• acute-phase response;
  • amyloidosis;
  • serum amyloid A;
  • transcriptional regulation


  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The serum amyloid A (SAA) family comprises a number of differentially expressed apolipoproteins, acute-phase SAAs (A-SAAs) and constitutive SAAs (C-SAAs). A-SAAs are major acute-phase reactants, the in vivo concentrations of which increase by as much as 1000-fold during inflammation. A-SAA mRNAs or proteins have been identified in all vertebrates investigated to date and are highly conserved. In contrast, C-SAAs are induced minimally, if at all, during the acute-phase response and have only been found in human and mouse. Although the liver is the primary site of synthesis of both A-SAA and C-SAA, extrahepatic production has been reported for most family members in most of the mammalian species studied. In vitro, the dramatic induction of A-SAA mRNA in response to pro-inflammatory stimuli is due largely to the synergistic effects of cytokine signaling pathways, principally those of the interleukin-1 and interleukin-6 type cytokines. This induction can be enhanced by glucocorticoids. Studies of the A-SAA promoters in several mammalian species have identified a range of transcription factors that are variously involved in defining both cytokine responsiveness and cell specificity. These include NF-κB, C/EBP, YY1, AP-2, SAF and Sp1. A-SAA is also post-transcriptionally regulated. Although the precise role of A-SAA in host defense during inflammation has not been defined, many potential clinically important functions have been proposed for individual SAA family members. These include involvement in lipid metabolism/transport, induction of extracellular-matrix-degrading enzymes, and chemotactic recruitment of inflammatory cells to sites of inflammation. A-SAA is potentially involved in the pathogenesis of several chronic inflammatory diseases: it is the precursor of the amyloid A protein deposited in amyloid A amyloidosis, and it has also been implicated in the pathogenesis of atheroscelerosis and rheumatoid arthritis.


apolipoprotein A-I


acute-phase protein


acute-phase serum amyloid A


constitutive SAA


extracellular matrix


high-density lipoprotein




IL-1 receptor antagonist




glucocorticoid receptor


glucocorticoid-responsive element


glucocorticoid-responsive unit


low-density lipoprotein




minimally modified low-density lipoprotein


recombinant human A-SAA


recombinant mouse IL-1ra


SAS-binding factor


SAA-activating sequence


SAA enhancer factor 1


secretory phospholipase A2


tumor necrosis factor


yin and yang 1

The acute-phase response

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The acute phase response is the immediate set of host inflammatory reactions that counteract challenges such as tissue injury, infection and trauma. Its role is to isolate and neutralize pathogens and prevent further pathogen entry while minimizing tissue damage and promoting repair processes, thereby permitting host homeostatic mechanisms to rapidly restore normal physiological function [1,2]. The acute-phase response involves the induction of an inflammatory mediator cascade which is characterized by both local vascular effects and systemic, multiorgan effects. The latter include biosynthetic changes, particularly pronounced in the liver, which modify the profile of circulating plasma proteins. Although the acute-phase response has evolved as a survival mechanism for the short term, its maintenance over the longer term in cases of chronic inflammation may have negative clinical consequences.

Initiation of the inflammatory cascade occurs primarily through activated blood monocytes and tissue macrophages at the site of the inflammatory stimulus. Upon activation macrophages release a range of primary inflammatory mediators, the most important of which are members of the interleukin-1 (IL-1) and tumor necrosis factor (TNF) cytokine families. These cause the release of a range of secondary cytokines and chemokines (e.g. IL-6, IL-8 and monocyte chemoattractant protein) from local stromal cells. The chemotactic activities of some of these molecules draw leukocytes such as neutrophils to the inflammatory site, where they in turn release further pro-inflammatory cytokines [3]. Consequently, the cytokine cascade and the recruitment of immune effector cells is rapidly augmented locally to deal with both the underlying inflammatory stimulus and the cellular debris generated by any associated tissue damage.

One of the most intensively studied systemic responses to an acute inflammatory stimulus is the alteration in the hepatic biosynthetic profile of acute-phase proteins (APPs) (reviewed in [4,5]). Increased synthesis of the positive APPs which counteract the inflammatory challenge ensures that they attain the plasma concentrations at which they are maximally effective. The positive APPs may be subclassified as either major or minor according to their fold induction during the acute-phase response. Partially to permit synthetic capacity to be redirected to the increased production of the positive APPs, another distinct class of liver proteins, the negative APPs, are down-regulated during the acute-phase response.

In addition to cytokines and chemokines, other inflammatory mediators include glucocorticoids and growth factors. Glucocorticoids can stimulate the expression of some APPs directly; however, they usually act by enhancing the transcriptional induction that is principally driven by cytokines such as IL-1, TNF and IL-6. Concurrent with their participation in pro-inflammatory processes, IL-1, TNF and IL-6 contribute to the resolution of the acute-phase response by initiating negative feedback on their own production by inducing corticosteroids which, as well as enhancing the cytokine-driven upregulation of APP synthesis, inhibit further cytokine gene expression. This feedback acts via the cytokine-induced production of adrenocorticotrophic hormone which in turn stimulates synthesis of the corticosteroid, cortisol [6–8]. Among the various growth factors that modulate APP regulation, insulin can attenuate the induction of most APP genes by IL-1 and IL-6 type cytokines [9], and is an example of a factor that has the opposite regulatory input to that associated with glucocorticoids.

The serum amyloid a (saa) family

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The SAA family was originally considered to comprise only a single circulating precursor of the amyloid A protein from which its name is derived. The amyloid A protein is the principal component of the secondary amyloid plaques that may be deposited in major organs as an occasional consequence of chronic inflammatory disease (see below) [10]. The SAA family is now known to contain a number of differentially expressed apolipoproteins which are synthesized primarily by the liver and can be divided into two main classes based on their responsiveness to inflammatory stimuli. ‘Acute-phase’ serum amyloid A (A-SAA) is the archetypal vertebrate major APP. It is induced from resting levels by more than 1000-fold during inflammation to plasma concentrations that can exceed 1 mg·mL−1[2,11,12], implying an important, beneficial role in host defense. During inflammation, A-SAA associates predominantly with the third fraction of high-density lipoprotein (HDL3) [13] replacing apolipoprotein A-1 (ApoA-1) as the predominant apolipoprotein on this particle [14]. ‘Constitutive’ SAAs (C-SAAs) have been described in two species, human [15] and mouse [16]. Unlike the A-SAAs, the C-SAAs, which are at most minimally induced during the acute-phase response, are associated with both normal and acute-phase HDL [15–17].

SAA genes and proteins

Multiple SAA genes and proteins have been described for several mammalian species including human, mouse, hamster, rabbit, dog, mink, cow, sheep and horse. The high degree of conservation of the SAA genes and proteins that has been maintained through the evolution of eutherian mammals [18] extends to other vertebrates including marsupials [19] and fish [20] thereby providing further evidence that they are likely to have important biological functions. All of the SAA genes described to date share a four-exon three-intron organization which is characteristic of many other apolipoproteins [4]. The mature SAA proteins range in size from 104 to 112 amino acids and are derived from primary translation products with 18-amino acid leader peptides (Fig. 1).


Figure 1. The structure of human A-SAA protein. The 18-amino acid signal peptide is shown (−18 to 0) together with the 104 mature protein (1–104). The most frequently observed C-terminus of the amyloid A protein is indicated at residue 76. Regions of potential functional importance are indicated by underlining in color, while structurally important regions are indicated by shading/hatching; both are discussed in the text. Sites delimiting sequences encoded by exons 2/3 and 3/4 are indicated (exon 1 contains only 5′ untranslated region).

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The SAA genes of human and mouse, which comprise four and five members, respectively, are those that have been subject to the most comprehensive analyses (see below). In other mammalian species the SAA family members are less well defined. There are at least three transcribed A-SAA genes in dog [21], mink [22,23] and rabbit (for references, see [24,25,26,27]). In the case of horse, three SAA isoforms have been found in acute-phase serum [28,29]. There are also at least two A-SAAs in hamster [30,31], and one each in cow [32,33], sheep [34] and wallaby [19]. Rat mRNA has been identified in primary hepatocytes, by representational differential and Northern-blot analyses, in response to aflatoxin B1, a mutagenic and carcinogenic food contaminant (see below) [35]. A-SAA isoforms have also been detected in fox and goat sera [24]. Arctic char (a salmonid closely related to trout), is the most evolutionarily distant vertebrate for which an SAA has been described [20]. Computer-based analysis of their sequences and incorporation into phylogenetic trees indicates that both the wallaby and arctic char proteins are A-SAAs rather than C-SAAs, and in vivo studies of the salmonid mRNA have established that its hepatic expression is dramatically induced by inflammation [19,20]. Furthermore, the in vivo studies of the hepatic induction of A-SAA mRNA in arctic char challenged with a lethal inoculum of the pathogen Aeromonas salmonicida indicate that the level of synthesis is driven by pathogen load. This strongly suggests that the dramatic upregulation of A-SAA in response to inflammatory stimuli is common to all vertebrates and in addition suggests a shared protective biological function.

The four human and five mouse SAA family members were originally named in numerical progression according to the order in which they were identified. Consequently SAA nomenclature in the literature does not accurately define true homologs between the species [18]. In order to address this, a revised nomenclature for the SAA family has recently been adopted [36] and is used in this review (Table 1). The SAA genes of both the human and mouse lineages have remained in close physical linkage (Fig. 2) and it seems likely that the SAA genes will be similarly clustered in the genomes of other species [18]. The human SAA1, 2, 3 and 4 genes are within 150 kb of each other on chromosome 11p15.1 [37,38]. This region of the human genome is syntenic with proximal mouse chromosome 7 [39] to which the mouse SAA genes were originally mapped [40], and within which the position of the cluster, containing Saa1, 2, 3, 4 and 5 and spanning only 45 kb, has been refined [41,42].

Table 1. The SAA family. Nomenclature for the human and mouse SAA families is from the new guidelines of the SAA Subcommittee of the Amyloidosis Nomenclature Committee, 1999 [36]; square brackets represent the old nomenclature for clarity. The SAA family members of other species and their gene/protein numbers are as referred to in original reports.
GeneProteinAlleles (coding)Alleles (noncoding)References
 SAA1SAA1 (A-SAA)SAA1.1 [SAA1α]SAA1.1.1 [SAA1α1] SAA1.1.2 [SAA1α2] SAA1.1.3 [SAA1α3][280,281,282] [283] [44]
  SAA1.2 [SAA1β] [284]
  SAA1.3 [SAA1γ] [276,285]
  SAA1.4 [SAA1δ] [286]
  SAA1.5 [SAA1β] [287]
 SAA2SAA2 (A-SAA)SAA2.1 [SAA2α]SAA2.1.1 [SAA2α1] SAA2.1.2 [SAA2α2][282,287] [44]
  SAA2.2 [SAA2β] [288,146]
 SAA3Pseudogene  [37,55]
 SAA4SAA4 (C-SAA)  [15,288]
 Saa1 [Saa2]Saa1 [Saa2] (A-SAA)  [45]
 Saa2 [Saa1]Saa2 [Saa1] (A-SAA)  [45]
 Saa3Saa3  [45]
 Saa4 [Saa5]Saa4 [Saa5] (C-SAA)  [16]
 Saa-ps1 [Saa4]Pseudogene  [45]
 Saa2.2 [CE/J Saa]Saa2.2  [289]

Figure 2. A comparative map of the human and mouse SAA gene families. The human family spans 150 kb on chromosome 11p15.1 [38] while the mouse family spans 45 kb on chromosome 7p [42]; these regions of the human and mouse genome are syntenic. The old SAA nomenclature for the mouse SAA family is in square brackets beside the new (Table 1). The relative positions of the flanking genes lactate dehydrogenase A and C (LDHA and LDHC), tryptophan hydroxylase (TPH), myogenic factor 3 (MYOD1) and a member of the potassium channel family (KCNC1) are indicated in the case of the human cluster; in mouse, these genes all map to a 500-kb region containing the SAA genes but have not been fine-mapped relative to the SAAs. The position of an anonymous human marker, D11S18, is also shown. Arrows within SAA genes represent 5′ [RIGHTWARDS ARROW] 3′ orientation of the gene.

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The human SAA1 and SAA2 and the mouse Saa1 and Saa2 genes all encode A-SAAs. It is impossible to determine the interspecies evolutionary relationships of these individual SAA family members based on nucleotide or protein sequence comparisons alone [18]. However, their relative map positions and transcriptional orientations (Fig. 2) provide strong evidence that human SAA1 and mouse Saa1 (formerly Saa2) are evolutionary homologs, as are human SAA2 and mouse Saa2[Saa1][38,42]. Within each species these two genes have almost identical sequences and organizations, suggesting that the A-SAA genes have been subjected to recent gene conversion (i.e. homogenizing) events within each evolutionary lineage [18]. They are co-ordinately induced during the acute-phase response and their mature protein sequences share greater than 90% identity. In the case of human, both SAA1 and SAA2 are allelic. SAA1 has at least five alleles [43], of which three (SAA1.1, SAA1.2 and SAA1.3) encode distinct proteins, and two are neutral polymorphisms of SAA1.1 (Table 1). SAA2 has three alleles, SAA2.1, of which there is a HindIII polymorphism of neutral effect [44], and the distinct SAA2.2 (Table 1).

Mouse Saa3 is an expressed A-SAA gene although it is more diverged relative to the other A-SAA genes Saa1 and Saa2[45–47]. Its protein product shares only 63% and 65% identity with Saa1 and Saa2, respectively. Unlike the other mouse SAAs, expression of Saa3 mRNA is not principally hepatic [48–50] and the Saa3 protein has been identified as a secreted product of macrophages [51]. SAA3 genes and mRNAs have been reported in other species, e.g. rabbit, rat and hamster, in all of which expression occurs in a range of extrahepatic tissues [30,31,52,53]. In contrast, the human SAA3 gene, the presumptive evolutionary homolog of mouse Saa3[54], is a pseudogene because of a single base insertion in exon 3 which produces a frameshift and, consequently, generates a downstream stop signal at codon 43 [37,55]. No mRNA or protein product specified by human SAA3 has been identified. However, it is clear that human SAA3 protein, if it exists, cannot be an ‘intact’ SAA3 molecule with functions analogous to those of the SAA3 molecules in other species.

The fourth member of the mouse SAA family to be identified was a pseudogene, originally named Saa4[45], and now referred to as Saa-ps1[36]. It is highly disrupted and contains only sequences related to exons 3 and 4 of the functional SAA genes together with a 25-bp deletion in the exon 3 portion, which introduces an in-frame stop codon downstream. The most recently identified human and mouse SAA genes are those encoding the C-SAAs. The human SAA4 gene [15], and the mouse gene, originally called Saa5[16], are the only constitutively expressed SAA genes described to date. Based on their expression characteristics, sequences and positions within the human and mouse SAA gene clusters, they are considered to be evolutionary homologs [16,18,38,42]. Consequently mouse Saa5 is now referred to as Saa4[56]. The human SAA4 protein sequence shares only 53% and 55% identities with human SAA1 and SAA2, respectively [15], and mature mouse Saa4 has 48% amino acid identity with Saa1 and Saa3 [16], establishing that the C-SAAs constitute a distinct branch of the SAA family. Both the human SAA4 and mouse Saa4 genes encode precursor molecules of 130 amino acids from which 18-residue signal peptides are cleaved to yield 112-residue mature proteins. The products are therefore eight amino acids longer than the mature A-SAAs in these species because of the apparent ‘insertion’ of an octapeptide between residues 69 and 70 (Fig. 1). Within the octapeptide encoded by the SAA4 gene, there is a glycosylation site (NSS) that is differentially used to generate the 14-kDa unglycosylated and 19-kDa glycosylated forms of C-SAA observed in human serum [15]. A similarly positioned octapeptide is also present in the A-SAAs of carnivores (dog and mink) and perissodactyls (horse), and a nonapeptide occurs in artiodactyls (cow and sheep). The distribution of these sequence elements within the SAA family is an evolutionary paradox, the functional significance of which, if any, is unknown [15,18].

Structure of SAA proteins

Early work based on predictive methods suggested that A-SAA is likely to contain two regions of α-helix in addition to β-sheet regions [57] (Fig. 1), the latter of which are common to all amyloid proteins including amyloid A protein [58]. The likelihood of α-helix being present has recently been augmented by CD studies of a human SAA1-staphylococcal nuclease fusion protein [59]. However, to date, structural studies using advanced methods have not been reported. Nevertheless, several regions of mammalian A-SAA proteins that may be important in facilitating the beneficial role(s) A-SAA may play during inflammation, and/or its pathogenic behavior after over-expression during chronic inflammation, have been identified (Fig. 1).

It has clearly been established that A-SAA is the serum precursor of the amyloid A protein that is found in secondary amyloid deposits by tracking the fate of human A-SAA-HDL introduced into mouse in vivo models of amyloidosis [60,61]. Although the precise mechanism is poorly defined, proteolytic cleavage of A-SAA appears to play a central role in amyloid deposition. Such cleavage results in the generation of N-terminal peptides that are deposited as amyloid A protein; these are predominantly 76 residues long, but smaller and larger amyloid A proteins have also been reported [10,62,63]. The capacity of synthetic peptides of human and mouse A-SAA, encompassing amino acids 2–12 and 1–11, respectively, to form fibrils in vitro indicates that the amyloidogenic region is within the first 10–15 N-terminal amino acids [64]. Furthermore, site-directed mutagenesis of the eighth amino acid and deletion of the first 11 amino acids of recombinant human SAA1.1 (rhSAA1.1) results in a reduction in amyloid fibril formation [65].

A-SAA and C-SAA both associate with HDL, the former being the major HDL-associated apolipoprotein during the acute-phase response and the latter being the predominant, perhaps only, HDL-associated SAA under normal physiological conditions. The early proposition that the lipid-binding, as well as the amyloidogenic, region of A-SAA resides within the first 11 N-terminal amino acids [57] is consistent with subsequent findings that amyloid A protein which lacks the C-terminal 28 residues, can associate with HDL [66], and that epitopes defined by antibodies raised against N-terminal A-SAA peptides (residues 1–30) are masked when A-SAA is complexed with HDL [67]. Recently, more comprehensive studies have suggested that the epitopes of residues 31–39, 64–78 and 95–104 are exposed whether A-SAA is associated with HDL or not, and epitopes of residues 1–30, 40–63 and 79–94 (the hydrophobic regions of A-SAA) are masked either because of specific self-folding or dimerization; both monomeric and dimeric lipid-free A-SAA coexist in denaturing, non-denaturing, acidic and basic environments [68]. In addition, a reduction in binding to HDL is observed when the N-terminus is mutated by either deletion of the first 11 residues or substitution of the eighth residue (glycine) with aspartate [65].

The region between residues 29 and 42 in human A-SAA contains two elements, YIGSD and RGN, which are very similar to the distinct cell-binding domains of the two extracellular matrix (ECM) cell adhesive glycoproteins, laminin (YIGSR) and fibronectin (RGD), respectively. Synthetic peptides of these elements specifically inhibit the receptor-mediated adhesion of human T lymphocytes and mouse M-4 melanoma cells to laminin and fibronectin, respectively [69]. Amyloid A protein (residues 1–76) and rhA-SAA can also inhibit the adhesion of human T lymphocytes to these two glycoproteins. The primary structure of this rhA-SAA differs from rhSAA1.1, as it has an additional N-terminal methionine and substitutions at positions 60 and 71 which are found in SAA2.2. The above suggests that A-SAA may be able inhibit immune cell migration towards inflammatory sites and, perhaps, metastatic processes in vivo[69]. Whereas the YIGSD laminin-like site is found only in human SAA1 and SAA2, the RGN fibronectin-like site is present in all A-SAA and C-SAA proteins described to date (for sequences, see [18,19,20]).

The RGD sequence of the ECM adhesive proteins is also the target that facilitates the binding of some of the mediators released from activated platelets, e.g. fibrinogen, to the platelet integrin glycoprotein IIb–IIIa receptor which is essential in platelet aggregation. It has been suggested that the inhibition of platelet aggregation that can be achieved using A-SAA, residues 25–76 of A-SAA, or amyloid A protein may occur through the conserved RGN sequence [70,71]. However, it should be noted that not all RGD-containing proteins interact with the glycoprotein IIb–IIIa complex and some RGD sequences may not be functionally active. Further evidence of A-SAA involvement in platelet aggregation is provided by its modulation of the induction of prostaglandin I2, a potent antiaggregation agent; this is mediated by the first 14 N-terminal amino acids of A-SAA [72].

A putative calcium-binding sequence, GPGG, between residues 48 and 51 [57], is conserved in all SAA sequences identified to date except for mouse Saa4, in which the corresponding tetrapeptide is GSGG. Early reports that amyloid deposits are rich in calcium [73] and are sites at which Ca2+-dependent protein binding interactions occur [74] suggested that this peptide may be important in amyloidogenesis. Furthermore, serum amyloid P, the other major component of secondary amyloid deposits, can bind glycosaminoglycans and amyloid A protein in both Ca2+-dependent and Ca2+-independent manners [75]. However, a recent report in which the capacity of human SAA1 to enhance the production of thromboxane A2 and prostaglandins can clearly be demonstrated, provided evidence that this property is not Ca2+-dependent [76]. In this study, SAA1 was unable to bind Ca2+, and, in addition, antibodies specific for residues 40–63 of SAA1, which encompass the GPGG site, had no effect on SAA1-mediated thromboxane A2 and prostaglandin synthesis.

Although relatively few functional studies of the C-terminus of A-SAA have been performed, there is evidence that it may facilitate binding to neutrophils. A peptide corresponding to residues 77–104, but not peptides spanning other regions, competitively inhibits such binding [77]. Interestingly residues 77–104 are usually not present in amyloid A deposits, raising the possibility that they are specifically released after proteolysis of A-SAA and that they have an immune-related biological function. Recently A-SAA has also been shown to have glycosaminoglycan, i.e. heparin sulfate and heparin, binding activity at its C-terminus between residues 77 and 103 [78].

Expression and induction of saa

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The major site of A-SAA synthesis, like that of most other acute-phase proteins, is the liver. A-SAA mRNA can be dramatically induced by inflammatory stimuli and may become one of the most abundant hepatic mRNAs [12,46]. In stimulated mice, as much as 2.5% of the synthetic capacity of the liver may be directed to A-SAA protein synthesis [79], while in arctic char infected with a lethal dose of the pathogen A. salmonicida, the amount of hepatic A-SAA mRNA present can increase to as much as 10% of the total [20]. A-SAA is also catabolized in the liver [80], has a much shorter half-life of 1 day [81] and is cleared from the plasma more rapidly than other HDL apoproteins such as apolipoprotein A-I (ApoA-I) which has a half-life of 4–6 days [82,83]. During an acute-phase response or chronic inflammation, the capacity of the liver to degrade A-SAA decreases, by 14% and 31%, respectively [84], thereby contributing to the elevated circulating A-SAA levels observed under these conditions.

A-SAA mRNA and protein synthesis are induced in vivo during the inflammatory response to challenges such as tissue injury, infection and trauma in all vertebrate species (Fig. 3). These challenges, which can be experimentally produced using agents such as bacterial lipopolysaccharide (LPS), casein, turpentine, AgNO3 and surgery, induce the pro-inflammatory cytokine cascade. The principal cytokines involved in the induction of A-SAA are IL-1, TNF-α and IL-6 (see below). Other cytokines that may be involved either directly or indirectly in A-SAA induction include IL-2, interferon-γ (IFN-γ) and ciliary neutrophic factor [85–87]. In addition to IL-6 and ciliary neutrophic factor, four other IL-6 type cytokines, i.e. IL-11, leukemia inhibitory factor, oncostatin M and cardiotrophin-1, induce A-SAA when administered to mice [88]. Glucocorticoids, which are also released during inflammation, have been shown to enhance cytokine-induced A-SAA expression in several of the above studies (see below).


Figure 3. Induction of A-SAA during the acute-phase response. The flow diagram represents the induction of the acute-phase response in tissues by inflammatory stimuli leading to recruitment of macrophages and subsequent cytokine production. These cause changes in transcription factor availability in different tissues which result in increased A-SAA expression and consequently increased protein concentrations. The production of glucocorticoids by the adrenal cortex is also shown; these are upregulated by inflammatory cytokines and enhance A-SAA synthesis, but themselves downregulate the systemic acute-phase response. ACTH, corticotropin.

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Although the liver is the major site of APP synthesis, the extrahepatic tissue/cellular expression of a wide range of APPs has been documented [89]. In most mammalian species, SAA1 and SAA2 are predominantly synthesized in the liver while SAA3 is the main isoform expressed at extrahepatic sites [10]. In mouse, for example, the extrahepatic synthesis of Saa2 has only been definitively documented in the kidney and intestine, while that of Saa1 appears to be restricted to the kidney [49]. Saa3, however, is produced in a wide range of tissues and cell types including macrophages [48,50,51,90]. Mouse A-SAA mRNA has also been detected by in situ hybridization in unstimulated and LPS-stimulated intestinal and liver epithelial cells and in stimulated convoluted tubules of the kidney [91], and by Northern-blot analyses in both mouse bone marrow stromal cells following induction by either TNF or IL-1 [92] and the jejunal mucosa in response to LPS-induced endotoxemia [93]. This last study also showed, by immunohistochemistry, that A-SAA protein expression is mainly in the lamina propria cells of the mucosa and the intestinal lumen. In all of the last three studies, the detection methods used could not identify which specific A-SAA isoform or combination of isoforms was expressed. In contrast with constitutive human SAA4 (below), mouse Saa4 mRNA has not been found extrahepatically [16]. In rats, SAA3 mRNA, but not the SAA1/2 mRNA, has been found extrahepatically following LPS, but not turpentine, treatment in the lung, ileum and large intestine [53]. However, in a third rodent species, i.e. hamster, SAA1, SAA2 and SAA3 mRNAs have been found in a wide range of tissues including kidney, stomach, muscle, spleen, brain, heart, lung, intestine, ovary, testis and uterus [30,94].

In mink, extrahepatic SAA gene expression has also been reported in both unstimulated ovary, testis, brain and intestine and in LPS-induced lung, heart, kidney convoluted tubules and uterine endometrium epithelial cells. SAA expression was also observed in amyloidotic adrenal glands. Again the transcripts do not appear to be derived from either SAA1 or SAA2 but from a third, distinct, mRNA species [23,91]. Of the rabbit SAA members, only SAA3 mRNA has been found in a wide range of extrahepatic tissue and cell types [91,95], including, significantly, synovial fibroblasts (see below) [52]. The induction profiles of the three rabbit SAA mRNAs in the liver, and of rabbit SAA3 mRNA in other tissues, vary considerably with different inflammatory agents [95].

Although initial reports of extrahepatic SAA expression were mainly for species other than humans, human extrahepatic SAA expression has recently been documented. In humans, both A-SAA (SAA1 and SAA2) and C-SAA (SAA4) mRNAs are expressed in monocytic/macrophage cell lines, including THP-1 cells [96,97]. SAA mRNA has also been detected in various cell types in human atherosclerotic lesions (i.e. macrophages, endothelial and smooth muscle cells) and in adipocytes using an RNA probe with 81% and 71% homology to human SAA1/2 and SAA4, respectively [98]. Furthermore, cultured smooth muscle cells stimulated with IL-1 or IL-6 plus dexamethasone express SAA1, SAA2 and SAA4 mRNA [98], and both SAA1/2 and SAA4 proteins have been found in atherosclerotic lesions, particularly in foam cells [99]. SAA1/2 and SAA4 mRNAs have also been found in a wide range of non-hepatic cell lines using Northern-blot analysis [100]. More recently, a study employing non-radioactive in situ hybridization and immunohistochemical staining has confirmed the colocalization of extrahepatic SAA mRNA expression and protein production, in a wide range of histologically normal human tissues [101]. This was especially evident in the epithelium of the tissues but was also observed, to a lesser extent, in scattered lymphocytes and plasma cells in the stroma, and expression was also found in the endothelial cells lining blood vessels. More specifically, reverse transcription–PCR of breast (lobule and duct epithelium), large intestine, esophagus, kidney and spleen demonstrated expression of SAA1, SAA2 and SAA4 genes but not SAA3. These findings suggest a possible role for constitutively expressed A-SAA as an immunological defense molecule at local sites, thereby providing an immediate localized defense against inflammatory challenges during the time taken to mount a systemic response by increased hepatic synthesis.

APPs may be designated as either ‘type-1’ or ‘type-2’ according to their responsiveness to different classes of cytokines [1]. Type-1 APPs are induced by the IL-1-type cytokines which, in addition to IL-1α and IL-1β, include TNF-α and TNF-β. Type-1 APPs are generally synergistically induced when an IL-1-type cytokine is combined with an IL-6-type cytokine. Type-2 APPs are primarily induced by the IL-6-type cytokines which include IL-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor and cardiotrophin-1. These cytokines all elicit similar responses because of the association of their respective individual receptors with a common membrane-spanning signal-transduction molecule, gp130 (reviewed in [102]). Although IL-6-type cytokines are the main inducers of type-2 APPs, they can also stimulate most other APPs [1]. However, the induction of type-2 APPs by IL-6-type cytokines is not synergistically increased by IL-1-type cytokines; indeed, the latter generally have no effect on type-2 APP biosynthesis, and may even inhibit it.

A-SAA is classified as a type-1 APP as it can readily be induced by either IL-1β or TNF-α. Furthermore, each of these cytokines can synergize with IL-6 to dramatically increase A-SAA mRNA and protein synthesis in several human hepatoma cell lines including PLC/PRF/5, Hep3B and HepG2 [103–114]. In PLC/PRF/5 hepatoma cells, however, IL-6 alone only induces A-SAA mRNA and protein to levels that are barely detectable [108,115,116]. TNF-α induces A-SAA mRNA synthesis in mouse liver in vivo[103] and stimulates A-SAA protein synthesis in primary human hepatocytes [117]. Furthermore, there appears to be a significant correlation between TNF-α levels and A-SAA concentrations in patients with sepsis [118]. In PLC/PRF/5 cells the combination of IL-1β and IL-6 is as effective at inducing A-SAA mRNA and protein synthesis as a more complex physiological stimulus, monocyte-conditioned medium [115].

Some caution in assessing the physiological relevance of tissue culture studies should, however, be used as there is considerable heterogeneity between cell lines with respect to the induction of A-SAA by various cytokines. For example, in one study, IL-1β and IL-6, but not TNF-α, could each induce A-SAA protein synthesis in human primary hepatocytes; IL-6 was the most effective inducer but synergy with either IL-1β or TNF-α was not seen [116]. In contrast, others have reported considerable A-SAA protein induction in primary hepatocytes in response to IL-6 but not to IL-1β or TNF-α[119].

In addition to the heterogeneity that exists between the hepatic primary and established cell lines, all of the above investigations used simultaneous cytokine additions when combination treatments were analyzed. Such regimens do not mimic in vivo inflammatory situations where the sequential appearance of IL-1 followed by IL-6 [120,121] and TNF-α followed by IL-6 [118] have been demonstrated. Moreover, IL-6, which is induced by both IL-1 and TNF, suppresses both in vitro and in vivo production of these two cytokines [122]. Furthermore, stimulation with IL-1β before IL-6 is essential for maximal synergistic transcriptional induction of the human SAA2 promoter in vitro[123]. Although IL-6 is required to achieve maximal synergistic induction of SAA2, the reciprocal treatment, i.e. stimulation with IL-6 before IL-1β, results in significantly less synergistic activation of the SAA2 promoter. As well as temporal differences, variable cytokine levels have also been reported in human disease. For example, although A-SAA concentrations correlate with elevated TNF-α levels, IL-6 levels vary, in sepsis patients [118]. In contrast, in Castleman’s disease, which is characterized by abnormally high levels of IL-6 production and may lead to systemic reactive amyloidosis, both IL-6 and A-SAA serum levels are high, although both IL-1 and TNF are undetectable [124]. The above suggest that in vitro studies of A-SAA expression (see below), and other APP induction, using combinations of cytokines should be designed to accurately reflect the sequential mobilization of individual cytokines at different stages of the in vivo inflammatory cascade.

As mentioned above, A-SAA mRNA and protein synthesis can by induced by a variety of pro-inflammatory stimuli. Two of the principal inflammatory agents used in many of the studies of A-SAA expression engage different cytokines, i.e. LPS, which has systemic inflammatory effects, and turpentine, subcutaneous injection of which induces localized tissue damage. The major receptor for LPS on monocytes and neutrophils is CD14. Although interaction of LPS with CD14 leads to IL-1, TNF and IL-6 induction, this ligand receptor interaction does not appear to be necessary for the induction of APPs, including A-SAA, as they can be induced with LPS in CD14-deficient mice to levels comparable with those observed in wild-type mice [125]. Studies of cytokine knockout mice have been used to investigate the complexity of APP induction during the acute-phase response to LPS and turpentine [126–130].

Several studies of transgenic and knockout mice have underscored the complexity of the inflammatory processes involved in A-SAA induction. In response to LPS, IL-1β-deficient mice are able to mount an acute-phase response, including the induction of A-SAA, presumably as they retain a spectrum of cytokines, such as IL-1α, TNF-α and IL-6, that are quantitatively and qualitatively sufficient to produce a systemic signaling cascade as potent as that of wild-type mice [128]. In contrast, these mice do not produce A-SAA protein in response to subcutaneous turpentine injection, indicating that cytokines capable of substituting for IL-1β are not generated by this stimulus [129]. Similarly, IL-6-deficient mice are unable to mount a response to turpentine that is comparable with that of wild-type mice; in particular, these mice did not have elevated liver A-SAA mRNA or serum A-SAA protein levels. However, IL-6-deficient mice are not greatly compromised with respect to their capacity to mount an acute-phase response to LPS [126,127]. TNFα-deficient mice have no difference in serum A-SAA protein induction levels following turpentine treatment when compared with wild-type mice [130]. However, in either TNFα deficient or IL-6 deficient mice, A-SAA serum protein levels could not be induced by LPS to the levels seen in the wild-type, and in mice deficient in both TNFα and IL-6, A-SAA induction was almost completely absent. In contrast, A-SAA mRNA was enhanced after 24 h treatment with LPS in wild-type mice and those deficient in TNFα, IL-6 and TNFα plus IL-6, suggesting post-transcriptional regulation of A-SAA expression (see below). These results reinforce the likely mechanistic differences between an acute-phase reaction driven by infection as opposed to trauma.

IL-1α and IL-1β can each bind to two distinct receptors, IL-1RI and IL-1RII, both members of the Ig superfamily (reviewed in [131]), of which only the former is able to transduce a signal. A naturally occurring antagonist of IL-1, the IL-1 receptor antagonist (IL-1ra) (reviewed in [132]), inhibits IL-1 by direct competition for IL-1RI, which it binds more effectively than it does IL-1RII, resulting in no signal transduction [133–135]. Two forms of IL-1ra are produced by the same gene and differ only in their N-terminal sequences because of the use of alternative first exons [136]. The secreted form [137,138] is produced by monocytes, macrophages, neutrophils and fibroblasts, but apparently not by hepatocytes and endothelial cells [132]. The alternatively spliced intracellular form is a fully active protein [139,140], the precise function of which has yet to be elucidated. In mice undergoing experimentally induced inflammation the peak of secreted IL-1ra induction coincides with the highest APP concentrations achieved [141]. Consequently there is an integrated biological response during inflammation whereby downregulators of the acute-phase response are induced at the same time as the proinflammatory APPs. In addition to IL-1ra there is a second means of controlling IL-1 agonist activity, i.e. the IL-1RII which may serve as a membrane bound-trap or receptor decoy for IL-1 [142].

IL-1ra has been used to demonstrate the central role that IL-1 plays in A-SAA induction in several studies. It can effect a 75% reduction in the A-SAA protein synthesis induced by monocyte-conditioned medium in human hepatoma cells [109], and can specifically block the IL-1β-driven component of the synergistic transcriptional activation of the human SAA2 promoter by a combination of IL-1β and IL-6 (see below) [114]. In a BALB/c mouse model of casein-induced acute inflammation, hepatic A-SAA mRNA induction, at least initially, is entirely IL-1-dependent as recombinant mouse IL-1ra (rmIL-1ra), coadministered with the stimulus, completely abolishes the induction of Saa1 and Saa2 mRNA for up to 12 h [143]. In this model, very modest levels of circulating A-SAA protein are observed in the rmIL-1ra-treated mice, indicating that there is extrahepatic synthesis of A-SAA protein that is not exclusively driven by IL-1. Similarly in C57BL/6 mice in which silver nitrate has been used to induce an acute-phase response, coadministration of rmIL-1ra significantly reduces the magnitude of hepatic induction of Saa1 and Saa2 mRNA for up to 24 h [144]. In the latter case, however, rmIL-1ra intervention is qualitatively less effective at reducing serum Saa1 and Saa2 protein, most likely because the more extreme necrotizing nature of the stimulus probably mobilizes a more aggressive cytokine response that permits a significant level of A-SAA hepatic synthesis to occur.

Transcriptional regulation of saa

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

A number of studies of the human SAA2[112,145–149], mouse Saa3[150–153], rat SAA1[154–157] and rabbit SAA2[97,113,158–164] acute-phase promoters have been carried out to identify the cis-acting sequences and trans-activating transcription factors involved in the massive transcriptional upregulation of the A-SAAs following an acute-phase stimulus. In the A-SAA promoters of all four species, NF-κB and C/EBP transcription factor recognition sequences (reviewed in [165,166], respectively), which are involved in conferring cytokine responsiveness and/or cell specificity of expression on genes, have been located and characterized. Co-operation between members of these two transcription factor families, which associate with each other via their bZIP and Rel domains, respectively [167,168], appears to be important for the induction of A-SAA gene transcription. In addition, several other transcription factors have been implicated in the regulation of A-SAA gene expression (see below).

Human SAA2 promoter

Early experiments in which a human SAA2 genomic clone was transiently transfected into mouse L cells established that A-SAA expression is enhanced by IL-1β, TNF-α, IL-6 and IFN-γ[146,147]. Subsequent analysis of the activity retained by SAA2 promoter deletion constructs fused to a CAT reporter gene, together with electrophoretic mobility shift and footprinting assays in the human hepatic HepG2 cell line, identified several transcription-factor-binding sites (Fig. 4) [112,145,148]. A proximal NF-κB element, located between residues −91 and −82, is necessary for constitutive expression, and also for full responsiveness of the promoter to IL-1β and IL-6 when either is used alone. In addition, it is needed for the synergistic activation that is apparent when these two cytokines are used in combination. IL-1β can induce binding, maximal at 5–30 min after induction, of a NF-κB-like protein [145] that is similar to the constitutive H2TF1 transcription factor [KBF1 or p(50)2][169]. Although IL-6 does not directly induce transcription factor binding to this site, it has a reduced capacity to activate the SAA2 promoter when the site is mutated [112]. Thus it is likely that there is some co-operativity between transcription factors and/or target sites such that the IL-6-mediated effect interacts with the proximal NF-κB site.


Figure 4. Regulatory elements of mammalian A-SAA promoters. The relative positions of transcription-factor-binding regions of the human SAA2, mouse Saa3, rat SAA1 and rabbit SAA2 promoters are shown and are discussed in the text. The transcription start sites of the genes are indicated by +1 and an arrow. Transcription-factor-recognition sites are NF-kB, C/EBP, AP-2, SAS, which binds SAF, SEF-1 and YY1. In the case of the rabbit, Sp1 has been shown to affect the promoter through its association with SAF.

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A C/EBP site (−184 to −171), identified by methylation-interference footprinting, is also required for the full response to either IL-1β or IL-6 administered alone, and to generate the maximum synergistic activation that can be achieved by combining these two cytokines [112]. IL-6 alone and the IL-1β plus IL-6 combination both induce similar changes in the pattern of factor binding to this site from 15 min through to 16 h after treatment. Binding can be prevented by preincubation of IL-6-treated nuclear extracts with antiserum against C/EBPβ (NF-IL6), indicating that the transcription factors are C/EBPβ or members of the C/EBP family.

Nuclear extracts from cells treated with either IL-1β alone or IL-1β plus IL-6 can interact with synthetic NF-κB targets to form NF-κB bandshift complexes that can be detected within 15 min of treatment and that subsequently decrease quantitatively through to 16 h [112]. Antibody against the p50 subunit of NF-κB can supershift these complexes, establishing that they are at least partly composed of NF-κB p50. A constitutive factor that does not react with anti-p50 can also be detected; this is not affected by cytokine treatment but may compete with NF-κB for the site, as its binding is enhanced by mutating the site to preclude NF-κB binding. Co-transfection experiments with NF-κB subunit (p50, p52 and p65) expression vectors indicate that co-operativity between the p65 subunit and C/EBPβ are largely responsible for the synergistic induction of the SAA2 promoter. This may be due to a direct association between p65 and C/EBPβ, as has been demonstrated for p50 [167], or p65 may interact via endogenous p50 bound to the proximal NF-κB site.

More recently, electrophoretic mobility-shift assays with HepG2 nuclear extracts have shown that NF-κB p65 and p50, but not p52 or C-Rel, can bind the proximal NF-κB site in response to IL-1 and that C/EBPβ and C/EBPδ, but not CEBPα, bind the C/EBP site in response to IL-6 [149]. Co-expression of NF-κB p65 with C/EBPβ and C/EBPδ showed co-operative association of the former with both C/EBP proteins; this association appears to be through direct protein to protein interaction. Mutation analysis of NF-κB p65 suggests that the C-terminal regions of both the Rel homology and the activation domains are important for the interaction with C/EBPβ[149].

In addition to the NF-κB and C/EBP sites described above, the human SAA2 promoter has a distal NF-κB site between residues −635 and −626, which is in reverse orientation to the proximal NF-κB site, and a 3′ adjacent C/EBP site between residues −582 and −572 (145), both of which appear to lie in an upstream repressor element. Mutation of the distal NF-κB site permits a doubling of both constitutive and IL-1β-stimulated expression. A constitutive nuclear factor with presumptive repressor activity, which binds the region spanning the NF-κB and adjacent C/EBP sites, is displaced when an IL-1β-inducible NF-κB-like factor binds to the NF-κB site. The constitutive factor may be a member of a family of constitutive C/EBP-like negative factors that interfere with IL-1-inducible binding of positive NF-κB-like factors [170].

The kinetics of human A-SAA promoter engagement are being investigated by us using a construct containing 1.2 kb of the human SAA2 upstream region fused to a luciferase reporter gene [114,123]. When transiently transfected into HepG2 cells, the construct can be effectively stimulated by inflammatory mediators [114], in a manner that closely mimics that of the endogenous SAA2 gene in established hepatoma culture systems [112,115]. The level of induction of SAA2-promoter-driven transcription by the IL-1-type cytokines, IL-1β and TNF-α, increases with time. In contrast, IL-6 has its greatest effect at the early time points and induces progressively less transcription at later time points, thus establishing that the kinetics of promoter engagement by the IL-1-type and IL-6-type cytokines are quite distinct. Combined agonist treatments, i.e. IL-1β plus IL-6 and TNF-α plus IL-6, attain levels of transcriptional induction at least 10-fold higher than single cytokine treatments; however, the kinetics of transcriptional induction achieved are qualitatively similar to IL-6 alone (i.e. both are greater at early time points). This indicates that IL-6 is integral to the early massive synergy observed when present with the IL-1-type cytokines, and that it may be the dominant partner in driving changes in promoter activity [114]. To achieve maximal synergistic induction of the SAA2 promoter, the order of cytokine addition is important; IL-1β followed by IL-6 is much more effective than the reciprocal order [123]. Consequently, the relative timing of exposure of the SAA2 promoter to specific signals generated by individual cytokines constitutes an additional important level of control. Furthermore, on the evidence available to date, maximum in vitro effects are produced by addition of cytokines in the order that reflects their appearance in the in vivo inflammatory cascade (see above).

Mouse Saa3 promoter and the SAA enhancer factor (SEF-1)

The 350-bp region immediately upstream from the transcription-start site of the mouse Saa3 gene is sufficient for both cytokine-induced and cell-specific expression of the gene [150]. Within this section there is a proximal response element between residues −39 and −79 which is necessary for hepatic expression of the gene and which contains two C/EBP-binding sites (Fig. 4) [151]. There is also a distal response element between −128 and −169 that is required for responses to conditioned medium and IL-1α[150]. The distal response element consists of three binding regions, all of which are required for full cytokine induction. Two of these are C/EBP sites with different binding affinities and the third site binds a novel constitutive transcription factor, the SAA enhancer factor 1 (SEF-1) [152]. As in the case of the rabbit SAA2 promoter (see below), C/EBP-β and C/EBP-δ bind the mouse Saa3 C/EBP sites with highest affinity in the induced state. NF-κB also interacts with an atypical motif, between residues −155 and −164, within the region encompassing the distal response element [153]. NF-κB p65, but not p50, can transactivate the Saa3 promoter while coexpression of either with C/EBP family members results in efficient induction of the promoter, indicating that synergy between these two transcription factor families occurs in mouse Saa3 gene transcription [153].

Rat SAA1 promoter and the yin and yang (YY1) transcription factor

The rat SAA1 gene has five known cis-regulatory elements within the 304 bp immediately upstream of its transcription start site (Fig. 4) [154,155]. This region responds to conditioned medium and IL-1 in a cell-type-specific manner, i.e. transcription driven by the rat SAA1 promoter appears to be restricted to liver cells. The most distal three sites, which include a C/EBP site, are required to mediate a higher basal activity of the promoter but are not essential for cytokine responsiveness. A more proximal 66-bp section which contains the remaining two sites is sufficient to confer cytokine responsiveness in a no-cell-specific manner. This cytokine response unit contains a second C/EBP site, with a lower binding affinity than the upstream site, and a NF-κB recognition site; both of these are required for cytokine responsiveness and they interact co-operatively to induce gene expression [154,155].

A third transcription factor, YY1, binds the rat SAA1 cytokine response unit in hepatic cells [156]. YY1 is a zinc finger protein that is expressed ubiquitously in mammalian cells and either represses or activates gene transcription depending on the particular gene promoter and its cellular environment [171]. Both basal and cytokine-mediated rat SAA1 expression are repressed by YY1, which competes with NF-κB for overlapping binding sites in the cytokine response unit [156]. Rat SAA1 gene transcription appears to be determined by the ratio of YY1 to NF-κB rather than changes in the relative binding activity of YY1, as this remains constant after induction by conditioned medium. YY1 is always present in the nucleus and is therefore able to bind and repress the rat SAA1 promoter constitutively. In contrast, most of the cellular pool of NF-κB is held inactive in the cytoplasm in a complex with its inhibitor, IκB. After an inflammatory stimulus, IκB is phosphorylated and subsequently degraded to release NF-κB which can then translocate to the nucleus (reviewed in [165]). Consequently, the concentration of active NF-κB in the nucleus rises dramatically while YY1 levels remain constant, YY1 is displaced from the SAA1 promoter and the gene is transcriptionally induced. When signals originating with the underlying pro-inflammatory stimulus cease, active nuclear NF-κB levels drop to permit YY1 to re-establish its interaction with the promoter to repress transcription. In addition to direct DNA binding, YY1 may also function through interactions with other proteins; therefore, C/EBP, in addition to NF-κB, may be required to displace YY1 from the rat promoter in vivo.

Recently the tissue-specific transcription factor AP-2 has been shown to interact with two sites in the rat SAA1 promoter, one of which, like the YY1 site, overlaps the NF-κB site [157]. AP-2 acts as a dominant inhibitor of SAA1 gene transcription, as it can displace NF-κB from the promoter but not vice versa. As its expression is absent in liver cells and it appears to act as an inhibitor of SAA gene expression in non-hepatic cells, AP-2 may be a key component in defining tissue-specific SAA expression in response to various cytokine combinations under different pro-inflammatory conditions.

Rabbit SAA2 promoter and the SAA-activating sequence (SAS)-binding factor (SAF)

The rabbit SAA2 promoter has two adjacent functional C/EBP-binding elements in addition to an NF-κB element in its promoter region (Fig. 4). Initial studies showed C/EBP-β, C/EBPδ and NF-κB p65 to be involved in the cytokine induction of this gene in the liver [158,161,172]. Activated p65 and C/EBP form heterodimers that associate with the three binding elements to varying degrees; these heterodimers are more potent inducers of transcription than either factor alone and promote transcription via both NF-κB and C/EBP sites [161]. More recently it has been reported that the two pro-inflammatory agents, LPS and turpentine, differentially activate, both quantitatively and qualitatively, the transcription factors that interact with the rabbit SAA2 promoter [159]. Turpentine, the more potent inducer of A-SAA, activates higher levels of C/EBP for longer periods of time than LPS. However, it does not activate NF-κB, unlike LPS which activates p65. Increased levels of C/EBPβ and C/EBPδ and decreased levels of C/EBPα relative to those present in the uninduced state appear to be important in the induction of rabbit A-SAA [158,159]. This is consistent with the finding that, during an LPS-induced acute-phase response in 2-month-old mice, rapid dramatic increases in C/EBPβ and C/EBPδ mRNA expression are observed while C/EBPα is downregulated [173]. However, C/EBPα is known to play an important role in the acute-phase response of neonatal mice (0–3-h-old); wild-type mice injected with LPS exhibit a strong acute-phase response which includes increased A-SAA gene expression, which is not evident in LPS-injected C/EBPα knockout mice despite the latter having similar high levels of C/EBPβ and C/EBPδ mRNA and DNA-binding activities [174]. Consequently, age-dependent factors may determine the capacity of an individual to respond to any given stimulus and may turn out to be important in the pathologies associated with chronic inflammation.

Treatment with IL-6 causes different transcription factors to be induced in hepatic and non-hepatic cell lines [113]. In the human hepatic cell line HepG2, IL-6 only induces C/EBP-binding activity, particularly that of heterodimers of C/EBP-α and C/EBP-β[113]. In contrast, moderate constitutive levels of all C/EBP isoforms are present in untreated non-hepatic cells, i.e. rabbit lung fibroblasts and synoviocytes, but their binding activities are not induced after treatment with IL-6. Instead, the rabbit SAA2 promoter is activated by a novel, IL-6-responsive nuclear factor, SAF, which is present in the nuclei of a range of extrahepatic cells, including the above, as well as those of HepG2 cells. Mutational analyses have established that the rabbit SAA2 promoter region has three SASs, all of which are essential for the IL-6-mediated transcriptional induction of the gene by SAF in non-hepatic cells [113]. The absence of C/EBP-binding activity in non-hepatic cells prompted the suggestion that C/EBP binding at extrahepatic sites is prevented by an inhibitor such as CHOP-10. This protein has strong homology to the bZIP DNA-binding domain of C/EBP family members through which it can form heterodimers with both C/EBP-α and C/EBP-β, thereby preventing them from binding their target sequences and activating gene transcription [175].

SAF cannot be detected until one hour after IL-6 treatment; this, together with the fact that SAF induction can be prevented by cycloheximide, indicates that this factor is made de novo in response to an IL-6-derived signal. Subsequent phosphorylation appears to be a requirement for SAF function, as its capacity to induce transcription can be reduced by phosphatase and protein kinase inhibitors and increased by protein phosphatase inhibitors [113].

At least three SAF cDNAs have been cloned by screening a rabbit brain cDNA expression library with a synthetic DNA probe containing multiple SASs [162]. All three belong to a protein family which share C-terminal zinc finger domains. SAF-1 appears to be the homolog of human MAZ/ZF87 and mouse Pur-1. The former is involved in the regulation of c-myc and serotonin 1 A receptor and the latter in insulin gene expression. SAF-5 and SAF-8 are unique. All three isoforms have similar binding sequence specificities upon interaction with the SAS element but they have different tissue expression profiles. An SAF-8-specific cDNA probe can detect approximately equal amounts of SAF mRNA in mouse heart, brain, spleen, lung, liver and kidney, testis and skeletal muscle, while SAF-5 is predominantly expressed in skeletal muscle, and a SAF-1 non-specific cDNA probe detects varying levels of SAF mRNA in all of the above tissues. The DNA-binding activity of SAF increases both in response to LPS or IL-6 and through post-translational phosphorylation modification [162].

Another zinc finger transcription factor, Sp1, is involved in the induction of the rabbit SAA2 promoter in response to a range of mediators including LPS, minimally modified low-density lipoprotein (MM-LDL), IL-1 and IL-6 in extrahepatic cell lines [97,160,163,164]. The analysis of such cell lines may prove useful in understanding the potential role of A-SAA in the pathogenesis of disease. Human THP-1 cells express A-SAA and are useful for studying the impact of the acute-phase response on monocytes/macrophages, as they can be made to differentiate by incubation with LPS [96,99]. Such treatment of THP-1 cells, transfected with SAF cDNA, causes activation of the overexpressed SAF protein, which, in turn, transactivates a cotransfected SAS element–reporter gene construct [160]. Sp1 can interact with SAF by forming a stable heteromeric complex which has a higher affinity for the SAS element and is therefore a better transactivator of the SAA2 promoter than either SAF or Sp1 alone [160]. MM-LDL can also induce SAF-binding and C/EBP-binding activity in THP-1 cells, thereby upregulating A-SAA mRNA expression, while native unmodified LDL has little effect [164]. As might be predicted from the above, mutations in the SAF and C/EBP, but not the NF-κB, recognition sequences reduce the responsiveness of the rabbit SAA2 promoter to MM-LDL. Like LPS, MM-LDL treatment causes Sp1 and SAF to form a heteromeric complex with enhanced activity.

The activities of C/EBP family members are modified by treatment of THP-1 cells with MM-LDL [164]. Following such treatment, both the absolute amount and the activity of C/EBPα and C/EBPδ are increased, suggesting that there is de novo protein synthesis or import to the nucleus allied to functional enhancement. This contrasts with C/EBPβ and SAF which are subject to functional modulation without any apparent change in their concentration. As the pathogenesis of atheroscelerosis involves changes in macrophage phenotypes possibly in response to oxidized lipoproteins, the above cellular processes may contribute to the expression of A-SAA in atherosclerotic lesions.

Cultured rabbit synovial fibroblasts, i.e. HI682 cells, have provided a useful model for studying A-SAA expression in the context of degenerative joint diseases such as rheumatoid arthritis. These cells produce A-SAA in response to inflammatory mediators by a mechanism involving SAF and Sp1 activation [163]. A-SAA is found in the synovial fluid of human rheumatoid arthritis patients [176,177], and it can induce collagenase [27,52] and the matrix metalloproteinases 2 and 3 [178] in both human and rabbit rheumatoid synovial fibroblasts. In addition, constitutive SAA3 mRNA expression has been demonstrated in rabbit corneal fibroblasts [179]. IL-1β and IL-6, which are present at high levels in patients with rheumatoid arthritis, both individually induce A-SAA gene expression in rabbit synovial cells, and in combination have a synergistic inductive effect [163]. This is achieved through the induction of the transcription factors Sp1 and certain SAF isoforms, distinct from constitutive SAF isoforms, in synovial cells. As in THP-1 cells, Sp1 forms a heteromeric complex with SAF to bind the SAA2 promoter. Cytokine treatment causes Sp1 mRNA and protein levels to rise and mediates the post-translational modification of SAF; together these changes produce a net increase in DNA-binding activity. This process requires the cytokine-driven phosphorylation of Sp1 and, at least one of the SAF isoforms [163].


Naturally occurring glucocorticoids are steroid hormones produced by the adrenal cortex (Fig. 3) which affect intermediary metabolism (e.g. stimulation of glycogen deposition by the liver) and act as anti-inflammatory mediators (e.g. cortisone). Synthetic glucocorticoids such as dexamethasone, an analog of cortisol, are derivative steroid drugs and have proved to be potent suppressors of immune and inflammatory reactions; consequently they are widely used as therapeutic agents [180].

Glucocorticoid receptors (GRs) are part of an inactive cytoplasmic complex comprising heat shock proteins and immunophils [181,182]. When the GR is bound by glucocorticoids, it dissociates from the heat shock protein complex, and rapidly translocates to the nucleus where it acts as a composite transcription factor (reviewed in [183]). Glucocorticoids act via two main mechanisms. (a) The first is direct interaction with the promoters of genes, e.g. the IL-6 gene [184], that have a recognition site, the glucocorticoid-responsive element (GRE), to which the GR complex binds. GREs may be clustered with other transcription-factor-binding sites in so-called glucocorticoid-responsive units (GRUs) [185]. Although GRs are expressed in most cell types the specificity of cellular glucocorticoid responses are determined in part by the interaction of GRs with tissue-enriched transcription factors at the GRUs. (b) The other mechanism is indirect interaction with the promoters of genes that do not have GREs or GRUs, via their effect on transcription factors such as AP-1 and NF-κB [186–188]. Glucocorticoids can physically interact with AP-1 to prevent its binding to recognition sites in target genes such as those encoding collagenase and IL-2, thereby repressing transcription [189–192]. Activated GR can also interact directly with NF-κB subunits (p65, p50 and c-Rel) thus preventing them from binding DNA. In addition, dexamethasone reduces the levels of p65 that can be achieved in the nucleus in response to TNF-α by either destabilizing the protein or, more likely, sequestering it in the cytoplasm [193]. Dexamethasone can also cause increased transcription from the gene encoding IκBα, the cytoplasmic inhibitor of NF-κB [194–196]. Consequently, even under conditions that promote IκBα degradation, increased synthesis of IκBα stimulated by glucocorticoids can be sufficient to bind NF-κB and retain it in the cytoplasm.

Glucocorticoids are upregulated by both IL-1α/β and TNF-α, and also enhance the capacity of these cytokines to induce APP synthesis in the liver. After their synthesis, glucocorticoids participate in the control of the acute-phase response by providing negative feedback on cytokine production (Fig. 3). Dexamethasone is a potent inhibitor of both IL-1β[197] and IL-6 [184], and acts at both a transcriptional and post-transcriptional level.

Dexamethasone enhances the induction of A-SAA mRNA by pro-inflammatory cytokines and LPS-conditioned medium in both human [100,106,108] and mouse [198] cell culture systems. Furthermore, increased levels of A-SAA are found in the serum of mice treated with dexamethasone, IL-1β and IL-6 [199]. Deletion reporter constructs containing 1.2 and 1.1 kb of the human SAA2 and SAA4 promoters, respectively, are unresponsive to dexamethasone (C. M. Uhlar, unpublished observation), indicating that the GREs or other control elements affected by glucocorticoids are more than 1 kb upstream of the transcription-start sites. There are precedents for the existence of eukaryotic enhancers, including GRUs, located many kilobases from the gene they regulate. For example, full glucocorticoid induction of rat tyrosine aminotransferase depends on the co-operative interaction of two GRUs 2.5 and 5.5 kb upstream of the transcription-start site [200,201].

Post-transcriptional regulation of a-saa

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

Post-transcriptional mechanisms have also been implicated in the regulation of A-SAA. In early mouse studies, the 2000-fold LPS-mediated induction of mature hepatic A-SAA mRNA was not matched by the increase in the rate of transcription which, at 300-fold, was about an order of magnitude lower [46]. Furthermore, the absolute level of hepatic mRNA continued to accumulate even as the transcription rate was decreasing, i.e. the former peaked at 12 h whereas the latter peaked at 3 h. In a subsequent study, Saa3 mRNA levels in mouse liver-derived BNL cells stimulated with conditioned medium from mouse macrophage cells increased in the absence of any measurable effect on transcription [202]. Similar results have been obtained for human cells. In human Hep3B hepatoma cells treated with IL-1β plus IL-6, A-SAA transcription has been estimated to increase by only 23-fold during the time that mRNA accumulates to 1000 times normal levels [111]. In this study, the transcriptional peak was at 12 h whereas mRNA levels had not even begun to decline by 72 h. The disparity between transcriptional upregulation and the net effect on cytoplasmic mRNA concentration in these studies strongly suggests the involvement of post-transcriptional regulatory mechanisms such as increased mRNA stability.

Increased A-SAA mRNA synthesis does not inevitably result in proportionally increased A-SAA protein synthesis. In an early study, human PLC/PRF/5 hepatoma cells treated with monocyte-conditioned medium accumulated A-SAA mRNA [115]. From 3 h, the first time point at which it could be detected, A-SAA mRNA increased 76-fold by 24 h. Over the same time period, however, A-SAA protein synthesis only increased 4–6-fold, suggesting that the available pool of A-SAA mRNA was used efficiently early in the in vitro acute-phase response and progressively less efficiently over time. Similar results were obtained with IL-1β plus IL-6 treatment. The lower protein levels at later times do not appear to be due to either alterations in A-SAA protein export transit time or changes in the efficiency of translational initiation, but may be due to decreased A-SAA mRNA translational efficiency via a mechanism involving the slowing of ribosome migration [115].

The stability of a given mRNA and the efficiency with which it is translated may be dependent on poly(A) tail length [203–206]. In both mouse [207,208] and dog [21] models an acute-phase stimulus causes a rapid transcriptional upregulation of hepatic A-SAA which is closely followed by an increase in mRNA size because of a lengthened poly(A) tail. Although this hyperadenylation is not coincident with transcriptional modulation, it may be dependent on it or may be driven by the same underlying mechanism. A similar increase in poly(A) tail length is likely to occur in all mammals, including humans, after transcriptional upregulation in vivo; however, the first A-SAA mRNA that can be detected in cytokine-stimulated human hepatoma cells is the hyperadenylated form, thereby precluding a detailed analysis of the kinetics of, and relationships between, the above two processes in vitro[115]. After the appearance of ‘full length’ A-SAA mRNA in vivo or in vitro, a gradual reduction in A-SAA mRNA length, attributable to poly(A) tail shortening, has been observed in mouse, dog, human and arctic char [20,21, 115,207,208]. Moreover, in tissue culture studies using the transcriptional inhibitor actinomycin D, the intrinsic stability of A-SAA mRNA does not appear to be affected by poly(A) tail shortening (which is unaffected by treatment), suggesting that A-SAA mRNA degradation may require the de novo synthesis of a short-lived factor, e.g. a nuclease [111,115,209]. This speculation opens the intriguing possibility that cytokine induction may initiate a cascade of intracellular changes that control the post-transcriptional fate of A-SAA mRNA and the transcriptional products of other APP genes.

Deadenylation of A-SAA mRNA has been confirmed in a more recent study [210]. In unstimulated mice, the Saa1 poly(A) tail ranges from 20 to 30 adenines in size. However, during an azocasein-induced acute-phase response, this changes to 100–300 adenines after 8 h and to 20–70 adenines after 48 h. Trace amounts of decapped Saa1 mRNA can be detected in untreated, 8, 48 and 72 h samples, suggesting that decapping follows deadenylation and precedes degradation. This chronology is analogous to the situation in yeast where deadenylation at the 3′ end causes mRNA decapping at the 5′ end and subsequent 5′-3′ exonucleolysis (for references see [210]).

Saa function and disease associations

  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The A-SAA proteins have been highly conserved through evolution [18–20] and this, together with the dramatic induction of A-SAA expression in response to potentially life-threatening physiological challenges, suggests a critical protective role in the acute-phase response. A-SAA may play a basic role in pathogen defense, as its mRNA levels rise continuously in response to increasing pathogen load in arctic char infected with the furunculosis-associated pathogen, A. salmonicida; in the final fatal stages of disease, A-SAA mRNA may constitute as much as 10% of total hepatic mRNA [20]. Moreover, other inflammatory mediators also elicit a dose-dependent response in other species, e.g. in Syrian hamsters increasing doses of IL-1, IL-6 and TNF drive the synthesis of increasing levels of hepatic A-SAA mRNA [211].

The best documented clinical condition associated with sustained high expression of A-SAA is amyloid A amyloidosis. However, the range of clinically important functions that have been proposed for the SAA family members include some that may also have other negative consequences during chronic inflammation. The functions fall into pro-inflammatory, lipid transport/metabolism related and anti-inflammatory categories; however, current research is placing the emphasis on the first two.

Immune-related functions of A-SAA

There are two reported immune-related functions of A-SAA. First, A-SAA can induce ECM-degrading enzymes, such as collagenase, stromelysin, matrix metalloproteinases 2 and 3, which are important for repair processes after tissue damage [27,178,179]. However, prolonged expression of A-SAA, and the consequent long-term production of these enzymes, may play a role in degenerative diseases such as rheumatoid arthritis. Second, in vitro studies have provided compelling evidence that A-SAA can act as a chemoattractant for such immune cells as monocytes, polymorphonuclear leukocytes, mast cells and T lymphocytes [212–217]. If A-SAA has this property in vivo, its local production would result in the active recruitment of these cell types to inflammatory sites and the augmentation of local inflammation.

In addition to the capacity of A-SAA to induce ECM-degrading enzymes, two recent studies have reported induction of pro-inflammatory cytokines by A-SAA. In the first, rhSAA1.1 induced monocytic THP-1 cells to synthesize IL-1β, IL-1ra and soluble TNFR-II mRNA and protein but had no effect on the synthesis of soluble TNFR-I, IL-6 or TNF-α[215], while in the second, SAA–ECM complexes induced TNF-α secretion by human T lymphocytes [217]. The above suggest that A-SAA may, itself, have cytokine-like properties which would require cellular receptors or binding sites. Rabbit SAA3 can bind, both specifically and saturably to synovial fibroblasts, with an affinity of ≈ 20 nm and 800 000 binding sites per cell which is compatible with the existence of cellular receptors for SAA3 on these cells [218].

Chemoattractants, such as N-formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the chemokines, act by binding seven-transmembrane-spanning G–protein-coupled receptors [219]. Several recent papers have presented evidence that A-SAA binds to such a receptor [216,220,221]. The hallmark of fMetLeuPhe and chemokine interactions with their receptors on monocytes via G–protein-dependent pathways are Ca2+ mobilization and protein kinase C activation. A-SAA binding is characterized by some of the same phenotypic effects, i.e. protein kinase C appears to be involved in the A-SAA signaling pathways that result in monocyte recruitment, and, on binding, rhA-SAA transiently induces monocyte intracellular Ca2+ levels from extracellular sources. Therefore it is probable that a G-protein is involved in A-SAA chemotaxis [220]. Furthermore, as the chemotactic effects mediated by rhA-SAA are pertussis toxin sensitive, they probably involve the Gi subset of G-proteins [220]. Indeed the participation of a Gi-protein in A-SAA chemotaxis has subsequently been confirmed in another cell type, i.e. human mast cells which are normally resident in tissues but can accumulate at inflammatory sites [216].

Recently cross-desensitization experiments have established that the agonist fMetLeuPhe can bind to the same receptor as A-SAA [221]. Although rhA-SAA does not desensitize the Ca2+ mobilization induced by chemokines or fMetLeuPhe, fMetLeuPhe can affect rhA-SAA-induced Ca2+ mobilization; this suggests that A-SAA acts via a low-affinity fMetLeu Phe receptor. There are two seven-transmembrane-spanning G-protein-coupled receptors, FPR and FPRL1 (or lipoxin A4 receptor), to which fMetLeuPhe can bind with high and low affinity, respectively, to effect Ca2+ mobilization. The latter has also been reported to be a high-affinity receptor for the eicosanoid lipid metabolite, lipoxin A4 and is expressed in monocytes, neutrophils and hepatocytes [222]. In human embryonic kidney cells transfected with and expressing FPRL1, but not in cells expressing FPR, rhA-SAA can induce both pertussis-toxin-sensitive Ca2+ mobilization and migration. Furthermore, ligand-binding experiments show that rhA-SAA can specifically bind to such FPRL1-transfected cells (Kd = 64 nm and 42 000 binding sites per cell), in addition to monocytes and neutrophils, and is a more efficient agonist of this receptor than fMetLeuPhe [221].

Lipid-related functions of A-SAA

When A-SAA is released into the circulation it is incorporated into HDL [223,224], the class of lipoprotein particles that play an important role in the prevention of atherosclerosis by both mediating reverse cholesterol transport and inhibiting the lipid (LDL) oxidation that promotes foam cell formation [225,226]. Consequently, the association of A-SAA with HDL during acute inflammation may alter HDL metabolism and cholesterol transport and promote a pro-atherogenic phenotype [227–232]. There are two main hypotheses on the role that A-SAA plays in modulating cholesterol transport during inflammation (reviewed in [233]). One is that A-SAA alters reverse cholesterol transport to allow delivery of lipid, particularly cholesterol, via HDL to peripheral cells that may have an increased requirement for cholesterol to facilitate tissue regeneration at inflammatory sites [234,235]. The other is that A-SAA facilitates removal of the large quantities of cholesterol liberated at sites of tissue damage during inflammation [236,237].

In addition to its association with HDL, a number of lines of evidence implicate A-SAA in lipid metabolism/transport. A-SAA binds cholesterol and promotes its cellular uptake [238,239] and A-SAA-HDL particles have a higher affinity for macrophages and a lower affinity for hepatocytes than HDL [227,240]. Furthermore, several enzymes involved in cholesterol metabolism, including lecithin–cholesterol acyltransferase, group-IIA non-pancreatic secretory phospolipase A2 (sPLA2) and neutral cholesterol ester hydrolase, are affected by induction of A-SAA during the acute-phase response. Under normal physiologic conditions, ApoA-I is the main apolipoprotein of HDL (reviewed in [241]). However, during the acute-phase response, A-SAA becomes the major HDL-associated apolipoprotein and the particle becomes depleted of ApoA-I [14]. As HDL is the site of cholesterol esterification which occurs through the action of lecithin–cholesterol acyltransferase, which, in turn, requires ApoA-I as an activating cofactor [242], the relative lack of ApoA-I on A-SAA–HDL during inflammation may account for the positive correlation observed between plasma A-SAA and unesterified cholesterol levels, and the negative correlation with lecithin–cholesterol acyltransferase activity [228]. sPLA2 is made by vascular smooth muscle cells [243] and is also found in human atherosclerotic lesions [244]. It was initially suggested that sPLA2 would act on the HDL particle in a manner similar to that of hepatic lipase, hydrolyzing HDL phospholipids and redistributing cholesterol from the core to the surface, thereby facilitating its transfer to cell plasma membranes [245,246]. A recent study has found that human A-SAA–HDL is two- to threefold more susceptible to hydrolysis by sPLA2 than is normal HDL [247]. Furthermore A-SAA, but not C-SAA, enhances the activity of sPLA2[246], and overexpression of sPLA2 in transgenic mice can suppress HDL levels by 30% [234]. Consequently, the enhanced hydrolysis of acute-phase HDL by sPLA2 appears to be mediated by A-SAA itself. Another product of sPLA2 hydrolysis is arachidonic acid, which is the precursor of the pro-inflammatory eicosanoids. Human SAA1, but not apoA-I, can enhance the biosynthesis of the eicosanoids, thromboxane A2 and prostaglandins E2 and F in calcium-ionophore-stimulated monocytes [76]. As atherogenesis may be promoted by the accumulation of released products of arachidonic acid, e.g. lysophosphatidylcholine which causes membrane damage, and toxic oxygenated fatty acids, A-SAA and sPLA2 may be involved in the pathogenesis of this disease. Finally, neutral cholesterol ester hydrolase activity is increased in the presence of A-SAA favoring free cholesterol formation [237].

Anti-inflammatory roles of A-SAA

Almost two decades ago, A-SAA was implicated in the suppression of in vitro immune responses to antigens by affecting T cell–macrophage interactions and helper T lymphocyte function [248–250]. Human A-SAA was subsequently found to be a potent inhibitor of lymphocyte, HeLa and MRC5 cell function [251]. A potential feedback relationship between SAA and immunoregulatory cytokines was proposed based on the observation that A-SAA inhibits IL-1-induced and TNF-induced fever in mice [252]. IL-1 and TNF cause fever by inducing prostaglandin E2 synthesis in the hypothalamus; prostaglandin E2 production correlates directly with the magnitude of the fever [253]. Platelet aggregation has also been reported to be inhibited by A-SAA [70,71], and A-SAA modestly induces prostaglandin I2 which is also an antiaggregation agent [72]. As both platelets and the range of mediators released by them upon activation are involved in inflammatory and thrombotic processes, these findings suggest that A-SAA may act to down-regulate such pro-inflammatory events during the acute-phase response.

A-SAA has also been reported to bind to neutrophils and, like other apolipoproteins such as ApoA-I [254], inhibit the oxidative burst response, suggesting that it may help prevent oxidative tissue damage during inflammation [255,256]. However, this effect may be concentration-dependent; in a recent study, acute-phase concentrations of rhA-SAA could inhibit both directed neutrophil migration and degranulation [256], whereas the inhibition of respiratory burst was restricted to lower concentrations. These results suggest that A-SAA may produce quite different effects according to local concentration and that the anti-inflammatory effects intrinsic to this APP may be selective and specific rather than systemic.

A-SAA, amyloid A protein and amyloidosis

A-SAA is the serum precursor of amyloid A protein [60,61] which is the principal component of the amyloid deposits found in the heterogeneous group of disorders, the amyloid A amyloidoses [257]. One of these, reactive amyloidosis, is a well-documented occasional clinical consequence of chronic inflammation (e.g. rheumatoid arthritis) and recurrent acute inflammatory episodes (e.g. tuberculosis).

The predominant amyloid A protein type found in amyloidotic tissues corresponds to the N-terminal two thirds of A-SAA, i.e. the first 76 residues of mature human A-SAA [60]. However, both smaller and larger amyloid A protein types, 45–95 residues, have also been found [10,62, 63,258,259]. Multiple proteolytic cleavage events may be involved in the processing of A-SAA as it appears to be degraded first into an intermediate product with the same size and antigenic properties as amyloid A protein and is subsequently processed further. Amyloid A amyloidosis may therefore be the result of the incomplete digestion, and consequent accumulation, of amyloidogenic intermediate peptides of A-SAA.

A large number of cell-associated and serum proteases have been implicated in the degradation of A-SAA (Table 2). A-SAA is probably degraded after its disassociation from HDL, as full-length A-SAA can be found in amyloid fibrils [260–263]. Furthermore, lipid-free A-SAA can be degraded [264]in vitro to form fibrils [265]. In addition, A-SAA degradation in vivo is inhibited by lipoproteins, in particular HDL [80,266], and differences between the plasma clearance rates of A-SAA and ApoA-I also suggest that the former is not associated with HDL when it is degraded [83,266].

Table 2. Proteases involved in the degradation of A-SAA.
Cell-associated activities[290,291,292]
Serum serine proteases (thrombin, kallikrein and plasmin)[293,266]
Elastase, collagenase and stromelysin[293,295,296]
Cathepsin B[296]
Aspartate proteases and cathepsin D[297,298]
Cathepsin G[264]

Mouse A-SAA can bind to two of the major components of the basement membrane, i.e. laminin and type-IV collagen, with high and low affinity, respectively [267]. Its binding to laminin is inhibited by entactin, a protein that normally binds laminin. As the basement membrane matrix appears to be disrupted in the vicinity of amyloid deposits, these interactions further support the involvement of A-SAA as an active, rather than a passive, participant in the process of amyloidosis.

Inflammatory macrophages and reticuloendothelial cells have both been implicated in the formation of amyloid A protein and amyloid fibrils from A-SAA, each of which processes can occur intracellularly [268–271]. Mouse peritoneal macrophages can bind either HDL or A-SAA–HDL, which undergo receptor-mediated endocytosis and subsequent retro-endocytosis [272,273]. The binding of the latter probably involves heparin sulfate for which A-SAA has a binding site [78]. Mouse peritoneal macrophages can also endocytose exogenous mouse Saa1 and Saa2, which are transported to endosome–lysosomes and are partially degraded to products similar in mass to amyloid A [274].

Although derivatives of SAA1 and SAA2 proteins are both found in human and mouse amyloid A deposits [275–277], those from SAA1 predominate [62,277–279]. This bias in favor of SAA1 deposition is supported by in vitro studies in which rhSAA1 had greater amyloid fibril-forming potential than either rhSAA2 or rhSAA4 [265].


  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References

The highly conserved A-SAA proteins, which are all dramatically induced by various pro-inflammatory stimuli, have been identified in a wide range of vertebrate species. Their upregulation involves both transcriptional and post-transcriptional mechanisms. An intricate picture of the former is emerging with respect to cytokine-mediated induction and cell/tissue specificity. Many transcription factors that engage the A-SAA promoters have been identified, including members of the NF-κB and C/EBP families. These appear to be principally responsible for the synergistic induction of A-SAA observed in response to the IL-1-type, e.g. IL-1β and TNF-α, and the IL-6-type cytokines. Other transcription factors also regulate A-SAA expression; YY1 and AP-2 inhibit A-SAA gene transcription under various conditions, and SAF and Sp1 are involved in extrahepatic expression. It also appears that the kinetics of A-SAA gene induction in response to IL-1β and IL-6 is complex and depends critically on the order in which these mediators appear in the pro-inflammatory cytokine cascade.

The A-SAAs are highly conserved across evolutionarily distinct vertebrate species with respect to both their sequence and inductive capacity, and it is consequently generally assumed that they have a crucial, yet ill-defined, protective role during inflammation. The small size of A-SAA and the special circumstances under which it is induced make it likely that only one of the documented properties of A-SAA, i.e. immune or lipid transport/metabolism related, is that for which there is an over-riding need during the acute-phase response. Such a pre-eminent role would mandate the strong positive selection to which A-SAA has been subjected. The other properties attributed to A-SAA may merely reflect the consequences of the physiological adaptations made to facilitate its primary function. For example, the association of A-SAA with lipid, and the resulting impact on reverse cholesterol transport, may simply be due to the need to effectively sequester systemic pools of A-SAA before its targeted and/or timed release as an immune effector molecule. Future studies will more precisely define this and other aspects of A-SAA biology.


  1. Top of page
  2. Abstract
  3. The acute-phase response
  4. The serum amyloid a (saa) family
  5. Expression and induction of saa
  6. Transcriptional regulation of saa
  7. Post-transcriptional regulation of a-saa
  8. Saa function and disease associations
  9. Conclusions
  10. References
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  1. Note: serum amyloid A nomenclature used in this review follows the new guidelines from the SAA Subcommittee of the Amyloidosis Nomenclature Committee [Sipe, J.D. & Committee (1999) Amyloid: Int. J. Exp. Clin. Invest.6, 67–69] and Table 1.