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• W2772088864 abstract "Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation. Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation. Acute-phase serum amyloid A (SAA) is a family of acute-phase proteins that have been evolutionarily conserved for approximately 500 million years (1.Eklund K.K. Niemi K. Kovanen P.T. Immune functions of serum amyloid A.Crit. Rev. Immunol. 2012; 32: 335-348Crossref PubMed Scopus (168) Google Scholar, 2.Uhlar C.M. Burgess C.J. Sharp P.M. Whitehead A.S. Evolution of the serum amyloid A (SAA) protein superfamily.Genomics. 1994; 19: 228-235Crossref PubMed Scopus (123) Google Scholar). Teleologically, this conservation suggests an important role for SAA in host defense. The precise functions of the SAA family have not been defined, although it is suggested that SAA has antibacterial properties (3.Derebe M.G. Zlatkov C.M. Gattu S. Ruhn K.A. Vaishnava S. Diehl G.E. MacMillan J.B. Williams N.S. Hooper L.V. Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection.eLife. 2014; 3: e03206Crossref PubMed Scopus (88) Google Scholar) and can serve as a chemoattractant for monocytes and neutrophils (4.Badolato R. Wang J.M. Murphy W.J. Lloyd A.R. Michiel D.F. Bausserman L.L. Kelvin D.J. Oppenheim J.J. Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue infiltration of monocytes and polymorphonuclear leukocytes.J. Exp. Med. 1994; 180: 203-209Crossref PubMed Scopus (428) Google Scholar). SAA can also promote inflammation by inducing inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, as well as metalloproteinases in smooth muscle cells and macrophages (5.Furlaneto C.J. Campa A. A novel function of serum amyloid A: a potent stimulus for the release of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-8 by human blood neutrophil.Biochem. Biophys. Res. Commun. 2000; 268: 405-408Crossref PubMed Scopus (191) Google Scholar, 6.Webb N.R. De Beer M.C. Wroblewski J.M. Ji A. Bailey W. Shridas P. Charnigo R.J. Noffsinger V.P. Witta J. Howatt D.A. et al.Deficiency of endogenous acute-phase serum amyloid A protects apoE-/- mice from angiotensin II-induced abdominal aortic aneurysm formation.Arterioscler. Thromb. Vasc. Biol. 2015; 35: 1156-1165Crossref PubMed Scopus (31) Google Scholar, 7.Song C. Shen Y. Yamen E. Hsu K. Yan W. Witting P.K. Geczy C.L. Freedman S.B. Serum amyloid A may potentiate prothrombotic and proinflammatory events in acute coronary syndromes.Atherosclerosis. 2009; 202: 596-604Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 8.Lee H.Y. Kim M.K. Park K.S. Bae Y.H. Yun J. Park J.I. Kwak J.Y. Bae Y.S. Serum amyloid A stimulates matrix-metalloproteinase-9 upregulation via formyl peptide receptor like-1-mediated signaling in human monocytic cells.Biochem. Biophys. Res. Commun. 2005; 330: 989-998Crossref PubMed Scopus (114) Google Scholar, 9.Migita K. Kawabe Y. Tominaga M. Origuchi T. Aoyagi T. Eguchi K. Serum amyloid A protein induces production of matrix metalloproteinases by human synovial fibroblasts.Lab. Invest. 1998; 78: 535-539PubMed Google Scholar). SAA may act as a danger-associated molecular pattern molecule by interacting with multiple pattern recognition receptors, such as Toll-like receptors, CD36, and FPRL1 (1.Eklund K.K. Niemi K. Kovanen P.T. Immune functions of serum amyloid A.Crit. Rev. Immunol. 2012; 32: 335-348Crossref PubMed Scopus (168) Google Scholar). The pro-inflammatory functions of SAA should be seen in the context of recent data that SAA is an innate immune molecule capable of activating the NLR family pyrin domain containing 3 (NLRP3) inflammasome, although the precise molecular events underlying this activation are not completely understood (10.Ather J.L. Ckless K. Martin R. Foley K.L. Suratt B.T. Boyson J.E. Fitzgerald K.A. Flavell R.A. Eisenbarth S.C. Poynter M.E. Serum amyloid A activates the NLRP3 inflammasome and promotes Th17 allergic asthma in mice.J. Immunol. 2011; 187: 64-73Crossref PubMed Scopus (183) Google Scholar, 11.Migita K. Izumi Y. Jiuchi Y. Kozuru H. Kawahara C. Nakamura M. Nakamura T. Agematsu K. Masumoto J. Yasunami M. et al.Serum amyloid A induces NLRP-3-mediated IL-1beta secretion in neutrophils.PLoS One. 2014; 9: e96703Crossref PubMed Scopus (33) Google Scholar, 12.Niemi K. Teirila L. Lappalainen J. Rajamaki K. Baumann M.H. Oorni K. Wolff H. Kovanen P.T. Matikainen S. Eklund K.K. Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway.J. Immunol. 2011; 186: 6119-6128Crossref PubMed Scopus (203) Google Scholar, 13.Yu N. Liu S. Yi X. Zhang S. Ding Y. Serum amyloid A induces interleukin-1beta secretion from keratinocytes via the NACHT, LRR and PYD domains-containing protein 3 inflammasome.Clin. Exp. Immunol. 2015; 179: 344-353Crossref PubMed Scopus (34) Google Scholar). SAA has a strong association with cardiovascular disease, where it can serve as a predictor of mortality (14.Morrow D.A. Rifai N. Antman E.M. Weiner D.L. McCabe C.H. Cannon C.P. Braunwald E. Serum amyloid A predicts early mortality in acute coronary syndromes: A TIMI 11A substudy.J. Am. Coll. Cardiol. 2000; 35: 358-362Crossref PubMed Scopus (118) Google Scholar). The key question is whether SAA serves as more than a marker, but rather as a mediator of acute vascular syndromes (15.Filep J.G. El Kebir D. Serum amyloid A as a marker and mediator of acute coronary syndromes.Future Cardiol. 2008; 4: 495-504Crossref PubMed Scopus (13) Google Scholar). The SAA family comprises at least four isoforms that likely arose from gene duplication. In humans, two SAA isoforms (SAA1 and SAA2) are highly induced during an acute-phase response, with plasma levels increasing up to 1,000-fold or more. SAA1 and SAA2 are predominantly expressed in the liver, but also in extrahepatic tissues, and are found in plasma associated with HDL (16.Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar). The mouse homologs (SAA1.1 and SAA2.1) are similarly induced and share more than 90% amino acid identity (2.Uhlar C.M. Burgess C.J. Sharp P.M. Whitehead A.S. Evolution of the serum amyloid A (SAA) protein superfamily.Genomics. 1994; 19: 228-235Crossref PubMed Scopus (123) Google Scholar). The SAA4 isoform is an HDL-associated apolipoprotein that is ∼55% homologous to SAA1/2 and constitutively expressed at relatively low levels in both human and mouse liver. To date, no known immune or inflammatory activities have been ascribed to SAA4 (17.Whitehead A.S. de Beer M.C. Steel D.M. Rits M. Lelias J.M. Lane W.S. de Beer F.C. Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein.J. Biol. Chem. 1992; 267: 3862-3867Abstract Full Text PDF PubMed Google Scholar, 18.De Buck M. Gouwy M. Wang J.M. Van Snick J. Opdenakker G. Struyf S. Van Damme J. Structure and expression of different serum amyloid A (SAA) variants and their concentration-dependent functions during host insults.Curr. Med. Chem. 2016; 23: 1725-1755Crossref PubMed Scopus (129) Google Scholar) and its function is largely unknown. Mice express a third acute-phase SAA isoform, SAA3, which is not expressed in humans due to a premature stop codon in the human Saa3 gene (19.Kluve-Beckerman B. Drumm M.L. Benson M.D. Nonexpression of the human serum amyloid A three (SAA3) gene.DNA Cell Biol. 1991; 10: 651-661Crossref PubMed Scopus (101) Google Scholar). Mouse SAA3 is 69% homologous to SAA1.1/2.1 (2.Uhlar C.M. Burgess C.J. Sharp P.M. Whitehead A.S. Evolution of the serum amyloid A (SAA) protein superfamily.Genomics. 1994; 19: 228-235Crossref PubMed Scopus (123) Google Scholar) and is induced in the liver as well as extrahepatic tissues (20.Meek R.L. Benditt E.P. Amyloid A gene family expression in different mouse tissues.J. Exp. Med. 1986; 164: 2006-2017Crossref PubMed Scopus (144) Google Scholar), particularly adipocytes, and to a lesser extent in macrophages in inflammatory settings. Prior studies have also reported SAA3 expression in testis, spleen, intestine, and kidney in acute inflammatory states (21.Benditt E.P. Meek R.L. Expression of the third member of the serum amyloid A gene family in mouse adipocytes.J. Exp. Med. 1989; 169: 1841-1846Crossref PubMed Scopus (48) Google Scholar), as well as in intestinal epithelial cells (22.Reigstad C.S. Lunden G.O. Felin J. Backhed F. Regulation of serum amyloid A3 (SAA3) in mouse colonic epithelium and adipose tissue by the intestinal microbiota.PLoS One. 2009; 4: e5842Crossref PubMed Scopus (105) Google Scholar), lymphocytes, lymphoid follicles, and plasma cells of many normal tissues (23.Urieli-Shoval S. Cohen P. Eisenberg S. Matzner Y. Widespread expression of serum amyloid A in histologically normal human tissues. Predominant localization to the epithelium.J. Histochem. Cytochem. 1998; 46: 1377-1384Crossref PubMed Scopus (182) Google Scholar). In general, SAA3 is considered to be the “extrahepatic” SAA, exerting similar pro-inflammatory activities in mouse macrophages (24.Meek R.L. Eriksen N. Benditt E.P. Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages.Proc. Natl. Acad. Sci. USA. 1992; 89: 7949-7952Crossref PubMed Scopus (141) Google Scholar) and adipose tissue (21.Benditt E.P. Meek R.L. Expression of the third member of the serum amyloid A gene family in mouse adipocytes.J. Exp. Med. 1989; 169: 1841-1846Crossref PubMed Scopus (48) Google Scholar, 25.Chiba T. Han C.Y. Vaisar T. Shimokado K. Kargi A. Chen M.H. Wang S. McDonald T.O. O'Brien K.D. Heinecke J.W. et al.Serum amyloid A3 does not contribute to circulating SAA levels.J. Lipid Res. 2009; 50: 1353-1362Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) as human SAA1 and SAA2. Whether SAA3 contributes to systemic SAA levels in mice has been the focus of a limited number of studies, with equivocal results. Whereas SAA3 was determined to be a circulating SAA bound to HDL in mice undergoing an acute inflammatory response to lipopolysaccharaide (LPS) injection (24.Meek R.L. Eriksen N. Benditt E.P. Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages.Proc. Natl. Acad. Sci. USA. 1992; 89: 7949-7952Crossref PubMed Scopus (141) Google Scholar, 26.de Beer M.C. de Beer F.C. McCubbin W.D. Kay C.M. Kindy M.S. Structural prerequisites for serum amyloid A fibril formation.J. Biol. Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar), it was not detected in HDL in plasma from ob/ob mice with chronic low-grade inflammation (25.Chiba T. Han C.Y. Vaisar T. Shimokado K. Kargi A. Chen M.H. Wang S. McDonald T.O. O'Brien K.D. Heinecke J.W. et al.Serum amyloid A3 does not contribute to circulating SAA levels.J. Lipid Res. 2009; 50: 1353-1362Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The goals of the present study were to carefully define the degree to which SAA3 contributes to circulating SAA during an acute-phase response, the extent to which SAA3 is associated with HDL, and its cellular sources. C57BL/6 mice were obtained from Jackson Laboratory, Bar Harbor, ME. Mice deficient in SAA1.1 and SAA2.1 were bred to obtain a >99.9% C57BL/6 background (27.de Beer M.C. Webb N.R. Wroblewski J.M. Noffsinger V.P. Rateri D.L. Ji A. van der Westhuyzen D.R. de Beer F.C. Impact of serum amyloid A on high density lipoprotein composition and levels.J. Lipid Res. 2010; 51: 3117-3125Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Mice deficient in SAA3 have been described (28.den Hartigh L.J. Wang S. Goodspeed L. Ding Y. Averill M. Subramanian S. Wietecha T. O'Brien K.D. Chait A. Deletion of serum amyloid A3 improves high fat high sucrose diet-induced adipose tissue inflammation and hyperlipidemia in female mice.PLoS One. 2014; 9: e108564Crossref PubMed Scopus (53) Google Scholar). For simplicity, C57BL/6 mice, mice lacking SAA1.1/SAA2.1, and mice lacking SAA3 are referred to as SAA-WT, SAA1.1/2.1-DKO, and SAA3-KO, respectively. An acute-phase response was elicited by intraperitoneal injection of 25 or 100 μg LPS (Escherichia coli 0111:B4; Sigma Chemical Co.), then mice were humanely euthanized and plasma and tissue were obtained for analyses and preparation of HDL (LPS dose and duration for each experiment are indicated in the figure legends). Control animals were not injected. All studies were performed with the approval of the University of Kentucky or University of Washington Institutional Animal Care and Use Committee. Primary hepatocytes were isolated from SAA-WT and SAA1.1/2.1-DKO mice 6 h after injection with 25 μg LPS using a two-step perfusion method (29.Ji A. Wroblewski J.M. Cai L. de Beer M.C. Webb N.R. van der Westhuyzen D.R. Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI.J. Lipid Res. 2012; 53: 446-455Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Briefly, the liver was first perfused with Ca+2/Mg+2-free HBSS containing 10 mM glucose, 10 mM HEPES, and 0.3 mM EDTA and then with HBSS containing 0.05% collagenase type IV (C5138; Sigma), 1.3 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 10 mM HEPES. Hepatocytes were washed by repeated low speed centrifugation (50 g for 2 min) and cell viability (normally 90–95%) was assessed by trypan blue exclusion. Cells were plated onto 12-well plates precoated with rat tail collagen (354236; BD Biosciences, Bedford, MA) at a density of 2 × 105 cells/well and cultured at 37°C under 5% CO2 in Williams' Medium E (GIBCO) containing 10% fetal bovine serum (GIBCO), 2% penicillin-streptomycin, 1% sodium pyruvate, 1% l-glutamine, and 1% insulin-transferrin-selenium (GIBCO). Cells were cultured for 18 h after plating and then collected for RNA isolation. Total RNA was isolated from mouse livers and hepatocytes using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA from epididymal fat was isolated by RNeasy lipid tissue mini kit (7484; Qiagen). RNA samples (9 μg) were treated with DNase 1 (TURBO DNA-free™ kit (AM1907; Invitrogen) for 30 min at 37°C. RNA from liver, epididymal fat (0.5 μg), or cultured cells (0.1 μg) was reverse transcribed into cDNA using the reverse transcription system (4368814; Applied Biosystems). After 4-fold dilution, 5 μl were used as a template for real-time RT-PCR. Amplification was done for 40 cycles using Power SYBR Green PCR Master Mix kit (4367659; Applied Biosystems). Quantification of mRNA was performed using the ΔΔCT method and normalized to GAPDH for liver and hepatocytes or 36B4 for epididymal fat. Primer sequences are as follows: GAPDH (accession number NM_008084) 5′-CTC ATG ACC ACA GTC CAT GCC A-3′, 5′-GGA TGA CCT TGC CCA CAG CCT T-3′ 36B4 (accession number NM_007475) 5′-AAG CGC GTC CTG GCA TTG TCT-3′, 5′-CCG CAG GGG CAG CAG TGG T-3′ SAA1.1/2/1 (accession number NM_009117.3) 5′-CTC CTA TTA GCT CAG TAG GTT GTG-3′, 5′-CAC TTC CAA GTT TCT GTT TAT TAC CC-3; SAA3 (accession number NM_011315.3) 5′-TTT CTC TTC CTG TTG TTC CCA GTC-3′, 5′- TCA CAA GTA TTT ATT CAG CAC ATT GGG A-3′. Aliquots of plasma (7 μl) or HDL (13.5 μg) isolated from LPS-injected mice were subjected to isoelectric focusing (IEF), as previously described (26.de Beer M.C. de Beer F.C. McCubbin W.D. Kay C.M. Kindy M.S. Structural prerequisites for serum amyloid A fibril formation.J. Biol. Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar), using an Ampholine gradient consisting of 20% (v/v) Ampholine (pH 3–10), 40% (v/v) Ampholine (pH 4–6.5), and 40% (v/v) Ampholine (pH 7–9) (Pharmacia LKB Biotechnology Inc.). Electrofocused samples were subjected to immunochemical analysis, as described (26.de Beer M.C. de Beer F.C. McCubbin W.D. Kay C.M. Kindy M.S. Structural prerequisites for serum amyloid A fibril formation.J. Biol. Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar). The SAA isoforms were identified by immunochemical staining utilizing rabbit anti-mouse SAA antibody to identify SAA1.1 and SAA2.1 and rabbit anti-mouse SAA3 (a gift from Dr. Phillip Scherer, University of Texas Southwestern) to identify SAA3. Lipoproteins in 50 μl of plasma pooled from six mice injected with 100 μg LPS were separated by fast protein (FP)LC utilizing a Superose 6 column (17-5172-01; GE Healthcare, Uppsala, Sweden). The cholesterol content of 0.5 ml fractions was determined. SAA1.1/2.1 and SAA3 in plasma and FPLC fractions were determined by a mouse SAA ELISA kit (TP802M; Tridelta Development Ltd., Ireland) and a mouse SAA3 ELISA kit (EZMSAA3-12K; EMD Millipore, Danvers, MA), respectively. HDL from mice that were injected with 25 μg LPS were subjected to SDS-PAGE or IEF. Coomassie-stained bands corresponding to the apparent molecular mass of SAA separated by SDS-PAGE (∼12 kDa) or the isoelectric point (pI) of SAA3 separated by IEF (∼9.2) were excised, washed, and subjected to in-gel trypsin digestion (30.Kamelgarn M. Chen J. Kuang L. Arenas A. Zhai J. Zhu H. Gal J. Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS.Biochim. Biophys. Acta. 2016; 1862: 2004-2014Crossref PubMed Scopus (76) Google Scholar). The resulting tryptic peptides were extracted and subjected to LC-MS/MS using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The LC-MS/MS results were searched against the mouse portion of the UniProt database using a local Mascot server (version 2.3; Matrix Science) for protein identification, including methionine oxidation and cysteine carbamidomethylation as allowed side chain modifications. A false discovery rate of 1% was used in decoy search for the high-confidence peptides. Proteins with a score of at least 30 for single high-confidence peptides were considered positive identifications. UniProt protein names and identifier numbers are used throughout this work. Paraffin-embedded liver sections (8 μm thick) were fixed in 4% paraformaldehyde and incubated with 3% H2O2 for 10 min to quench endogenous peroxidases. After blocking endogenous biotin using an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA), slides were incubated overnight at 4°C with rabbit anti-mouse SAA (26.de Beer M.C. de Beer F.C. McCubbin W.D. Kay C.M. Kindy M.S. Structural prerequisites for serum amyloid A fibril formation.J. Biol. Chem. 1993; 268: 20606-20612Abstract Full Text PDF PubMed Google Scholar) (1:2,000 dilution). Sections were subsequently incubated with biotinylated anti-rabbit IgG and avidin-peroxidase according to the vendor's instructions (Vectastain Elite ABC kit; Vector Laboratories). Immunoreactivity was visualized using a DAB kit (K3468; Dako North America, Inc., Carpinteria, CA), followed by counterstaining with hematoxylin. Immunoreactivity was visualized using the red chromogen, 3-amino-9-ethyl carbazole (Vector Laboratories). Images were captured on a Nikon ECLIPSE 80i microscope with the aid of NIS-Elements BR 4.00.08 software. In situ hybridization was performed essentially as described (31.Li Y. Ji A. Weihe E. Schafer M.K. Cell-specific expression and lipopolysaccharide-induced regulation of tumor necrosis factor alpha (TNFalpha) and TNF receptors in rat dorsal root ganglion.J. Neurosci. 2004; 24: 9623-9631Crossref PubMed Scopus (101) Google Scholar). For PCR amplification of liver cDNA, the following SAA gene-specific primers were used: murine SAA1.1/2.1 (NM_009117/NM_011314) forward primer: 5′-ctcctattagctcagtaggttgtg-3′, reverse primer: 5′-cacttccaagtttctgtttattaccc-3′ and murine SAA3 (NM_011315) forward primer: 5′-tttctcttcctgttgttcccagtc-3′, reverse primer: 5′-tcacaagtatttattcagcacattggg-3′. PCR was performed using 5 μl cDNA in a total volume of 50 μl containing 0.2 μM primer mix, 1× PCR buffer, 3.0 mM MgCl2, 200 μM dNTP mixture, and 1 U GoTaq Flexi DNA polymerase (Promega) and the following program: 2 min at 95°C, 40 cycles at (45 s at 95°C, 45 s at 56°C, and 45 s at 72°C), and a 10 min extension at 72°C. The PCR products were subcloned into pGEM-T (Promega) and their sequence identity was confirmed by double-stranded DNA sequencing. Plasmid constructs were linearized by SphI and NdeI for SAA1.1/2.1 and SAA3. Riboprobes in antisense and sense orientation were generated from linearized plasmid constructs by in vitro transcription using the appropriate RNA polymerases and 35S-UTP in a volume of 10 μl containing 1 μg of linearized plasmids. After 90 min incubation at 37°C, 10 U of RNase-free DNase I were added to the reactions and incubated for another 15 min. After adding RNase-free water to 20 μl and 20 μl sodium carbonate buffer [80 mM NaHCO3 and 120 mM Na2CO3 (pH 10.2)] for limited hydrolysis, the reaction was incubated at 60°C for the appropriate time [t = (Lo – Lf)/K × Lo × Lf, where Lo is the cDNA length, Lf is the expected length of the probes (i.e., 250 bp), and K is 0.11], as described (31.Li Y. Ji A. Weihe E. Schafer M.K. Cell-specific expression and lipopolysaccharide-induced regulation of tumor necrosis factor alpha (TNFalpha) and TNF receptors in rat dorsal root ganglion.J. Neurosci. 2004; 24: 9623-9631Crossref PubMed Scopus (101) Google Scholar). The reaction was stopped by adding 2 μl of 10% acetic acid. The probes were purified using Micro Bio-Spin P-30 columns (Bio-Rad). For in situ hybridization histochemistry, frozen 8 μm-thick liver sections were fixed in 4% paraformaldehyde for 60 min. After three washes in PBS and incubation in 0.4% Triton X-100 for 10 min, the slides were rinsed in deionized water and transferred to 0.1 M triethanolamine (pH 8.0). After 1 min incubation, acetic anhydride was added under strong stirring to a final concentration of 0.25% (v/v) and further incubated for 10 min. The slides were washed for 10 min in PBS and rinsed in deionized water prior to dehydration in 50% and 70% ethanol. Radioactive probes were diluted to 5 × 104 dpm/μl in hybridization solution [600 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA-Na2, 0.05% tRNA, 20 mM dithiothreitol, 1× Denhardt's solution, 10% dextran sulfate, 100 μg/ml sonicated salmon sperm DNA, and 50% formamide]. Prehybridized sections were incubated with 100 μl hybridization solution containing radioactive RNA probes at 60°C in a formamide humid chamber box for 16 h. After a series of washes, the sections were dehydrated in 50% and 70% ethanol. For autoradiographic detection, slides were coated with Kodak NTB-2 nuclear emulsion (Eastman Kodak, Rochester, NY) and exposed at 4°C for 5–30 days in the dark. Slides were developed in Kodak D19 solution and counterstained with cresyl violet. Photographic documentation was performed under bright-field illumination using a Nikon ECLIPSE 55i microscope with the aid of NIS-Elements BR 4.13.04 software. Data are expressed as mean ± SD. Results for Fig. 2, Fig. 3 were analyzed by one-way ANOVA followed by a Bonferroni posttest. Statistical analysis for Fig. 4D was performed using the two-tailed Student's t-test for unpaired data. P < 0.05 was considered statistically significant. All statistical analyses were carried out using GraphPad Prism 5.Fig. 3SAA protein in plasma. A. SAA isoforms in plasma of untreated SAA-WT and LPS-injected (100 μg LPS) SAA-WT and SAA3-KO mice were separated by IEF utilizing an Ampholine gradient, as described in the Materials and Methods, and pressure-blotted onto nitrocellulose. The SAA isoforms were identified by immunochemical staining utilizing rabbit anti-mouse SAA antibody to identify SAA1.1 and SAA2.1 (a) and rabbit anti-mouse SAA3 to identify SAA3 (b). B: SAA3 and SAA1.1/2.1 in the plasma of control SAA-WT and LPS-injected (100 μg LPS) SAA-WT, SAA3-KO, and SAA1.1/2.1-DKO mice were measured by ELISA utilizing kits specific for the mouse SAA isoforms. Values are the mean ± SD, n = 3–6. ****P < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4SAA3 association with HDL. Pooled plasma from six SAA-WT mice injected with 100 μg LPS was separated by FPLC and the total cholesterol (TC) content of eluted fractions was measured to determine the lipoprotein profile (y axis of panels A and B). The SAA1.1/2.1 content of FPLC fractions (A) and SAA3 content of FPLC fractions (B) were measured by isoform-specific ELISAs. C: HDL (d 1.063–1.21) was isolated by sequential density gradient ultracentrifugation from the plasma of SAA-WT mice injected with 25 μg LPS. Aliquots of plasma (7 μl) and HDL (13.5 μg) were subjected to IEF utilizing an Ampholine gradient, as described in the Materials and Methods, to separate the SAA isoforms, followed by pressure-blotting onto nitrocellulose. Equivalent amounts of HDL-cholesterol were loaded in each lane. SAA1.1 and SAA2.1 were detected with a rabbit anti-SAA antibody (a) and SAA3 was detected by an anti-mouse SAA3 antibody (gift of Dr. Phillip Scherer, University of Texas Southwestern) (b). D: Immunopositive bands from C were quantified by densitometric scanning and the values for isolated HDL were expressed relative to the corresponding values for plasma, which were arbitrarily given a value of 1. **P < 0.01, ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The classical murine acute-phase reactants, SAA1.1 and SAA2.1, are expressed primarily in the livers of mice and are dramatically upregulated following an inflammatory stimulus (20.Meek R.L. Benditt E.P. Amyloid A gene family expression in different mouse tissues.J. Exp. Med. 1986; 164: 2006-2017Crossref PubMed Scopus (144) Google Scholar). In contrast to SAA1.1 and SAA2.1, SAA3 is generally thought to be mainly expressed extra-hepatically, predominantly by adipocytes, but also by macrophages (21.Benditt E.P. Meek R.L. Expression of the third member of the serum amyloid A gene family in mouse adipocytes.J. Exp. Med. 1989; 169: 1841-1846Crossref PubMed Scopus (48) Google Scholar, 24.Meek R.L. Eriksen N. Benditt E.P. Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages.Proc. Natl. Acad. Sci. USA. 1992; 89: 7949-7952Crossref PubMed Scopus (141) Google Scholar). However, the relative expression of the various mouse acute-phase SAA isoforms in tissues and plasma has not been adequately documented, partly due to the lack of validated reagents that distinguish SAA1.1/2.1 and SAA3. The recent development of mice deficient in SAA1.1/2.1 (27.de Beer M.C. Webb N.R. Wroblewski J.M. Noffsinger V.P. Rateri D.L. Ji A. van der Westhuyzen D.R. de Beer F.C. Impact of serum amyloid A on high density lipoprotein composition and levels.J. Lipid Res. 2010; 51: 3117-3125Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) or SAA3 (28.den Hartigh L.J. Wang S. Goodspeed L. Ding Y. Averill M. Subramanian S. Wietecha T. O'Brien K.D. Chait A. Deletion of serum amyloid A3 improves high fat high sucrose diet-induced adipose tissue inflammation and hyperlipidemia in female mice.PLoS One. 2014; 9: e108564Crossref PubMed Scopus (53) Google Scholar) allo" @default.
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