transcriptional regulation of serum amyloid a gene expression* · 0 1986 by the american society of...

0 1986 by The American Society of Biological THE JOURNAL OF BIOLOGICAL CHEMISTRY

Chemists, Inc. Val. 261, No. 18, Issue of June 25, pp. 84534461,1986

Printed in U.S.A.

Transcriptional Regulation of Serum Amyloid A Gene Expression* (Received for publication, September 30,1985)

Clifford A. Lowell$, Robert S. Stearman$, and John F. Morrow From the Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Serum amyloid A (SAA) is a plasma apolipoprotein produced by the liver in response to inflammatory stimuli. The murine SAA gene family is made up of three genes, SAA,, SAA2, and SAA3, plus apseudogene. The SAA, and SAAz genes are highly homologous while the SAA3 gene has diverged substantially from the other two genes. Using small fragments from the cloned genes, we have analyzed the expression of each gene in the SAA family. Within 12 h after endotoxin administration, total liver SAA mRNA increases by 2000-fold, reaching approximately 20,000 transcripts/cell. Each gene accounts for approximately one- third of total SAA mRNA transcripts at this time. The increase is specific, since the levels of the mRNAs encoding albumin and apolipoprotein A-I in liver decrease 2-fold by 24 h. This correlates with a 2-fold decrease of the serum concentrations of these two proteins as well as their in vitro protein synthesis in primary hepatocytes. SAA1+2 mRNAs maintain their maximum levels until 36 h after lipopolysaccharide administration, while SAAJ mRNA is degraded to 20% its maximal level. As assayed by in vitro transcription in isolated hepatocyte nuclei, total SAA gene transcription increases at least 300-fold during the inflammatory response. The transcription rates of the individual SAA genes are similar during the initial stages of this response, reaching peak levels at 3 h. A comparison of the rates of SAA gene transcription and SAA mRNA accumulation suggests that SAA mRNA levels are regulated during the acute phase response by increased transcription and mRNA stabilization.

Infection, inflammation, or tissue injury initiates a process called the acute phase response in mammals. The acute phase response produces changes in the expression of a number of genes in mammalian liver. The best studied of these are the acute phase reactants (Koj, 1974; Kushner, 1982). In mice, serum amyloid A (SAA’) is the predominant acute phase

* This work was supported in part by Grant 5POlCA16519-09 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Recipient of Medical Scientist Training Program Grant 2T32GM07309 from the National Institutes of Health.

$ Present address: Dept. of Biophysics, Johns Hopkins School of Medicine, Baltimore, MD 21205.

The abbreviations used are: SAA, serum amyloid A; LPS, lipopolysaccharide; bp, base pairs; kb, kilobase pairs; EGTA, [ethylene- bis(oxyethylenenitrilo)]tetraacetic acid; UTS, untranslated segment; Pipes, piperazine-N,l\l’-bis(Z-ethanesulfonic acid); Hepes, 4-(2-hy- droxyethy1)-1-piperazineethanesulfonic acid; HDL, high density lipoprotein; SAAI+2, experiments which cannot distinguish between the SAA, and SAAz genes and, therefore, measure the sum of both genes; apo-A-I, apolipoprotein A-I.

protein. Its serum concentration increases 1000-fold (up to 1 mg/ml in the serum) after a single injection of bacterial LPS (Gorevic et al., 1978; Benson and Aldo-Benson, 1979). SAA is a component of HDL particles and makes up a large percentage of total HDL protein during the acute phase response (Benditt et al., 1979). The large increase in hepatic SAA synthesis appears to be regulated by increased production of SAA mRNA (Morrow et al., 1981). Thus, the acute phase induction of SAA mRNA is an excellent model system to study differential gene expression in response to a specific stimulus.

We have isolated several X phage recombinants carrying murine SAA genes (Lowell et al., 1986). Analysis of these phage and hybridization with murine DNA revealed that the SAA gene family consists of three genes and a pseudogene. Two of the genes (SAA, and SAA,) have 96% sequence homology over a region of 3215 bp, which includes the coding sequences, the introns, and 288 bp of 5’ plus 443 bp of 3‘ flanking DNA. It has been proposed that this extensive homology is the result of gene conversion. The nucleotide sequences of cDNA clones derived from the SAA, and SAAz genes have recently been published (Yamamoto and Migita, 1985). The cDNA sequences are in agreement with our exon sequences and directly confirm the locations of our exon boundaries. The third SAA gene (SAAS) has diverged substantially from the other two genes and i s partially homologous in the translated sequences of the exons. We have analyzed the expression of the individual SAA genes using gene-specific probes constructed from their 3”terminal exons. Quantitative measurement of total SAA mRNA levels in normal and treated animals reveals that SAA mRNA increases approximately 2000-fold to become one of the most abundant liver mRNAs. The increase in total SAA mRNA is regulated, in part, by an increase in nuclear transcription from the three SAA genes. SAA gene transcription increases at least 300-fold. However, comparison of the rate of total SAA gene transcription with the rate of total SAA mRNA accumulation suggests that SAA mRNA levels may also be regulated by post-transcriptional stabilization.

EXPERIMENTAL PROCEDURES

Induction of SAA mRNA by LPS, Preparation of RNA and RNA Blot Hybridization with Gene-specific Probes-Fifty pg of LPS (Sigma, L-2630) dissolved in 4% normal serum was injected intraperitoneally into male BALB/c mice (20-30 g) to induce acute inflammation. Livers were removed at various times after LPS administration, frozen in liquid N2, and RNA prepared by homogenization in guani- dinium thiocyanate as described (Chirgwin et al., 1979).

For detection of SAA mRNA in normal mice, the insert of the SAAs cDNA clone pRS48 (Stearman et al., 1982) was gel purified and labeled by the random oligo-primer technique of Feinberg and Vogel- stein (1983). The reaction was carried out using 50 ng of DNA for 4 h and the unincorporated [ W ~ ~ P ] ~ C T P separated from the labeled DNA by Sephadex G-50 chromatography. Using this procedure, a total of 2 X 10’ cpm was incorporated into 50 ng of the SA& cDNA

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8454 Regulation of Serum Amyloid A Gene Expression

insert fragment. Plasmid cDNA clones for apo-A-I, p1804 (Ertel- Miller et al., 1983) and albumin, prAlb576 (which is related to pAlbI of Kioussis et al., 1979) were labeled by nick translation to a specific activity of approximately 2 X 10' cpmlpg.

SAA gene-specific clones constructed in M13 vectors were labeled using the probe-primer (New England Biolabs). Probe-primer was annealed to the M13 clone as done for normal sequencing, then extended by adjustment to 0.5 mM dATP, dGTP, dTTP, and 100 pCi of [32P]dCTP in normal DNA sequencing buffer (Sanger et al., 1980) followed by addition of 1 unit of Klenow fragment. The reaction was incubated at 30 "C for 1h h, and the labeled M13 clone was separated from unincorporated nucleotides by Sephadex G-50 chromatography.

In each lane, 15 pg of total liver RNA or varying amounts of SAA gene DNA fragments (1-15 ng of approximately 550-bp fragments denatured by heating in formamide) were separated by electrophoresis on 1.1% agarose/formaldehyde gels and transferred to nitrocellulose as described (Maniatis et al., 1982). Hybridizations were done with 5 X lo7 cpm of probe (0.5 pg) in 50% formamide, 0.9 M NaCl, 50 mM Tris, pH 7.8, 5 mM EDTA, 0.2% sodium dodecyl sulfate, 20 pg/ml each denatured salmon sperm DNA, poly(rA) and -(rC), and 0.04% each bovine serum albumin, Ficoll, and polyvinylpyrrolidone for 48 h at 42 "C. Filters were washed in 50 mM NaC1,50 mM Tris, pH 8.0, 5 mM EDTA at 50 "C. For each experiment, at least four differently timed autoradiographic exposures (done at -70 "C using preflashed Kodak XAR-5 film with DuPont Lightning Plus intensifying screens (Laskey, 1980)) were obtained. The intensity of bands on the autoradiographs was quantitated by densitometry using a Joyce-Loebl microdensitometer (Laskey and Mills, 1975). Four separate hybridization experiments were performed.

Preparation of Singk-stranded Probes and SI Protection Map- ping-Probes used for S1 protection were 3'-end labeled by cleaving SAA subclones with EcoRI, filling in the ends with DNA polymerase I Klenow fragment and [w3'P]dATP (3000 Ci/mmol), followed by the isolation of EcoRI/XbaI fragments. The specific activity of these probes was generally 4 X lo6 cpm/pmol end. To facilitate hybridization of probes to SAA mRNA, single-stranded 32P-end-labeled fragments were prepared. Labeled fragments were denatured and hybridized in solution to an M13 clone containing the sense mRNA strand of the EcoRI/XbaI fragment from the 4th exon of SAAI. Hybridiza- tions were carried out with a 2-fold molar excess of single-stranded M13 phage DNA in 0.2 M NaC1, 0.1 M Tris, pH 8.0, at 60 "C for 1 h. The M13 clone hybridized with the 3'-32P-end-Iabeled DNA strand was separated from the unlabeled nonhybridized strand and reannealed double-stranded fragment by gel filtration chromatography on a Bio-Gel A-50m (Bio-Rad) column (0.75 X 30 cm) in 0.2 M NaCl, 10 mM Tris, pH 8.0, 0.5 mM EDTA at room temperature with a flow rate of 0.1 ml/min. The M13 DNA annealled with the 32P-end-labeled fragment is excluded from the column bed and elutes in the void volume. These fractions containing 32P counts were pooled and denatured by addition of NaOH to 0.1 N. The DNAs were then rechro- matographed on the same column, adjusted to 0.1 N NaOH, 0.5 mM EDTA, to separate the 32P-end-labeled DNA strand, which is in- cluded, from the M13 phage DNA which is excluded.

Single-stranded probes were hybridized to SAA mRNA under conditions of approximately 3-fold DNA excess. Approximately 5 X lo5 cpm (0.13 pmol) was hybridized to 2.5 pg of poly(A+) liver RNA purified from normal and LPS-induced animals in 64% formamide, 10 mM Pipes, pH 6.8,l mM EDTA, and 0.36 M NaCl at 50 "C for 6 h in a volume of 50 pl. The hybridizations were then adjusted to S1 buffer conditions (0.25 M NaC1, 30 mM N d c , pH 4.6, 1 mM ZnSOd, 5 mM dithiothreitol, and 100 pg/ml denatured salmon sperm DNA in a volume of 300 $1) and digested with varying amounts of S1 nuclease for 1 h. The S1 digests were stopped by addition of EDTA and the protected fragments concentrated by ethanol precipitation. The protected 32P-DNA fragments were resolved on urea/polyacrylamide gels and autoradiographed as above.

Elongation of Nascent RNA Chains in Isolated Nuclei and Hybrid- ization of 32P-RNA to SAA DNA-Liver nuclei were purified for in vitro transcription experiments using a modification of the method described by Schibler et al. (1983). Liver tissue (usually 2 g pooled from 2 mice) was homogenized in 20 ml of 0.3 M sucrose, 15 mM Hepes, pH 7.5, 60 mM KC1, 3 mM MgCl,, 0.15 mM spermine, 0.5 mM spermidine, 14 mM p-mercaptoethanol, 1 mM EDTA, and 0.5 mM EGTA. The homogenate was filtered through cheesecloth and layered over a 10-ml cushion of 30% sucrose in the above buffer and spun for 10 min at 2500 rpm in a Sorvall HB4 rotor a t 4 "C. The crude nuclear pellet was resuspended in 4 ml of 2 M sucrose in the above buffer with 0.1 mM EDTA and 0.1 mM EGTA, layered over a 1-ml cushion

of the same 2 M sucrose solution, and centrifuged for 1 h at 36,000 rpm in a Beckman SW 50.1 rotor a t 4 "C. The clean nuclear pellet was resuspended in 1 ml of nuclear storage buffer (50% glycerol, 20 mM Tris, pH 8.0,75 mM NaCl, 0.5 mM EDTA, 0.85 mM dithiothreitol, 0.125 mM phenylmethylsulfonyl fluoride, and 3 mM MgCl,), then sedimented by spinning for 2 min in an Eppendorf centrifuge at 4 "C, and resuspended in 300 p1 of nuclear storage buffer at 4 "C to an average concentration of 2-3 X 10' nuclei/ml. Nuclei were stored at -70 "C for up to 4 weeks without loss of total RNA polymerase activity. The DNA of liver nuclei prepared in this manner was completely intact indicating that the presence of M%+ did not activate endogenous nucleases (in contrast to the observations of Schibler et al., 1983). However, failure to include EGTA (or the addition of Ca'+ ions) did result in activation of endogenous nucleases which rapidly degraded the nuclear DNA.

The in vitro elongation reactions were carried out using approximately 4 x lo7 nuclei/reaction. Nuclei in storage buffer were centrifuged briefly in an Eppendorf centrifuge and resuspended in 90 pl of reaction buffer (25% glycerol, 20 mM Tris, pH 8.0, 150 mM KCl, 2 mM dithiothreitol, 4 mM MnC12, 3 mM MgCl,, 0.1 mM EDTA, 3 mM EGTA, 1 mM each of ATP, CTP, GTP, 10 mM creatine phosphate, 100 units/ml placental ribonuclease inhibitor (Bolton Biologicals), and 0.1 mM phenylmethylsulfonyl fluoride. The in vitro elongation reactions were initiated by addition of 10 pl of [c~-~'P]UTP (600 Ci/ mmol at 1 mCi/ml) and carried out for 15 min at 26 "C. Under these conditions the bulk of the nuclear DNA remained greater than 20 kb after the in vitro transcription reaction. Final concentrations of 0.5% Sarkosyl, 1 mg/ml heparin, or 2 pg/ml u-amanitin were added to the nuclei before addition of [CP~~PJUTP. Labeled RNA was extracted from the nuclei using the trichloroacetic acid precipitation method described by Groudine et al. (1981). The length of the nascent ["PI RNA synthesized (as judged by electrophoresis or glyoxlated RNA in agarose gels) ranged from 50-500 nucleotides with an average size of 200 nucleotides. This is in good agreement with the average size of nascent t3'P]RNA made in isolated nuclei from other tissues (Schibler et al., 1983; Cleveland and Havercroft, 1983).

Southern blots (Southern, 1975) were used to quantitate the amount of SAA nascent [32P]RNA produced in isolated nuclei. Sub- cloned SAA DNA was digested with various restriction enzymes, separated by electrophoresis on 1% agarose gels and transferred to nitrocellulose as described (Pearson et al., 1981). The appropriate amount of plasmid DNA was used in each digest to produce approximately 150 ng of SAA DNA fragments averaging 1.5 kb to ensure that hybridizations would be in DNA excess. Nascent [32P]RNA (preheated to 80 "C for 5 min) was hybridized to the filters using the same conditions described for hybridization of gene-specific probes. Filters were hybridized at least 48 h and washed in a buffer containing 10 mM NaC1, 25 mM Tris, pH 7.5, 5 mM EDTA, and 0.2% sodium dodecyl sulfate a t 65 "C for 10 min. Filters were autoradiographed, and the [32P]RNA hybridization was quantitated as described above. The [32P]RNA produced by in vitro transcription from untreated mouse nuclei gave background levels of hybridization. In order to calculate a minimum estimate of increased transcription of the SAA genes after LPS injection, we have assigned background hybridization a relative value of 1. Materials-Restriction/modification enzymes, M13 cloning vec-

tors, and bacterial host strains were obtained from Bethesda Research Laboratories or New England Biolabs and used as recommended by the supplier. S I nuclease and DNase I were supplied by Boehringer Mannheim. Cloning of gel-purified SAA restriction fragments into M13 vectors, transformation of bacterial host strain K12 JM103, and preparation of single-stranded or replicative form recombinant DNA was carried out according to Sanger et al. (1980). Radioactive nucleotides were from New England Nuclear or Amersham Corp.

RESULTS

Detection of SAA mRNA in Normal Liver-In order to gauge the increase in SAA mRNA levels during inflammation it was important to determine the level of SAA mRNA in normal liver. This has been difficult because SAA mRNA in normal mice is undetectable using in vitro translation and hybridization to total RNA (Morrow et al., 1981). To determine if the SAA genes are transcribed in normal animals, liver poly(A+) RNA was purified from individual normal mice and hybridized with the SAA, cDNA clone labeled to very

Regulation of Serum Amyloid A Gene Expression a455

high specific activity (approximately 4 X lo9 cpm/pg). Since the SAA, cDNA clone contains conserved coding sequences (Stearman, 1982; Yamamoto and Migita, 1985)) it hybridizes with mRNA from all three SAA genes. Even with this high specific activity probe, SAA mRNA is undetectable in total normal liver RNA (Fig. 1, lane I ) but can be detected in normal poly(A+) RNA (lane 8). In contrast, SAA mRNA is easily detectable in as little as 2 pg of total RNA isolated from LPS-treated animals (lane 2). Poly(A+) RNA isolated from two other normal animals had a similar (within 2-fold) amount of SAA mRNA.

In order to compare the hybridization intensities of the SAA mRNA in normal versus LPS-treated mice it is necessary to control for variability in the enrichment of poly(A+) RNA in each preparation. This was done by determining the enrichment of albumin mRNA in the poly(A+) RNA preparations: if poly(A+) RNA makes up 2% of total RNA (Young et al., 1976; Lewin, 1980) then the maximal enrichment of albumin mRNA in a poly(A+) preparation is 50-fold. An albumin cDNA clone, prAlb576 (Kioussis et al., 1979) was hybridized to varying amounts of poly(A+) and total RNA from normal animals and the hybridization intensities were compared by densitometry. This revealed that each poly(A+) RNA preparation was 30-50-fold enriched for albumin mRNA (data not shown). Knowing the purity of the poly(A+) RNA used, a comparison of the hybridization intensities of SAA mRNA in normal and LPS-treated animals (Fig. 1) demonstrated that the SAA mRNA level is increased approximately 2000-fold within 12 h after LPS administration.

All Three SAA Genes Are Expressed-To determine which SAA genes are expressed in mouse liver after LPS administration, probes from the 3’ UTS segments of the genes, cloned in M13 vectors, were hybridized to blots of liver RNA. Two probes were used one which hybridizes to SAA,,, mRNAs and one which hybridizes to only SAA, mRNA. The SAA,+,- specific insert is 112 nucleotides long while the SAA,-specific probe is 92 nucleotides, so that the two probes hybridize with similar rates to their respective mRNAs. We confirmed that neither probe was capable of hybridizing to the heterologous gene by incubating each with duplicate filters containing SAA,, SAAz, and SAA3 cloned DNA.

These probes were hybridized to filters containing electro- phoretically fractionated total liver RNA isolated from normal and LPS-treated animals. With both probes, SAA mRNA is

1 2 3 4 5 6 7 8

FIG. 1. Levels of SAA mRNA in normal mice. Total or poly(A+) liver RNA isolated from untreated mice or 12 h after LPS administration was separated by electrophoresis in 1.1% agarose/ formaldehyde gels, transferred to nitrocellulose, and hybridized with 50 ng (2 X 10’ cpm) of the SAA3 cDNA insert fragment labeled by the method of Feinberg and Vogelstein (1983). Lane 1, 15 pg of total RNA from normal mice; lanes 2-5,2,4,8, or 16 pg of total RNA from LPS-treated animals; lanes 6 4 5 , 10, or 20 pg of poly(A+) RNA from a single untreated mouse. Hybridization and washing conditions are described under “Experimental Procedures.” Only the SAA mRNA band (M, approximately 700 nucleotides) is shown. The hybridization intensities of poly(A+) RNA from two other individual normal mice were approximately the same as shown in lanes 6-8.

undetectable in total normal mouse liver RNA (Fig. 2, lane 1). It reaches peak levels 12 h after LPS administration (lane 5 and panel C). Levels of SAA1+2 mRNA remain elevated 36 h after LPS administration (panel A, lane 7), then drop sharply before 48 h (panel A, lane 8). SAA3 mRNA levels drop

A B 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

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3 6 9 1 2 24 36 48 L

Time After LPS Administration (hrs) FIG. 2. Analysis of SAA mRNA by hybridization. Total liver

RNA (15 pgllane) was separated by electrophoresis in 1.1% agarose/ formaldehyde gels and transferred to nitrocellulose. Panel A was hybridized with the probe specific for RNA from the SAAl and SAAz genes; panel B was hybridized with the SAA3-specific probe. Lane 1, normal liver RNA; lanes 2-8, liver RNA isolated 3, 6,9, 12,36, or 48 h, respectively, after LPS administration. The probe specific for SAA, and SAAz mRNAs was constructed by cloning the AvaII/XhoI fragment from the 3’ UTS of the SAA, gene into the SmaI site of the M13 vector mp8. The SAA3 probe contained the AvaIIPstI fragment from the 3’ UTS of the SAA3 cDNA plasmid cloned into the PstI and SmaI sites of the M13 mpll vector. Inserts were confirmed by DNA sequencing. Probes were labeled using the probe-primer (New Eng- land Biolabs) and hybridized as described under “Experimental Pro- cedures.” Varying amounts of the respective SAA gene DNAs were electrophoresed in other lanes of the same gel which contained the RNA. These SAA DNA restriction fragments had approximately the same molecular weights as SAA mRNA. They served as hybridization intensity standards for comparison of SAA1+z and SAA3 mRNA hybridization (Pearson et al., 1981). Both filters were hybridized with the same amount of probe in the same manner and exposed for 38 h each. Panel C, the lower line (squares) represents SAA3 mRNA, while the upper line (circles) represents mRNAs. The hybridization intensities of the SAAl+, mRNA, SAA3 mRNA, and the SAA DNA standards from at least 4 differently timed autoradiographic exposures for each experiment were quantitated using a Joyce-Loebl microdensitometer. A standard curve of hybridization intensity versus amount of DNA fragment standard was constructed for each autoradiographic exposure using a least-squares fit of either first or second order equations. The standard curves were used to estimate the amount of SAA mRNA/15 pg of total liver RNA at each time point. This number was converted to transcripts/cell by assuming a total of 33 pg of total RNA/hepatocyte (Galau et al., 1977). The results shown are the average of 4 separate hybridization experiments; errors are the standard error of the mean.


continuously after peaking at 12 h (panel B, lanes 6-8). SAA DNA fragments were present on the same filters as hybridization standards. Standard curves of the hybridization intensities were generated to estimate the number of SAA transcripts/hepatocyte. We calculate the SAA mRNA level reaches about 20,000 transcripts/hepatocyte at 12 h after LPS administration. This level corresponds to 1-2% of the liver poly(A+) RNA. Therefore, SAA mRNAs are among the most abundant mRNAs in the liver when fully induced. Fig. 2 shows that SAA mRNAs decrease in size with increasing time after LPS administration. This may result from degradation of the poly(A) tails, since it has been found that the poly(A) sequences on numerous mRNAs decrease with mRNA aging (Brawerman and Diez, 1975).

Quantitation of repeated hybridizations reveals that there is twice as much RNA from the SAA, and SAA, genes together as from SAA, at 12-h peak levels. The 2:1 ratio was confirmed by hybridization of a third M13 probe which contains a 115- bp fragment from the highly conserved region of exon 3 of the SAA, gene. This probe will hybridize under thesecondi- tions to mRNA derived from any of the three SAA genes. The sum of the hybridization intensities observed with the two gene-specific probes at the peak time point was equal to the signal obtained with the conserved sequence probe (data not shown). We conclude that the SAAl and SAAz genes together account for two-thirds of the total SAA mRNA, while the SAA, gene mRNA represents the other third.

SAA, and SAA, mRNA can be distinguished using S1 nuclease because the SAAl gene has a 3-bp insertion (CTT) within the 3' UTS compared to the SAAz gene. A restriction fragment probe from the SAA, gene hybridized to SAAz mRNA will have a 3-nucleotide loop of unhybridized DNA (Fig. 3A). A DNA probe from the SAA, gene hybridized to SAA, mRNA will have a 3-nucleotide loop in the mRNA. The relative enzymatic activity of S1 nuclease on these two different substrates is not known. Therefore, this experiment is only qualitative for the presence of SAA, and SAA, mRNAs. S1 nuclease cleaves these nucleic acid loops and gives the predicted size fragments (Fig. 3B). With both probes three major sets of bands are seen. The upper bands (390 bp) are reannealed full length probes. The middle bands (326 or 323 bp) are the S1 nuclease-protected fragments which result from hybridization of the DNA probes to their homologous mRNA, while the lower bands (172 bp) are from hybridization to heterologous mRNA. The weaker bands at 256 and 267 bp result from SI cleavage at 2 nucleotide mismatches in the heterologous mRNA/DNA hybrids. These results indicate that both the SAA, and SAA, genes are transcribed and, from the lengths of the homologous mRNA/DNA hybrids, proc- essed to mature mRNA.

Apolipoprotein A-I and Albumin mRNA Levels during Acute Inflammation-The mRNA levels of two other major proteins produced by the liver, apo A-I and albumin, were investigated to determine whether the expression of these genes is affected during the acute phase response. Apo A-I is the major apolipoprotein in HDL particles and together with SAA apolipoprotein makes up the majority of the protein content of HDL during acute inflammation (Hoffman and Benditt, 1982a). Previous results from Hoffman and Benditt (1982b) indicated that the rate of apo A-I secretion from primary hepatocytes, isolated 3 h after LPS administration, is the same as from normal hepatocytes. Baumann et al. (1983a) found primary hepatocytes isolated 24 h after initiation of acute inflammation have a decreased rate of apo-A-I synthesis. Albumin mRNA levels, as well as serum albumin protein levels, have been found to decrease 2-%fold during acute inflammation

(Ricca et al., 1981; Kushner, 1982). The levels of apo-A-I and albumin mRNA were determined

by hybridizing the cDNA clones p1804 (Ertel-Miller et al., 1983) and prAlb576, respectively, to filters containing total liver RNA isolated from either normal or LPS-treated mice. As shown in Fig. 4, the amount of apo-A-I mRNA decreases gradually; the levels at 3 h after LPS administration and in normal liver are similar. By 24 h, it decreases to approximately 40% of its normal level. At 48 h after LPS administration, the amount of apo-A-I mRNA increases to approximately 150% of the normal level. These results may explain the observations of Hoffman and Benditt (198213) versus Bau- mann et al. (1983a) since the former group investigated apo- A-I synthesis when its mRNA level is similar to the normal amount, while the latter group used primary hepatocytes at a time when the apo-A-I mRNA level is reduced.

The albumin mRNA level also decreases gradually, reaching 45% of the normal level at 24 h after LPS administration. In contrast to apo-A-I mRNA, it remains at this reduced level through 48 h (Fig. 4). The relative decrease in albumin mRNA observed here is in good agreement with previous estimates (Ricca et al., 1981; Morrow et al., 1981).

SAA Gene Transcription in Isolated Hepatocyte Nuclei-To investigate whether the increase in SAA mRNA level during acute inflammation is regulated by increased transcription of SAA genes, we used a nuclear transcription assay (McKnight and Palmiter, 1979). The relative rate of [,'P]RNA synthesis in vitro has been shown to reflect accurately the relative transcription of specific gene sequences in vivo (Derman et al., 1981). In nuclear transcription systems, it is unlikely that RNA chain initiation occurs (Gariglio et al., 1981; Groudine et al., 1981), so chain elongation resulting from the steady- state distribution of endogenous RNA polymerases along each gene is measured. Previous results using this assay system indicated that total SAA gene transcription increased at least 10-fold over background in LPS-treated animals (Stearman et al., 1982). We have extended these previous results by measuring separately the transcription from the SAA1+, and SAA, genes. This was done by hybridizing [32P]RNA to South- ern blots containing SAA,, SAA2, or SAA, DNA fragments. Because of the high nucleotide sequence homology between the SAA, and SAA, genes, nascent transcripts from one gene will cross-hybridize with restriction fragments from the other. The relative amounts of [32P]RNA hybridizing t o SAA,,, or SAA, genes were quantitated by densitometry.

Hepatocyte nuclei isolated from either normal animals or 6 h after LPS administration were used to synthesize [,'P]RNA using [a-32P]UTP. The total amount of [,'P]RNA synthesized was similar in nuclei from normal or LPS-treated animals. About 2 X IO7 cpm of [32P]RNA was incubated with each Southern blot of subcloned SAA genomic DNA (Fig. 5). The [,,P]RNA made in nuclei from untreated animals gave background levels of hybridization to SAA exon-specific fragments. Only the [32P]RNA made in nuclei from LPS-treated animals gave detectable hybridization to DNA fragments containing SAA exon sequences. Hybridization was observed to SA& exon-specific fragments as well as SAAI and SAAz exon-specific fragments. In addition, the two restriction fragments containing repeated DNA elements (at approximately 4.5 and 10 kb) hybridized to [32P]RNA from both normal and LPS-treated animals.

To obtain a quantitative estimate for the relative amounts of [,*P]RNA hybridization to SAA1, SAAZ, or SAA3 DNA, we confirmed that the hybridization conditions used were in DNA excess and were driven to completion. This was done by hybridizing [,'P]RNA synthesized in nuclei from LPS-

A SAA 1 E X

Regulation of Serum Amyloid A Gene Expression

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* 390 bp 387 bp

S A A l m R N A S A A 2 m R N A S A A l m R N A A S A A 2 mRNA

IS’ I.‘ x 326 bp -172 bp -172 bp * 323 bp

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FIG. 3. S1 nuclease mapping of SAAl and SAAz mRNA. A, restriction map of SAA, and SAA2. The 4th exons of the SAA, and SAA2 genes are shown with the large filled box representing the translated region and the thinner box representing the 3’ UTS of each gene. The EcoRI (E) and XbaI ( X ) sites used are shown along with the sizes of the probe fragments, the structures of the hybrids with homologous or heterologous SAA mRNA, and the predicted sizes of the S1-protected fragments. Since the SAAZ gene has a 3-bp deletion within the 3’ UTS, heterologous mRNA/DNA hybrids have a 3-bp loop in either the RNA or DNA strand. B, poly(A+) RNA (2.5 pg) from LPS-treated animals (12 h) was hybridized with 5 X lo5 cpm (0.13 pmol) of the single-stranded EcoRI/XbaI fragment, 3’-end labeled at the EcoRI site, as described under “Experimental Procedures.” Hybrids were digested with increasing amounts of S1 nuclease and resolved on a 6% polyacrylamide-urea gel. Lane M, molecular weight markers, pBR322 plasmid DNA digested with HpaII and 3’-end labeled lune I , SAAl EcoRI/XbaI fragment alone; lunes 2-5, SAA, EcoRI/XbaI fragment hybridized with poly(A+) RNA and digested with 200, 500, 1000, or 1500 units of S1 nuclease/pg of nucleic acid lune 6, SAAz EcoRI/XbaI fragment alone; lunes 7-10, SAAz EcoRI/XbaI fragment hybridized with poly(A+) RNA and digested with 200, 500, 1000, or 1500 units of S1 nuclease/pg of nucleic acid.

treated animals with different amounts of SAA DNA for varying lengths of time. The unbound [32P]RNA was then re- hybridized to a second set of filters containing SAA DNA. Hybridization for 48 h with approximately 150 ng of the SAA gene fragments was sufficient to ensure that over 95% of the SAA-specific [32P]RNA had hybridized (data not shown). To calculate the relative increase in SAA gene transcription in LPS-treated animals, the hybridization to pBR322 DNA (shown in lane 5 ) was used to estimate the background [32P] RNA hybridization/kb. Hybridization to pBR322 DNA was assigned a relative value of 1-fold/kb as [32P]RNA transcription from normal animals gave background hybridization. Thus, the relative increases in SAAl+2 and SAA3 transcription

after LPS administration are minimum estimates. The amount of [32P]RNA hybridization to SAA3 DNA/kb (using the SAA, gene-specific fragment shown in lane 1) was at least 200-fold/kb over background while the [,‘P]RNA hybridization to SAA, and SAA, DNA (using the SAA,,, gene-specific fragment in lane 2) was at least 400-fold/kb over background. Therefore, total nuclear SAA transcription in this assay is undetectable in normal mice and increases a t least 300-fold/ kb over background at 6 h after LPS administration.

SAA Gene Transcription in the Presence of a-Amanitin, Sarkosyl, or Heparin-To confirm that the SAA transcription being measured is carried out by RNA polymerase 11, liver cell nuclei isolated from LPS-treated animals were used to


A B 1 2 3 4 5 6 7 1 2 3 4 5 6 7

W f ; 520- FIG. 4. Levels of apolipoprotein A-I and albumin mRNA

during acute inflammation. Total liver RNA (15 pg) isolated from either normal or LPS-treated animals was separated by electrophoresis on 1.1% agarose/formaldehyde gels, transferred to nitrocellulose, and hybridized with nick-translated p1804 (panel A ) or prAlb576 (panel B). Lane 1 , total RNA from normal mice; lanes 2-7, total RNA isolated 3, 6, 9, 12,24, and 48 h after LPS administration. Molecular weight markers are from both the 18 S rRNA and from pBR322 digested with HinfI.

synthesize [,'P]RNA in the presence of 2 pg/ml a-amanitin, a concentration which inhibits RNA polymerase 11, but not RNA polymerase I or I11 (Kedinger et al., 1970; Lindell et al., 1970). The addition of a-amanitin decreased the total incor- poration of [c~-~~P]UTP into RNA by approximately 50%, about the same decrease observed by others (McKnight and Palmiter, 1979; Cleveland and Havercroft, 1983). About 2 X 10" cpm of [32P]RNA made in these nuclei in the presence of a-amanitin were incubated with filters containing subcloned SAA DNA. No hybridization to the SAA exon-containing DNA fragments was observed (Fig. 6D) as compared to the hybridization seen from untreated nuclei (Figs. 5B and 6E). The presence of a-amanitin did not affect synthesis of ["PI RNA which hybridizes to fragments containing repeated DNA sequences, as these sequences are most likely transcribed by RNA polymerase I11 (Elder et al., 1981; Sharp, 1983; Bennett et al., 1984).

The effects of Sarkosyl and heparin on the transcription of SAA genes in normal and LPS-treated animals were also investigated. Sarkosyl and heparin remove histone and non- histone proteins from DNA, but do not affect previously initiated RNA polymerase molecules (Ferencz and Seifart, 1975; Gariglio, 1976). Thus, if RNA polymerases are present on the SAA genes in normal mice but are prevented from elongating by proteins bound to SAA DNA, removal of these proteins should stimulate SAA transcription in normal nuclei. The stimulation of repressed P-globin transcripts in Sarkosyl- treated chicken erythrocyte nuclei has been observed (Gariglio et al., 1981). The effect of heparin, which also acts as an RNase inhibitor (Cox, 1976), was tested to address the pos- sibility that SAA genes were transcribed in normal nuclei but that the transcripts were rapidly degraded during the labeling period (15 min). As shown in Fig. 6, addition to normal nuclei or those after LPS treatment with either of these compounds produced no substantial change in the [32P]RNA hybridization patterns compared to control nuclei. Therefore, it is unlikely that the differences in the amount of [32P]SAA RNA synthesized in normal nuclei and nuclei after LPS are due to rapid RNA degradation or blocked RNA polymerase 11.

To confirm that the SAA RNA synthesis is the result of transcription of the coding strand of the SAA genes, ["PI RNA was hybridized with single-stranded M13 clones containing the coding or noncoding strands of the SAA2 or SAA, genes (data not shown). The results indicated that [32P]RNA made in LPS nuclei hybridized strongly to both SAA, and SAA, clones containing the coding strand but that hybridi-

A 1 2 3 4 5 1 2 3 4 5 IB

9.4- 6.6- 4.4-

2.3- 2.0- 1.6-

0.5- FIG. 5. Hybridization of nascent [32P]RNA to SAA DNA.

Subclones of SAA DNA were digested with various enzymes, separated by electrophoresis on l% agarose gels, denatured, and transferred to nitrocellulose filters. The filters were hybridized with nascent [3ZP]RNA (2 X 10' cpm each) synthesized in liver nuclei isolated from either untreated mice (panel A ) or LPS-treated mice (panel B ) 6 h postinjection. The digests used were as follows: lane 1 , subclone pSA415, the BarnHI fragment which contains exons 1 and 2 of the SAA3 gene cloned into pBR322, digested with BarnHI; lane 2, subclone mSA33 RI/S, the EcoRI/SmI fragment which contains exons 1 and 2 of the SAAZ gene cloned into M13 replicative form, digested with PstI; lane 3, subclone pSA30, containing all of the SAA, gene and flanking interspersed repeated elements, digested with PstI; lane 4, subclone pSA7, which contains introns 2 and 3 and exons 3 and 4 of the SAA3 gene, digested with XbaI; lane 5, pBR322 digested with EcoRI. These digests were used because the SAA DNA fragments are all approximately the same size, between 1.0 and 1.8 kb, and each digest separates the SAA gene sequences from the repeated DNA sequences. The large fragments seen in lanes 3 and 4 contain both SAA gene sequences and repeated DNA sequences. The SAA3 and SAA, fragments in lanes 1 and 2 do not cross-hybridize under these conditions, so [32P]RNA which hybridizes to these fragments is specifically transcribed from either the SAA3 or the SAA,,, genes, respectively. In contrast, the SAAl and SAA3 fragments in lanes 3 and 4 contain 278 bp of coding sequence which does cross-hybridize; however, the majority of [32P]RNA hybridizing to these fragments represents primary transcripts from the introns and 3' flanking regions which do not cross-hybridize. The molecular weight markers are from X DNA digested with Hind111 and pBR322 digested with HinfI.

zation to clones containing the noncoding strand was equal to hybridization observed with the mp8 vector alone. The coding strand SAA, clone hybridized considerably more ["PI RNA than the coding strand SAAs clone as the high nucleotide homology of the SAAl and SAA2 genes allows nascent RNA from either gene to hybridize to the M13 SAA2 clone.

Time Course of SAA Gene Transcription-To examine the time course of SAA gene transcription during acute inflammation, liver nuclei were isolated at various times after LPS administration. The nuclei then synthesized [32P]RNA which was hybridized to Southern blots containing subcloned SAA DNA. The amount of [32P]RNA hybridizing to fragments specific for the SAA,,, or the SAA, genes was quantitated by densitometry. Fig. 7 shows the relative amount of transcription from the SAA genes as a function of time after LPS

Regulation of Serum Amyloid A Gene Expression 8459

A B C D 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

E F G 1 2 3 4 1 2 3 4 1 2 3 4

Time After LPS Administration ( h n ) FIG. 7. Time course of SAA gene transcription after LPS

administration. Liver nuclei isolated at the indicated time points after LPS administration were used for in vitro transcription, and the nascent [3ZP]RNA was hybridized to separate filters containing the SAA DNA digests shown in Fig. 5. The amount of [32P]RNA hybridized to the SAA1+2 or SAA3 gene specific fragments was quantitated, per kb length of DNA, by densitometric scanning of multiple autoradiographic exposures. The hybridizations were all done under identical conditions of DNA excess and with equal amounts of ["PI RNA (2 X lo7 cpm). The data is plotted as a percentage of the amount of [3ZP]RNA hybridization to SAAI+Z DNA using 3-h nuclei. Each data point is the average value derived from 3 to 5 separate hybridization experiments using nuclei preparations from liver tissue pooled from 2 mice. At least 4 differently timed autoradiographs were quantitated, per experiment, to ensure that the exposures were within the linear range of the x-ray film. The errors are the standard error of the means.

FIG. 6. Effect of a-amanitin, Sarkosyl, and heparin on transcription of SAA genes in isolated nuclei. Digests of subcloned SAA DNA were separated by electrophoresis on agarose gels, denatured, and transferred to nitrocellulose. The filters were hybridized with nascent [32P]RNA made in liver nuclei, from either normal or LPS-treated animals (6 h post-LPS administration), that were incubated with either a-amanitin, Sarkosyl, or heparin. In each panel lunes 1-4 are the same digests as lanes 1 4 in Fig. 5. All filters were hybridized with 2 X lo7 cpm of [32P]RNA. The hybridization of ["PI RNA made in normal nuclei is shown in panel A. Panels B and C show the hybridization of [32P]RNA made in normal nuclei incubated in either 0.5% Sarkosyl or 1 mg/ml heparin, respectively. The hybridization of [32P]RNA made in nuclei from LPS-treated animals is shown in panel E. Panels D, F, and G show the hybridization of ["PI RNA made in LPS nuclei incubated in either 2 pg/ml a-amanitin, 0.5% Sarkosyl, or 1 mg/ml heparin, respectively.

administration. These results indicate that peak levels of both SAA,,, and SAA3 gene transcription occur 3 h after LPS administration. At this peak time, SAA, transcription is approximately 50% of SAA1+, transcription (normalized per kb of DNA), i.e. two-thirds of the total SAA gene transcripts are from the SAAl and SAAz genes and one-third is from the SAA, gene. Later than 3 h, SAA,,, transcription makes up greater than two-thirds of total SAA transcription. By 24 h after LPS administration, the SAAs gene transcription is undetected and only SAA, and SAA, genes are transcribed, though at 25% of their maximal level. All three genes are transcriptionally inactive by 36 h after LPS treatment. As a control, the transcription of the 18 S ribosomal RNA genes over this same time course was also measured (data not shown). In contrast to SAA transcription, 18 S rDNA transcription is constant over this time period.

DISCUSSION

The Three SAA Genes Are Coordinately Expressed-The induction of total SAA mRNA in liver tissue during the acute phase response was estimated to be approximately 2000-fold

by comparing the amount of SAA mRNA in normal and LPS- treated mice. Since SAA mRNA is present at low levels in normal animals, SAA genes may be transcribed prior to induction. In this regard, it is interesting that the 5'-end regions of the genes are undermethylated, and in the liver the methylation state of the genes does not change after LPS administration (Stearman et al., 1982).

At peak levels, SAA,,, mRNA makes up two-thirds of the total SAA mRNA, while SAAs mRNA makes up the other third. Since both SAAl and SAA, are expressed (Fig. 3), it is likely that each SAA gene is transcribed to produce one-third of the total SAA mRNA. Equal expression of the SAA, and SAA, genes is supported by the similar amounts of these polypeptides found in serum (Hoffman et al., 1984). The mRNAs transcribed from all three SAA genes are coordinately induced after LPS administration.

SAA mRNA levels reach approximately 20,000 transcripts/ cell and are among the most abundant liver transcripts during acute inflammation. Hastie and Bishop (1976) estimate that there are only 10 mRNA species in liver with more than 12,000 transcripts/cell. For example, the mRNA encoding albumin (Derman et al., 1981) and the major urinary proteins (Tse et al., 1978) make up approximately 22,000 and 16,000 transcripts/cell (assuming 2 x lo5 mRNAs/hepatocyte; Hastie and Bishop, 1976). The liver mRNA level for the major acute phase protein in rats, al-acid glycoprotein, is also abundant: approximately 7,400 transcripts/cell (Baumann et al., 1983b).

Total SAA gene transcription increases at least 300-fold/ kb during acute inflammation as assayed by in vitro transcription in isolated hepatocyte nuclei (Fig. 5 ) . Since the amount of SAA transcription in untreated animals is at the background level of detection, it is likely that a 300-fold/kb increase in SAA transcription is a minimum estimate. The increase in SAA gene transcription is probably due to de novo initiation of RNA polymerase 11, rather than the release of previously initiated RNA polymerase I1 molecules as neither


Sarkosyl nor heparin affected SAA gene transcription (Fig. 6). The time course of SAA gene transcription demonstrated that the SAA1+, and SAA, genes are maximally transcribed at 3 h after LPS administration. They make up two-thirds and one-third, respectively, of the total SAA transcription (Fig. 7). Given the high degree of nucleotide sequence homology of the SAA, and SAA, genes and similar serum concentrations of the SAAl and SAA, polypeptides (Hoffman et al., 1984), it is likely that each gene is transcribed at a similar rate. At peak levels of both SAA transcription and SAA mRNA accumulation, each gene appears to make up approximately one-third of the total SAA gene expression. Protein encoded by the SAA, gene has not been found; it was not purified with the SAA, and SAA, polypeptides in HDL (Hoff- man et al., 1984).

The nuclear transcription assay was also used to determine the transcriptional domains of the SAA genes by examining the amounts of [32P]RNA which hybridized to regions 5' and 3' of the SAA genes (data not shown). These results demonstrated that there is no detectable transcription of sequences upstream from the cap sites of the SAA genes, except from repeated sequences. The regions beyond the 3' termini of the genes are transcribed, as has been found with numerous other genes (Hofer and Darnell, 1981; Nevins et al., 1980).

SAA mRNA Levels May Be Regulated by Both Increased Transcription and RNA Stabilization-While the kinetics of mature SAA mRNA accumulation and SAA gene transcription indicate that all three genes are coordinately induced, it is important to note that the maximal rates of mRNA accumulation and gene transcription are not synchronous (Fig. 8). The rate of total SAA gene transcription peaks at 3 h after LPS administration, while the maximal rate of mRNA accumulation, as defined by the slope of the mRNA accumulation curve (Fig. 8), occurs between 9 and 12 h. In fact, the rate of SAA transcription is decreasing when the rate of SAA mRNA accumulation is increasing. The delayed accumulation of mature SAA mRNA is probably not due to accumulation of RNA intermediates, because hybridization of gene-specific probes to total liver RNA failed to show high molecular weight species (Fig. 2 A ) . Moreover, studies of adenovirus transcription have shown that processing of primary transcripts to

Time After LPS Administration (hrs) FIG. 8. SAA gene transcription versus SAA mRNA accu-

mulation. The sum of SAA,,, and SA& gene transcription (from Fig. 7) is plotted with the sum of the SAAl+* and SAA, mRNA levels (from Fig. 2) at each time point after LPS administration. The total SAA gene transcription (open triangles) is plotted as a percentage of the transcription at 3 h while the amount of total SAA mRNA (closed triangles) is plotted as transcripts/cell. The errors are the sum of the standard error of the mean for the values at each time point.

mature mRNAs occurs very rapidly (Nevins and Darnell, 1978). A possible explanation for the delayed accumulation of SAA mRNA (despite the high transcriptional activity from the genes) is that a large proportion of SAA transcripts synthesized during the first 6 h of induction are degraded. Between 9 and 12 h the rate of degradation of the SAA transcripts may decrease allowing more mature SAA mRNA to accumulate. Unlike SAA gene expression, the peak rate of transcription occurred synchronously with the maximal rate of mRNA accumulation during the induction of ovalbumin, conalbumin, metallothionein, and prolactin genes (McKnight and Palmiter, 1979; Durnam and Palmiter, 1981; Murdoch et al., 1983). Therefore, SAA mRNA levels may be regulated by increased transcription of the SAA genes, as well as subse- quent mRNA stabilization.

Rat oll-acid glycoprotein shows a similar pattern of mRNA accumulation during the acute phase as SAA mRNA (Ricca et al., 1981; Baumann et al., 1983b). Four h after initiation of inflammation, the al-acid glycoprotein mRNA level is low but rapidly increases to its peak level at 12 h. It has been suggested that multiple effector systems may be regulating crl-acid glycoprotein mRNA induction in vivo (Baumann et al., 1983b). The increase in al-acid glycoprotein mRNA levels caused by glucocorticoids appears to be by stabilization of nascent transcripts in vitro (Vannice et al., 1984).

The SAA1+, and SAA, mRNA levels also demonstrate different stabilities. The SAA1+, mRNA level remains constant through 36 h after LPS administration while the SAA, mRNA level has decreased to 20% of its maximum level (Fig. 2). After 36 h, SAA1+2 mRNA is rapidly degraded implying an abrupt change in mRNA stability. Cessation of hormonal stimulation of the liver may result in the rapid degradation of the SAA1+, mRNAs. Changes in liver mRNA stability after cessation of hormonal stimulation have been reported for vitellogenin and very low density lipoprotein mRNAs (Wis- kocil et al., 1980).

SAA mRNA levels may be regulated at both transcriptional and post-transcriptional levels. All three genes have the necessary regulatory DNA sequences for induction of transcription, but SAA3 mRNA is less stable than SAA1+2 mRNAs. This phenomenon can be investigated by fusion of SAA promoters and gene regions to marker genes, like chloram- phenicol acetyltransferase, followed by transfection into cul- tured cells. The role of continued hormonal stimulation for both the maintenance of SAA transcription and the stabilization of SAA mRNA can be investigated in vitro by repeated hormonal stimulation of hepatocytes.

Acknowledgments-We wish to thank Drs. Thomas Kelly, Jeff Corden, and David Potter for critical review of this work. We also thank Cynthia Peltzman for expert technical assistance, and Gerry Hunsicker and Holly Porter for manuscript preparation.

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