The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in P

Availableonlineatwww.sciencedirectcomJOURNALOf° SciencedirectGENETICS ANDGENOMICSELSEVIERJ. Genet Genomics 35(2008)153-161The ethanol response gene Cab45 can modulate the impairmentelicited by ethanol and ultraviolet in pc12 cellsYunfeng Zhu, Quanli Wang, Wangru Xu, Sha Liab Department of Transfusion in Beijing 307 Hospital, Fengtai District, Beying 100071, ChinaReceived for publication 15 October 2007: revised 27 November 2007: accepted 7 December 2007AbstractHigh consumption of ethanolic beverages facilitates neurodegeneration, but the mechanism of this process still remained elusive. Sup-pression subtractive hybridization(SSH)is a technique for detection of rare transcripts, With SSH approach, we identified one ethanolresponse gene Cab45, which was down-regulated by ethanol with time-dependent manner in B104 cells. The full-length sequence ofCab45 gene was obtained by 5-RACE(5 Rapid Amplification of cDNA Ends) for the first time in rat. Based on the sequence of deducedamino acid of rat Cab45, the alignment was conducted with its counterparts in different species and displayed a high conservation. Usingdifferent tissues in rat and cell lines, Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation.Given that ethanol facilitates some cell differentiation, we hypothesize that Cab45 is involved in ethanol-mediated differentiation. withtransient transfection, the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells. Ethanol treatment andUV exposure were conducted subsequently and cell proliferations were detected by MTT (Methyl Thiazolyl Tetrazolium) approach. Itrevealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells. As a member of newalcium binding protein family, the exact role of Cab45 still remains unclear.Keywords: ethanol; Cab45: calcium-binding protein; gene identification; subtractive hybridizationIntroductionneurodegeneration in infant rat and mouse brain(Young etaL., 2003). The cause of neurodegeneration by ethanol re-As a major human disease risk factor, ethanol is in- mains to be elusive and considerable research attention hasvolved in many diseases concerned with energy and sub- been focused on identifying the endogenous and environ-strate metabolism, e.g, arteriosclerosis, dementia, diabetes, mental factors that contribute to its etiologyand conditions associated with Zn metabolism(Kim et al.The positive association between ethanol consumption2004; Suter, 2004). High consumption of ethanolic bever- and neurodegeneration was highlighted by recent studies: 1)ages increases the risk of certain cancers(Doll and Peto, ethanol induces apoptotic neurodegeneration during1981; Mufti, 1993; Tonnesen et al, 1994)and facilitates synaptogenesis period of development in rodents(Olney etneurodegeneration(Fein and Sclafani, 2004; Luchsinger et al., 2002); 2)ethanol selectively decreases Purkinje cellal, 2004). It has been reported that ethanol exposure leads expression of tyrosine-kinase B(TrkB)and tyrosine-kinaseto impairments of the cerebra and cerebellum in different C (TrkC)receptors following exposures within postnatalareas, such as agranular insular cortex, anterior piriform days 4-6. These results suggest that ethanol may inducecortex, perirhinal cortex, lateral entorhinal cortex, and the loss of中国煤化 Neurotrophic regulatemporal dentate gyrus, and triggers widespread apoptotic tion atH102); 3)using a dualmechaCNMHGmethyl-D-aspartatesponding author Fax: +86-10-5112 8448.(MDA) glutamate receptors and excessive activation ofmailaddresszhuyf2004@163.comGABA (A)receptors, ethanol can trigger widespreadYunfeng Zhu et al. /Joumal of Genetics and Genomics 35 (2008)153-161apoptotic neurodegeneration in the developing rat forebrain. Neuron growth factor (NGF) treatment for PC12In humans, vulnerability coincides with the period of synaptogenesis and extends from the sixth month of gestation PC12 cells were cultured in Dulbecco's modified Ea-to several years after birth (Ikonomidou et al., 2000); 4)by gle's medium(DMEM)supplemented with 5% horse se-suppressing neuronal activity through altering glutamate rum and 10% fetal calf serum(Hyclone, South Logan, UT,nd GABA transmission, ethanol causes millions of nerve USA)(ean et al, 2005). For experiments, the PC12 cellscells to commit death in the developing rodent brain. This were incubated with serum-free medium(Dulbecco'pro-apoptotic effect of ethanol provides a likely explana- modified Eagle's medium containing 2 mg/mL bovine se-tion for the diminished brain size and lifelong neurobehavrum albumin, 1 ug/mL insulin, 2 ug/mL transferin, 30ioral disturbances associated with the human fetal ethanol nmol/L Na,SO, and 10 mmol/L HEPES, pH 7. 4)before-syndrome(Farber and olney, 2003)hand for 12 h and followed by treatment with NGF(30Based on the phenomenon of ethanol involvement in the ng/mL). The differentiation of PC12 cells induced by NGFprocess of neurodegeneration, we tried to identify ethanol was evaluated by synapse outgrowthresponse genes using suppressed subtractive hybridization(SSH) method. This method offers two major advantages,Suppressing subtractive hybridizationi.e., the inclusion of all the sequences in the cDNA pooL,and the equalization of rare and abundant messages fol-Using a commercial kit( Clontech Laboratories, Palolowing the initial hybridization(Duguid and DinauerAlto, CA, USA), the subtractive hybridization was per-1990). With this method, we identified an ethanol response formed in B104 cells(rat neuroblastoma cell). Brieflygene, the counterpart of the mouse and human Cab45 gene B104 cells were treated with ethanol (0.2%)for 6 daysin rat. The time course of expression exhibits a time-de- Total RNas derived from both ethanol treated B104 andpendent down-regulation by ethanol treatment. Based oncontrol were isolated by Trizol regent. mRNAs were pre-vestigated Cab45 expression using cell model of nge pared by polya tract kit (Promega, Madison, WL, USA)(Neuron Growth Factor)-induced PC12 cells and rat cerebellus with different postnatal days. with the stratagemRsa I and in which tester cdNa was then ligated withof up-regulation and down-regulation, Cab45 was demon- adaptors provided by the kit. Hybridization was conductedstrated to modulate the impairment elicited by ethanol or between the tester and the driver, followed by nest PCRUV in PC12 cellsamplification with the specific primers provided by the(primerl for the first amplification and nested primer2R for the second amplification). PCR productsMaterials and methodsligated with the TA vectors pCR4-TOPO (Invitrogen,Carlsbad, CA, USA) to generate recombinant plasmid Hi-Cell culture and treatmentbrary (pCR4/SSH). After being transformed into TOP10cells(Invitrogen, Carlsbad, CA, USA), the clones wereB104 cells were routinely grown in RPMI1640 supple- cultured for further identificationmented with 2 mmolL L-glutamine, 4.5 gL glucose, 10mmol/L HEPES, I mmol/L sodium pyruvate, 1.5 g/L so- Reverse dot blot hybridizationdium bicarbonate, 7.2 mg/L insulin, 100 ug/mL Penicillin/Strepmycin, and 10% fetal bovine serum Cultures were Filters preparationincubated at 37C with a saturating humidity and 5% coThe transformed clones were picked up randomly,atmosphere. Medium was changed every 3 daysseeded in 96-well plates filled with 100 AL LB broth(con-taining 50 ug/mL penicillin) per well beforehand, and cul-Ethanol exposure for B104 cellstured at 37C for 6 h. PCR amplification was conducted foreach clone with the primers of Nested primer I and 2R andGiven to the volatility of ethanol, the sealed containers condition was carried out at 95C for 4 min followed by 30were used for maintaining ethanol concentration in me- cycles of 94 C for 30 s, 55C for 30 s, and 72C for I min.dium(Adickes et al., 1988; Luo and Miller, 1996). Briefly, pCr products were denatured in 0.6 mol/L Naoh contain-the amount of ethanol was calculated in terms of the vol- ing 0.5% bromophenol blue and then transferred onto Ny-ume of medium in dish and of water-bath in container(e. g. tran中国煤化工ot- blotting apparatus0.2%), respectively. A small volume of CO2 was injected (SchIinto the container before sealing. The ethanol-containing TwoCNMHGampshire, Germany)water bath was changed daily to maintain the ethanol con-centration. Cultures were maintained at 37.C in a saturat- Labeled cdNA library preparationing humidity and 5%cO2 atmospherBoth labeled cDNA libraries(driver and tester)wereYunfeng Zhu et al. Journal of Genetics and Genomics 35 (2008)153-161prepared by reverse transcription with [PJ-a-dCTP incor- Cincinnati, OH, USA), total RNAs from different tissuesporation(Zhu et al, 2001). In brief, the reaction mixture(35 and cells were extracted For each sample, 10 ug RNA (perHL)was prepared with the following components: mRNA, lane)was fractionated by agarose gel electrophoresis, fol-oligo-dT(10 umol/L), 50x dNTP (10 mmol/L dATP, 10 lowed by transferring into a Nytran membrane and fixingmmolL dGTP, 10 mmolL dTTP, and 100 umol/L dCTP), with ultraviolet cross-linkingreverse transcriptase (M-MLV, 200 U/HL, BRl, rockville,MD, USA),["P]H-a-dCTP(3,000 CimmolL, ICN Bio. Probe preparationmedicals, Irvine, CA, USA)and reverThe probes for rat Cab45 and cyclophilin(intermal stan-The mixture was incubated at 42%C for 90 min and then at dard)were prepared by incorporation of [PI-a-dCTP in75%C for 10 min. After adding I HL RNase A(20 ng/L)and process of PCR amplitication In brief, the PCR reactionI Al RNase H(I U/HL), the mixture was incubated at 37oc mixture was prepared with following componentsfor 45 min Unincorporated nucleotides were removed using[PI-a-dCTP (3,000 Ci/mmoLL), templates(subtractivea G-50 sephadex spin column and the probe activity wasDNA clones or cyclophilin recombinant plasmid, 20detected by scintillation counterng/mL), 50x dNTP(10 mmol/L dATP, 10 mmolL dGTP10 mmolL dTTP, and 100 umouL dCTP), Taq dna po-Hybridizationlymerase(5 U/HL), and the PCR primers(provided by theWith two same filters and hybridization buffer (UL kit). PCR amplification was conducted at 95C for 3 minTRAhybM Buffer, Austin, TX, USA), pre-hybridization followed by 30 cycles of 94 C for 30s, 55%C for 30 s, andwas conducted at 42 C for 30 min Isotope labeled cDNA 72C for 2 min. The PCR products labeled withlibraries(1x10 cpm/mL)derived from driver and tester [P]-a-dCTP were purified by G-50 column(Promega,were denatured(100 C for 5 min)and immediately added Madison, WI, USA)to remove unincorporated nucleotidesto their pre-hybridization solution, respectively. After The probe activity was detected by scintillation counter.overnight incubation at 42.C, the filters were washedbriefly with solution 1( 2x SSC, 0. 1% SDS)for three times Blotting and hybridization(5 min each) at 42C followed by three additional washesHybridization was conducted with the same procedure as(15 min each) with solution 2(0. 1x SSC, 0. 1% SDS)atdescribed in reverse dot blot. The filters were processed42C.Hybridization signals were visualized with Phos. with Phosphor Imager and subsequently the probes werephorlmager( Molecular Dynamics, Houston, TX, USA) stripped off immediately by incubating with the solution ofand analyzed by Image QuaNT (Molecular Dynamics, 50% formamide and 2x SSPE at 65C for I h. The sameSunnyvale, CA, USA)filter was re-hybridized with intermal-standard probeedure. The values of signalsFull-length gene preparationwere analyzed with Image QuaNT software.The full-length gene of Cab45 in rat was prepared by 5 Rat cerebellum cell preparationRACE (5 Rapid Amplification of cDNA Ends, Life Technologies, Grand Island, NY, USA). In brief, threeAccording to"The Guide or The Care and Use of Labo-atory Animals in US", the rats with different post-natalgene-specific primers(termed GSPL, GSP2, and GSP3) days as well as adult were sacrificed and their cerebellumwere designed based on known DNA sequence With GSPIprimer,cDNA was prepared by reverse transcription and (Dutton et al, 1981). In brief, the rats with differentpoly-dc tail was added with terminal deoxynucleotidyl post-natal days and adult were anesthetized and decapitatedtransferase. With GSP2 and a bridged anchor primer (pro- rapidly. Whole cerebellum was moved out and subsevided by the kit), PCR amplification was performed and quently rolled on frosted-glass plate to strip off its meningeproducts were ligated with TA cloning vector (pCR4- Covering with a drop of solution IH(NaCl 3.8 mmolLTOPO, Carlsbad, CA, USA)to generate recombinant KCI 5.0 mmol/L, HEPES 25.0 mmol/L, NaHCO3 4.2plasmids PCR4-TOPO/ Cab45. After being transfected into mmol/L, NaH, PO, H, 0 1.0 mmol/L, Phenol red 0.01%TOP10 cells(Invitrogen, Carlsbad, CA, USA), the positive bovine serum albumin 0.3%, and MgSO4 solution 0.04%),clones were identified by PCR amplification with GSP3 the cerebellum was chopped into 400 um tissue blocksprimer and bridged anchor primer中国煤化工 sin solution(25mgtrypsinNorthern blot hybridizationCNMHGfoT I5 min with oc.-- was neutralized byadding equal amount of diluted DNase/trypsin inhibitorTotal RNa isolationsolution. Through a 9-inch Pasteur pipette, tissues wereWith TRI reagent (Molecular Research Center, INC, dissociated and settled clumps were removed by centrifuYunfeng Zhu et al. Journal of Genetics and Genomics 35(2008)153-161gation. Carefully underlaid cell suspension with 4% bSA was confirmed by sequencingsolution in 15 mL centrifuge tube, which formed a distinctinterface between the heavier/clear 4% BSA and the Transient transfectionlighter/cloudy cell suspension. The pellet cells were colected by brief centrifugation(50-100 g for 5 min)PC12 cells were cultured in Dulbecco's modified Ea-re-suspended with RPMI-1640 medium included asgle's medium(MEM)supplemented with 10% horse se-component as described aboverum and 5% fetal calf serum(Hyclone, South Logan, UT,USA). The transient transfection was performed followingConstruction of recombinant plasmidsmanufacturer's instructions of LipofectAMINE 2000(Irvitrogen, Carlsbad, CA, USA). Briefly, PC12 cells wereFor Cab45 up-regulation: Based on Cab45 full-length seeded in 24-well plate with the concentration of 1-2x 105gene and multi-clone site of pcDNA3. 1/HisA, both up- cells/mL and cultured overnight(about 95%confluence).stream and downstream primers of Cab45 were designed For each well, I Hg DNA and 2 HL LipofectAMINE 2000and synthesized(upstream primer: 5-CGGGATCCTGAT. reagent were added into 50 HL OPTI-MEM I mediumGGTCTGGCTGGTGGCA-3, BamH I site is underlined spectively followed by mixing and then incubated at roand start code is in bold; downstream primer: 5-CGGAAT temperature for 20 min. One hundred microliters of theTCTCAGAACTCTTCATGCAC-3 EcoR I site is under- mixture were added to each well with gentle rocking andlined and reverse complement sequence of stop code is in cultured overnight.bold). With the template of cDNA of B104 cells, PCR am-plification was conducted at 95 C for 3 min followed by 30 Ethanol and UV treatment for transfected PC12 cellscycles of 94@C for 30 s, 55.C for 30 s, and 72oC for 2 min.Being digested with EcoR I /BamH L, PCR products andEthanol treatmentspcDNA3. I/Hisa were ligated to generate a recombinantTransfected PC12 cells and control(parental cells)wereplasmid pcDNA3. 1/Cab45. After being transformed into treated by ethanol at concentrations of 0.05%,0.1%,0.15%TOPIO cells, the recombinant plasmid was confirmed byand 0. 2% for 48 h with the same procedure describedabove. The proliferations in different cells were evaluatedFor Cab45 down-regulation: Using the software of by MTT testsiRNA Target Finder(Ambion, Austin, TX, USA), RNAi UVtreatment(RNA interference)sequence for Cab45 wasTransfected PC12 cells and control(parental cells)weretwo candidate sequences(5-AAAGACAGGAAGAAAG- treated by UV exposure for 10 s and 20 s under a 40 WattAGTTT-3'and 5'-AAACTCTTTCTTCCTGTC-3) were UV light followed by culturing for additional 24 hThechosen. Based on the instruction of psilencer 2. 1-06/neo proliferations in different cells were evaluated by mtTkit(Clontech, Palo Alto, CA, USA), the hairpin structure test.of these rNaiwith BamH I cohesive site in 5 end: 5 -GATCCAAAGA- Statistical analysisCAGGAAGAAAGAGTTTTTCAAGAGAAAACTCTTT-CTTCCTGTCTTTTTTTTTA-3, anti-sense strand 1 withDifferences among groups were tested using one-wayHind Ill cohesive site in 5'end: 5-AGCTTAAAAAAAAA. analysis of variance(ANOVA). Differences with P<0.05GACAGGAAGAAAGAGTTTTCTCTTGAAAAACTCTwere considered as statistically significant. In the casesTTCTTCCTGTCTTTG-3'; sense strand 2 with Bamh I where significant differences were detected, specificcohesive site in 5' end: 5-GATCCAAACTCTTTCTTC-post-hoc comparisons between groups were examined byCTGTCTTCAAGAGAGACAGGAAGAAAGAGTTTTTStudent-Newman-Keuls tests(Altman et al., 1988)TTTTA-3 anti-sense strand 2 with Hind Ill cohesive sitein 5'end: 5-AGCTTAAAAAAAAACTCTTTCTTCCTGE:ResultsCTCTCTTGAAGACAGGAAGAAAGAGTTTG-3, hair. Resultspin structure is underlined). With gradient annealing(from ldentification of ethanol response genes in cells100C to 37 C), two pairs of double-strand RNAi sequences were formed and followed by ligating with the中国煤化工es were obtained andpsilencer2. 1-U6/neo digested prior with BamH I /Hind Ill conYHCNMHGpresented pattern isto generate recombinant plasmid psilencer2. 1/Cab45- shown in Fig. 1. The time-course expression of the candi-RNAi-l and psilencer2. 1/Cab45-RNAi-2. After being date clones was further investigated by Northern blottingtransformed into TOP10 cells, the recombinant plasmid analysis afterwards. One clone, 5Bll, exhibited a timeYunfeng Zhu et al. /Joumal of genetics and Genomics 35(2008)153-161dependent downregulation by ethanol for long-term treat. To investigate the conservation of Cab45, the deductivement even though a slight up-regulation initially(Fig. 2). amino acid of Cab45 rat was aligned with its counterpart inThe expressions of 5Bll and cyclophilin(intermal standard) mouse and human. The homologies are 95% with mousewere quantified by Image QuaNT software. After being and 89% with human(Fig 3). Especially, the six EF-handnormalized by the values of cyclophilin in different motifs(110-121, 149-160, 208-219, 245-256, 290-301time-points, the relative values of clone 5Bll were calcu- 326-337)in Cab45 were highly conservative, which arelated and plotted against treating time( fig. 2). After being 100% homology between rat and mouse, and 93% betweensequenced and checked by gen Bank database, clone 5Bll rat and human respectivelydisplayed a high homology (99%)with mouse Cab45a(calcium binding protein with 45 kDa). Thus, clone 5Bll Cab45 is expressed ubiquitouslywas considered as a counterpart of mouse Cab45 in rat.Based on the fact of other members in Cab45 familyCharacterization of Cab45 genely have a ubiquitous expression, we investigated the dis-tribution of Cab45 with rat tissues. As expected, a ubiqui-Because the full-length sequence of Cab45 was not de- tous expression of Cab45 was observed in different tissuesermined in rat, we prepared it by 5'RACE. Based on the of rat(Fig 4)character of flank sequence of start code(Kozak, 1997).the sequence combined from the clones of 5B11 and Cab45 is involved in the regulation of cell differentiationPCR4-TOPO/Cab45 is considered as full-length sequenceof Cab45 in rat(GenBank/EMBL accession no. AF405545)With the differentiation models of NGF-treated PC12EthanolFig 1. The clones derived from SSH were confirmed by reverse dot blot hybridization. The DNAs derived from of subtractive clones was prepared by PCRmplification and coated onto Nytran membrane by Minifold I dot-blotting apparatus. For each patten, two exacted membranes were made. cDNAs de-rived from driver and tester were prepared by reverse transcription with ["PI-a-dCTP incorporation. After hybridizations, the signals were processed withPhosphorlmager and analyzed with Image QuaNT software. A representative pattern including clone 5B11 is shown(solid arrow: up-regulation by ethanol;blank arrow: down-regulation by ethanol)1.846914Ct Et Ct Et Ct Et Ct Et Ct Et Ct Et Ct Et他 Cyelophilin1/41346914daysThe time course of ethanol treatmentFig. 2. The time course of expression of clone 5B1lwas investigated by Northen blo中国煤化工As derived from differenttime points treated by ethanol in B104 cells were extracted and fractionated with 1.0%lone 5Bll an(intemal standard)were prepared by PCR with["PI-a-dCTP incorporation. After hyCNMHwith Phosphorethanol treatment(0.2%); Ct: control (free of ethanol treatment). Triplicate experiments were carried out independently. (B)The values ofclone SBll and cyclophilin in different time-points were quantified by Image QuaNT. After normalization by the value of cyclophilin, the relatiof 5Bll in ethanol-treated B104 cells to control were calculated and plotted against treating time. The data for statistics analysis was derived from threeindependent experiments(mean*S E M)Yunfeng Zhu et al. Joumal of Genetics and Genomics 35(2008)153-161Mouse 1 /VW-LVAMTFROESLCGLAAHGLWFLGLVLLMDATARPANHSSTRERAANREENETMPPDHumanWPWVANAsRWGP减cDA5 ARPANHSSTREPVANREEN玩Consensus 1 mvw 1VAMtsRq sLcGLAahgLWfLGlVLLMDAtARPANHSstRERaANReENEIiPPD60 HI NGVKLEMDGHLNKDFHQEVFIGKDMDGFDEDSEPRRSRRKLMVI FSKVDVNTDRRIConsensus 61 HLNGVKLEMDGHLNkdFHQEVFLGKDmdGFDEDsEPRRSRRKIMVIESKVDVNTDRrISA120VKFLASKMouse120 KEMQHWIMEKTAEHFQEAVKENKLHFRAVDEDGDGHVSWDEYKVKFLASKGHNEREIAEAHuman121 KEMO暑暑配 TFRAVDPDGDGHVSWDEYKVKFLASKGH巨K以DConsensus 121 KEMQhWIMEKTAEHFQEAvkEnKlHFRAVDPDGDGHVSWDEYKVKFLASKGHnErEiAdA180K计了E心东刈APA下E[EFFP5PK180 IKNHEELKVDEETQEVLGNLRDRWYQALNPPADLLLTEDEFLSFLHPEHSRGMLKEMVKE81国心EK江W? PPADLLLTEEEFLSFTHPEHSR的KConsensus 181 IknhEELKVDEETQEVLgNLrDRWYQADnPPADLLLTEdEFLSFLHPEHSRGMLKEMVKELPEFISLPVGTVENQOGQDI DDNWVKDRKKEFEELIDSNHDGIV'IMFRDL DODGDKQLSLPE FISLPVGTVENQQGQDIDDNWVKDRKKEFEELIDSNHUGIVI241 [VRDI DODGDKQLSYPEFISLPVGTVENQQGQDIDDNWVKDRKKEFEELIDSNHDGIConsensus 241 IVRDLDQDGDKQLS1PEFISLPVGTVENOGQDIDDNWVKDRKKEFEELIDSNHDGIVT'nMouseHmam301EA比A长AN小EEKConsensus 301 EELEnYMDPMNEYNALNEAKQMIAlADENQNHHLEPEEiLKYSEFFTGSKLmDYARnVHEConsensus 361 EFFig 3. The conservation of rat Cab45 was investigated among different species. The full-length sequence of rat Cab45 gene was prepared by 5RACE(Gene Bank/EMBL accession number: AF405545)and its amino acids sequence was deduced. The conservation of Cab45 was investigated by aligningwith the amino acid sequences among rat, mouse and human, which displayed a homology of 95% between rat and mouse as well as 89% between rat andhuman. The high homology in six EF-hand motifs labeled by rectangle line(110-121, 149-160, 208-219, 245-256, 290-301, 326-337)was revealed that100% between rat and mouse and 93% between rat and human.we conducted followed experiments. The differentiation ofPC12 cells was induced by NGF and evaluated by syn-apses growth. The expression of Cab45 was investigatedThe distribution of Cab45 was investigated in different tissues of by Northem blot in the process of differentiation. As exNorthem blot. Total RNAs were isolated from different tissues of pected, following the progression of differentiation, Cab45adult rat and fractionated with 1.0% agarose/fomaldehyde gel. The probe exhibent down-regulationfor Cab45 was prepared by PCR with ["Pj-a-dCTP incorporation. After中国煤化工his concentration forhybridization,the signals were visualized with Phospholmager. Lane 1: cellliver, lane 2: spleen; lane 3: heart; lane 4: testis; lane 5: intestine; lane 6:CN MH GMTT. which did notfat; lane 7: colon; lane 8: kidney; lane 9: lungshow any effect on cell death(data not shown).Given that the cell differentiation is still underway incells and cerebellums of rat with different post-natal days, cerebellums of rats after birth, we investigated Cab45 exfeng Zhu et al. /Journal of Genetics and Genomics 35(2008)153-161pression by Northem blot with these rat cerebellums(de- ized by intemal standard gene factin. As expected, Cab45rived from different post-natal days). The result indicated was up-regulated in pcDNA3. 1/Cab45 transfected PC12that the down-regulation of Cab45 was consistent with cells and down-regulated in psilencer2. 1/ Cab45-RNAi-lprogression of differentiation(Fig. 5). This suggests that transfected PC12 cells, respectively(Fig. 6). Ethanol treatCab45 is probably involved in the differentiation of rat ment and UV exposure were conducted subse quently andcerebellumindicated that the over-expression of Cab45 modulated theimpairment elicited by ethanol(P<0.05)as well as 10s UV123456山exposure(P<0.05)in transfected PC12 cells, whereas longCahstime UV exposure(20 s)didn't show any protection(Fig. 7)Easa42-Oclophilin--44Accordantly, down-expression of Cab45 intensified the impairment elicited by(Fig. 7)but not significantly by ethanol treatment(P>0.05,Fig. 5. Cab45 expression was investigated in the process ofation data not shown), in transfected PC12 cells.of NGF-induced PCl2 cells and cerebellums of ratdifferentpost-natal days. Total RNAs were extracted from NGF-induced PC12 cellwith different time points and cerebellums of rats with different postnataldays followed by fractionating with 1.0% agarose/fomaldehyde gel. TheDiscussionprobes for Cab45 and Cyclophilin were prepared by PCR with["Pl-a-dCTP incorporation. After hybridization, the signals were visual-By means of SSH, we identified an ethanol responseized with Phospholmager. Triplicate experiments were carried out inde- gene Cab45 in B104 cells. The time course of expressionpendently. A: NGF-induced PCI2 cells. Lanes 1, 2,3,4, and 5: control exhibited a time-dependent down-regulation of Cab45 by(free of NGF treatment), day 1, day 2, day 3, and day 7. B: The cerebel-lums of rats with different post-natal days, Lanes 1, 2, 3, 4, 5, and 6: ethanol for long-term treatment. The slight up-regulation ofpostnatal day 3, postnatal day 6, postnatal day 9, postnatal day 12, post- Cab45 by initial ethanol treatment may be attributednatal day 18, and adult.stress response, which is a common character for ethanolresponse gene. The full-length sequence of Cab45 in ratCab45 can modulate the impairment elicited by ethanol was prepared for the first time and its conservation wasand UVinvestigated with deduced amino acid sequence in differentspecies. The EF-hand motif, which is common feature foOwing that ethanol can down-regulate Cab45, we hy. calcium binding protein, are characterized in Cab45 andpothesized that up-regulation of this gene may modulate the revealed high conservativeness. It is noteworthy that the kmpairment elicited by ethanol or other risk factors. We per- (H) DEL as terminal amino acids in other members of thisformed over-expression of Cab45 in PC12 cells by transient family is replaced with HEEF in Cab45. The amino acid oftransfection with recombinant plasmid of pcDNA3. 1/ Cab45. K(H)DEL is considered as signal peptide for ER-local-To consolidate further, down-regulation of Cab45 wasalso ization, so the peptide of HEEF in Cab45 may account forperformed with recombinant plasmid of psilencer2. 1/ its Golgi-localization. Given to its high conservation andCab45-RNAi-I and psilencer2. 1/Cab45-RNAi-2 in PC12 ubiquitous distribution, we speculated that Cab45 is in-cells. The up-regulation and down-regulation of Cab45 in volved in some important functions.the transfected cell was confirmed by RT-PCR, and normal- Based on the fact that ethanol can affect cell differentiaCans中国煤化工Fig. 6. Cab45 expressions were detected in the transfected PC12 cells by RT-PCR. TeCNMHGnsfected cells and parentcells respectively followed by reverse transcription for cDNA preparation. The PCR amplification was conducted with specific primers of Cab45 andactin respectively, which designed a size of 441 bp in Cab45 and 221 bp in p-actin. A: Up-regulation of Cab45. B: Down-regulation of Cab45. Lane M:NA ladder(2,000 bp, 1,000 bp, 750 bp, 500 bp, 250 bp, and 100 bp); lane 1: parent cells(PC12 cells ) lane 2: pcDNA3. I/Cab45 transfected PC12 cells;lane 3: psilencer2. 1/ Cab45-RNAi-l transfected PC12 celYunfeng Zhu et al. /Journal of Genetics and Genomics 35 (2008)153-161a pcDNA3. 1/Cab45psilencer2. 1/Cab45-RNAiMock080.0005%0.10%0.15%0.20%The time of uv treatmentEthanol concentrationThe time of uv treatmentFig. 7. Cab45 was investigated for its function with transfected PC12 cells treated by ethanol and UV exposure. Different transfected cells and parent cells(PC1 cells)were treated by ethanol and UV exposure, respectively. Their proliferations were evaluated by MTT test afterwards. After being normalized bythe value of parental cells, the relative MTT values in different transfected PC12 cells were calculated and plotted against their treatments. The data forstatistics analyses were derived from three independent experiments. A: Cab45 up-regulation(pcDNA3. 1/Cab45 transfection)and mock(pcDNA3. 1/HisAtransfection)in PC12 cells. B: Cab45 down-regulation (psilencer 2 1/CaA45-RNAi-1 transfection)and mock(psilencer 2. 1/neo transfection)in PC12 cells.tion(Santillano et aL., 2005), we hypothesize that Cab45 is (Nixon and Saito, 1994; Muller and Koch, 1998; Jelligerinvolved in the process of differentiation. The down-regu- 1999). Compared with classical calcium binding proteinslation of Cab45 in the process of differentiation using the ( Scherer et al., 1996; Koivu et al., 1997; Honore andmodels of PC12 cells and the cerebellums of rats with dif- Vorum, 2000), some members in this new calcium bindingferent post-natal days suggested that Cab45 is involved in protein family were considered to involve in the regulationthe regulation of neuron cell. The function of rat Cab45 of calcium homeostasis in endoplasmic reticulum(Yabe etwas investigated with the stratagem of its up-regulation al., 1997). Recently, it is reported that a Golgi-localizedand down-regulation by transient transfection with recom- protein Pmrlp, a counterpart of Cab45 in yeast, played anbinant plasmids in PC12 cells. Following treatment by important role in calcium homeostasis(Bates et al., 2005;ethanol and UV exposure, the proliferations of different Paschen and Mengesdorf, 2005). We hypothesize thattransfected cells were investigated by MTT approach. To Cab45 probably determine retention of soluble proteins inour knowledge, it is the first time to demonstrate that the Golgi compartment through calcium regulation. Aber45 can modulate the impairments elicited byrant expressions of this family were demonstrated to have aand short time UV exposure in PC12 cells. The impairment close relationship with many diseases (Liu et al, 1997:elicited by UV exposure for long time may beyond the Vorum et al, 1998). For example, reticulocalbin was in-capacity of Cab45 protection and that damage elicited by volved in the process of breast cancer invasion( Chen et altransfection itself contribute to cell death will be exhibited. 1995), and ERC-55 participated carcinogenesis elicited bySo, this is the reason that the MTt values in pcDNA3. 1/ HPV and vitamin D-dependent nuclear receptor transloca-Cab45 transfected PC12 cells is lower than it in parent tion and neuron transmitters(Vorum et al., 1999). Caluells for 20 s UV exposure. To explore the mechanism of menin contributed to immunity defense and degenerationCab45 regulation further, we employed yeast two-hybrid of the neuron system elicited by amyloid P (Vorum et alapproach to identify its interaction protein in vivo. Unex- 2000; Uccelletti et al., 2005). However, Cab45 was notpectedly, Cab45 showed a strong transactivation function, determined to contribute to some diseases yetwhich couldn't be alleviated until addition of 25 mmol/LIn conclusion, the ethanol response gene Cab45 was3-AT, a specific suppressor for auto-transactivation, into identified and its full-length sequence was prepared in ratthe medium(data not shown). This suggested that Cab45 for the first time. In the process of cell differentiation,probably had a transactivation domain itself or can interact Cab4with some transcriptional factors in yeast.As a member of new calcium binding protein family pairH中国煤化工 h time-dependent-can modulate the im-CNMHGtime uv exposure in( Cab45, reticulocalbin, ERC-55 and calumenin), Cab45 PC12. Apparently, Cab45 contributes to the regula tion ofwas characterized by the same features, e.g., six EF-hand neuron damage and senescence elicited by ethanol andmotifs and localization in secretory pathway( Golgi lumen) other factors. This suggests that Cab45 maybe involved inYunfeng Zhu et al. /Journal of Genetics and Genomics 35(2008)153-161neuron-conceming disease progress and further investiga- Light, K E, Brown, D P. Newton, B W Belcher, SM, and Kane, CJtion is also needed to understand its clinical application.(2002). Ethanol-induced alterations of neurotrophin receptor expres-sion on Purkinje cells in the neonatal rat cerebellum. Brain Res. 924:71-81Liu, Z, BAcknowledgementsreticulocalbin in the highly invasive cell line, MDA-MB-435, versusthe poorly invasive cell line, MCF-7. Biochem. Biophys. Res. Com-mun.231:283-289tional Institutes of Health (No NIAAA AAl2968 and NCI Luchsinger, J a Tang, Mx, Siddiqul M: Shea, S, and Mayeux g.CA 90385). We appreciated of Dr Jia Luo(Associate pro-540-546fessor in Department of Microbiology Immunology, Luo, J, and Miller, MW.(1996). 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