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The Journal of Immunology, 1999, 163: 4537-4545.
Copyright © 1999 by The American Association of Immunologists

Posttranscriptional Regulation of Acute Phase Serum Amyloid A2 Expression by the 5'- and 3'-Untranslated Regions of Its mRNA1

Daniel B. Longley2,*, Diana M. Steel3,* and Alexander S. Whitehead4,{dagger}

* Department of Genetics, Trinity College, Dublin, Ireland; and {dagger} Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human acute-phase serum amyloid A protein (A-SAA) is a major acute phase reactant, the concentration of which increases dramatically as part of the body’s early response to inflammation. A-SAA is the product of two almost identical genes, SAA1 and SAA2, which are induced by the pro-inflammatory cytokines, IL-1 and IL-6. In this study, we examine the roles played by the 5'- and 3'-untranslated regions (UTRs) of the SAA2 mRNA in regulating A-SAA2 expression. SAA2 promoter-driven luciferase reporter gene constructs carrying the SAA2 5'-UTR and/or 3'-UTR were transiently transfected into the HepG2 human hepatoma cell line. After induction of chimeric mRNA with IL-1ß and IL-6, the SAA2 5'- and 3'-UTRs were both able to posttranscriptionally modify the expression of the luciferase reporter. The SAA2 5'-UTR promotes efficient translation of the chimeric luciferase transcripts, whereas the SAA2 3'-UTR shares this property and also significantly accelerates the rate of reporter mRNA degradation. Our data strongly suggest that the SAA2 5'- and 3'-UTRs each play significant independent roles in the posttranscriptional regulation of A-SAA2 protein synthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Injury, trauma, and infection all lead to inflammation, a defensive reaction, the primary role of which is to eliminate pathogens, promote tissue repair, and return the host to optimal normal function. The systemic and metabolic changes that take place during the early stages of inflammation are collectively known as the acute phase response (1, 2). Among these changes are increases in the plasma concentrations of a number of acute phase proteins (APPs)5, which are classified as either moderate or major, depending on the extent to which they are induced.

The serum amyloid A (SAA) family is a heterogeneous group of 12- to 14-kDa apolipoproteins that are synthesized primarily, but not exclusively, in the liver (3, 4). Acute-phase SAA (A-SAA) is a major APP, because its concentration increases by up to 1000-fold during the acute phase response (5). Human A-SAA is the product of two hyperinducible genes, SAA1 and SAA2, which share 95% overall nucleotide identity in their exon, intron, and promoter regions (6, 7), and which are coordinately regulated (1). SAA3 is a pseudogene (8, 9). SAA4 encodes a constitutively expressed SAA protein, which is not induced during the acute phase response, shares only 55% amino acid identity with the A-SAA proteins (3), and has a distinct gene sequence (8). All four genes are in close linkage on chromosome 11p15.1 (10).

Although the precise physiological function of A-SAA is unclear, its massive and rapid induction in response to inflammatory stimuli is strongly suggestive of a vital short-term protective role. A-SAA associates with the third fraction of high density lipoprotein (HDL3) (11, 12) and may be involved in delivering HDL3 to sites of inflammation, thereby facilitating tissue repair (13, 14). However, a number of immune-related activities, for example that of a chemotactic factor that promotes transmigration of monocytes into sites of inflammation, also have been proposed (15, 16, 17).

During chronic inflammation, caused by conditions such as rheumatoid arthritis, A-SAA concentrations remain elevated for prolonged periods. There are a number of known and potential clinical consequences of such long-term over-production of A-SAA. A-SAA is the circulating precursor of the amyloid A (AA) protein that is deposited as insoluble ß-sheet fibrils in a number of organs during the ultimately fatal, inflammation-associated disease reactive amyloidosis (18, 19). Furthermore, as A-SAA replaces apolipoprotein AI, which is required for normal cholesterol metabolism (20), as the principal apolipoprotein associated with HDL3, its prolonged association with this lipoprotein fraction may disrupt reverse cholesterol transport and thereby contribute to the development of atherosclerosis. This event could provide, at least in part, a molecular explanation for the increased mortality from cardiovascular disease observed in patients with inflammatory conditions (21). The factors controlling the regulation of A-SAA synthesis are, therefore, of considerable clinical importance.

Previous studies using the human hepatoma cell line PLC/PRF/5 have established that A-SAA mRNA and protein synthesis are mediated by pro-inflammatory cytokines and that, of these, IL-1 and IL-6 are together sufficient to generate an optimal response (22, 23). The induction of A-SAA protein synthesis is reflected by an enormous increase in mRNA concentrations that is thought to be primarily the result of increased transcription (23, 24); however, posttranscriptional events also appear to play a vital role (23) and provide the cell with an additional level at which A-SAA expression may be fine-tuned.

The 5'- and 3'-untranslated regions (UTRs) of many eukaryotic mRNAs are involved in posttranscriptional regulation of gene expression (25). Elements within UTRs can have significant effects on the translational efficiency and/or the stability of mRNAs and can therefore modulate the levels of protein produced. In this paper, we have examined how the 5'- and 3'-UTRs of the SAA2 mRNA modulate the expression of luciferase reporter constructs in the human hepatoma cell line HepG2, following stimulation with IL-1ß and IL-6. Previous studies in experimental animal models have indicated that inflammatory stimuli up-regulate the synthesis of both mRNA and protein for each of the major A-SAAs in a qualitatively and quantitatively similar manner (26). As the 5'- and 3'-UTRs of SAA1 and SAA2 mRNA are almost identical (8), it is likely that their respective biologic functions in both APPs will be directly comparable and that these elements in either mRNA will be representative of human A-SAA 5'- and 3'-UTRs.

We establish that both the SAA2 5'- and 3'-UTRs are intrinsically important for the control of A-SAA2 expression. The SAA2 5'-UTR promotes translation of chimeric reporter transcripts in HepG2 cells treated with IL-1ß alone, IL-6 alone, or IL-1ß and IL-6 in combination, and this effect increases in magnitude as the time postcytokine stimulus increases. In cells treated with IL-1ß and IL-6 simultaneously, the SAA2 3'-UTR elevates the translational efficiency of chimeric mRNAs but also accelerates their rate of degradation. We find no evidence of interactions between the SAA2 5'- and 3'-UTRs that modulate either translation or mRNA stability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression vectors

The pGL-2 promoter plasmid (Promega, Madison, WI) containing the luciferase reporter gene driven by the SV40 promoter was modified for this study as follows. An EagI site was introduced immediately 3' of the SV40 promoter by oligonucleotide-directed mutagenesis using the original method of Kunkel et al. (27). This permitted excision of the SV40 promoter by restriction enzyme digestion at this site and the endogenous BglII site at the 5' end. The SAA2 promoter (700 bp), amplified by PCR with the introduction of BglII and EagI sites at the 5' and 3' ends, respectively, was directionally ligated between the two sites in the vector made available by the excision of the SV40 promoter.

The SAA2 5'-UTR was prepared for cloning by annealing complementary sense and antisense oligonucleotides, each of which was flanked by HindIII restriction site sequences at their 5' and 3' ends. The double-stranded product was digested with HindIII, gel purified, and cloned in both the sense (i.e., forward) and antisense (i.e., reverse) orientations into a unique HindIII site between the SAA2 promoter and the luciferase coding region of the modified pGL-2 plasmid. Oligonucleotide-directed mutagenesis was used to delete the vector sequences between the SAA2 promoter and the sense and antisense SAA2 5'-UTRs, and between the UTRs and the luciferase start codon. The antisense SAA2 5'-UTR construct was further modified by oligonucleotide-directed mutagenesis of the 3 bp at its 3' end, from CTG->ACC, to create a Kozak consensus around the start codon of this construct. These manipulations generated the sense 5'/Luc 3' and anti-Koz 5'/Luc 3' constructs (see Fig. 1Go, B and E).



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  		FIGURE 1. Schematic diagrams of the SAA2 gene and the mRNA it transcribes and of the SAA2/luciferase reporter gene constructs. The figures show the spatial organization of the genetic elements but are not drawn to scale. A, The structures of the SAA2 gene and its mRNA. B, The sense 5'/Luc 3' construct. C, The anti-Koz 5'/SAA2 3' construct. D, The sense 5'/SAA2 3' construct. E, The anti-Koz 5'/Luc 3' construct. Ex, exon; In, intron; SAA2 Pro, proximal 700 bp of the SAA2 promoter; sense 5'-UTR, sense strand of the SAA2 mRNA 5'-UTR; anti-Koz 5'-UTR, antisense strand of the SAA2 5'-UTR, modified to include the Kozak consensus sequence; SAA2 3'-UTR, 3'-UTR of the SAA2 mRNA; Luc 3'-UTR, 3'-UTR of the luciferase reporter gene mRNA in the pGL-2 promoter plasmid; and Luc coding region, coding region of the luciferase reporter gene in the pGL-2 promoter plasmid.

 
The SAA2 3'-UTR plus 750 bp of 3'-genomic sequence were amplified by PCR with the introduction of NsiI and SalI sites at the 5' and 3' ends, respectively. NsiI sites were introduced immediately 3' to the luciferase stop codons of the sense 5'/Luc 3' and anti-Koz 5'/Luc 3' constructs by oligonucleotide-directed mutagenesis. The luciferase reporter 3'-UTR of each construct was excised by restriction enzyme digestion at the introduced NsiI site and the endogenous SalI site, leaving sites into which the SAA2 3'-UTR plus 3'-genomic region was ligated, thereby generating the anti-Koz 5'/SAA2 3' and sense 5'/SAA2 3' constructs (see Fig. 1Go, C and D).

Cell culture

Human HepG2 cells (European Collection of Animal Cell Cultures, Salisbury, U.K.) were maintained in 5% CO2/37°C in DMEM (Life Technologies, Scotland, U.K.) with 10% bovine calf serum, supplemented with 1 mM sodium pyruvate, 0.5x MEM nonessential amino acids, 2 mM L-glutamine, and 0.05 mg/ml gentamicin. Recombinant human IL-1ß (1.81 x 107 U/mg) was obtained from the National Cancer Institute (Frederick, MD). Recombinant human IL-6 (5.1 x 105 U/mg) was a gift from the Genetics Institute (Cambridge, MA).

Transfections and cytokine treatments

The following transfection protocol was applied to each luciferase reporter construct. Luciferase reporter plasmid (10 µg), together with 10 µg of pMJH.20RSV ß-galactosidase (ß-gal) reporter plasmid (gift of Dr. Mary Weiss, Pasteur Institute, Paris, France), were transfected by electroporation into 7.5 x 106 HepG2 cells resuspended in 250 µl of electroporation medium (DMEM supplemented with 1 mM sodium pyruvate and 0.5x nonessential amino acids). The latter contains the ß-gal gene driven by the Rous sarcoma virus (RSV) promoter and controls for differences in transfection efficiency and cell numbers between and within treatment groups. Electroporations were performed in 0.4-cm electroporation cuvettes (Invitrogen, Leek, The Netherlands) at 1000 µF and 250 V, with the external circuit of the electroporator set for infinite resistance (electroporator II, Invitrogen). The transfected cells were transferred into 37°C culture medium and aliquotted, 1 ml/35-mm-diameter well, onto 6-well plates (Falcon, Becton Dickinson, Cowley, U.K.). After 20 h, the medium was replaced with fresh medium containing either 10 ng/ml IL-1ß, 10 ng/ml IL-6, or 10 ng/ml IL-1ß + 10 ng/ml IL-6. For the analysis of SAA2 promoter activity, when only the anti-Koz 5'/Luc 3' construct was transfected, the cells were assayed after 1.5, 3, 6, 12, 24, 48, and 72 h. For the analysis of the SAA2 UTRs, when all four constructs were simultaneously transfected into identical aliquots of HepG2 cells, the cells were assayed after 3, 6, 12, and 24 h. Each treatment was performed in duplicate at each time point. The results presented in this report are representative of those obtained in several separate transfection series.

Luciferase and ß-gal readings

After cytokine treatment, cells were washed with warm 1x PBS (Life Technologies/BRL) and incubated for 15 min at room temperature in 0.5 ml of 1x reporter lysis buffer (Promega). Cell lysates were harvested, and the luciferase and ß-gal activities were measured using Promega assay systems. The luciferase readouts were taken on a TD-20e Luminometer (Turner Designs, Sunnyvale, CA), and the ß-gal readings were determined by measuring the absorbance of the terminated reactions at 420 nm on a spectrophotometer (CE 2020, Cecil Instruments, Cambridge, U.K.). The ß-gal readings were used to control the luciferase readouts for variations in the number of transfected cells harvested per sample. For each sample, the ratio of luciferase to ß-gal readings (Luc/ß-gal, arbitrary units) was calculated to determine the luciferase activity per unit number of transfected cells. For each timed treatment, duplicate samples were analyzed, and the average Luc/ß-gal value was determined.

Measurement of chimeric luciferase mRNA levels

To detect the various chimeric luciferase mRNA species by Northern blotting, it was necessary to scale up the transfection protocol described above. For each construct, two 50-µg aliquots of luciferase reporter plasmid were transfected into two 7.5 x 106 aliquots of HepG2 cells at 250 V and 1000 µF and the aliquots pooled in 24 ml of 37°C culture medium. The transfected cells (4-ml aliquots) were plated into each of four 100-mm-diameter tissue-culture dishes (Falcon) and 1-ml aliquots into each of eight 35-mm wells of 6-well plates. After 20 h, the medium was replaced with fresh medium containing 10 ng/ml IL-1ß + 10 ng/ml IL-6, and the cells were incubated for 3, 6, 12, and 24 h. Total RNA was extracted from the cells on the 100-mm-diameter dishes using the LiCl/urea method (28), and the cells on the 6-well plates were lysed with 1x reporter lysis buffer and assayed for luciferase and ß-gal as above.

The RNA samples were treated with 3 U of RQ1 RNase-free DNase (Promega) in 30 µl of 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2, 1 mM DTT, and 30 U of RNasin ribonuclease inhibitor (Promega) at 37°C for 1.5 h, before being phenol/chloroform extracted twice. Duplicate 30-µg aliquots of each RNA sample were size-fractionated on a 1% agarose formaldehyde gel, transferred to nitrocellulose filters (Hybond-N, Amersham, Buckinghamshire, U.K.), and baked at 80°C for 2 h. The resulting Northern blots were hybridized overnight at 45°C in 5x SSC, 5x Denhardt’s, 0.2% SDS, 0.05% sodium pyrophosphate, 100 µg/ml tRNA, 0.1 mM EDTA, and 50% formamide with an [{alpha}-32P]-radiolabeled DNA probe complementary to the first 590 bp of the luciferase coding region. The membranes were washed twice for 30 min at 45°C in 2x SSC, 0.1% SDS, and 0.05% sodium pyrophosphate. The intensities of the hybridization bands on two exposures of autoradiographs were measured on an Pharmacia LKB (Piscataway, NJ) Ultrascan XL laser densitometer. 28S and 18S ribosomal bands from ethidium bromide-stained gels were scanned to confirm equal loading of the RNA samples in the Northern blot gels.

DNA probe labeling

The DNA probe complementary to the first 590 bp of the luciferase coding region was generated by restriction enzyme digestion of the sense 5'/SAA2 3' construct with NcoI and EcoRI. The probe was labeled with [{alpha}-32P]dCTP (Amersham) to a specific radioactivity of ~1.3 x 109 dpm/µg using an oligonucleotide-labeling kit (Pharmacia). The labeled probe was separated from unincorporated dNTPs using Biospin 6 chromatography columns (Bio-Rad, Hercules, CA) and denatured at 100°C for 10 min before being added to the hybridization solution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Luciferase reporter constructs

Four luciferase reporter constructs were generated to facilitate investigation of the respective roles played by the 38-nt 5'-UTR and 138-nt 3'-UTR of the SAA2 mRNA in posttranscriptional control of A-SAA2 protein expression (Fig. 1Go, B–E). All four constructs (sense 5'/Luc 3', anti-Koz 5'/SAA2 3', sense 5'/SAA2 3', and anti-Koz 5'/Luc 3') transcribe the luciferase coding region and contain the 700 bp of promoter region that are immediately upstream of the 5'-UTR in the SAA2 gene. These 700 bp have been previously shown to be sufficient to permit the full synergistic induction of SAA2 promoter activity by IL-1ß and IL-6 in HepG2 cells (24).

Fig. 1GoA is a schematic diagram showing the structure of the SAA2 gene and the mRNA it transcribes. The four reporter constructs are named according to the UTRs that they generate: the sense 5'/Luc 3' construct (Fig. 1GoB) transcribes the sense (i.e., forward) strand of the SAA2 5'-UTR plus the luciferase reporter 3'-UTR; the anti-Koz 5'/SAA2 3' construct (Fig. 1GoC) transcribes a modified version of the antisense (i.e., reverse) strand of the SAA2 5'-UTR together with the SAA2 3'-UTR; the sense 5'/SAA2 3' construct (Fig. 1GoD) transcribes the sense strand of the SAA2 5'-UTR plus the SAA2 3'-UTR; and the anti-Koz 5'/Luc 3' construct (Fig. 1GoE) transcribes the modified antisense SAA2 5'-UTR and the luciferase reporter 3'-UTR. The modification in the sequence of the antisense strand of the SAA2 5'-UTR in the two anti-Koz 5' constructs is the introduction of a Kozak consensus sequence immediately upstream of the luciferase start codon. The Kozak consensus acts as a positional marker that increases the efficiency with which the 43S ribosomal scanning complex initiates translation at the start codon (29, 30).

The antisense strand of the SAA2 5'-UTR is a good control for studying the regulatory capacity of the SAA2 5'-UTR, because it is the same size as the sense strand and has the same GC content and, therefore, is likely to form RNA secondary structures with similar free energies. This is an important consideration as strong secondary structures within 5'-UTRs can have potent effects on translational efficiency (31). Furthermore, because it has no endogenous equivalent in HepG2 cells, the antisense sequence is unlikely to contain any biologically active elements and is therefore likely to be neutral in its effect on luciferase synthesis. However, as the sense strand of the SAA2 5'-UTR contains the functionally important Kozak consensus element, we modified the antisense SAA2 5'-UTR sequence to control for the contribution of the Kozak consensus per se to the translation of transcripts containing the sense SAA2 5'-UTR. Analyses comparing the readouts from the Kozak-containing antisense SAA2 5'-UTR construct and its unmodified precursor established that the presence of the consensus increases luciferase reporter readout by between 1.5- and 2-fold in HepG2 cells (data not shown).

The luciferase 3'-UTR acts as a control for the effect of the SAA2 3'-UTR on luciferase protein synthesis. Because the luciferase reporter 3'-UTR has no endogenous equivalent in human cells, we considered it unlikely that it would actively modulate either translation or mRNA stability in response to cytokine treatment. Therefore, 750 bp of the 3'-genomic region immediately downstream of the SAA2 3'-UTR were included in the anti-Koz 5'/SAA2 3' and sense 5'/SAA2 3' constructs (Fig. 1Go, C and D) so that 3'-end processing of the chimeric mRNAs would be as similar as possible to that of native SAA2 mRNA. Also, the inclusion of the 750 bp of 3'-genomic sequence ensured that the constructs containing the SAA2 3'-UTR would have a similar formula weight to those containing the 940 nt luciferase reporter 3'-UTR (Fig. 1Go, B and E), thereby favoring similar transfection efficiencies of all four constructs under analysis.

SAA2 promoter activity

To define the regulatory activity of the SAA2 promoter in the absence of modulation by the SAA2 UTRs, the anti-Koz 5'/Luc 3' construct (Fig. 1GoE) was transfected into human HepG2 hepatoma cells along with a ß-gal reporter cotransfection control plasmid. After incubation with IL-1ß alone, IL-6 alone, or IL-1ß plus IL-6 in combination for 1.5, 3, 6, 12, 24, 48, and 72 h, the cells were assayed for luciferase and ß-gal activities. The ß-gal readings control for variations in the number of transfected cells assayed in each sample; therefore, we calculated the ratio of luciferase to ß-gal readouts (Luc/ß-gal, arbitrary units) to determine luciferase activity per unit number of transfected cells. For each cytokine treatment, duplicate samples were analyzed at each time point and the results plotted (Fig. 2Go, A and B).



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  		FIGURE 2. Cytokine stimulation of SAA2 promoter activity. A, Single cytokine stimulation of SAA2 promoter activity determined by measuring the luciferase activity per unit number of transfected cells (Luc/ß-gal, arbitrary units) generated by the anti-Koz 5'/Luc 3' construct in HepG2 cells treated with IL-1ß or IL-6 for 1.5, 3, 6, 12, 24, 48, and 72 h. B, SAA2 promoter activity after the single cytokine treatments depicted in the above compared with its activity after stimulation by treatment with IL-1ß and IL-6 simultaneously. The summed Luc/ß-gal values of the single cytokine treatments ((IL-1ß alone) + (IL-6 alone)) are also included to illustrate the synergistic induction of SAA2 promoter activity after cotreatment with IL-1ß and IL-6.

 
A comparison of the single cytokine treatments (Fig. 2GoA) reveals that IL-1ß and IL-6 generate radically different induction profiles. After treatment with IL-1ß, the readouts increase gradually, peaking at 24 h and decreasing slowly thereafter, so that at 72 h the readout is still approximately one-half the peak value. In contrast, the readouts after IL-6 treatment peak after only 3 h and then decrease rapidly, with the Luc/ß-gal value at 12 h already less than one-third the peak value. Thus, the kinetics of SAA2 promoter induction following the two single cytokine treatments are quite distinct. Furthermore, for the majority of the time course (from 6 to 72 h), SAA2 promoter activity is higher after IL-1ß treatment than after IL-6 treatment, thereby supporting the classification of SAA2 as a type-1 APP, i.e., one which is induced more strongly by IL-1-type cytokines than IL-6-type cytokines (32).

The synergistic nature of the induction of SAA2 promoter activity by IL-1ß and IL-6 previously described by us (23, 24) is depicted in Fig. 2GoB; at each time point poststimulus, the readout from cells treated with these two cytokines in combination is higher than the sum of the readouts generated by each individually. The degree of synergy is greatest from 1.5 to 6 h, when the Luc/ß-gal values achieved after dual cytokine treatment are approximately nine times higher than the summed single cytokine values ((IL-1ß alone) + (IL-6 alone)). After the 6-h time point, the extent of the synergy decreases gradually, so that by 48 and 72 h the effect is only approximately one-half as strong as it was at the early time points (Fig. 2GoB).

Following dual cytokine treatment, the shape of the readout profile (Fig. 2GoB) is similar to that generated by treatment with IL-6 alone (Fig. 2GoA). Each rapidly reaches a peak at 3 h and then decreases, although the rate of decrease is somewhat less during the dual treatment. Therefore, although both IL-1ß and IL-6 are required to produce the synergistic activation of the SAA2 promoter, the kinetics of this induction are more strongly influenced by IL-6 than IL-1ß, consistent with our previously published findings (24).

Effects of the SAA2 5'- and 3'-UTRs on luciferase readout

Cotreatment of human hepatoma cell lines with IL-1ß and IL-6 has previously been shown to be sufficient to achieve maximum induction of both A-SAA mRNA and A-SAA protein synthesis (23). All four luciferase reporter constructs (Fig. 1Go) together with the ß-gal control plasmid were transiently transfected into identical aliquots of HepG2 cells. The cells were incubated with IL-1ß alone, IL-6 alone, or IL-1ß plus IL-6 for 3, 6, 12, and 24 h and assayed for luciferase and ß-gal activities. For each construct, duplicate samples were analyzed and the average Luc/ß-gal ratios calculated as above.

As the Luc/ß-gal values represent luciferase activity per unit number of transfected cells, the values generated by the four constructs can be directly compared. To isolate the effects of the SAA2 5'- and 3'-UTRs, alone and in combination, on luciferase activity, the Luc/ß-gal values of the sense 5'/Luc 3', anti-Koz 5'/SAA2 3', and sense 5'/SAA2 3' constructs at each time point were divided by those of the anti-Koz 5'/Luc 3' control construct at the equivalent time points to generate "relative readout" values. For example, dividing the Luc/ß-gal values from the sense 5'/Luc 3' construct with those from the anti-Koz 5'/Luc 3' control defines the capacity of the SAA2 5'-UTR to modulate luciferase readout over time. The relative readout values in Fig. 3Go, A–C, are from one transfection series but are representative of those obtained from several experiments.



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  		FIGURE 3. The effects of the SAA2 5'- and 3'-UTRs on luciferase activity. The influence of the SAA2 UTRs was determined in HepG2 cells treated with IL-1ß alone, IL-6 alone, and IL-1ß plus IL-6 in combination. The relative readout values were generated by dividing the Luc/ß-gal values from the sense 5'/Luc 3', anti-Koz 5'/SAA2 3', and sense 5'/SAA2 3' constructs by those from the anti-Koz 5'/Luc 3' control construct. These values isolate the effects of the SAA2 5'- and 3'-UTRs, alone and in combination, on luciferase activity. A, Relative readouts generated by the sense 5'/Luc 3' construct (determines the effect of the SAA2 5'-UTR). B, Relative readouts generated by the anti-Koz 5'/SAA2 3' construct (determines the effect of the SAA2 3'-UTR). C, Relative readouts generated by the sense 5'/SAA2 3' construct (determines the effect of the presence of both SAA2 UTRs on the same transcript).

 
Fig. 3GoA depicts the relative readouts generated by the sense 5'/Luc 3' construct after the three different cytokine treatments. It shows that, 3 h after treatment with IL-1ß and IL-6 in combination, the SAA2 5'-UTR confers a 1.35-fold enhancement of readout, an advantage that increases to 3.3- and 3.1-fold at 12 and 24 h, respectively. The relative readouts generated after both single cytokine treatments follow a very similar pattern, indicating that the effects of all three treatments are essentially the same, each modifying cellular biosynthesis to confer an advantage on the SAA2 5'-UTR with respect to readout.

The relative readouts from the anti-Koz 5'/SAA2 3' construct after each cytokine treatment are, like those generated by the sense 5'/Luc 3' construct, all greater than unity (Fig. 3GoB), establishing that the SAA2 3'-UTR also has a positive effect on luciferase readout. The magnitude of this effect, however, is smaller than that of the SAA2 5'-UTR. After IL-1ß treatment, the enhancement in readout conferred by the SAA2 3'-UTR is essentially constant, between 1.5 and 1.75. Similarly, the IL-6 treatment produces little variation, with the relative readout decreasing slightly from a value of 1.4 at 3 h, to 1.1 at 12 h, and then increasing again to 1.5 at 24 h. Between the 3- and 12-h time points of the dual cytokine treatment, the relative readout decreases by 42% from 1.9 to 1.1 and remains at approximately the same level until 24 h. Therefore, the early (3 h) positive effect of the SAA2 3'-UTR on readout is significantly diminished at the later (12 and 24 h) time points of the combined treatment with IL-1ß and IL-6.

For the sense 5'/SAA2 3' construct (Fig. 3GoC), the relative readouts are essentially equal (within experimental error) to the sum of the values from the sense 5'/Luc 3' and anti-Koz 5'/SAA2 3' constructs (Fig. 3Go, A and B), indicating that the net effect of the presence of both SAA2 UTRs is additive. Therefore, we find no evidence that cooperative interactions between these regions of the SAA2 mRNA influence luciferase synthesis. After the dual cytokine treatment (12 and 24 h), the relative readouts from the sense 5'/SAA2 3' construct are significantly lower than those generated after either of the single cytokine treatments (Fig. 3GoC). This supports the finding outlined above for the anti-Koz 5'/SAA2 3' construct, that after simultaneous treatment with IL-1ß and IL-6, the SAA2 3'-UTR mediates an effect that reduces the level of readout at the last two time points.

Effect of the SAA2 UTRs on chimeric mRNA levels

To determine the extent to which the temporal effects of the SAA2 5'- and 3'-UTRs on cytokine driven luciferase readout are contingent upon changes in chimeric luciferase mRNA levels, we quantified RNA from transfected cells that had been subjected to timed treatments with IL-1ß and IL-6 in combination. To generate detectable levels of chimeric mRNA, the cells were transfected with 100 µg of each plasmid (i.e., 10 times more than in the luciferase readout experiments described above). The ß-gal control plasmid was cotransfected as before, and duplicate subaliquots of the transfected cells were assayed for luciferase and ß-gal activities at each of the four time points.

Northern blots of duplicate RNA samples from each construct at each time point were generated and hybridized with a DNA probe complementary to the first 590 bp of the luciferase coding region (Fig. 4GoA). The size difference between the mRNAs is due to the relative lengths of their 3'-UTRs: the SAA2 3'-UTR is 138 nt long, compared with the 940-nt luciferase reporter 3'-UTR, with the result that the corresponding mRNAs are 1829 and 2631 nt long, respectively, (not including the poly(A) tails).



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  		FIGURE 4. The impact of the SAA2 5'- and 3'-UTRs on mRNA levels. A, Chimeric luciferase mRNA transcribed from the sense 5'/SAA2 3', sense 5'/Luc 3', anti-Koz 5'/SAA2 3', and anti-Koz 5'/Luc 3' constructs in HepG2 cells 3, 6, 12, and 24 h after cotreatment with IL-1ß and IL-6. *, Chimeric transcripts carrying the 138-nt SAA2 3'-UTR; **, transcripts carrying the 940-nt luciferase reporter 3'-UTR. RNA (30 µg) were size fractionated on a 1% agarose formaldehyde gel, transferred to a nitrocellulose filter, and baked at 80°C for 2 h. The resulting Northern blot was hybridized with an [{alpha}-32P]-radiolabeled DNA probe complementary to the first 590 bp of the luciferase coding region. 28S and 18S ribosomal bands from ethidium bromide-stained gels were scanned to confirm equal loading of the RNA samples in the Northern blot gel (data not shown). B, Effect of the SAA2 UTRs on chimeric mRNA levels in HepG2 cells cotreated with IL-1ß plus IL-6. The chimeric mRNA band intensities were determined by densitometry, and the average values for duplicate samples were calculated. The average band intensities at each time point were divided by the corresponding ß-gal readings to control for differences between the transfection efficiencies of the four constructs. The ß-gal-controlled mRNA band intensities of the sense 5'/SAA2 3', sense 5'/Luc 3', and anti-Koz 5'/SAA2 3' constructs at each time point were divided by those generated by the anti-Koz 5'/Luc 3' construct, to give the relative [mRNA] values. These values monitor the effect of the presence of the SAA2 5'- and 3'-UTRs, alone and in combination, on mRNA concentration. C, The concurrent relative readouts generated during this time course. D, The relative translational efficiencies (i.e., readout per unit mRNA) of the three constructs, determined by dividing the relative readouts (C) by the relative [mRNA] values (B).

 
The chimeric mRNA band intensity of each sample was determined by densitometry, and the average values for duplicate samples were calculated. To control for differences between the transfection efficiencies of the four constructs, the average band intensities at each time point were divided by the corresponding ß-gal readouts. Then, to assess the effect of the presence of one or both of the SAA2 UTRs on mRNA concentration, the ß-gal controlled mRNA band intensities of the sense 5'/SAA2 3', sense 5'/Luc 3', and anti-Koz 5'/SAA2 3' constructs at each time point were divided by those of the anti-Koz 5'/Luc 3' control, generating the "relative [mRNA]" values plotted in Fig. 4GoB.

The relative [mRNA] values of the sense 5'/Luc 3' construct vary between ~0.9 and 1.0 during the time course (Fig. 4GoB), which establishes that the concentrations of chimeric transcripts generated by the sense 5'/Luc 3' and anti-Koz 5'/Luc 3' constructs are very similar at each time point. Therefore, the sense and antisense strands of the 5'-UTR have approximately the same net effect on chimeric mRNA levels, indicating that the SAA2 5'-UTR does not actively modulate either mRNA stability or SAA2 promoter activity in cells stimulated with IL-1ß plus IL-6.

In contrast, the SAA2 3'-UTR has a dramatic effect on chimeric mRNA levels (Fig. 4GoB). The relative [mRNA] values of the anti-Koz 5'/SAA2 3' construct decrease from a value of 0.8 after 3 h of the dual treatment, to 0.25 and 0.35 after 12 and 24 h, respectively, which indicates that the SAA2 3'-UTR renders the chimeric mRNA unstable relative to the control.

The relative levels of the sense 5'/SAA2 3' mRNA decrease over time in a manner that is indistinguishable to that observed for the anti-Koz 5'/SAA2 3' mRNA (Fig. 4GoB), confirming that the SAA2 3'-UTR alone accelerates mRNA turnover and does not interact with the SAA2 5'-UTR to further modify mRNA stability. In addition, the nearly identical profiles generated by these two constructs support the conclusion reached above from the sense 5'/Luc 3' construct that the SAA2 5'-UTR does not influence chimeric mRNA concentrations.

The concurrent luciferase and ß-gal readings were processed as before to generate the relative readouts plotted in Fig. 4GoC. The positive effect of the SAA2 5'-UTR on luciferase readout increases as the time course proceeds, which is consistent with the data depicted in Fig. 3GoA. The SAA2 3'-UTR has a stronger positive influence on readout at the 3 and 6 h time points of this transfection series than in the series discussed above (IL-1ß + IL-6 treatment, Fig. 3GoB), as the relative readout from the anti-Koz 5'/SAA2 3' construct is slightly higher at these time points than in the previous time course. Furthermore, the decrease in relative readout between the early and late time points is somewhat more pronounced, with a reduction of 55% from 2.75 at 3 h to 1.25 at 24 h. The relative readouts generated by the sense 5'/SAA2 3' construct are again attributable to the approximate summed effects of the individual UTRs.

To evaluate the differential contributions of the SAA2 5'- and 3'-UTRs to translation per unit of mRNA, we divided the relative readout values (Fig. 4GoC) by the corresponding relative [mRNA] values (Fig. 4GoB). The resulting "relative translational efficiency" values are plotted in Fig. 4GoD. The relative translational efficiency of the sense 5'/Luc 3' transcript is 1.5 times higher than that of the control transcript at 3 h poststimulus, increasing to more than three times higher at the 12- and 24-h time points (Fig. 4GoD). This result clearly illustrates that the increase in relative readout from this construct (Fig. 4GoC) is due to the increasing efficiency with which its mRNA is translated over time compared with the control anti-Koz 5'/Luc 3' mRNA.

The relative translational efficiency of the anti-Koz 5'/SAA2 3' construct is ~4 at all time points poststimulus (Fig. 4GoD), indicating that the SAA2 3'-UTR elevates translation by an approximately constant 4-fold over control levels throughout the time course. Comparing the relative [mRNA] and relative readout values of the anti-Koz 5'/SAA2 3' construct (Fig. 4Go, B and C), it is clear that the decreases in each over time occur in parallel, i.e., the reduction in relative readout is approximately proportional to the reduction in the relative [mRNA]. Therefore, the reduction in the relative readout from the anti-Koz 5'/SAA2 3' construct over time is entirely attributable to decreasing mRNA levels and is not due to decreasing translational efficiency.

The sense 5'/SAA2 3' transcript was translated with just over four times the efficiency of the control transcript at the 3- and 6-h time points, an advantage that increased to ~9-fold at the two later time points (Fig. 4GoD). These changes are consistent with the translational enhancement mediated independently by the 5'-UTR and 3'-UTR being essentially additive (within experimental error) when these elements are both present on an mRNA species.

Thus, in HepG2 cells cotreated with IL-1ß and IL-6, both the SAA2 5'- and 3'-UTRs increase translational efficiency, with the SAA2 3'-UTR also causing accelerated mRNA turnover. When both UTRs are present on the same transcript, these processes occur independently and the net effect on protein synthesis is additive. Treatment with either IL-1ß alone or IL-6 alone produced the same increases in relative readout from the sense 5'/Luc 3' construct as those elicited by the combined cytokine treatment (Fig. 3GoA), which strongly suggests that the increases during the single cytokine time courses are also due to increasing translational efficiency mediated by the SAA2 5'-UTR. However, compared with the dual cytokine treatment, the single cytokine treatments do not cause the same reduction in relative readout from the SAA2 3'-UTR constructs at the 12 and 24 h time points (Fig. 3Go, B and C). Therefore, it appears that IL-1ß and IL-6 are both required to accelerate the turnover of the SAA2 3'-UTR transcripts that underlies these decreases in relative readout.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Posttranscriptional mechanisms make an important contribution to the overall control of gene expression. These processes often involve more-or-less specific recognition of sequences and structural elements in mRNA molecules by an enormous array of protein factors (25). The fate of mature mRNA can be regulated by controlling either its stability or its translation. Posttranscriptional mechanisms are particularly likely to participate in effecting synthetic changes in dynamic processes such as the acute phase of inflammation, in which there are rapid changes in the circulating concentrations of a limited subset of serum proteins. Indeed, previous studies have indicated that such mechanisms do operate to regulate the synthesis of a number of APPs, including A-SAA (23, 33, 34).

We have established that the SAA2 5'-UTR has a potentially very important influence on SAA2 gene expression. The sense and antisense orientations of the SAA2 5'-UTR have approximately equal net effects on the steady state levels of chimeric luciferase mRNAs in HepG2 cells cotreated with IL-1ß and IL-6, which implies that the SAA2 5'-UTR does not affect either mRNA half-life or transcription. However, the sense orientation of the SAA2 5'-UTR increases luciferase readout from HepG2 cells treated with IL-1ß alone, IL-6 alone, or IL-1ß and IL-6 in combination, an effect that becomes more pronounced over time. This result suggests that transcripts containing the SAA2 5'-UTR are translated with increasingly higher efficiency in IL-1ß- and/or IL-6-treated HepG2 cells, at least up to 24 h poststimulus.

Interactions between proteins and mRNA 5'-UTRs can decrease the efficiency of translation by interfering with the interaction of the 43S ribosomal pre-initiation complex with the mRNA, e.g., the interaction between the iron responsive element and iron regulatory protein in the 5'-UTRs of the ferritin mRNAs (35). However, some protein/5'-UTR interactions appear to act as tissue- or cell-specific signals to enhance translation of particular transcripts (36), and it is possible that the SAA2 5'-UTR is subject to this mode of direct regulation by a factor induced or activated during the acute phase response. Another possibility is that the SAA2 5'-UTR is very efficient at initiating translation compared with other 5'-UTRs, a property which may become more prominent and confer particular translational advantage when translational initiation factors become depleted (as may be the case in the later stages of induction with potent transcriptional activators such as IL-1ß and IL-6). Under the latter model, chimeric mRNAs with the sense SAA2 5'-UTR would compete more efficiently for translation initiation factors than those carrying the antisense 5'-UTR. Regardless of the mechanism by which the SAA2 5'-UTR confers translational advantage, the high translational efficiency of transcripts carrying this element is clearly of biological importance in that it favors the rapid synthesis of the high concentrations of A-SAA protein that are required during the early stages of the acute phase response.

Previous studies raised the possibility that A-SAA mRNA degradation requires de novo synthesis of a short-lived factor, e.g., a nuclease (23, 37). In this study, we found that 12 and 24 h after cotreatment of HepG2 cells with IL-1ß and IL-6, transcripts carrying the SAA2 3'-UTR are at much reduced levels relative to control transcripts. This finding suggests that the presence of this element promotes accelerated mRNA degradation after the first few hours of induction. However, the presence of the SAA2 3'-UTR also confers an approximately constant 4-fold enhancement of translational efficiency relative to the control, establishing that the SAA2 3'-UTR is also of central importance to the rate of A-SAA2 protein synthesis. The binding of cytoplasmic trans-acting factors to 3'-UTRs can have potent effects on both mRNA stability and translation (38). Treatment of HepG2 cells with IL-1ß and IL-6 in combination may stimulate activation or de novo synthesis of a factor, or factors, that interact with the SAA2 3'-UTR to produce the effects described above. Our data strongly suggest that the 3'-UTR is centrally important for SAA2 mRNA turnover, possibly via provision of a target site for endonucleolytic cleavage. Such substrate specific cleavage events are known to lead to rapid degradation of other mRNA species (39, 40). Experiments using SAA2 3'-UTR deletion constructs to identify the regions of the 3'-UTR that are involved in accelerating mRNA turnover are in progress.

When luciferase readout is derived from a chimeric transcript carrying both the SAA2 5'- and 3'-UTRs, the net effect appears to be a combination of the individual effects imposed by each region. That the composite readout is essentially additive indicates that there is little or no interaction between the SAA2 5'- and 3'-UTRs under the experimental conditions used.

The magnitude and rapidity of A-SAA induction in response to an acute inflammatory stimulus indicate that this apolipoprotein plays a very important role in survival (1). If the above findings accurately reflect the behavior of endogenous SAA2 mRNA during the in vivo acute phase response, then several conclusions may be drawn regarding the overall control of A-SAA expression. The previously reported transcriptional up-regulation of the A-SAA genes, leading to the accumulation of A-SAA mRNA (23, 24), together with the translational enhancement conferred by the SAA2 mRNA 5'- and 3'-UTRs that is documented here, clearly act in concert to augment protein synthesis from an early time poststimulus. This is entirely compatible with the presumed biological requirement for high absolute amounts of A-SAA in the early stages of the inflammatory response. Intuitively, the role of the 3'-UTR in promoting SAA2 mRNA degradation runs counter to the drive toward high protein synthetic capacity discussed above. However, the effect is most pronounced in the later stages of the induction protocols used by us, suggesting that the 3'-UTR may act, perhaps in concert with other factors, to rapidly reduce the amount of SAA2 mRNA available to the cellular biosynthetic machinery at a time when sufficient A-SAA protein has been made. There are theoretically sound physiological reasons for strictly limiting the synthesis of large quantities of A-SAA protein to the period of inflammatory challenge when it is most needed. Its absence from normal serum indicates that it is not a constitutive product that is required under normal conditions (11). That A-SAA may, in fact, be pathogenic when expressed at high levels over prolonged periods is clear from its known identity as the serum precursor of the amyloid A fibrils that are deposited in secondary amyloidosis (18, 19). In addition, the long-term association of A-SAA with HDL may compromise reverse cholesterol transport, with potentially atherogenic consequences (1, 20).

This study provides evidence in support of three different processes by which A-SAA2 synthesis is controlled (summarized in Fig. 5Go): 1) transcriptional up-regulation driven by pro-inflammatory cytokines that is mediated by the A-SAA2 promoter; 2) translational enhancement during the early and later stages of the acute phase response that is mediated by both the 5'-UTR and 3'-UTR of the SAA2 mRNA; and 3) clearance of the SAA2 mRNA during the later stages of the acute phase response that is mediated by its 3'-UTR. All three findings require cellular stimulation with pro-inflammatory cytokines but probably act through quite different pathways and mechanisms. The magnitude and duration of A-SAA synthesis following an inflammatory stimulus is likely to be dependent on the extent to which each of the above three regulatory features of the cellular response is engaged.



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  		FIGURE 5. Summary of the three different processes involved in the control of A-SAA2 expression. Transcriptional up-regulation of SAA2 mRNA synthesis mediated through the synergistic action of IL-1ß and IL-6 on the SAA2 promoter, allied to efficient translation mediated through the SAA2 mRNA UTRs, permits high levels of A-SAA2 protein to be rapidly synthesized in response to the acute phase stimulus. Once the requirement for A-SAA2 protein synthesis has passed, SAA2 mRNA is rapidly cleared, an effect which appears to be mediated through its 3'-UTR.

 
A-SAA is emerging as an excellent model system for the analysis of cytokine driven changes in gene expression. The data presented here extend our working knowledge of this system from the well-documented factors governing transcription to the areas of mRNA stability and translational efficiency. In addition, the involvement of the SAA2 mRNA 5'- and 3'-UTRs in the regulation of this potentially pathogenic molecule increases the number of targets for the development of therapeutic strategies to down-regulate A-SAA synthesis during chronic inflammation.


   Acknowledgments
 
We thank Dr. Derval J. Gaughan and Dr. Clarissa M. Uhlar for their advice and insight.


   Footnotes
 
1 This work was supported by EU Commission Demonstration Project Contract BMH-CT9–0505 and by Forbairt Basic Research Grant SC/96/312. D.M.S. was supported by a Wellcome Trust Postdoctoral Fellowship. Back

2 Current address: Department of Oncology, Queen’s University, Belfast, BT9 7AB, Northern Ireland. Back

3 Current address: Foley, Hoag and Eliot LLP, 1 Post Office Square, Boston, MA 02109. Back

4 Address correspondence and reprint requests to Dr. Alexander S. Whitehead, Department of Pharmacology and Center for Pharmacogenetics, University of Pennsylvania School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104. E-mail address: Back

5 Abbreviations used in this paper: APP, acute phase protein; A-SAA, human acute phase serum amyloid A protein; SAA1–4, human serum amlyloid A genes; UTR, untranslated regions of mRNA; Luc/ß-gal, ratio of luciferase readout to ß-galactosidase readout; HDL3, the third fraction of high density lipoprotein. Back

Received for publication March 26, 1999. Accepted for publication August 2, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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