAA1 and SAA2 gene Constitutive and NF-␬B-like proteins in the regulation of the serum amyloid A expression may provide the means to modulate the relative pro- gene by interleukin 1. Cytokine 3:380.

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