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Identification of cAMP analogue inducible genes in RAW264 macrophages
Biochimica et Biophysica Acta 1492 (2000) 385^394
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Identi¢cation of cAMP analogue inducible genes in RAW264 macrophages Yukihiro Takahashi 1;2 , Masaaki Miyata 1;3 , Ping Zheng, Takayuki Imazato 4 , Andrew Horwitz, Jonathan D. Smith * Laboratory of Biochemical Genetics and Metabolism, Box 179, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 9 March 2000; received in revised form 1 May 2000; accepted 5 May 2000
Abstract RNA was isolated from RAW264 cells treated with or without 8-Br-cAMP and the differential display and subtractive hybridization methods were performed. One hundred and twenty-five differentially displayed bands were identified. Upon Northern blot analysis, only three of these bands were confirmed as cAMP inducible mRNAs, named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1^3). The cI-3 probe was identical to a previously known gene, gly96. Using the novel cI-1 and cI-2 partial cDNAs as probes, a mouse macrophage cDNA library was screened and the two full length genes were cloned, sequenced, and characterized as encoding large hydrophobic proteins. One hundred and fifteen partial cDNA clones from a subtractive hybridization library were also screened by Northern blot and 64 were found to be cAMP inducible. Of these, 45 represented 31 known unique genes in the GenBank nr database (cI-4^34), and 19 clones representing 15 unique sequences were not in the nr database (cI-35^49). One of the previously known genes was ABC1, the Tangier disease gene, which was identified from four independent partial cDNAs. ABC1 was upregulated in RAW cells by cAMP, concurrent with the cAMP induction of lipid efflux to apolipoprotein A1. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Di¡erential display; Subtractive hybridization; Cholesterol e¥ux; ABC1
1. Introduction The presence of monocyte-macrophage derived foam cells in the arterial intima is the earliest visible stage of atherosclerosis [1]. Since cholesterol loading of macrophages does not lead to scavenger receptor downregula-
Abbreviations : cI-n, cAMP inducible gene number n; apo, apolipoprotein ; DMEM, Dulbecco's modi¢ed Eagle's medium ; DGGB, DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% bovine serum albumin * Corresponding author. Fax: +1-212-327-7165; E-mail : [email protected] 1 These authors contributed equally to this work. 2 Present address: First Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-0042, Japan. 3 Present address: First Department of Internal Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. 4 Present address: Division of Biological Sciences, School of Science, Hokkaido University, kita-10, nishi-8, Sapporo 060-0810, Japan.
tion, these cells continue to take up modi¢ed lipoproteins as long as the substrate is present. Thus, cholesterol e¥ux is an important pathway to unload these cells and either inhibit the progression or lead to the regression of atherosclerosis. E¥ux of cholesterol from macrophages in vitro occurs by at least two distinct pathways. The ¢rst, as characterized by Rothblat and others, occurs as cholesterol di¡uses from a membrane with a high cholesterol to phospholipid ratio to an acceptor particle, like high density lipoprotein, with a lower cholesterol to phospholipid ratio [2]. This pathway is thought to be mediated by the scavenger receptor SR-BI protein [3]. The second pathway, as characterized by Yokoyama and others, occurs as cholesterol and phospholipids are transferred from cells to lipid free apolipoproteins (apo), or synthetic peptide analogues [4,5]. One apparent protein mediator of this pathway is the ABC1 gene, recently identi¢ed by several labs as the Tangier disease gene [6^9]. Fibroblasts from Tangier disease subjects are de¢cient in lipid e¥ux to apoA1 [10,11]. We previously reported that cAMP analogues induce apoE and apoA1 binding and uptake and promote cholesterol e¥ux from the RAW264 macrophage cell line to these apo acceptors [12]. This pathway is associated
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 3 3 - 0
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with coated pit endocytosis of apoA1 and its resecretion as a nascent lipoprotein [13]. Thus a study was performed to identify cAMP inducible genes that might be playing a role in this pathway. RNA di¡erential display and subtractive hybridization were used to identify cAMP inducible genes. Upon Northern blot con¢rmation, di¡erential display yielded only three cAMP inducible cDNAs, while subtractive hybridization was much more e¤cient yielding 64 inducible cDNAs. Both screens identi¢ed known genes and genes that were not present in the GenBank nr database. We obtained the full length cDNA clones for two of the unknown genes and characterize their mRNA and protein sequence, as well as the time course of their induction by cAMP. ABC1, the Tangier disease gene, was identi¢ed by four independent partial cDNAs as a cAMP inducible gene in RAW264 cells. Antisense transfections have shown that ABC1 plays a role in lipid e¥ux to apoA1 [6,9]. The current catalog of cAMP induced genes may be helpful in elucidating other genes that play a role in the cAMP mediated e¥ux of lipids to apoA1 in RAW264 cells. 2. Materials and methods 2.1. Cell culture The RAW264.7 cell line, derived from murine macrophages, was obtained from American Type Culture Collection and cultured in Dulbecco's modi¢ed Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For cAMP treatment, subcon£uent cells were incubated with or without 0.1^0.3 mM 8-Br-cAMP (Sigma) in DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% bovine serum albumin (Sigma, A6003) (referred to as DGGB) for 24 h. 2.2. RNA isolation Total RNA for di¡erential display and Northern blot analysis was isolated from RAW264.7 cells treated with or without 0.1 mM 8Br-cAMP for 24 h in DGGB by the standard guanidinium thiocyanate method [14]. For di¡erential display, possible DNA contamination was removed by treating with RNase free DNase I (Boehringer Mannheim) for 30 min at 37³C. Poly(A) RNA was extracted with the FastTrack 2.0 Kit (Invitrogen) from RAW264.7 cells treated for 24 h with 50 Wg/ml acetylated low density lipoprotein and with or without 0.3 mM cAMP (referred to as +cAMP RAW and 3cAMP RAW mRNA, respectively). The A260 /A280 ratio of the puri¢ed poly(A) RNA exhibited ranged from 1.8 to 2.0 and its integrity and equalization were con¢rmed by comparing their glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels by Northern blot analysis.
2.3. Di¡erential display Di¡erential display was performed by the method described by Liang and Pardee with minor modi¢cation [15]. Primer sets for di¡erential display were obtained from Operon Technologies. Reverse transcription to generate cDNA was performed using 400 ng of DNase I treated RNA in 1Ureverse transcription bu¡er, 10 mM dithiothreitol, 20 WM dNTP, and 1 WM of 3P primer mixture (T11AG, T11CG, T11GG). The solution was heated to 75³C for 5 min, cooled on ice, and 20 units of RNase inhibitor (Life Technologies), and 400 units of reverse transcriptase (SuperScript II, Life Technologies) were added. After incubation at 42³C for 90 min, the mixture was heated to 85³C for 5 min prior to storage at 320³C. To amplify the reverse transcriptase products, PCR was performed in duplicate with the cDNA from control or cAMP treated cells (above) in PCR bu¡er containing 2 WM of dNTP, 0.4 Wl of [35 S]dATP (Amersham), 1 WM of the 3P primer mixture, 1 WM separately of each of 26 speci¢c arbitrary decamers (5P primer), and 1 unit of Taq DNA polymerase (Perkin Elmer). PCR reactions were performed as follows : 1 cycle of 94³C for 2 min, 40 cycles of 94³C for 45 s, 35³C for 1 min and 72³C for 1 min, and 1 cycle of 72³C for 5 min. Denaturing loading bu¡er (United States Biochemical) was added and heated to 85³C for 5 min prior to loading onto 6% polyacrylamide sequence gels. Gels were run at 100 V, dried, and exposed directly to Kodak XAR-5 ¢lm at 380³C. Bands which appeared more prominently in lanes from cAMP treated cells were cut out and eluted by boiling in 100 Wl water for 10 min. DNA was recovered from the supernatant by ethanol precipitation in the presence of glycogen as a carrier. The recovered DNA was suspended in 10 Wl of TE (10 mM Tris^HCl,1 mM EDTA) and reampli¢ed in PCR bu¡er containing 20 WM of dNTP, 1 WM of the original 3P and 5P primers, and 1 unit of Taq DNA polymerase. PCR consisted of 40 cycles of 94³C for 1 min, 40³C for 1 min, and 72³C for 1 min, followed by a ¢nal extension at 72³C for 5 min. PCR products were analyzed by electrophoresis in 1.5% agarose gels stained with ethidium bromide, and then puri¢ed by phenol^chloroform extraction and ethanol precipitation. The puri¢ed DNA products were used as probes for Northern blot analysis and were subcloned into pCR2.1 using the TA cloning kit (Invitrogen). 2.4. Northern blot analysis Radioactive probes were generated using Amersham Megaprime labeling kit and [32 P]dCTP (Amersham). Alternatively, non-radioactive probes were prepared by PCR using the DIG system (Boehringer Mannheim). 10^15 Wg of total RNA, denatured in a formamide loading bu¡er, was run on 1.2% agarose formaldehyde gels. RNA was transferred to membranes and crosslinked with UV light.
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For tissue distribution expression analysis, total RNA was isolated from mouse liver, spleen, kidney, brain, and bone marrow derived macrophages. Bone marrow was isolated from femurs and cultured for 2 weeks in the presence of Lcell conditioned medium, as a source of macrophage colony stimulating factor, as previously described [16].
Sequences were scanned for the presence of the other nested primer denoting the end of cDNA insert, and the traces were examined to delete sequence at the 5P and 3P ends where the signal to noise ratio was poor.
2.5. Full length cDNA isolation and characterization
DNA and protein database searches were performed at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) using the blastn and blastp programs with the GenBank nr and dbEST databases.
Using the subcloned di¡erential display products as probes, a mouse macrophage cDNA phagemid library (Stratagene, catalog #937306) was screened by ¢lter lifts. After tertiary screening, independent cDNA clones were treated with an exonuclease III/mung bean nuclease deletion kit (Stratagene) and sequenced in both directions. The longest open reading frame was determined and translated into amino acid residues. The predicted proteins were subjected to Kyte^Doolittle hydrophobicity and Goldman^ Engelman^Steitz transfer free energy analyses using LaserGene software (DNAstar Inc.). The protein sequences were also analyzed by the SOSUI program to predict the number of transmembrane helices (http://azusa.proteome. bio.tuat.ac.jp/sosui/) [17]. 2.6. Subtractive cDNA library construction and analysis Using poly(A) mRNA from cAMP and control treated RAW cells, a library of cAMP induced cDNAs was constructed using the PCR-Select cDNA subtraction kit (Clontech). In this study, cDNA from +cAMP RAW cells was used as a tester and cDNA from 3cAMP RAW cells as a driver. These cDNAs were then digested with RsaI. The tester cDNA was divided into two portions and each was ligated with a di¡erent cDNA adapter (adapter 1 or adapter 2R). The ¢rst round of hybridizations consisted of an excess amount of driver cDNA added to each of the two tester cDNAs (adapter 1 and adapter 2R samples) at a driver:tester ratio of 30:1. The two samples from the ¢rst hybridization were mixed and freshly denatured driver cDNA was added for the second hybridization round, in which new hybrid molecules corresponding to di¡erentially expressed cDNA with di¡erent adapters on each end were formed. These hybrids were ampli¢ed exponentially by PCR (27 cycles). A secondary PCR ampli¢cation was then performed (10 cycles) using nested primers to enrich for di¡erentially expressed sequences. This combination of 27 cycles in the ¢rst PCR and 10 cycles in the second PCR was selected after checking the subtraction e¤ciency using GAPDH and cI-1 (see Section 3) as negative and positive cAMP induced cDNA controls, respectively. The PCR products were cloned into pT-Adv vector (Clontech). The subtractive cDNA library was obtained after transformation into DH5K Escherichia coli strain. Single pass automated £uorescent sequencing of clones was performed by the Rockefeller University Protein and DNA Technology Center using one of the nested primers.
2.7. Nucleotide and protein sequence comparisons
3. Results 3.1. Di¡erential display and Northern blot con¢rmation To identify cAMP inducible genes in RAW264 macrophages, mRNA di¡erential display was performed with total RNA isolated from RAW264 cells treated with or without 0.1 mM 8-Br-cAMP for 24 h. PCR ampli¢cations were performed with 26 primer combinations using duplicate RNA samples from control and cAMP treated cells. One hundred and twenty-¢ve PCR products were found in higher levels from the cAMP treated samples compared to the control samples. To con¢rm the gene regulation patterns observed in the di¡erential display assay, the 125 selected PCR products were cut from the gels, reampli¢ed,
Fig. 1. Di¡erential display of three genes. Di¡erential mRNA display was performed to compare patterns for RAW264 cells treated with or without 0.1 mM 8-Br-cAMP for 24 h. PCR ampli¢cations were performed with 26 primer combinations using duplicate RNA samples from control (3) and 8-Br-cAMP treated cells (+). The arrows point to the middle of the segment of the gel excised which gave rise to partial cDNA clones named cI-1, cI-2, and cI-3.
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Fig. 2. Northern blot con¢rmation of cAMP induced mRNAs. The PCR products of Fig. 1 were cut from the gels, reampli¢ed, and together with an ABC1 probe were hybridized with Northern blots prepared with 10 Wg of total RNA from control (3) and 8-Br-cAMP treated (+) RAW264 cells. The dramatic cAMP induction of speci¢c RNA molecules by Northern blot analysis was observed with the cI-1, cI-2, cI-3, and ABC1 probes. Marks to the left of the blots represent the migration of the 28S (top) and 18S (bottom) rRNA bands, detected by ethidium bromide staining.
and used to probe Northern blots prepared with RNA from control and cAMP treated RAW264 cells. Most of the probes gave very low or undetectable levels of expression in RNA from either control or treated cells. Some probes detected RNA expression on the Northern blots, but revealed no di¡erence between the control and treated cells. Three of the probes yielded RNA expression patterns that were robustly upregulated upon cAMP treatment, which we named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1^3). Fig. 1 shows the upregulation of bands corresponding to cI-1, cI-2, and cI-3 in the di¡erential display assay. Fig. 2 shows the dramatic induction of speci¢c RNA molecules by Northern blot analysis using the cI-1, cI-2, and cI-3 probes on RNA from 8-Br-cAMP treated and control RAW264 cells. 3.2. Characterization and analysis of cI-1, cI-2, and cI-3 cDNAs The PCR ampli¢ed fragments of cI-1, cI-2, and cI-3 were cloned into plasmid pCR2.1 and completely sequenced on both strands. The results revealed that all fragments were £anked by the sequences corresponding to the particular 3P and 5P primers used in the di¡erential display assay. The sizes of the cI-1, cI-2, and cI-3 partial cDNA fragments were 282, 384, and 466 bp, respectively. Their nucleotide sequences were analyzed by searching for homologies against the GenBank nr database. Clone cI-3 was identical to the gly96 gene (accession number X67644), which had been characterized as an immediate early serum responsive gene from mouse ¢broblasts [18].
At that time no characterized full length genes were identical to clones cI-1 and cI-2. Therefore, a mouse macrophage cDNA library (poly(dT) primed) was screened using cI-1 and cI-2 probes to obtain the full length cDNA. Nine independent phagemid cDNA clones were isolated with the cI-1 probe and eight independent cDNA clones were isolated with the cI-2 probe. All of the cDNA clones were sequenced on both strands. The cDNA clones contained varying lengths of 5P sequence and several clones of each con¢rmed the longest 5P extension and are thus presumed to represent the full length cDNA. The full length cDNA clones for cI-1 was 2361 bp and the sequence has been deposited in GenBank, accession number AF121080. Using the full length cI-1 sequence, database searches revealed that the human version of this gene has been sequenced as part of the human genome project and located to human chromosome 11q12.2 (accession number AC004126). The similarity with the human genomic sequence extends from nucleotide 597 to 2338 of the mouse cI-1 cDNA with four small gaps, and covers at least nine exons over 14 kb. The 3P end of the cI-1 cDNA had several strong matches in the dbEST database, the strongest was a 91% identity over 562 bp with a rat expressed sequence tag (EST; accession number AI172497). There were many additional similarities to other ESTs including mouse, rat, and human putative amino acid/oligopeptide transporters. The Northern blot of RNA from cAMP treated RAW264 cells con¢rms that the size of the endogenous cI-1 mRNA is approximately 2.3 kb (Fig. 2). There were two classes of the full length cI-2 cDNAs, which varied at their 3P end. Five independent clones had the longer 3P end yielding a cDNA of 3131 bp, while three independent clones had the shorter 3P end yielding a cDNA of 2849 bp. Up to this earlier termination site, the sequences of these two classes of cDNAs were identical. This variation at the 3P end is apparently due to the use of an alternative polyadenylation site resulting in premature termination of transcription. The sequence of the full length cI-2 cDNA has been deposited in GenBank, accession number AF121081. There were no signi¢cant matches of this sequence to the GenBank nr database, while the dbEST database revealed many sequence similarities with the strongest showing 100% identity over 439 bp to a mouse EST (accession number AA673901). The Northern blot of RNA from cAMP treated RAW264 cells con¢rms that the size of the endogenous cI-2 mRNA is approximately 3.1 kb (Fig. 2). The tissue distribution of expression of cI-1, cI-2, and gly96 mRNAs was determined in mouse liver, spleen, kidney, lung, brain, and bone marrow derived macrophages by Northern blot analysis using full length cDNAs as probes (Fig. 3). A 2.3 kb mRNA was detected with the cI-1 probe, which was expressed highly in bone marrow derived macrophages, and weakly in spleen and lung. A 3.1 kb mRNA was detected with the cI-2 probe, which was
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Fig. 3. Tissue distribution of cI-1, cI-2, and gly96. Total RNA was obtained from mouse liver, spleen, kidney, brain, and bone marrow derived macrophages. 10 Wg of RNA was loaded on each lane and the blots were probed with cI-1 (left), cI-2 (center), and gly96 (right) cDNAs. Marks to the left of the blots represent the migration of the 28S (top) and 18S (bottom) rRNA bands.
expressed highly in bone marrow derived macrophages with very weak expression also in spleen. The macrophage cI-2 band appeared to be a doublet indicating that both 3P terminations are used. Gly96 (cI-3) expression was previously reported to be strongest in uterus, testis, and lung, and was also found in heart, ovary, adrenals, stomach and kidney [18]. Of the tissues screened in the current study, gly96 was expressed highest in macrophages and lung, and upon overexposure was also detected in kidney, spleen, and brain. 3.3. Predicted structures of protein encoded by cI-1 and cI-2 The full length cI-1 cDNA contained an open reading frame of 578 amino acid residues with no obvious signal peptide which encodes a protein with a predicted molecular mass of 64 kDa and a basic pI of 8.83. The predicted amino acid sequence has been deposited in GenBank (accession number AAD24570). The best match in the protein database was with the rat PHT1 histidine/peptide transporter (accession number 2208839) [19] which had 40% protein sequence identity over 562 residues. This membrane bound transporter also contains no signal sequence [19]. There were numerous other weaker matches with other plant and animal transporters. The Kyte^Doolittle hydrophobicity plot and Goldman^Engelman^Steitz transfer free energy plot for the predicted cI-1 protein revealed 43% hydrophobic residues, with 10^12 membrane spanning domains, suggestive of an integral membrane protein (Fig. 4). SOSUI software predicted 11 transmembrane helices [17]. The full length cI-2 cDNA contained an open reading frame of 501 amino acid residues, which was not a¡ected by the 3P truncation observed in several cDNA clones.
There was no obvious signal peptide for this protein with a predicted molecular mass of 55 kDa and an acidic pI of 6.17. The predicted amino acid sequence has been deposited in GenBank (accession number AAD24571). The best match in the protein database was with the Arabidopsis glycerol 3-phosphate permease (accession number 2245113) [20] which had 43% protein sequence identity over 478 residues. The strongest mammalian identity was with the human glucose 6-phosphate translocase (accession number 2765461) with 22% identity over 254 amino acids [21]. The Kyte^Doolittle hydrophobicity plot and Goldman^Engelman^Steitz transfer free energy plot for the predicted cI-2 protein revealed 42% hydrophobic residues, with numerous membrane spanning domains, also suggestive of an integral membrane protein (Fig. 4).
Fig. 4. Hydrophobicity and free energy plots of the predicted cI-1 and cI-2 proteins. The predicted proteins of cI-I (top) and cI-2 (bottom) were subjected to Kyte^Doolittle hydrophobicity (A) and Goldman^Engelman^Steitz free energy (B) analyses, using LaserGene software (DNAstar). In the Kyte^Doolittle analysis, positive is hydrophilic and negative is hydrophobic. In the Goldman^Engelman^Steitz analysis, a positive value is predictive of a membrane spanning non-polar K-helical region.
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Fig. 5. Time course of cI-1, cI-2, and gly96 induction by cAMP in RAW cells. Total RNA was isolated from untreated cells (0), and from cells 2, 4, 8, and 24 h after treatment with 0.3 mM 8-Br-cAMP. 10 Wg of total RNA was loaded onto each lane. (A,C,E) Northern blots probed with cI-1, cI-2, and gly96, respectively. (B,D,F) Scans of the cI-1, cI-2, and gly96 blots, respectively, normalized to expression at 24 h.
SOSUI analysis predicted 13 membrane spanning helices [17].
3.5. Subtractive hybridization and Northern blot con¢rmation
3.4. Time course of cAMP induction of cI-1, cI-2, and gly96 in RAW264 cells
RNA from control and 8-Br-cAMP treated RAW cells was used with the PCR-Select cDNA subtraction kit (Clontech) to enrich the cDNA pool with cAMP induced mRNA. The resulting pool of cDNA was reampli¢ed and cloned into pT-Adv. We picked 140 colonies from agar plates, but the cDNA library we created could be used to plate thousands of additional colonies. One hundred and ¢fteen clones were used to successfully make probes for Northern blot analysis, the remainder either having no insert, or being poorly ampli¢ed or sequenced. Sixty-four of these probes yielded 8-Br-cAMP induced mRNAs on Northern blots, with some having very low or undetectable expression in the control mRNA, and others having signi¢cant basal expression further induced by 8-Br-cAMP treatment (Tables 1 and 2).
The time course of mRNA expression of cI-1, cI-2 and gly96 was analyzed by Northern blots (Fig. 5). Very low levels of cI-1 mRNA were detected constitutively in RAW264 cells and treatment with 0.3 mM 8-Br-cAMP led to a slow induction which increased over time to V40-fold by 24 h. Expression of cI-2 mRNA was barely detected constitutively in RAW264 cells, and the mRNA was also induced slowly by 8-Br-cAMP over time to reach V50-fold higher levels by 24 h. The gly96 mRNA was also barely detectable constitutively, but it was induced rapidly such that by 2 h of 8-Br-cAMP treatment there was s 50-fold induction of the mRNA, and V80-fold induction by 24 h of treatment.
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3 + 3 + 3 + + 3 3 3 3 3 + + 3 3 3 + 3 +
cI-15 cI-16 cI-17 cI-18 cI-19 cI-20 cI-21 cI-22 cI-23 cI-24 cI-25 cI-26 cI-27 cI-28 cI-29 cI-30 cI-31 cI-32 cI-33 cI-34
+8-Br-cAMP
+++ ++ + +++ + ++ ++ + +++ + + + ++ ++ + + + +++ + ++
++ +++ ++ ++
+ ++ +++ ++ +++ +++ ++
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 2 1 1
4 4 3 2 2 2 2
Number of independent clones mouse ABC1 mouse transferrin mouse CD14 mouse cI-1 mouse CREM mouse MAC1 K (3PUTR) mouse cys rich transmembrane protein mouse heat shock protein mouse retrotransposon BVL-1 mouse gal/NAG lectin mouse diacylglycerol acetyltransferase mouse NIK kinase mouse protein tyrosine phosphatase mouse Q-glutamyltransferase (Tgm2) mouse ornithine decarboxylase mouse centromere protein A mouse BLR1 (gpcr) mouse cAMP phosphodiesterase mouse NF-ATc mouse TRAF1 rat G protein L6 mouse CDC10 mouse PAF-AH human KIAA0966 mouse galactosyltransferase 3 rat syntaxin 13 mouse diacylglycerol kinase mouse protein kinase Myak-L mouse TNF receptor family 9 human paxillin mouse calmodulin
Best match in nr database
MMU88984 MMMPTPPES AF076928 MUSODC AF012709 MMBRL1 AF208023 AF087434 MUSTRAF1A AF051155 MMU223786 AF030884 AB023183 AF142671 AF044581 AF085219 AF01070 NM011612 HSU14588 MMCALMOD
MUSHSPE MMBVL1 S36676 AF078752
MMABC1 MUSLLT MMCD14R AF121080 MUSCREM MMMAC1A MMMSGLYP
Match identi¢er
663 267 708 158
536 239 447 720 735 157 555 bp bp bp bp
bp bp bp bp bp bp bp
96%, 499 bp 98%, 138 bp 99%, 302 bp 99%, 404 bp 99%, 316 bp 97%, 203 bp 95%, 665 bp 97%, 543 bp 98%, 360 bp 88%, 459 bp 97%, 629 bp 94%, 235 bp 87%, 388 bp 100%, 138 bp 95%, 460 bp 100%, 422 bp 93%, 687 bp 98%, 136 bp 88%, 154 bp 98%, 457 bp
97%, 95%, 98%, 99%,
98%, 99%, 99%, 99%, 99%, 96%, 99%,
Identity
Sequence submitted to GenBank only if clone has additional sequence information not contained in nr or dbEST database.
+ + 3 3
cI-11 cI-12 cI-13 cI-14
a
3 3 + 3 3 + +
38Br-cAMP
Relative expression
cI-4 cI-5 cI-6 cI-7 cI-8 cI-9 cI-10
Clone name
Table 1 Known cAMP inducible genes from subtractive hybridization
98%, 389 bp
93%, 490 bp
99%, 425 bp
AA451022
AA789908
98%, 332 bp
99%, 377 bp
Identity
W10902
AA509708
AI57394
Better EST match
AW561897 AW561898
AW561896
AW561895
AW561894
AW561893
726 161
637
479
510
621
Accession number for Submitted clone (bp) cI clonea
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Table 2 Unknown cAMP inducible genes from subtractive hybridization Clone name
Relative expression
Best EST match
Identity
Sequence length (bp)a
Accession number for cI clone
Notesb
AI181281 W18456 AA451508 AA718566 AI851938 AW124964 AA896604 AA250100 AW209647 AA218439 AI225382 AI155594 AA283608 AA278893 none
99%, 328 bp 93%, 449 bp 99%, 238 bp 96%, 436 bp 97%, 83 bp 99%, 236 bp 100%, 405 bp 99%, 157 bp 98%, 66 bp 99%, 303 bp 100%, 391 bp 99%, 407 bp 84%, 232 bp 84%, 346 bp
798 739 677 743 166 694 514 161 147 542 615 535 726 525 753
AW561899 AW561900 AW561901 AW561902 AW561903 AW561904 AW561905 AW561906 AW561907 AW561908 AW561909 AW561910 AW561911 AW561912 AW561913
3 clones 2 clones 2 clones
38Br-cAMP +8-Br-cAMP cI-35 cI-36 cI-37 cI-38 cI-39 cI-40 cI-41 cI-42 cI-43 cI-44 cI-45 cI-46 cI-47 cI-48 cI-49 a b
+ 3 + + 3 + 3 3 3 + + 3 3 3 +
+++ +++ ++ ++ ++ ++ ++ + + ++ +++ +++ ++ + ++
human human
For clones with more than one matching sequence, the length of the contig is entered. Unless speci¢ed, best matches were with mouse ESTs.
3.6. Characterization of cDNA clones The 64 cAMP inducible partial cDNAs were sequenced and compared to the GenBank nr and dbEST databases. Forty-¢ve represented 31 known proteins in the nr database (cI-4^34), with more than one independent clone occurring for nine of these proteins (Table 1). ABC1, the Tangier disease gene, and transferrin were both identi¢ed by four independent cDNAs, and were the most frequently identi¢ed clones. The Northern blot con¢rmation of ABC1 induction by 8-Br-cAMP is included in Fig. 2. cI1 was identi¢ed by two independent clones; however, neither cI-2 or cI-3 (gly96) was identi¢ed in this screen, indicating that the 64 clones are not a complete set of the cAMP induced genes in RAW264 cells. For more than half of these inducible cDNAs the contiguous length of identity with these known genes extended for over 300 bp, with a range of 134^735 bp. For six of these inducible cDNAs, there were better matches with the dbEST database than for the nr database, and these identities are listed in Table 1. Six cDNAs also had signi¢cant additional regions of sequence, contained internally or at the end, not matching either database, and thus we have submitted these sequences to dbEST of GenBank and include their accession numbers in Table 1. The vast majority of the cDNAs with highest similarity to known mouse genes had s 95% identity to these genes. Less than perfect identity is primarily due to ambiguous nucleotides in our sequence data which were obtained in a single pass on one strand only. Many of the known genes upregulated by cAMP treatment of RAW264 cells are involved in various signal transduction pathways such as CREM, cAMP phosphodiesterase, calmodulin, diacylglycerol acetyltransferase, diacylglycerol kinase, NIK kinase, Myak-L kinase, protein tyrosine phosphatase, BLR1 (a G protein coupled receptor), G protein L6, TNF receptor family 9, TNF
receptor associated factor, and the transcription factor NF-AT. Nineteen of the 64 clones (cI-35^49) had no strong identity with sequences in the GenBank nr database, and they were analyzed for similarity to the dbEST database (Table 2). All but three of these sequences had strong matches with mouse ESTs, while two had their highest match with human ESTs and one had no strong match with any EST. Three of the sequences were represented by more than one independent clone. The average length of sequence obtained from these cDNAs was s 550 bp, and they were all longer than the region of identity with their corresponding best match EST from the database, thus all of these sequences have been submitted to dbEST of GenBank, and their accession numbers are included in Table 2. 4. Discussion We performed the di¡erential display and subtractive hybridization methods in order to identify cAMP inducible genes from RAW cells. The di¡erential display protocol was highly ine¤cient in detecting cAMP inducible genes. Of the 125 bands that were initially selected after di¡erential display, probes derived from only three of the bands gave the expected cAMP inducible pattern upon Northern blot analysis. Perhaps random selection of any 125 cDNA clones would have led to an equivalent detection of cAMP inducible genes. The use of 40 cycles during RT-PCR and ampli¢cation of the di¡erentially expressed bands may have also led to some PCR artifacts decreasing the e¤ciency of this procedure to detect true cAMP inducible cDNAs. Most of the probes gave undetectable expression on Northern blots, and some gave detectable but not inducible expression. It is possible that some of the
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probes giving undetectable expression on Northern blots are in fact cAMP inducible, and that more sensitive detection methods would yield the discovery of low abundance, yet cAMP inducible, genes. Of the three cAMP inducible genes discovered by di¡erential display, the cI-3 cDNA clone was identical to a previously characterized mouse cDNA called gly96 [18]. Gly96 was identi¢ed as an immediate early serum inducible gene in ¢broblasts [18]. In addition to serum treatment of serum starved cells, FGF, PDGF, acute treatment with phorbol esters, or combined treatment with cholera toxin and phosphodiesterase inhibitors all led to gly96 induction in 3T3 ¢broblasts, implying that multiple signal transduction pathways, including the cAMP and protein kinase C pathways, can induce gly96 [22]. The serum induction of gly96 mRNA was extremely rapid but transient, with the peak transcription levels occurring 10^20 min after serum induction [18]. This is in contrast to the sustained gly96 mRNA expression that we observed by 8-Br-cAMP treatment of RAW264 cells. The gly96 mRNA is 1127 nucleotides and encodes a protein of 153 amino acids with one predicted transmembrane domain [18]. The gly96 protein is glycosylated and has a short half-life, consistent with a protein whose expression can be rapidly up- or downregulated. The rat version of this gene, called PRG1, has also been identi¢ed as a gene from a pancreatic carcinoma transiently inducible by a pituitary peptide that activates adenylate cyclase [23]. Compared to the chronic induction of gly96 by 8-Br-cAMP in RAW264 cells, the transient induction of gly96/PRG1 in ¢broblasts and a pancreatic cell line and by serum and peptide hormones might be attributable to (1) these reagents leading to only transient increases in cAMP, or (2) di¡erent cAMP mediated regulation of gly96 mRNA production and turnover in RAW264 macrophages compared to these other cell types. The function of gly96 is not known. The other two partial cDNAs, cI-1 and cI-2, obtained from the di¡erential display were not identical to known genes. Thus, we isolated and sequenced full length cDNA clones and determined that both encoded hydrophobic proteins with multiple predicted transmembrane domains. Expression of both of these genes was largely con¢ned to macrophages. cI-1 is similar at the DNA and protein level to several amino acid, dipeptide, or oligopeptide transporters, and the human gene has been partially sequenced and is located on chromosome 11. The cI-2 cDNA had some DNA and protein similarity to several transporters of phosphorylated carbohydrates. The protein was most closely related to the glycerol 3-phosphate permease from Arabidopsis. Compared to the di¡erential display method, the subtractive hybridization method was extremely e¤cient yielding a library of cDNAs highly enriched with cAMP induced clones, with 56% (64 of 115) of the cDNAs con¢rmed inducible by Northern blot analysis. We identi¢ed 35 known proteins as cAMP inducible, many of which are
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involved in signal transduction. One of the cAMP inducible genes discovered was ABC1, the Tangier disease gene [6^9]. We con¢rmed that ABC1 mRNA is induced by 8Br-cAMP treatment of RAW264 cells, which is consistent with it playing a role in the cAMP induced lipid e¥ux to apoE and apoA1 [12,13]. Others have now claimed that antisense transfections of ABC1 inhibit lipid e¥ux to apolipoproteins [6,9]. However, it is not clear if other cAMP inducible genes also play a role in this pathway. Thus, we are initiating similar functional studies to con¢rm the role of ABC1 in this lipid e¥ux pathway and test the role of additional cAMP inducible genes in this pathway by making stable cell lines expressing antisense cDNA constructions from the cAMP regulated genes that we discovered in the present study. In conclusion, we have identi¢ed 48 di¡erent cDNAs that were upregulated in RAW264 cells by a 24 h treatment with 8-Br-cAMP (cI-1^49, with cI-1 and cI-7 representing the same gene). Thirty-one of these 48 clones (cI3^34, except for cI-7) represent recognized gene products, and all, except for one, of the remaining cDNAs are similar to mouse, rat, or human ESTs. The two full length cAMP inducible cDNAs that were completely sequenced (cI-I and cI-2) have been submitted to GenBank and can be found in the nr database, and 21 additional cAMP inducible partial cDNAs have been submitted to the dbEST database. Acknowledgements This research was supported by an Established Investigatorship from the American Heart Association (to J.D.S.) and by Grant PO1 HL54591 from the National Institutes of Health. Y.T. was supported, in part, by a grant from the Ito Foundation.
References [1] R. Ross, New Engl. J. Med. 340 (1999) 115^126. [2] G.H. Rothblat, F.H. Mahlberg, W.J. Johnson, M.C. Phillips, J. Lipid Res. 33 (1992) 1091^1097. [3] Y. Ji, B. Jian, N. Wang, Y. Sun, M.L. Moya, M.C. Phillips, G.H. Rothblat, J.B. Swaney, A.R. Tall, J. Biol. Chem. 272 (1997) 20982^ 20985. [4] H. Hara, S. Yokoyama, J. Biol. Chem. 266 (1991) 3080^3086. [5] J.F. Oram, S. Yokoyama, J. Lipid Res. 37 (1996) 2473^2491. [6] A. Brooks-Wilson, M. Marcil, S.M. Clee, L.H. Zhang, K. Roomp, D.M. van Dam, L. Yu, C. Brewer, J.A. Collins, H.O. Molhuizen, O. Loubser, B.F. Ouelette, K. Fichter, K.J. Ashbourne-Exco¡on, C.W. Sensen, S. Scherer, S. Mott, M. Denis, D. Martindale, J. Frohlich, K. Morgan, B. Koop, S. Pimstone, J.J. Kastelein, J.J. Genest, M.R. Hayden, Nature Genet. 22 (1999) 336^345. [7] S. Rust, M. Rosier, H. Funke, J. Real, Z. Amoura, J.C. Piette, J.F. Deleuze, H.B. Brewer, N. Duverger, P. Dene£e, G. Assmann, Nature Genet. 22 (1999) 352^355. [8] M. Bodzioch, E. Orso¨, J. Klucken, T. Langmann, A. Bo«ttcher, W. ë zcu«ru«Diederich, W. Drobnik, S. Barlage, C. Bu«chler, M. Porsch-O
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[18] C.H. Charles, J.K. Yoon, J.S. Simske, L.F. Lau, Oncogene 8 (1993) 797^801. [19] T. Yamashita, S. Shimada, W. Guo, K. Sato, E. Kohmura, T. Hayakawa, T. Takagi, M. Tohyama, J. Biol. Chem. 272 (1997) 10205^ 10211. [20] M. Bevan, I. Bancroft, E. Bent, K. Love, H. Goodman, C. Dean, R. Bergkamp, W. Dirkse, S.M. Van Staveren, W. Stiekema, L. Drost, P. Ridley, S.A. Hudson, K. Patel, G. Murphy, P. Pi¡anelli, H. Wedler, E. Wedler, R. Wambutt, T. Weitzenegger, T.M. Pohl, N. Terryn, J. Gielen, R. Villarroel, N. Chalwatzis et al., Nature 391 (1998) 485^ 488. [21] I. Gerin, M. Veiga-da-Cunha, Y. Achouri, J.F. Collet, S.E. Van Schaftingen, FEBS Lett. 419 (1997) 235^238. [22] L.F. Lau, D. Nathans, Proc. Natl. Acad. Sci. USA 84 (1987) 1182^ 1186. [23] H. Schafer, A. Trauzold, E.G. Siegel, U.R. Folsch, W.E. Schmidt, Cancer Res. 56 (1996) 2641^2648.
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Identi¢cation of cAMP analogue inducible genes in RAW264 macrophages Yukihiro Takahashi 1;2 , Masaaki Miyata 1;3 , Ping Zheng, Takayuki Imazato 4 , Andrew Horwitz, Jonathan D. Smith * Laboratory of Biochemical Genetics and Metabolism, Box 179, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Received 9 March 2000; received in revised form 1 May 2000; accepted 5 May 2000
Abstract RNA was isolated from RAW264 cells treated with or without 8-Br-cAMP and the differential display and subtractive hybridization methods were performed. One hundred and twenty-five differentially displayed bands were identified. Upon Northern blot analysis, only three of these bands were confirmed as cAMP inducible mRNAs, named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1^3). The cI-3 probe was identical to a previously known gene, gly96. Using the novel cI-1 and cI-2 partial cDNAs as probes, a mouse macrophage cDNA library was screened and the two full length genes were cloned, sequenced, and characterized as encoding large hydrophobic proteins. One hundred and fifteen partial cDNA clones from a subtractive hybridization library were also screened by Northern blot and 64 were found to be cAMP inducible. Of these, 45 represented 31 known unique genes in the GenBank nr database (cI-4^34), and 19 clones representing 15 unique sequences were not in the nr database (cI-35^49). One of the previously known genes was ABC1, the Tangier disease gene, which was identified from four independent partial cDNAs. ABC1 was upregulated in RAW cells by cAMP, concurrent with the cAMP induction of lipid efflux to apolipoprotein A1. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Di¡erential display; Subtractive hybridization; Cholesterol e¥ux; ABC1
1. Introduction The presence of monocyte-macrophage derived foam cells in the arterial intima is the earliest visible stage of atherosclerosis [1]. Since cholesterol loading of macrophages does not lead to scavenger receptor downregula-
Abbreviations : cI-n, cAMP inducible gene number n; apo, apolipoprotein ; DMEM, Dulbecco's modi¢ed Eagle's medium ; DGGB, DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% bovine serum albumin * Corresponding author. Fax: +1-212-327-7165; E-mail : [email protected] 1 These authors contributed equally to this work. 2 Present address: First Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-0042, Japan. 3 Present address: First Department of Internal Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. 4 Present address: Division of Biological Sciences, School of Science, Hokkaido University, kita-10, nishi-8, Sapporo 060-0810, Japan.
tion, these cells continue to take up modi¢ed lipoproteins as long as the substrate is present. Thus, cholesterol e¥ux is an important pathway to unload these cells and either inhibit the progression or lead to the regression of atherosclerosis. E¥ux of cholesterol from macrophages in vitro occurs by at least two distinct pathways. The ¢rst, as characterized by Rothblat and others, occurs as cholesterol di¡uses from a membrane with a high cholesterol to phospholipid ratio to an acceptor particle, like high density lipoprotein, with a lower cholesterol to phospholipid ratio [2]. This pathway is thought to be mediated by the scavenger receptor SR-BI protein [3]. The second pathway, as characterized by Yokoyama and others, occurs as cholesterol and phospholipids are transferred from cells to lipid free apolipoproteins (apo), or synthetic peptide analogues [4,5]. One apparent protein mediator of this pathway is the ABC1 gene, recently identi¢ed by several labs as the Tangier disease gene [6^9]. Fibroblasts from Tangier disease subjects are de¢cient in lipid e¥ux to apoA1 [10,11]. We previously reported that cAMP analogues induce apoE and apoA1 binding and uptake and promote cholesterol e¥ux from the RAW264 macrophage cell line to these apo acceptors [12]. This pathway is associated
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 1 3 3 - 0
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with coated pit endocytosis of apoA1 and its resecretion as a nascent lipoprotein [13]. Thus a study was performed to identify cAMP inducible genes that might be playing a role in this pathway. RNA di¡erential display and subtractive hybridization were used to identify cAMP inducible genes. Upon Northern blot con¢rmation, di¡erential display yielded only three cAMP inducible cDNAs, while subtractive hybridization was much more e¤cient yielding 64 inducible cDNAs. Both screens identi¢ed known genes and genes that were not present in the GenBank nr database. We obtained the full length cDNA clones for two of the unknown genes and characterize their mRNA and protein sequence, as well as the time course of their induction by cAMP. ABC1, the Tangier disease gene, was identi¢ed by four independent partial cDNAs as a cAMP inducible gene in RAW264 cells. Antisense transfections have shown that ABC1 plays a role in lipid e¥ux to apoA1 [6,9]. The current catalog of cAMP induced genes may be helpful in elucidating other genes that play a role in the cAMP mediated e¥ux of lipids to apoA1 in RAW264 cells. 2. Materials and methods 2.1. Cell culture The RAW264.7 cell line, derived from murine macrophages, was obtained from American Type Culture Collection and cultured in Dulbecco's modi¢ed Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For cAMP treatment, subcon£uent cells were incubated with or without 0.1^0.3 mM 8-Br-cAMP (Sigma) in DMEM supplemented with 50 mM glucose, 2 mM glutamine, and 0.2% bovine serum albumin (Sigma, A6003) (referred to as DGGB) for 24 h. 2.2. RNA isolation Total RNA for di¡erential display and Northern blot analysis was isolated from RAW264.7 cells treated with or without 0.1 mM 8Br-cAMP for 24 h in DGGB by the standard guanidinium thiocyanate method [14]. For di¡erential display, possible DNA contamination was removed by treating with RNase free DNase I (Boehringer Mannheim) for 30 min at 37³C. Poly(A) RNA was extracted with the FastTrack 2.0 Kit (Invitrogen) from RAW264.7 cells treated for 24 h with 50 Wg/ml acetylated low density lipoprotein and with or without 0.3 mM cAMP (referred to as +cAMP RAW and 3cAMP RAW mRNA, respectively). The A260 /A280 ratio of the puri¢ed poly(A) RNA exhibited ranged from 1.8 to 2.0 and its integrity and equalization were con¢rmed by comparing their glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA levels by Northern blot analysis.
2.3. Di¡erential display Di¡erential display was performed by the method described by Liang and Pardee with minor modi¢cation [15]. Primer sets for di¡erential display were obtained from Operon Technologies. Reverse transcription to generate cDNA was performed using 400 ng of DNase I treated RNA in 1Ureverse transcription bu¡er, 10 mM dithiothreitol, 20 WM dNTP, and 1 WM of 3P primer mixture (T11AG, T11CG, T11GG). The solution was heated to 75³C for 5 min, cooled on ice, and 20 units of RNase inhibitor (Life Technologies), and 400 units of reverse transcriptase (SuperScript II, Life Technologies) were added. After incubation at 42³C for 90 min, the mixture was heated to 85³C for 5 min prior to storage at 320³C. To amplify the reverse transcriptase products, PCR was performed in duplicate with the cDNA from control or cAMP treated cells (above) in PCR bu¡er containing 2 WM of dNTP, 0.4 Wl of [35 S]dATP (Amersham), 1 WM of the 3P primer mixture, 1 WM separately of each of 26 speci¢c arbitrary decamers (5P primer), and 1 unit of Taq DNA polymerase (Perkin Elmer). PCR reactions were performed as follows : 1 cycle of 94³C for 2 min, 40 cycles of 94³C for 45 s, 35³C for 1 min and 72³C for 1 min, and 1 cycle of 72³C for 5 min. Denaturing loading bu¡er (United States Biochemical) was added and heated to 85³C for 5 min prior to loading onto 6% polyacrylamide sequence gels. Gels were run at 100 V, dried, and exposed directly to Kodak XAR-5 ¢lm at 380³C. Bands which appeared more prominently in lanes from cAMP treated cells were cut out and eluted by boiling in 100 Wl water for 10 min. DNA was recovered from the supernatant by ethanol precipitation in the presence of glycogen as a carrier. The recovered DNA was suspended in 10 Wl of TE (10 mM Tris^HCl,1 mM EDTA) and reampli¢ed in PCR bu¡er containing 20 WM of dNTP, 1 WM of the original 3P and 5P primers, and 1 unit of Taq DNA polymerase. PCR consisted of 40 cycles of 94³C for 1 min, 40³C for 1 min, and 72³C for 1 min, followed by a ¢nal extension at 72³C for 5 min. PCR products were analyzed by electrophoresis in 1.5% agarose gels stained with ethidium bromide, and then puri¢ed by phenol^chloroform extraction and ethanol precipitation. The puri¢ed DNA products were used as probes for Northern blot analysis and were subcloned into pCR2.1 using the TA cloning kit (Invitrogen). 2.4. Northern blot analysis Radioactive probes were generated using Amersham Megaprime labeling kit and [32 P]dCTP (Amersham). Alternatively, non-radioactive probes were prepared by PCR using the DIG system (Boehringer Mannheim). 10^15 Wg of total RNA, denatured in a formamide loading bu¡er, was run on 1.2% agarose formaldehyde gels. RNA was transferred to membranes and crosslinked with UV light.
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For tissue distribution expression analysis, total RNA was isolated from mouse liver, spleen, kidney, brain, and bone marrow derived macrophages. Bone marrow was isolated from femurs and cultured for 2 weeks in the presence of Lcell conditioned medium, as a source of macrophage colony stimulating factor, as previously described [16].
Sequences were scanned for the presence of the other nested primer denoting the end of cDNA insert, and the traces were examined to delete sequence at the 5P and 3P ends where the signal to noise ratio was poor.
2.5. Full length cDNA isolation and characterization
DNA and protein database searches were performed at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) using the blastn and blastp programs with the GenBank nr and dbEST databases.
Using the subcloned di¡erential display products as probes, a mouse macrophage cDNA phagemid library (Stratagene, catalog #937306) was screened by ¢lter lifts. After tertiary screening, independent cDNA clones were treated with an exonuclease III/mung bean nuclease deletion kit (Stratagene) and sequenced in both directions. The longest open reading frame was determined and translated into amino acid residues. The predicted proteins were subjected to Kyte^Doolittle hydrophobicity and Goldman^ Engelman^Steitz transfer free energy analyses using LaserGene software (DNAstar Inc.). The protein sequences were also analyzed by the SOSUI program to predict the number of transmembrane helices (http://azusa.proteome. bio.tuat.ac.jp/sosui/) [17]. 2.6. Subtractive cDNA library construction and analysis Using poly(A) mRNA from cAMP and control treated RAW cells, a library of cAMP induced cDNAs was constructed using the PCR-Select cDNA subtraction kit (Clontech). In this study, cDNA from +cAMP RAW cells was used as a tester and cDNA from 3cAMP RAW cells as a driver. These cDNAs were then digested with RsaI. The tester cDNA was divided into two portions and each was ligated with a di¡erent cDNA adapter (adapter 1 or adapter 2R). The ¢rst round of hybridizations consisted of an excess amount of driver cDNA added to each of the two tester cDNAs (adapter 1 and adapter 2R samples) at a driver:tester ratio of 30:1. The two samples from the ¢rst hybridization were mixed and freshly denatured driver cDNA was added for the second hybridization round, in which new hybrid molecules corresponding to di¡erentially expressed cDNA with di¡erent adapters on each end were formed. These hybrids were ampli¢ed exponentially by PCR (27 cycles). A secondary PCR ampli¢cation was then performed (10 cycles) using nested primers to enrich for di¡erentially expressed sequences. This combination of 27 cycles in the ¢rst PCR and 10 cycles in the second PCR was selected after checking the subtraction e¤ciency using GAPDH and cI-1 (see Section 3) as negative and positive cAMP induced cDNA controls, respectively. The PCR products were cloned into pT-Adv vector (Clontech). The subtractive cDNA library was obtained after transformation into DH5K Escherichia coli strain. Single pass automated £uorescent sequencing of clones was performed by the Rockefeller University Protein and DNA Technology Center using one of the nested primers.
2.7. Nucleotide and protein sequence comparisons
3. Results 3.1. Di¡erential display and Northern blot con¢rmation To identify cAMP inducible genes in RAW264 macrophages, mRNA di¡erential display was performed with total RNA isolated from RAW264 cells treated with or without 0.1 mM 8-Br-cAMP for 24 h. PCR ampli¢cations were performed with 26 primer combinations using duplicate RNA samples from control and cAMP treated cells. One hundred and twenty-¢ve PCR products were found in higher levels from the cAMP treated samples compared to the control samples. To con¢rm the gene regulation patterns observed in the di¡erential display assay, the 125 selected PCR products were cut from the gels, reampli¢ed,
Fig. 1. Di¡erential display of three genes. Di¡erential mRNA display was performed to compare patterns for RAW264 cells treated with or without 0.1 mM 8-Br-cAMP for 24 h. PCR ampli¢cations were performed with 26 primer combinations using duplicate RNA samples from control (3) and 8-Br-cAMP treated cells (+). The arrows point to the middle of the segment of the gel excised which gave rise to partial cDNA clones named cI-1, cI-2, and cI-3.
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Fig. 2. Northern blot con¢rmation of cAMP induced mRNAs. The PCR products of Fig. 1 were cut from the gels, reampli¢ed, and together with an ABC1 probe were hybridized with Northern blots prepared with 10 Wg of total RNA from control (3) and 8-Br-cAMP treated (+) RAW264 cells. The dramatic cAMP induction of speci¢c RNA molecules by Northern blot analysis was observed with the cI-1, cI-2, cI-3, and ABC1 probes. Marks to the left of the blots represent the migration of the 28S (top) and 18S (bottom) rRNA bands, detected by ethidium bromide staining.
and used to probe Northern blots prepared with RNA from control and cAMP treated RAW264 cells. Most of the probes gave very low or undetectable levels of expression in RNA from either control or treated cells. Some probes detected RNA expression on the Northern blots, but revealed no di¡erence between the control and treated cells. Three of the probes yielded RNA expression patterns that were robustly upregulated upon cAMP treatment, which we named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1^3). Fig. 1 shows the upregulation of bands corresponding to cI-1, cI-2, and cI-3 in the di¡erential display assay. Fig. 2 shows the dramatic induction of speci¢c RNA molecules by Northern blot analysis using the cI-1, cI-2, and cI-3 probes on RNA from 8-Br-cAMP treated and control RAW264 cells. 3.2. Characterization and analysis of cI-1, cI-2, and cI-3 cDNAs The PCR ampli¢ed fragments of cI-1, cI-2, and cI-3 were cloned into plasmid pCR2.1 and completely sequenced on both strands. The results revealed that all fragments were £anked by the sequences corresponding to the particular 3P and 5P primers used in the di¡erential display assay. The sizes of the cI-1, cI-2, and cI-3 partial cDNA fragments were 282, 384, and 466 bp, respectively. Their nucleotide sequences were analyzed by searching for homologies against the GenBank nr database. Clone cI-3 was identical to the gly96 gene (accession number X67644), which had been characterized as an immediate early serum responsive gene from mouse ¢broblasts [18].
At that time no characterized full length genes were identical to clones cI-1 and cI-2. Therefore, a mouse macrophage cDNA library (poly(dT) primed) was screened using cI-1 and cI-2 probes to obtain the full length cDNA. Nine independent phagemid cDNA clones were isolated with the cI-1 probe and eight independent cDNA clones were isolated with the cI-2 probe. All of the cDNA clones were sequenced on both strands. The cDNA clones contained varying lengths of 5P sequence and several clones of each con¢rmed the longest 5P extension and are thus presumed to represent the full length cDNA. The full length cDNA clones for cI-1 was 2361 bp and the sequence has been deposited in GenBank, accession number AF121080. Using the full length cI-1 sequence, database searches revealed that the human version of this gene has been sequenced as part of the human genome project and located to human chromosome 11q12.2 (accession number AC004126). The similarity with the human genomic sequence extends from nucleotide 597 to 2338 of the mouse cI-1 cDNA with four small gaps, and covers at least nine exons over 14 kb. The 3P end of the cI-1 cDNA had several strong matches in the dbEST database, the strongest was a 91% identity over 562 bp with a rat expressed sequence tag (EST; accession number AI172497). There were many additional similarities to other ESTs including mouse, rat, and human putative amino acid/oligopeptide transporters. The Northern blot of RNA from cAMP treated RAW264 cells con¢rms that the size of the endogenous cI-1 mRNA is approximately 2.3 kb (Fig. 2). There were two classes of the full length cI-2 cDNAs, which varied at their 3P end. Five independent clones had the longer 3P end yielding a cDNA of 3131 bp, while three independent clones had the shorter 3P end yielding a cDNA of 2849 bp. Up to this earlier termination site, the sequences of these two classes of cDNAs were identical. This variation at the 3P end is apparently due to the use of an alternative polyadenylation site resulting in premature termination of transcription. The sequence of the full length cI-2 cDNA has been deposited in GenBank, accession number AF121081. There were no signi¢cant matches of this sequence to the GenBank nr database, while the dbEST database revealed many sequence similarities with the strongest showing 100% identity over 439 bp to a mouse EST (accession number AA673901). The Northern blot of RNA from cAMP treated RAW264 cells con¢rms that the size of the endogenous cI-2 mRNA is approximately 3.1 kb (Fig. 2). The tissue distribution of expression of cI-1, cI-2, and gly96 mRNAs was determined in mouse liver, spleen, kidney, lung, brain, and bone marrow derived macrophages by Northern blot analysis using full length cDNAs as probes (Fig. 3). A 2.3 kb mRNA was detected with the cI-1 probe, which was expressed highly in bone marrow derived macrophages, and weakly in spleen and lung. A 3.1 kb mRNA was detected with the cI-2 probe, which was
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Fig. 3. Tissue distribution of cI-1, cI-2, and gly96. Total RNA was obtained from mouse liver, spleen, kidney, brain, and bone marrow derived macrophages. 10 Wg of RNA was loaded on each lane and the blots were probed with cI-1 (left), cI-2 (center), and gly96 (right) cDNAs. Marks to the left of the blots represent the migration of the 28S (top) and 18S (bottom) rRNA bands.
expressed highly in bone marrow derived macrophages with very weak expression also in spleen. The macrophage cI-2 band appeared to be a doublet indicating that both 3P terminations are used. Gly96 (cI-3) expression was previously reported to be strongest in uterus, testis, and lung, and was also found in heart, ovary, adrenals, stomach and kidney [18]. Of the tissues screened in the current study, gly96 was expressed highest in macrophages and lung, and upon overexposure was also detected in kidney, spleen, and brain. 3.3. Predicted structures of protein encoded by cI-1 and cI-2 The full length cI-1 cDNA contained an open reading frame of 578 amino acid residues with no obvious signal peptide which encodes a protein with a predicted molecular mass of 64 kDa and a basic pI of 8.83. The predicted amino acid sequence has been deposited in GenBank (accession number AAD24570). The best match in the protein database was with the rat PHT1 histidine/peptide transporter (accession number 2208839) [19] which had 40% protein sequence identity over 562 residues. This membrane bound transporter also contains no signal sequence [19]. There were numerous other weaker matches with other plant and animal transporters. The Kyte^Doolittle hydrophobicity plot and Goldman^Engelman^Steitz transfer free energy plot for the predicted cI-1 protein revealed 43% hydrophobic residues, with 10^12 membrane spanning domains, suggestive of an integral membrane protein (Fig. 4). SOSUI software predicted 11 transmembrane helices [17]. The full length cI-2 cDNA contained an open reading frame of 501 amino acid residues, which was not a¡ected by the 3P truncation observed in several cDNA clones.
There was no obvious signal peptide for this protein with a predicted molecular mass of 55 kDa and an acidic pI of 6.17. The predicted amino acid sequence has been deposited in GenBank (accession number AAD24571). The best match in the protein database was with the Arabidopsis glycerol 3-phosphate permease (accession number 2245113) [20] which had 43% protein sequence identity over 478 residues. The strongest mammalian identity was with the human glucose 6-phosphate translocase (accession number 2765461) with 22% identity over 254 amino acids [21]. The Kyte^Doolittle hydrophobicity plot and Goldman^Engelman^Steitz transfer free energy plot for the predicted cI-2 protein revealed 42% hydrophobic residues, with numerous membrane spanning domains, also suggestive of an integral membrane protein (Fig. 4).
Fig. 4. Hydrophobicity and free energy plots of the predicted cI-1 and cI-2 proteins. The predicted proteins of cI-I (top) and cI-2 (bottom) were subjected to Kyte^Doolittle hydrophobicity (A) and Goldman^Engelman^Steitz free energy (B) analyses, using LaserGene software (DNAstar). In the Kyte^Doolittle analysis, positive is hydrophilic and negative is hydrophobic. In the Goldman^Engelman^Steitz analysis, a positive value is predictive of a membrane spanning non-polar K-helical region.
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Fig. 5. Time course of cI-1, cI-2, and gly96 induction by cAMP in RAW cells. Total RNA was isolated from untreated cells (0), and from cells 2, 4, 8, and 24 h after treatment with 0.3 mM 8-Br-cAMP. 10 Wg of total RNA was loaded onto each lane. (A,C,E) Northern blots probed with cI-1, cI-2, and gly96, respectively. (B,D,F) Scans of the cI-1, cI-2, and gly96 blots, respectively, normalized to expression at 24 h.
SOSUI analysis predicted 13 membrane spanning helices [17].
3.5. Subtractive hybridization and Northern blot con¢rmation
3.4. Time course of cAMP induction of cI-1, cI-2, and gly96 in RAW264 cells
RNA from control and 8-Br-cAMP treated RAW cells was used with the PCR-Select cDNA subtraction kit (Clontech) to enrich the cDNA pool with cAMP induced mRNA. The resulting pool of cDNA was reampli¢ed and cloned into pT-Adv. We picked 140 colonies from agar plates, but the cDNA library we created could be used to plate thousands of additional colonies. One hundred and ¢fteen clones were used to successfully make probes for Northern blot analysis, the remainder either having no insert, or being poorly ampli¢ed or sequenced. Sixty-four of these probes yielded 8-Br-cAMP induced mRNAs on Northern blots, with some having very low or undetectable expression in the control mRNA, and others having signi¢cant basal expression further induced by 8-Br-cAMP treatment (Tables 1 and 2).
The time course of mRNA expression of cI-1, cI-2 and gly96 was analyzed by Northern blots (Fig. 5). Very low levels of cI-1 mRNA were detected constitutively in RAW264 cells and treatment with 0.3 mM 8-Br-cAMP led to a slow induction which increased over time to V40-fold by 24 h. Expression of cI-2 mRNA was barely detected constitutively in RAW264 cells, and the mRNA was also induced slowly by 8-Br-cAMP over time to reach V50-fold higher levels by 24 h. The gly96 mRNA was also barely detectable constitutively, but it was induced rapidly such that by 2 h of 8-Br-cAMP treatment there was s 50-fold induction of the mRNA, and V80-fold induction by 24 h of treatment.
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3 + 3 + 3 + + 3 3 3 3 3 + + 3 3 3 + 3 +
cI-15 cI-16 cI-17 cI-18 cI-19 cI-20 cI-21 cI-22 cI-23 cI-24 cI-25 cI-26 cI-27 cI-28 cI-29 cI-30 cI-31 cI-32 cI-33 cI-34
+8-Br-cAMP
+++ ++ + +++ + ++ ++ + +++ + + + ++ ++ + + + +++ + ++
++ +++ ++ ++
+ ++ +++ ++ +++ +++ ++
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 2 1 1
4 4 3 2 2 2 2
Number of independent clones mouse ABC1 mouse transferrin mouse CD14 mouse cI-1 mouse CREM mouse MAC1 K (3PUTR) mouse cys rich transmembrane protein mouse heat shock protein mouse retrotransposon BVL-1 mouse gal/NAG lectin mouse diacylglycerol acetyltransferase mouse NIK kinase mouse protein tyrosine phosphatase mouse Q-glutamyltransferase (Tgm2) mouse ornithine decarboxylase mouse centromere protein A mouse BLR1 (gpcr) mouse cAMP phosphodiesterase mouse NF-ATc mouse TRAF1 rat G protein L6 mouse CDC10 mouse PAF-AH human KIAA0966 mouse galactosyltransferase 3 rat syntaxin 13 mouse diacylglycerol kinase mouse protein kinase Myak-L mouse TNF receptor family 9 human paxillin mouse calmodulin
Best match in nr database
MMU88984 MMMPTPPES AF076928 MUSODC AF012709 MMBRL1 AF208023 AF087434 MUSTRAF1A AF051155 MMU223786 AF030884 AB023183 AF142671 AF044581 AF085219 AF01070 NM011612 HSU14588 MMCALMOD
MUSHSPE MMBVL1 S36676 AF078752
MMABC1 MUSLLT MMCD14R AF121080 MUSCREM MMMAC1A MMMSGLYP
Match identi¢er
663 267 708 158
536 239 447 720 735 157 555 bp bp bp bp
bp bp bp bp bp bp bp
96%, 499 bp 98%, 138 bp 99%, 302 bp 99%, 404 bp 99%, 316 bp 97%, 203 bp 95%, 665 bp 97%, 543 bp 98%, 360 bp 88%, 459 bp 97%, 629 bp 94%, 235 bp 87%, 388 bp 100%, 138 bp 95%, 460 bp 100%, 422 bp 93%, 687 bp 98%, 136 bp 88%, 154 bp 98%, 457 bp
97%, 95%, 98%, 99%,
98%, 99%, 99%, 99%, 99%, 96%, 99%,
Identity
Sequence submitted to GenBank only if clone has additional sequence information not contained in nr or dbEST database.
+ + 3 3
cI-11 cI-12 cI-13 cI-14
a
3 3 + 3 3 + +
38Br-cAMP
Relative expression
cI-4 cI-5 cI-6 cI-7 cI-8 cI-9 cI-10
Clone name
Table 1 Known cAMP inducible genes from subtractive hybridization
98%, 389 bp
93%, 490 bp
99%, 425 bp
AA451022
AA789908
98%, 332 bp
99%, 377 bp
Identity
W10902
AA509708
AI57394
Better EST match
AW561897 AW561898
AW561896
AW561895
AW561894
AW561893
726 161
637
479
510
621
Accession number for Submitted clone (bp) cI clonea
Y. Takahashi et al. / Biochimica et Biophysica Acta 1492 (2000) 385^394 391
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Table 2 Unknown cAMP inducible genes from subtractive hybridization Clone name
Relative expression
Best EST match
Identity
Sequence length (bp)a
Accession number for cI clone
Notesb
AI181281 W18456 AA451508 AA718566 AI851938 AW124964 AA896604 AA250100 AW209647 AA218439 AI225382 AI155594 AA283608 AA278893 none
99%, 328 bp 93%, 449 bp 99%, 238 bp 96%, 436 bp 97%, 83 bp 99%, 236 bp 100%, 405 bp 99%, 157 bp 98%, 66 bp 99%, 303 bp 100%, 391 bp 99%, 407 bp 84%, 232 bp 84%, 346 bp
798 739 677 743 166 694 514 161 147 542 615 535 726 525 753
AW561899 AW561900 AW561901 AW561902 AW561903 AW561904 AW561905 AW561906 AW561907 AW561908 AW561909 AW561910 AW561911 AW561912 AW561913
3 clones 2 clones 2 clones
38Br-cAMP +8-Br-cAMP cI-35 cI-36 cI-37 cI-38 cI-39 cI-40 cI-41 cI-42 cI-43 cI-44 cI-45 cI-46 cI-47 cI-48 cI-49 a b
+ 3 + + 3 + 3 3 3 + + 3 3 3 +
+++ +++ ++ ++ ++ ++ ++ + + ++ +++ +++ ++ + ++
human human
For clones with more than one matching sequence, the length of the contig is entered. Unless speci¢ed, best matches were with mouse ESTs.
3.6. Characterization of cDNA clones The 64 cAMP inducible partial cDNAs were sequenced and compared to the GenBank nr and dbEST databases. Forty-¢ve represented 31 known proteins in the nr database (cI-4^34), with more than one independent clone occurring for nine of these proteins (Table 1). ABC1, the Tangier disease gene, and transferrin were both identi¢ed by four independent cDNAs, and were the most frequently identi¢ed clones. The Northern blot con¢rmation of ABC1 induction by 8-Br-cAMP is included in Fig. 2. cI1 was identi¢ed by two independent clones; however, neither cI-2 or cI-3 (gly96) was identi¢ed in this screen, indicating that the 64 clones are not a complete set of the cAMP induced genes in RAW264 cells. For more than half of these inducible cDNAs the contiguous length of identity with these known genes extended for over 300 bp, with a range of 134^735 bp. For six of these inducible cDNAs, there were better matches with the dbEST database than for the nr database, and these identities are listed in Table 1. Six cDNAs also had signi¢cant additional regions of sequence, contained internally or at the end, not matching either database, and thus we have submitted these sequences to dbEST of GenBank and include their accession numbers in Table 1. The vast majority of the cDNAs with highest similarity to known mouse genes had s 95% identity to these genes. Less than perfect identity is primarily due to ambiguous nucleotides in our sequence data which were obtained in a single pass on one strand only. Many of the known genes upregulated by cAMP treatment of RAW264 cells are involved in various signal transduction pathways such as CREM, cAMP phosphodiesterase, calmodulin, diacylglycerol acetyltransferase, diacylglycerol kinase, NIK kinase, Myak-L kinase, protein tyrosine phosphatase, BLR1 (a G protein coupled receptor), G protein L6, TNF receptor family 9, TNF
receptor associated factor, and the transcription factor NF-AT. Nineteen of the 64 clones (cI-35^49) had no strong identity with sequences in the GenBank nr database, and they were analyzed for similarity to the dbEST database (Table 2). All but three of these sequences had strong matches with mouse ESTs, while two had their highest match with human ESTs and one had no strong match with any EST. Three of the sequences were represented by more than one independent clone. The average length of sequence obtained from these cDNAs was s 550 bp, and they were all longer than the region of identity with their corresponding best match EST from the database, thus all of these sequences have been submitted to dbEST of GenBank, and their accession numbers are included in Table 2. 4. Discussion We performed the di¡erential display and subtractive hybridization methods in order to identify cAMP inducible genes from RAW cells. The di¡erential display protocol was highly ine¤cient in detecting cAMP inducible genes. Of the 125 bands that were initially selected after di¡erential display, probes derived from only three of the bands gave the expected cAMP inducible pattern upon Northern blot analysis. Perhaps random selection of any 125 cDNA clones would have led to an equivalent detection of cAMP inducible genes. The use of 40 cycles during RT-PCR and ampli¢cation of the di¡erentially expressed bands may have also led to some PCR artifacts decreasing the e¤ciency of this procedure to detect true cAMP inducible cDNAs. Most of the probes gave undetectable expression on Northern blots, and some gave detectable but not inducible expression. It is possible that some of the
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probes giving undetectable expression on Northern blots are in fact cAMP inducible, and that more sensitive detection methods would yield the discovery of low abundance, yet cAMP inducible, genes. Of the three cAMP inducible genes discovered by di¡erential display, the cI-3 cDNA clone was identical to a previously characterized mouse cDNA called gly96 [18]. Gly96 was identi¢ed as an immediate early serum inducible gene in ¢broblasts [18]. In addition to serum treatment of serum starved cells, FGF, PDGF, acute treatment with phorbol esters, or combined treatment with cholera toxin and phosphodiesterase inhibitors all led to gly96 induction in 3T3 ¢broblasts, implying that multiple signal transduction pathways, including the cAMP and protein kinase C pathways, can induce gly96 [22]. The serum induction of gly96 mRNA was extremely rapid but transient, with the peak transcription levels occurring 10^20 min after serum induction [18]. This is in contrast to the sustained gly96 mRNA expression that we observed by 8-Br-cAMP treatment of RAW264 cells. The gly96 mRNA is 1127 nucleotides and encodes a protein of 153 amino acids with one predicted transmembrane domain [18]. The gly96 protein is glycosylated and has a short half-life, consistent with a protein whose expression can be rapidly up- or downregulated. The rat version of this gene, called PRG1, has also been identi¢ed as a gene from a pancreatic carcinoma transiently inducible by a pituitary peptide that activates adenylate cyclase [23]. Compared to the chronic induction of gly96 by 8-Br-cAMP in RAW264 cells, the transient induction of gly96/PRG1 in ¢broblasts and a pancreatic cell line and by serum and peptide hormones might be attributable to (1) these reagents leading to only transient increases in cAMP, or (2) di¡erent cAMP mediated regulation of gly96 mRNA production and turnover in RAW264 macrophages compared to these other cell types. The function of gly96 is not known. The other two partial cDNAs, cI-1 and cI-2, obtained from the di¡erential display were not identical to known genes. Thus, we isolated and sequenced full length cDNA clones and determined that both encoded hydrophobic proteins with multiple predicted transmembrane domains. Expression of both of these genes was largely con¢ned to macrophages. cI-1 is similar at the DNA and protein level to several amino acid, dipeptide, or oligopeptide transporters, and the human gene has been partially sequenced and is located on chromosome 11. The cI-2 cDNA had some DNA and protein similarity to several transporters of phosphorylated carbohydrates. The protein was most closely related to the glycerol 3-phosphate permease from Arabidopsis. Compared to the di¡erential display method, the subtractive hybridization method was extremely e¤cient yielding a library of cDNAs highly enriched with cAMP induced clones, with 56% (64 of 115) of the cDNAs con¢rmed inducible by Northern blot analysis. We identi¢ed 35 known proteins as cAMP inducible, many of which are
393
involved in signal transduction. One of the cAMP inducible genes discovered was ABC1, the Tangier disease gene [6^9]. We con¢rmed that ABC1 mRNA is induced by 8Br-cAMP treatment of RAW264 cells, which is consistent with it playing a role in the cAMP induced lipid e¥ux to apoE and apoA1 [12,13]. Others have now claimed that antisense transfections of ABC1 inhibit lipid e¥ux to apolipoproteins [6,9]. However, it is not clear if other cAMP inducible genes also play a role in this pathway. Thus, we are initiating similar functional studies to con¢rm the role of ABC1 in this lipid e¥ux pathway and test the role of additional cAMP inducible genes in this pathway by making stable cell lines expressing antisense cDNA constructions from the cAMP regulated genes that we discovered in the present study. In conclusion, we have identi¢ed 48 di¡erent cDNAs that were upregulated in RAW264 cells by a 24 h treatment with 8-Br-cAMP (cI-1^49, with cI-1 and cI-7 representing the same gene). Thirty-one of these 48 clones (cI3^34, except for cI-7) represent recognized gene products, and all, except for one, of the remaining cDNAs are similar to mouse, rat, or human ESTs. The two full length cAMP inducible cDNAs that were completely sequenced (cI-I and cI-2) have been submitted to GenBank and can be found in the nr database, and 21 additional cAMP inducible partial cDNAs have been submitted to the dbEST database. Acknowledgements This research was supported by an Established Investigatorship from the American Heart Association (to J.D.S.) and by Grant PO1 HL54591 from the National Institutes of Health. Y.T. was supported, in part, by a grant from the Ito Foundation.
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