AngRem104, an Angiotensin II–Induced Novel Upregulated Gene in Human Mesangial Cells, Is Potentially Involved in the Regulation of Fibronectin Expression
Xiubin Liang,
Hong Zhang,
Anyu Zhou and
Haiyan Wang
Renal Division of Peking University First Hospital, Institute of Nephrology, Peking University, Beijing, China.
Correspondence to Dr. Hong Zhang, Renal Division of Peking University, First Hospital, Institute of Nephrology, No. 8 Xi Shi Ku Street, Peking University, Beijing, 100034, China. Phone: 86-10-66171122 ext. 2388; Fax: 86-10-66170758;
ABSTRACT. Accumulation of extracellular matrix (ECM) in theglomerular mesangium is a common feature of many progressiverenal diseases. Angiotensin II (AngII) plays important rolesin the proliferation of glomerular mesangial cells (MC) as wellas the synthesis of ECM such as fibronectin (FN) and collagens.However, the precise molecular signals responsible for theseeffects are unknown. To explore possible molecule mechanismof ECM accumulation related to AngII, suppression subtractivehybridization (SSH) was performed to screen and identify upregulatedgenes induced by AngII in cultured human MC. A novel gene, AngRem104(GenBank accession number, AF367870), was isolated. The full-lengthcDNA of AngRem104 is 1690 bp, and it contains a 1041-bp openreading frame (ORF) encoding 347 amino acid residues with apredicted molecular mass of 37.2 kD. AngRem104 widely expressedin human heart, placenta, liver, muscle, kidney, and pancreas.Moreover, AngRem104 was found in human glomeruli and tubuleby in situ hybridization. In human MC, the upregulation of AngRem104induced by AngII was time-dependent, and it was dose-dependentlyblocked by AngII type 1 receptor antagonist (AT1RA), Losartan.The subcellular localization detected by AngRem104-pEGFP fusionprotein revealed that AngRem104 was a nuclear protein. Interestingly,when AngRem104 was overexpressed by transfection of its senseconstruct, cDNA Microarray showed that two of the ECM-relatedgenes, i.e., human mRNA for FN and integrin--1 (FN receptor),dramatically upregulated their expressions. Furthermore, AngRem104could regulate the expression of FN induced by AngII, whichwere detected by RT-PCR and quantitative real-time PCR, whenAngRem104 was overexpressed. It is concluded that AngRem104is a novel human gene potentially involved in the regulationof FN induced by AngII in human MC. These findings may providenew insights into mechanisms of glomerular sclerosis associatedwith AngII. E-mail: hongzh@bjmu.edu.cn
Angiotensin II (AngII), the main factor of the rennin-angiotensinsystem (RAS), plays multiple roles involved in the progressionof renal disease, including the proliferation of glomerularcells as well as tubular hypertrophy, which may finally resultin glomerular sclerosis and tubulointerstitial fibrosis (1–4).In experimental models and clinical trials, the growing evidencesrevealed that the angiotensin type 1 receptor antagonist (AT1RA)as well as angiotensin-converting enzyme inhibitor (ACEI) notonly decrease BP but also retard the progression of renal injury(5,6). Our previous study also revealed that the level of renallocal AngII is correlated with the progression of chronic puromycinnephropathy (7). Mesangial cells (MC), as the major intrinsiccells in glomeruli, are one of the targets of AngII in variousrenal diseases. Many investigators have revealed that AngIIaccelerate the proliferation of glomerular MC as well as thesynthesis of extracellular matrix (ECM) protein, such as fibronectin(FN) and collagens (2,8–11). However, the precise molecularmechanisms of ECM accumulation induced by AngII are still ambiguous.It is one of the effective ways to explore the molecules involvedin glomerular cell proliferation and matrix expansion. In thepresent study, we applied suppression subtractive hybridization(SSH) to screen and identify upregulated genes in human MC inducedby AngII, and a novel gene, termed AngRem104 (AngII-relatedgene in mesangial cells), was identified and characterized.
Cell Lines and Cell Culture
Primary human MC was obtained from Clonetics Corp. (Clonetics,San Diego, CA), and COS-1 (monkey renal blastocyte) was purchasedfrom ATCC (American Type Culture Collection, Manassas, VA).The human MC was cultured in RPMI 1640 medium (Life Technologies,Baltimore, MD) supplemented with 10% heat-inactivated fetalbovine serum (Life Technologies BRL, Grand Island, NY), 100U/ml penicillin, 100 µg/ml streptomycin, 12.5 mM Hepesbuffer, 2 mM L-glutamine, and insulin (5 µg/ml)–transferrin(5 µg/ml)–sodium selentin (5 ng/ml) media supplement(Sigma). COS-1 cells were grown in high glucose (4500 mg/L)Dulbecco modified Eagle’s medium (DMEM; Life Technologies)containing 10% heat-inactivated fetal bovine serum, 100 u/mlpenicillin, 100 µg/ml streptomycin, and 12.5 mM Hepesbuffer. All cells were maintained in 5% CO2 at 37°C.
Suppression Subtractive Hybridization (SSH)
SSH was performed with Clontech PCR-select cDNA SubtractionKit (Clontech, Palo Alto, CA) following the manufacturer’sprotocol. In brief, RNA prepared from human MC stimulated byAngII (Sigma) (10-6 M, 24 h) was used as tester, whereas RNAprepared from the human MC without AngII stimulation was usedas driver. Double-stranded cDNAs were synthesized from 2 µgof mRNA as the tester or the driver and digested with RsaI,respectively. Two types of adapter, provided by the manufacturer,were independently ligated to the tester cDNA. First and secondhybridizations were performed, and the resulting annealed materialwas amplified by PCR. The final PCR products were subtractiveproducts, which presented the differentially expressed genesin tester population compared with driver population.
Reverse Northern Blot Analysis
Reverse Northern blot was performed to screen the overexpressedgenes in tester population. The SSH subtracted final productswere subcloned into pGEM-T easy vector (Promega, Madison, WI).After transformation, bacterial colonies were randomly pickedand plasmid clones with inserts were purified using a plasmidpurification system (Qiagen, Hilden, Germany). The cDNA insertswere released by EcoRI and subjected to 2% agarose gel electrophoresis,denatured with 1.5 M NaCl + 0.5 N NaOH, neutralized with 0.5M Tris-HCL (pH 7.4) + 1.5 M NaCl, and transferred to nylon membranes.The transferred DNAs were hybridized with [-32P]-dCTP-labeledtester and driver cDNA populations independently, which reflectthe mRNA expression with or without AngII stimulation. The overexpressedclones were picked up and sequenced by automated DNA sequenceanalyzer (ABI PRISM 310, Genetic Analyzer, Perkin-Elmer, FosterCity CA). The homology search was performed by NCBI online server.
Northern Blot Analysis
Northern blots were prepared as described previously (12). TotalRNA was isolated from human MC without or with AngII stimulationusing Trizol reagent (Life Technologies), respectively. Thirtymicrograms of total RNA was subjected to 2.2 M formaldehyde1% agarose gel electrophoresis and capillary transferred tothe Hybond N+ nylon membranes (Amershan, Arlington Heights,IL). cDNA inserts of differentially expressed genes were digestedwith EcoRI, purified by QIAEX II Kit (Qiagen), and radiolabeledwith [-32P]-dCTP using the random primer labeling system (Promega).The membranes were hybridized with -32P-radiolabeled cDNA probes(1 x 106 cpm/ml) at 42°C in 50% formamide, 5x SSC (salinesodium citrate), 1x Denhardt solution, 50 mM sodium phosphate(pH 7.0), and 200 µg/ml salmon sperm DNA for 24 h. Filterswere washed at the high stringency condition (four times atroom temperature for 15 min in 1x SSC/0.1% SDS, followed bytwo times at 50°C in 0.1x SSC/0.1% SDS). The membranes werealso hybridized with a glyceraldehydes-3-phosphate (GAPDH) asan internal control. To detect the distribution of AngRem104in normal tissues, a commercial human Multiple Tissue Expression(MTE) Northern blot panel (Clontech) was hybridized with 32P-labeledfull-length AngRem104 cDNA according to the above hybridizationprocedure.
Isolation of Full-Length cDNA and Nucleotide Sequencing of AngRem104
The 5'- and 3'-RACE reactions were performed using SMART RACEcDNA Amplification kit (Clontech). Double-stranded cDNAs weresynthesized from 1.0 µg of mRNA and subjected to rapidamplification of 5'- and 3'-cDNA ends (RACE) performed withprimers (5'-CAG ATC AAC GGG TAT GGG ATG CTG-3' and 5'-GTT GCTTTT GAG CCG GCA CAA CAG-3'). The 5'- and 3'-RACE products weresubcloned into the pGEM-T Easy vector (Promega) and sequencedby automated DNA sequence (ABI PRISM 310 Analyzer). The fulllength of AngRem104 was amplified by end-to-end PCR using upstreamand downstream primers (5'-ACG CGG GGA GGA ACA AGG TTC CT-3'and 5'-GGC AAT TTA GTA GAT TAA TTC AAA GCC-3'). At least fourdifferent clones were sequenced to ensure the fidelity of Taqpolymerase.
Homology and Nucleotide Sequencing Analysis
Analysis of full-length sequence of AngRem104 was performedby GENETYX-WIN (Software Development, Tokyo, Japan). The homologysearch was performed on the BLAST program through NCBI Database(http://www.ncbi.nlm.nih.gov/BLAST/) and Celera discovery system.Proteomics tools of the Expert Protein Analysis System (ExPASy)server of the SWISS Institute of Bioinformatics (http://cn.expasy.org/tools/)were used to analyze the primary sequence and motifs of theAngRem104 protein.
In Situ Hybridization
For in situ hybridization, biotin-labeled sense and antisenseprobes spanning residues 795–1130 of AngRem104 cDNA (Seeprobe sequence, Figure 2) were produced by in vitro transcriptionusing T7 or SP6 polymerase, respectively, following the manufacturer’sprotocol (Promega). Human renal specimens were prepared andfixed in 4% paraformaldehyde in PBS buffer. Tissues were embeddedin paraffin. Four-micrometer-thick sections were prepared andmounted on RNase-free glass slides. Sections were dewaxed andthen prehybridized for 3 h. Sections were then incubated with100 µl hybridization buffer (50% formamide, 10% dextransulfate, 1x Denhardt’s solution, 4x SSC, 500 µg/mlsalmon sperm DNA, 200 µg/ml yeast tRNA, 10 mM DTT) containing10 µg/ml biotin-labeled probes at 42°C for 20 h. Biotin-labeledsense probe was severed as the control. After hybridization,sections were washed with 2x SSC and buffer (100 mM Tris-HCl,pH7.5, 150 mM NaCl), blocked with goat serum, and then incubatedwith streptavidin-HRP. Unbound HRP was removed by washing withbuffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2).Finally, the biotin-labeled hybrids were detected by DAB.
Figure 2. Nucleotide and predicted amino acid sequences of the AngRem104 cDNA. AngRem104 cDNA full-length is 1690 bp. It contains a 1041-bp open reading frame flanked by 5'- and 3'-untranslated regions. Kozak sequence was in the box. The poly A signal (ACTAAA) is observed at the 3'-untranslated region and is indicated by double underline. The probe used for in situ hybridization is indicated by bold underline.
Generation of AngRem104 Eukaryotic Expression Plasmid Constructs
The full-length coding sequence of AngRem104 was amplified byPCR. 5'-EcoRI site (GAATTC) and 3'-BamHI site (GGATCC) wereintroduced using sense primer (5'-CGG AAT TCA TGT CAG ATT ATAATC CTG ATG-3') and antisense primer (5'-CGG GAT CCA CTG TAGAGG AGT CCT AGG-3'). The PCR amplified products were digestedwith EcoRI and BamHI, agarose gel-purified, and ligated intoEcoRI-BamHI-digested pEGFP-N1 expression vector (Clontech) togenerate recombinant plasmid, AngRem104-pEGFP-N1 (13). Meanwhile,the sense and antisense full-length coding sequence of AngRem104,which was amplified by PCR using sense primer (5'-ATG TCA GATTAT AAT CCT GAT G-3') and antisense primer (5'-ACT GTA GAG GAGTCC TAG G-3'), was ligated into pcDNA3.1/V5-His-TOPO vector(Invitrogen, San Diego, CA) following the manufacturer’sprotocol to construct the mammalian expression vector. All constructswere sequenced to ensure proper in-frame ligation and Taq polymerasefidelity. Constructs were subcloned, and the plasmid cDNAs werepurified by Midi plasmid preparation kit (Qiagen).
Expressive Analysis of Eukaryotic Constructs
COS-1 cells (ATCC) were cultured at 60 to 80% confluence andplated into six-well plates, grown overnight, and transfectedtransiently with the AngRem104-pEGFP-N1 or pEGFP-N1 plasmidDNA by LipofectAMINT 2000 Reagent (Invitrogen) according tothe manufacturer’s instructions. Subcellular localizationof AngRem104-pEGFP was observed at different time points witha fluorescence microscope after transfection (14). The humanMC (Clonetics) was also transfected transiently with the sense-,the antisense-AngRem104-pcDNA3.1/V5-His-TOPO, and the pcDNA3.1/V5-His-TOPOplasmid DNA by Lipofect-AMINT 2000 Reagent, respectively. Foreach transfection, 10 µl of lipofectin reagent and 1 µgplasmid DNA were used. -gal staining kit (Invitrogen) was usedto evaluate efficiency of each transfection.
Western Blot Analysis
The sense-, antisense-AngRem104-pcDNA3.1/V5-His-TOPO, and pcDNA3.1/V5-His-TOPOplasmid DNA were transfected into human MC, and human MC werecollected 48 h after transfections. Protein preparation wasperformed according to the manufacturer’s instructions(Santa Cruz Biotechology, Santa Cruz, CA). The extracted proteins(5 µg) were subjected to 10% polyacrylamide gel electrophoresisin the presence of sodium dodecyl sulfate (SDS-PAGE), and transferredelectrophoretically onto a nitrocellulose membrane (Bio-Rad,Hercules, CA). The transfected protein from human MC was probedwith anti-V5-HRP-antibody (Invitrogen). The reaction bands weredetected by enhanced chemiluminescence (Pierce Chemical, Rockford,IL).
cDNA Microarray
1 x 107 human MC was transfected with the sense-AngRem104-pcDNA3.1/V5-His-TOPOor the antisense AngRem104-pcDNA3.1/V5-His-TOPO plasmid DNA.After 48 h, total RNA was isolated from the sense AngRem104-or antisense AngRem104-transfected human MC using Trizol reagent(Life Technologies). Two micrograms of each Poly(A)+ RNA werepurified using Oligotex Direct mRNA Midi kit (Qiagen) and werethen submitted for human 4000 genes microarray analysis (BioStarUnited Gene Tech, Shanghai, PRC). The microarray was scannedat a resolution of ten microns to detect Cy3 and Cy5 fluorescence.The images were analyzed using GenePix pro 3.0 software. Thearea surrounding each element image was used to calculate alocal background, which was then subtracted from the total elementsignal. Background subtracted element signals were used to calculateCy3:Cy5 ratios. The average of the resulting total Cy3 and Cy5signal gave a ratio that was used to balance or normalize thesignals.
RT-PCR and Quantitative Real-Time RT-PCR
Reverse transcription and PCR (RT-PCR) were performed with RT-PCRsystem (Promega), and amplification of an approximately 1 Kbsegment for AngRem104 and a 500 bp segment for FN resulted.In brief, the RT reactions were started with 2 µg of RNA.PCR reaction was performed with the upstream and downstreamprimers, which sequences were listed as above. The upstreamprimer of FN was 5'-TGG AAC TTC TAC CAG TGC GAC-3', and downstreamprimer 5'-TGT CTT CCC ATC ATC GTA ACA C-3'. Amplification reactionswere carried out at 94°C 1 min, at 55°C 1 min, and at72°C 1 min for 30 cycles in a PTC-100 Programmable ThermalCycler (MJ Research Inc, Watertown, MA). Amplification reactionsof 300-bp and 500-bp segments of GAPDH were used as control.
The expressions of FN were also analyzed by quantitative real-timePCR using iCycler iQ Multi-Color Real Time PCR Detection System(Bio-Rad, Hercules, CA). RNA from human mesangial cells wasreverse transcribed using RT-PCR system (Promega) and subjectedto real-time PCR using TaqMan Universal PCR Master kit. Amplificationconditions were a single cycle at 50°C for 2 min and 95°Cfor 10 min, followed by 40 cycles at 95°C for 30 s, 55°Cfor 30 s, and 30 s at 60°C using FN probe and primers designedusing Primer Express software 1.0 (PE Applied Biosystems). Theforward primer was 5'-TCC TTG CTG GTA TCA TGG CAG-3', and thereverse primer was 5'-AGA CCC AGG CTT CTC ATA CTT GA-3'. Anoligonucleotide probe of FN, labeled with a fluorescence tagat 5'-end and a quenching molecule at the 3'-end was 5'-/56-FAM/CCACGT GCC AGG ATT ACC GGC TAC AT/36-TAMRA/-3'. The PCR productof FN was 76 bp. The probe and primers of -actin used as aninternal control. The forward primer was 5'-ATT GCC GAC AGGATG CAG AA-3', and reverse primer was 5'-GCT GAT CCA CAT CTGCTG GAA-3', and the probe was 5'-/56-FAM/CAA GAT CAT TGC TCCTCC TGA GCG GA/36-TAM/-3'. The PCR product of -actin was 150bp. For quantitative assessment of FN and -actin, a calibrationcurve with a plasmid containing the FN target sequence and aplasmid containing -actin target sequence were used, respectively.The standard curve was obtained by serial dilutions range from0.1 to 12.8 ng of a linearized plasmid obtained by cloning thetarget FN and -actin sequence into a PCR4-TOPO vector (Invitrogen),respectively. The final quantity of FN was shown as the ratioof FN (ng) to -actin (ng).
Statistical Analyses
All experiments were performed in triplicate. Continuous variables,expressed as mean ± SD, were compared by ANOVA. Multiplicativeterms were included for evaluation of interactions among explanatoryvariables. The Student-Newman-Keuls procedure was used to evaluatepairwise comparisons. P < 0.05 defined statistical significance.
Isolation and Sequencing Analysis of the Full-Length AngRem104 cDNA
To screen and identify genes, which are involved in the accumulationof ECM induced by AngII in human MC, the differences betweenwith and without AngII stimulation (10-6 M, 24 h) in human MCmRNA populations were analyzed by SSH. The subtracted cDNAswere subcloned, and 120 clones were randomly picked up for differentialexpression screening using Reverse Northern blot. Fifty-fiveclones verified upregulation in tester human MC (data not shown).Twenty of the fifty-five upregulated clones were sequenced,and homology search was performed by NCBI online sever. On thebasis of the BLAST search through NCBI Database (http://www.ncbi.nlm.nih.gov/BLAST/),one novel clone, Clone 104 (referred to as AngRem104; GenBankaccession number, AF367870), whose up-regulated expression wasconfirmed by analysis of Northern blot (Figure 1), was isolated.To obtain the full-length cDNA of AngRem104, 3'- and 5'-RACE-PCRwere performed and an approximately 1.7-Kb cDNA was obtainedand sequenced. The human AngRem104 cDNA full-length was 1690bp. It contained a 1041-bp open reading frame (ORF) flankedby of 5'- and 3'-untranslated regions. The nucleotide sequence(CTGATGT) surrounding the initiation codon at 90–92ntreasonably fits a Kozak consensus site in 5'-untranslated regions,suggesting that it is a site of translation initiation (15).A poly(A) tail with a potential polyadenylation signal (ACTAAA)at the 3'-untranslated region was present. The ORF encodes 347amino acid residues with a predicted molecular mass of 37.2kD (Figure 2). The nucleotide sequence of AngRem104 did notmatch any genome sequences on the BLAST search through NCBIDatabase and Celera Discovery System. However, it has 33% identitieswith nucleocapsid phosphoprotein associated with RNA polymerase(NC-001906) at amino acid level on the BLAST search throughNCBI Database. There was a common conserved domain between nucleocapsidphosphoprotein and AngRem104. It spanned amino acid residues224 to 316 of AngRem104 protein. This conserved domain is essentialfor the activity of the RNA polymerase complex. Proteomics toolsin Expert Protein Analysis System (ExPASy) server of the SWISSInstitute of Bioinformatics (http://cn.expasy.org/tools/) wereused to perform the analysis of primary sequence and motif analysisof AngRem104 protein. The predicted protein has a calculatedisoelectric point of 4.95. No amino terminal (N-terminal) signalpeptide, transmembrane domain, peroxisomal targeting signal(C-terminal), and membrane retention motifs were found; however,the Reinhardt method released that the subcellular localizationof protein AngRem104 was predicted to localize at the cellularnucleus.
Figure 1. Northern blot analysis of AngRem104 mRNA in human mesangial cells (MC) induced with (T, tester) and without (D, driver) Angiotensin II (AngII; 10-6 M). Thirty micrograms of total RNA from T and D were separated by agarose gel electrophoresis and transferred to the Hybond N+ nylon membrane. The membrane was hybridized with -32P-radiolabeled AngRem 104 probe. The same membrane was also hybridized with human GAPDH probe, which was as an internal control. The expression of AngRem104 mRNA was significantly upregulated in AngII-induced human MC.
AngRem104 mRNA Is Upregulated in Human MC Induced by AngII and Blocked by AT1RA (Losartan)
To examine the effect of AngII on AngRem104 expression, humanMC was stimulated with AngII (10-6 M) and the mRNA expressionsof AngRem104 were then detected at 6 h, 12 h, 24 h, and 48 hafter AngII treatment by Northern blot analysis. The expressionof AngRem104 was significantly increased at 6 h in AngII-inducedhuman MC, and the increasing expression was maintained up to48 h after AngII treatment compared with control samples (Figure 3A).To detect whether AngII could specifically regulate theexpression of AngRem104 in human MC, a block experiment wasperformed using one of AT1RA, Losartan (Merck & Co Inc,Whitehouse Station, NJ), in serial concentrations (10-4 M, 10-5M, 10-6 M). The result showed that the upregulated expressionof AngRem104 induced by AngII was dose-dependent blocked byLosartan (Figure 3B).
Figure 3. Northern blot analysis of AngRem104 expression induced by AngII and blocked by Losartan in human MC. Northern blot procedure was performed as described in Materials and Methods. (A) The mRNA expressions of AngRem104 were detected by Northern blot after human MC was stimulated by AngII (10-6 M) for 6, 12, 24, and 48 h. (B) Losartan-blocking experiment was performed with serial concentrations from 10-4 M to 10-6 M. The expression of AngRem104 was significantly increased at 6h after AngII stimulation and the increasing maintained to 48 h, and the upregulated expression of AngRem104 induced by AngII was dose-dependently blocked by Losartan. *P< 0.05 versus control.
AngRem104 Is a Nuclear Protein, and Its mRNA Is Widely Expressed in Human Tissues and Localized in Glomeruli and Tubule in Human Kidney Tissue
To determine the distribution of AngRem104 mRNA in human normaltissues, human Multiple Tissue Expression (MTE) Northern blotswere analyzed. A single transcript of approximately 1.7-Kb AngRem104mRNA was widely expressed in human heart, liver, muscle, kidney,and pancreas. However, three bands, 5.0-Kb, 4.6-Kb, and 1.7-Kb,were detected in human placenta. Weak signal was detected inhuman lung and brain. The transcripts size (1.7 kb) is consistentwith the full-length of AngRem104 sequenced by RACE-PCR (Figure 4).Furthermore, distribution and localization of AngRem104in human kidney tissue were detected by in situ hybridization.It indicated that the AngRem104 mRNA signals were detectableon glomerular and tubular cells (Figure 5). The subcellularlocalization of AngRem104 was investigated using expressed AngRem104-pEGFPin transfected COS-1 cells. Fluorescence microscopy showed thatthe AngRem104-pEGFP fusion protein was localized in both cytoplasmand nucleus of transfected COS-1 cells, but concentrated innucleus; meanwhile, GFP diffusely expressed in cytoplasm ofGFP only transfected COS-1 cells (Figure 6).
Figure 4. Northern blot analysis of the distribution of AngRem104 in human multiple tissues. A commercial human Multiple Tissue Expression (MTE) panel (Clontech) was hybridized with 32P-labeled full-length AngRem104 cDNA. A single transcript of approximately 1.7-Kb AngRem104 mRNA was widely expressed in human heart, liver, muscle, kidney, and pancreas, whereas, in human placenta, three bands, i.e., 5.0 kb, 4.6 kb, and 1.7kb, were detected. Weak signal was detected in human lung and brain.
Figure 5. In situ hybridization analysis of AngRem104 in human kidney tissue. In situ hybridization was performed to detect the distribution and localization of AngRem104 in human kidney tissue. It indicated that AngRem104 mRNA signals were detected on glomeruli (A) and tubule (B). Magnification, x200 in A and B.
Figure 6. Subcellular localization of AngRem104 in COS-1 cells. AngRem104-pEGFP construct was transfected into COS-1 cells, and the expression of fusion protein was detected by the fluorescence microscopy. AngRem104-pEGFP fusion protein was localized in both cytoplasm and nucleus of COS-1 cells, but it was concentrated in nucleus (B and D). However, GFP was diffusely expressed in cytoplasm of COS-1 cells transfected with pEGFP-N1 (A and C). Magnifications: x100 in A and B; x400 in C and D.
Fibronectin Is the Upregulated Gene Related to AngRem104 Overexpression
To investigate the function of AngRem104, we constructed sense-and antisense-AngRem104 mammalian expression vectors (sense-and antisense-AngRem104-pcDNA3.1/V5-His-TOPO constructs) andperformed transfection in human MC. The -gal staining showedthat the transfection efficiency reached peak (45%) at 48 hafter Eukaryotic constructs had been transfected into humanMC. The alternative expressions of sense- and antisense-AngRem104in transfected human MC were detected by RT-PCR and Westernblot, respectively. It revealed that AngRem104 was overexpressedin both mRNA (Figure 7A) and protein levels (Figure 7B) whenhuman MC was transfected with sense-AngRem104, whereas its expressionwas suppressed when human MC was transfected with antisense-AngRem104.In addition, we employed to cDNA Microarray technique to detectthe differentially expressed genes related to the overexpressionof AngRem104, which caused transfection of the sense- and antisense-AngRem104constructs into human MC. The analysis of cDNA microarray showedthat the expressions of 94 genes were upregulated, and two geneswere downregulated when AngRem104 was overexpressed (data notshown). Interestingly, two of the ECM-related genes, human mRNAfor FN and integrin--1 (FN receptor), were dramatically overexpressedin the top of all upregulated genes. RT-PCR was performed toconfirm the expression of FN regulated with sense- and antisense-AngRem104constructs transfection. Figure 8 shows that sense-AngRem104construct significantly enhanced expression of FN in human MCcompared with the antisense-AngRem104 construct transfection,as well as with the control cells (no construct transfection);addition of AngII (10-6 M) in cultured human MC transfectedwith antisense-AngRem104 construct couldn’t affect theexpression of FN. Quantitative real-time PCR was also used todetect the different expression of FN under the conditions describedas above. The standard curves of FN and -actin generated a meanslope of -3.25 and -3.38, respectively. The result of quantitativereal-time PCR also showed that sense-AngRem104 construct significantlyenhanced expression of FN in human MC, and then addition ofAngII (10-6 M) in cultured human MC transfected with antisense-AngRem104construct couldn’t affect the expression of FN (Figure 9).
Figure 7. The expressions of sense- and antisense-AngRem104 constructs in human MC. Sense- and antisense-AngRem104 constructs were transfected into human MC, and the level of mRNA and protein expression was detected by RT-PCR (A) and Western blot (B), respectively. Western Blot analysis of the expression of AngRem104 protein probed with anti-V5-HRP-antibody. (A) AngRem104 was overexpressed in human MC transfected with sense AngRem104 at both 24 and 48 h after transfection (lanes 2 and 6). Lanes 1 and 5 were negative controls. Lanes 4 and 8 were vector controls. Meanwhile, weak signal was detected in human MC transfected with antisense-AngRem104 (lanes 3 and 7). (B) The approximately 42-kD and 5-kD bands represent sense-AngRem104-pcDNA3.1/V5-His fusion protein (lane 2) and pcDNA3.1/V5-His protein (lane 4), respectively. No signals were detected in negative control (lane 1) and transfected with antisense-AngRem104 transfection (lane 3).
Figure 8. Fibronectin (FN) mRNA expression was upregulated by AngRem104 overexpression. FN mRNA expression in human MC was detected by RT-PCR. The expression of GAPDH served as an internal control. FN was overexpressed in human MC transfected with sense-AngRem104 (lane 2) as well as induced by AngII (lane 5), whereas negative control (lane 1), vector control (lane 6), and antisense-AngRem104 construct transfection (lane 3) only showed weak signals, respectively. However, there was no significantly effect to FN expression with addition of AngII (10-6 M) in human MC transfected with antisense-AngRem104 (lane 4). * P< 0.05 versus control.
Figure 9. FN mRNA expression was detected by quantitative real-time PCR. The expression of -actin served as an internal control. FN was overexpressed in human MC transfected with sense-AngRem104 (lane 2) as well as induced by AngII (lane 5), whereas negative control (lane 1), vector control (lane 6), and antisense-AngRem104 construct transfection (lane 3) only showed weak signals, respectively. However, there was no significant effect to FN expression with addition of AngII (10-6 M) in human MC transfected with antisense-AngRem104 (Lane 4). * P< 0.05 versus control.
AngII has been known as a critical factor for the progressionof chronic renal diseases (16). It has also been well knownthat AngII stimulates glomerular MC to produce ECM such as FNand type I and III collagen (11,17,18), The synthesis of extracellularprotein in MC plays an important role in mesangial expansionand pathogenesis of glomerulosclerosis (19). AngII affects cellularbehavior by binding to its cell surface receptor (20). ACEIand AT1RA have been found to ameliorate the glomerulosclerosis/interstitialfibrosis and retard the progression of chronic renal diseases(21,8,22). In addition, the growing evidences have suggestedthat the role of AngII in glomerulosclerosis is partially throughthe regulation of downstream genes, such as transforming growthfactor- (TGF-) and plasminogen-activator inhibitor type-1 (PAI-1)(23,24,17). However, the information at hand is insufficientto elucidate the precise molecular mechanism for AngII producingECM in MC. To explore possible molecule mechanism of ECM accumulationrelated to AngII, we performed SSH to screen and identify upregulatedgenes induced by AngII in cultured human MC (25). In the presentstudy, the novel gene, AngRem104, was isolated and characterized.The expression of AngRem104 was upregulated in human MC inducedby AngII, and the overexpression was dose-dependently blockedby Losartan. It indicated that AngII regulates the expressionof AngRem104 in human MC through AT1R.
To explore the functional clues for AngRem104, cDNA Microarraywas carried out. The results showed that there were 94 upregulatedgenes and two downregulated genes when AngRem104 was overexpressed.Interestingly, two of the ECM-related genes, i.e., human mRNAfor FN and integrin--1 (FN receptor), were dramatically overexpressed.Furthermore, we proved that the overexpression of FN inducedby AngII could be regulated by AngRem104. Therefore, it revealedthat AngRem104 might relate to the regulation of the expressionof FN. From literature, FN regulates many cellular functionsafter directly binding to integrins (26,27). Accumulation ofFN could be inhibited by anti-integrin antibodies (28,29). Theresult of our cDNA microarray showed that the upregulation ofintegrin--1 was accompanied by the overexpression of FN itself.FN is a high-molecular mass adhesive glyocoprotein implicatedin a wide variety of cellular properties, including cell adhesion,differentiation, proliferation, migration, and apoptosis (30).It has been reported that the expression of FN could be regulatedby many molecules, such as TGF- (31,32), cAMP (32), epidermalgrowth factor (EGF), PDGF (33), and interferon- (IFN-) (34).In addition, various cells transfected with oncogenes, e.g.,src, ras, mos, and EIA, could lead to the decreased expressionof FN (35). However, the mechanism responsible for productionand accumulation of FN remains poorly understood. The identificationand functional study of AngRem104 might open a new investigativeview for the regulation mechanism of FN.
How does AngRem104 regulate the expression of FN? Accordingto the results from transfection of COS-1 cells with AngRem104-pEGFP-N1constructs in our study, it was shown that AngRem104 is a nuclearprotein, which is consistent with the predicted result of bioinformatics.It suggested that AngRem104 might play a role in the regulationof gene transcription in the nucleus. Therefore, possible mechanismfor expression of FN upregulated by AngRem104 might includethe binding with some response elements of FN promoter in theprocess of transcriptional regulation, or altering nuclear RNAsplicing and nuclear stability in the process of posttranscriptionalregulation. Northern blot analysis of distribution of mRNA inhuman normal tissues indicated that AngRem104 widely expressesin a variety of tissues, and perhaps additional isoforms existsin human placenta. It could be postulated that AngRem104 mighthave physiologic or pathologic effects on various human tissues.
Our work proves that AngRem104 is a novel human gene. It codesa nuclear protein that relates to the expression of FN. Theexpression of AngRem104 was upregulated by AngII in human MC.AngRem104 might conceivably be one of the key molecules involvedin the pathologic process related to the accumulation of FNinduced by AngII. Further investigation for the function ofAngRem104, particularly the regulating pathway on AngII, AngRem104,and FN, will provide insight into the molecular mechanisms ofAngII on glomerular sclerosis.
Acknowledgments
This work was supported by the National Natural Science Foundationof P.R.China (Grant No. 39970929) and the Foundation of PekingUniversity Center of Human Disease Genomics (Grant No. A-21).We thank Dr. Yanling Zhang (Peking University Health ScienceCenter, Beijing, China) for her technical assistance on in situhybridization. We would like to thank Dr. Edward J. Benz Jr.and Dr. Shu-Ching Huang (Dana-Farber Cancer Institute, HarvardMedical School, Boston, MA) for their helpful comments on themanuscript.
Wolf G: Angiotensin II: A pivotal factor in the progression of renal diseases. Nephrol Dial Transplant 14 [Suppl 1]: 42–44, 1999[Free Full Text]
Egido J: Vasoactive hormones and renal sclerosis. Kidney Int 49: 578–597, 1996[Medline]
Wolf G: Molecular mechanisms of angiotensin II in the kidney: Emerging role in the progression of renal disease: beyond haemodynamics. Nephrol Dial Transplant 13: 1131–1142, 1998[Abstract/Free Full Text]
Wolf G, Neilson EG: Cellular biology of tubulointerstitial growth. Curr Top Pathol 88: 69–97, 1995[Medline]
Nakaya H, Sasamura H, Kitamura Y, Amemiya T, Konishi K, Hayashi M, Saruta T: Effects of angiotensin inhibitors on renal injury and angiotensin receptor expression in early hypertensive nephrosclerosis. Hypertens Res 22: 303–312, 1999[Medline]
Burnier M, Brunner HR: Angiotensin II receptor antagonists and the kidney. Curr Opin Nephrol Hypertens 3: 537–545, 1994[CrossRef][Medline]
Li J, Zhou C, Yu L, Wang H: Renal Protective effects of blocking the intrarenal renin-angiotensin system. Hypertens Res 22: 223–228, 1999[Medline]
Weiss RH, Ramirez A: TGF- and angiotensin II-induced mesangial matrix protein secretion is mediated by protein kinase C. Nephrol Dial Transplant 13: 2804–2813, 1998[Abstract/Free Full Text]
Wolf G, Haberstroh U, Neilson EG: Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. Am J Pathol 140: 95–107, 1992[Abstract]
Ray PE, Bruggeman LA, Horikoshi S, Aguilera G, Klotman PE: Angiotensin II stimulates human fetal mesangial cell proliferation and fibronectin biosynthesis by binding to AT1 receptors. Kidney Int 45: 177–184, 1994[Medline]
Gomez-Garre D, Ruiz-Ortega M, Ortego M, Largo R, Lopez-Armada MJ, Plaza JJ, Gonzalez E, Egido J: Effects and interactions of endothelin-1 and angiotensin II on matrix protein expression and synthesis and mesangial cell growth. Hypertension 27: 885–892, 1996[Abstract/Free Full Text]
Zhang H, Wada J, Hida K, Tsuchiyama Y, Hiragushi K, Shikata K, Wang H, Lin S, Kanwar YS, Makino H: Collectrin, a collecting duct-specific transmembrane glycoprotein, is a novel homolog of ACE2 and is developmentally regulated in embryonic kidneys. J Biol Chem 276: 17132–17329, 2001[Abstract/Free Full Text]
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 263: 802–805 1994[Abstract/Free Full Text]
Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P: Green fluorescent protein as a reporter of gene expression and protein localization. Biotechniques 19: 650–655 1995[Medline]
Kozak M: Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266: 19867–19870, 1991[Free Full Text]
Kim S, Iwao H: Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal disease. Pharmacol Rev 52: 11–34, 2001
James LR, Ingram A, Ly H, Thai K, Cai L, Scholey JW: Angiotensin II activates the GFAT promoter in mesangial cells. Am J Physiol Renal Physiol 281: F151–F162, 2001[Abstract/Free Full Text]
Kagami S, Kuhara T, Okada K, Kuroda Y, Border WA, Noble NA: Dual effects of angiotensin II on the plasminogen/plasmin system in rat mesangial cells. Kidney Int 51: 664–671, 1997[Medline]
Edwards RM, Aiyar N: Angiotensin II receptor subtypes in the kidney. J Am Soc Nephrol 3: 1643–1652, 1993[Abstract]
Zhou A, Yu L, Li J, Zhang J, Wang H: Renal protective effects of blocking the intrarenal renin-angiotensin system: angiotensin II type I receptor antagonist compared with angiotensin-converting enzyme inhibitor. Hypertens Res 23: 391–397, 2000[Medline]
Ruiz-Ortega M, Egido J: Angiotensin II modulates cell growth-regulated events and synthesis of matrix proteins in renal interstitial fibroblasts. Kidney Int 52: 1497–1510, 1997[Medline]
Motojima M, Kakuchi J and Yoshioka T: Association of TGF- signaling in angiotensin II-induced PAI-I mRNA upregulation in mesangial cells: role for PKC. Biochim Biophys Acta 1449: 217–216, 1999[Medline]
Chung KY, Agarwal A, Uitto J, Mauviel A: An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta. J Biol Chem 271: 3272–3278, 1996[Abstract/Free Full Text]
Liang X, Zhang H, Zhou A, Hou P, Wang H: Screening and identification of the up-regulated genes in human mesangial cells exposed to angiotensin II. Hypertens Res 26: 225–235, 2003[CrossRef][Medline]
Akiyama SK, Yamada SS, Chen WT, Yamada KM: Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol 109: 863–875, 1989[Abstract/Free Full Text]
Darribere T, Guida K, Larjava H, Johnson KE, Yamada KM, Thiery JP, Boucaut JC: In vivo analyses of integrin beta 1 subunit function in fibronectin matrix assembly. J Cell Biol 110: 1813–1823, 1990[Abstract/Free Full Text]
Fogerty FJ, Akiyama SK, Yamada KM, Mosher DE: Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (51) antibodies. J Cell Biol 111: 699–708, 1990[Abstract/Free Full Text]
Dean DC: Expression of the fibronectin gene. Am J Respir Cell Mol Bio 1: 5–10, 1989
Blatti SP, Foster DN, Ranganathan G, Moses HL Getz MJ: Introduction of fibronectin gene transcription and mRNA is a primary response to growth factor stimulation of AKR-2B cells. Proc Natl Acad Sci 85: 1119–1123, 1988[Abstract/Free Full Text]
Dean DC, Newby RF, Bourgeois S: Regulation of fibronectin biosythesis by dexamethasone, transforming growth factor beta and cyclic AMP in human cell lines. J Cell Biol 106: 2159–2170, 1988[Abstract/Free Full Text]
Kaiura TL, Itoh H, Kubaska SM3rd, McCaffrey TA, Liu B, Kent KC: The effect of growth factors, cytokines, and extracellular matrix proteins on fibronectin production in human vascular smooth muscle cells. J Vasc Surg 31: 577–584, 2000[CrossRef][Medline]
Czaja MJ, Weiner FR, Eghbali M, Giambrone MA, Eghbali M, Zern M: Differential effects of -inferferon on collegen and fibronectin gene expression. J Biol Chem 262: 13348–13351, 1986
Setoyama C, Liau G, de Crombrugghe B: Pleiotropic mutants of NIH 3T3 cells with altered regulation in the expression of both type I collagen and fibronectin. Cell 41: 201–209, 1985[CrossRef][Medline]
Received for publication June 4, 2002.
Accepted for publication February 28, 2003.
This article has been cited by other articles:
X. Liang, K. W. Peters, M. B. Butterworth, and R. A. Frizzell 14-3-3 Isoforms Are Induced by Aldosterone and Participate in Its Regulation of Epithelial Sodium Channels
J. Biol. Chem.,
June 16, 2006;
281(24):
16323 - 16332.
[Abstract][Full Text][PDF]
P. J. M. Jack, D. B. Boyle, B. T. Eaton, and L.-F. Wang The Complete Genome Sequence of J Virus Reveals a Unique Genome Structure in the Family Paramyxoviridae
J. Virol.,
August 15, 2005;
79(16):
10690 - 10700.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-1 (FN receptor), dramatically upregulated their expressions. Furthermore, AngRem104 could regulate the expression of FN induced by AngII, which were detected by RT-PCR and quantitative real-time PCR, when AngRem104 was overexpressed. It is concluded that AngRem104 is a novel human gene potentially involved in the regulation of FN induced by AngII in human MC. These findings may provide new insights into mechanisms of glomerular sclerosis associated with AngII. E-mail: hongzh@bjmu.edu.cn 
-32P]-dCTP-labeled tester and driver cDNA populations independently, which reflect the mRNA expression with or without AngII stimulation. The overexpressed clones were picked up and sequenced by automated DNA sequence analyzer (ABI PRISM 310, Genetic Analyzer, Perkin-Elmer, Foster City CA). The homology search was performed by NCBI online server. 
(IFN-
