THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2002 by The … · morphological changes observed in the female reproductive tract tissues of SSAT transgenic mice. In the present study, the

Altered Levels of Growth-related and Novel Gene Transcripts inReproductive and Other Tissues of Female Mice OverexpressingSpermidine/Spermine N1-Acetyltransferase (SSAT)*

Received for publication, January 26, 2001, and in revised form, November 7, 2001Published, JBC Papers in Press, November 14, 2001, DOI 10.1074/jbc.M100751200

Seok Hong Min‡, Rosalia C. M. Simmen‡, Leena Alhonen§, Maria Halmekyto¶, Carl W. Porter¶,Juhani Janne§, and Frank A. Simmen‡�

From the ‡Interdisciplinary Concentration in Animal Molecular and Cell Biology, Genetics Institute, and Department ofAnimal Sciences, University of Florida, Gainesville, Florida 32611-0910, the ¶Grace Cancer Drug Center, Roswell ParkCancer Institute, Buffalo, New York 14263, and the §A. I. Virtanen Institute for Molecular Sciences, University of Kuopio,P. O. Box 1627, FIN-70211 Kuopio, Finland

Overexpression of SSAT (polyamine catabolic en-zyme) in female mice results in impaired ovarian follicu-logenesis and uterine hypoplasia. To identify the molec-ular basis for this, the gene expression profiles in uterusand ovary and for comparison, liver and kidney, fromnon-transgenic (NT) and SSAT transgenic (ST) micewere compared. The mRNA abundance for lipoproteinlipase and glyceraldehyde-3-phosphate dehydrogenasewas elevated in all four ST (>NT) tissues. The transla-tion initiation factor-3 subunit 5 mRNA, and transcriptsrelated to endogenous murine leukemia provirus (MLV-related) and murine retrovirus-related sequences(MuRRS) were decreased in ST tissues. A novel calmod-ulin-related mRNA was strongly induced in ST liver andkidney. SSAT overexpression was associated with in-creased levels of IGF-binding protein-2 (IGFBP-2) in theuterus and ovary, and a reduction in IGFBP-3 mRNAlevels in the uterus. Exogenous spermidine and sperm-ine elevated endogenous IGFBP-2 and SSAT mRNAabundance, whereas, putrescine stimulated IGFBP-2mRNA abundance and transfected IGFBP-2 gene pro-moter activity in human (Hec-1-A) uterine cells. Sp1 andBTEB1 mRNAs that encode transcription factors for theIGFBP-2 gene also were induced in some ST tissues. Thedata suggest that SSAT and polyamines are importantfor the control of molecular pathways underlying repro-ductive tract tissue growth, phenotype, and function.

The polyamines putrescine, spermidine, and spermine areubiquitous components of cells. Although many of their specificfunctions are still unclear, these polycationic molecules areessential for cell proliferation and differentiation (1, 2). Theintracellular levels of polyamines are tightly regulated by thecells’ growth status (3), which in turn, is dependent on meta-bolic pathways that mediate their cell synthesis, degradation,

and/or excretion. Ornithine decarboxylase (ODC)1 is the firstrate-limiting enzyme in polyamine biosynthesis and has beenthe subject of intense scrutiny in the last decade, due to itspossible involvement in proliferative disorders including can-cer. Development of several drugs, notably difluromethyl orni-thine, which inhibits ODC, results in a depletion of the cellularpolyamine pool and a decrease in cell proliferation (4, 5). Al-though inhibitors of polyamine synthesis are potential candi-dates for cancer chemotherapy, the results of clinical trialshave not always met expectations. Furthermore, recent studieswith transgenic mice have shown that life-long overexpressionof ODC or other polyamine biosynthetic enzymes does notincrease the incidence of spontaneous tumors (6–8). The ab-sence of marked phenotypic changes in these mice may beattributable to the relatively minor changes observed in higherpolyamine pools, although an accumulation of putrescine intissues was observed.

The increase in putrescine in transgenic mice overexpressingODC or other polyamine anabolic enzymes suggests that acounter-regulatory mechanism, such as activation of the cata-bolic pathway, may maintain polyamine homeostasis in vivo.Spermidine/spermine N1-acetyltransferase (SSAT) is the rate-limiting enzyme in polyamine catabolism that, together withpolyamine oxidase, back-converts spermine and spermidine ul-timately to putrescine, a function that is presumed to preventcellular toxicity due to polyamine excess (9). Recently, therehas been growing interest in this pathway as a target formanipulating polyamine pools to control cell proliferation. In-deed, several polyamine analogues, particularly the compoundN,N�-bis(ethyl)spermine, have been developed and shown todown-regulate ODC, and more importantly, to up-regulateSSAT. In a number of cell lines (10–12), the induction of SSATis closely associated with the anti-proliferative action of thesedrugs. This linkage appears to be mediated by the depletion ofspermidine and spermine, as facilitated by SSAT, together withthe inability of analogues to substitute for the depleted naturalpolyamines in functions associated with cell proliferation.

Although polyamine analogues may provide an effective wayto regulate SSAT and hence, cell growth processes, applicationof these drugs in an in vivo context is complicated, partly

* This work was supported by National Institutes of Health GrantsHD21961 (to R. C. M. S. and F. A. S.), CA76428 (to C. W. P. and J. J.),and CA-16056 (to C. W. P.) and by the Florida Agricultural ExperimentStation. This is Florida Agricultural Experiment Station publicationSeries No. R-08404. The costs of publication of this article were de-frayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AY061807-AY061811.

� To whom correspondence should be sent. Tel.: 352-392-5590; Fax:352-392-5595; E-mail: [email protected].

1 The abbreviations used are: ODC, ornithine decarboxylase; CALM-Rel, calmodulin-related; eIF-3, eukaryotic translation initiation fac-tor-3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGFBP, in-sulin-like growth factor-binding protein; KLF, Kruppel-like family;LPL, lipoprotein lipase; MLV, murine leukemia provirus; MuRRS, mu-rine retrovirus-related DNA sequence; SSAT, spermidine/spermine N1-acetyltransferase; FFA, free fatty acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 5, Issue of February 1, pp. 3647–3657, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 3647

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

because of their interference with the polyamine syntheticpathway and transport (13, 14). Recently, the Porter and Jannelaboratories (15) generated SSAT overexpressing transgenicmice to further define a role for SSAT in polyamine metabolismin vivo. As expected, these mice exhibit profound changes intissue polyamine pools, including a large accumulation of pu-trescine, the appearance of N1-acetylspermidine, and in certaintissues a decrease in spermidine and spermine. These changesoccur despite simultaneous increases in ODC and other poly-amine anabolic enzyme activities. The magnitude of changes inthe polyamine levels was much more prominent than thoseobserved in transgenic mice overexpressing ODC, further em-phasizing the prominent role of SSAT in maintaining poly-amine homeostasis. Disturbances in polyamine pools due tooverexpression of SSAT lead to marked phenotypic changes aswell, including permanent hair loss at an early age, skin wrin-kling, loss of subcutaneous fat, and in females, an underdevel-oped uterus and abnormal ovaries. Interestingly, the latter twotissues of normal and SSAT transgenic mice did not differ withrespect to tissue spermidine and spermine contents, whereas,the SSAT overexpressors had markedly elevated putrescinecontent in female reproductive organs. These collective resultsdemonstrated the utility of SSAT overexpression as a means tomodulate polyamine pools in tissues, for the purposes of un-raveling the role(s) of polyamines in normal and abnormalcellular proliferation, differentiation, and apoptosis.

Although transgenic mice provide a useful model for identi-fying metabolic and biological consequences of altered poly-amine pools, molecular mechanisms underlying these changesremain elusive. Given the fact that polyamines are polycationsat physiological pH and thus, can interact with negativelycharged molecules such as DNA and RNA, it is highly likelythat deregulation of polyamine pools may affect expression ofmultiple genes which could explain in part, the phenotypic andmorphological changes observed in the female reproductivetract tissues of SSAT transgenic mice. In the present study, thegene expression profiles in uterus and ovary, and for compar-ison, in liver and kidney, of SSAT overexpressing mice wereevaluated relative to those of their normal, non-transgeniccounterparts. A number of distinct genes, some of which areknown to be associated with growth regulation (IGFBP-2,IGFBP-3, and Kruppel-like (KLF) transcription factors) andsome of which are novel, were identified to exhibit markedalterations in mRNA levels during SSAT overexpression, sug-gesting that SSAT and/or polyamines are crucial for the controlof molecular pathways underlying reproductive tract tissuegrowth, phenotype and function.

EXPERIMENTAL PROCEDURES

Animals and Tissue Collection—Transgenic mice systemically over-expressing the polyamine catabolic enzyme SSAT were previously gen-erated using standard pronuclear microinjection techniques as de-scribed in detail elsewhere (15). Members of the UKU165b line weremaintained as a breeding colony by mating transgenic males withnon-transgenic female BALBc X DBA/2 mice, since transgenic femaleswere infertile. Transgenic animals acquired normal first hair but lost itat the age of 3 to 4 weeks, allowing them to be identified withoutgenotyping. Transgenic and non-transgenic female mice were killed at20 weeks by cervical dislocation and various tissues removed and placedimmediately in liquid nitrogen.

RNA Extraction—Total cellular RNA was extracted using TRIzolreagent (Life Technologies, Grand Island, NY) according to the manu-facturers recommendations. RNA samples were freed of contaminatingDNA by treatment with DNase I.

mRNA Differential Display (ddRT-PCR)—Differential display re-agents and primers (HIEROGLYPHTM) were purchased from GENO-MYX Corp., Foster City, CA. An equal amount of RNA from the uterus,ovary, liver, and kidney of ST (n � 4) and NT (n � 4) mice were pooledwithin tissue but kept separate between groups. DNA-free total RNA (2�g/tissue sample) was subjected to reverse transcription using an-

chored 3� oligo(dT) primer sets (5�-T12NM-3�, where NM � GA, GC, GG,GT, CA, CC, or CG, primers 1–7, respectively). Following reverse tran-scription, one-tenth of this reaction (2 �l) was used in a PCR amplifi-cation reaction (20 �l) containing 400 �M of each dNTP, 2.5 �Ci of[�-33P]dATP, and two primers: 4 �M T12 oligonucleotide (above) and 4�M of an arbitary decamer, M13r-ARP1 (5�-CGACTCCAAG-3�) or M13r-ARP2 (5�-GCTAGCATGG-3�). These reactions also contained 1 unit ofAmpliTaq DNA polymerase (PerkinElmer Life Sciences, Norwalk, CT).The PCR was performed with 25 cycles of 15 s denaturation at 92 °C,30 s annealing at 46 °C, and 2 min extension at 72 °C.

Re-amplification and Subcloning of cDNA Fragments—PCR prod-ucts from 14 different primer combinations (seven anchored primersand two arbitrary decamers) for each of eight tissues (ST, NT: uterus,ovary, liver, and kidney) were separated in nondenaturing 4.5% poly-acrylamide sequencing gels and visualized by autoradiography. Bandsexhibiting differential expression in a given tissue between ST and NTgroups were excised from the dried gels, transferred into polypropylenetubes, and re-amplified using the appropriate primers, except that[�-33P]dATP was omitted. Each PCR reaction (20 �l) was electrophore-sed in an agarose gel to confirm amplification of a single product. ThePCR product was subcloned into TOPOTM TA vector (Invitrogen, Carls-bad, CA) and used for Northern analysis. Once confirmed as represent-ing a differentially expressed transcript by Northern blot, each cDNAclone was sequenced and the final sequence compared with those inGenBankTM (www.ncbi.nlm.nih.gov/BLAST/).

Northern Blot Analysis—Total cellular RNA (20 �g) was fractionatedin a 1.5% formaldehyde-agarose gel and transferred to a Biotrans nylonmembrane by downward capillary transfer using the TurboBlottingsystem (Schleicher and Schuell, Keene, NH). Nylon membranes werecross-linked by exposure to a UV light source for 1.5 min and thenbaked at 85 °C for 25 min. Blots were pre-hybridized in ULTRAhybTM

(Ambion, Austin, TX) at 42 °C for 2 h. Hybridization was carried outovernight in the same buffer containing a cDNA fragment that waslabeled with [�-32P]dCTP by nick translation (Amersham Biosciences,Inc., Piscataway, NJ). The same procedure was used for labeling ofcDNA inserts (or PCR products) representing porcine SSAT, porcineIGF-I, rat IGF-II, rat IGFBP-2, human IGFBP-3, rat acid labile sub-unit, human Sp1, human BTEB1 (KLF9), and human BTEB2 (KLF5)mRNAs. The membranes were washed twice at 42 °C for 15 min with2 � SSC, 0.1% SDS, then twice with 0.1 � SSC, 0.1% SDS using thesame conditions. After a final wash, the membrane was subjected toautoradiography using intensifying screens at �80 °C. The relativechanges in mRNA levels were quantified by use of a Gel Documentation& Analysis System (Alpha Innotech Corp., San Leandro, CA). Thefilters were stripped of radioactive probe between hybridizations bywashing twice for 45 min in 1% SDS at 95 °C and were stored at 4 °Cuntil further use.

DNA Sequence Analysis—Sequencing of cloned cDNA fragments wascarried out by the DNA Sequencing Core Facility of the Interdiscipli-nary Center for Biotechnology Research at the University of Florida.

Western Ligand Blot of IGF-binding Proteins—Samples of uterus,ovary, liver, and kidney from ST and NT mice were homogenized in 4volumes of 0.01 M sodium phosphate (pH 7.4), 0.15 M NaCl. Homoge-nates were centrifuged at 20,000 � g for 1 h. The pellet (microsomalmembranes) was solubilized in RIPA (0.1 M sodium phosphate (pH 7.2),0.01 M EGTA, 0.01 M EDTA, 0.01 M NaF, 1% sodium deoxycholate, 0.1%sodium dodecyl sulfate, 0.001 M phenylmethylsulfonyl fluoride, 200kallikrein units/ml of aprotinin) and centrifuged to remove insolublematerial. Proteins in solubilized microsomal membrane fractions werequantified by the Bradford procedure (Bio-Rad Laboratories, RockvilleCenter, NY), with bovine serum albumin as standard. Samples (30 �gof protein) were subjected to nonreducing SDS-PAGE gel (4.0% stackinggel and 12.5% separating gel) and transferred onto a nitrocellulosemembrane using a Bio-Rad Transfer unit at 200 W for 3 h. The nitro-cellulose membrane was washed three times with 1 � TBS (10 mM Tris,160 mM NaCl, pH.7.4) for 5 min each and then blocked in 1% Blotto (1 �TBST (TBS containing 1% Tween 20) � 1% Carnation non-fat dry milk)at 4 °C overnight. The nitrocellulose membrane was washed for 10 minin TBST and incubated with 1.0 � 106 cpm of 125I-rhIGF-II per 5 ml ofbuffer (TBST � 1% bovine serum albumin) overnight with gentle rock-ing at 4 °C. After several washes with TBST, the membrane was air-dried and exposed to BioMax film (Eastman Kodak Co., Rochester, NY)with an intensifying screen at �80 °C for 10 days. The identity of eachIGFBP was assigned according to its estimated molecular weight (16).

Cell Culture—Human Hec-1-A endometrial carcinoma cell line(American Type Culture Collection, Manassas, VA) was cultured inMcCoy’s 5A medium with 10% fetal bovine serum and maintained at37 °C in an atmosphere of 5% CO2, 95% air. For polyamine treatments,

SSAT-modulated Genes in Female Reproductive Tissues3648

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Hec-1-A cells were plated into 6-well plates (�200,000 cells/ml) andallowed to grow in serum-containing medium until confluent. Cellswere then incubated in serum-free medium for 24 h, at which time theyreceived fresh serum-free medium also containing 400 �M putrescine,spermidine, or spermine. RNA was extracted 24 h after polyamineaddition.

IGFBP-2 Promoter Reporter Construct—An IGFBP-2 promoter con-struct containing 1397 bp of DNA 5� to the translation initiation codon(the latter located within exon 1) of the porcine IGFBP-2 gene fused tothe luciferase reporter gene was used in transient transfectionexperiments.

SSAT Mammalian Expression Vector—The entire coding region ofporcine SSAT (17) was subcloned into the EcoRI site of the pIND vector(Invitrogen Corp.). The orientation (sense or antisense) of the resultingconstructs was confirmed by restriction digestion and nucleotide se-quence analysis. Plasmid DNAs were purified using the Qiagen MaxiKit (Qiagen, Chatsworth, CA).

Transient Transfection and Luciferase Assays—Hec-1-A cells wereplated in 6-well plates and grown until 60–70% confluent. The IGFBP-2promoter/luciferase reporter construct was co-transfected with theSSAT sense or antisense expression vectors and their effects on IG-FBP-2 promoter activity determined under serum-free conditions.Transfections were performed using LipofectAMINE (Invitrogen, Rock-ville, MD), following the manufacturers suggested procedure. Cellswere harvested 48 h after transfection and whole cell extracts wereanalyzed for luciferase activity in an Autolumat Luminometer (EG&G,Berthold, Germany). Results from transfection analysis were normal-ized for protein content of cellular extracts, expressed as LSM � S.E.M.,and compared using the GLM procedures of the SAS statistical pack-age. Similar statistical analyses were performed on Northern blot data,which were first corrected for loading by use of corresponding 18 Sribosomal RNA intensity.

RESULTS

Differential Display Analysis of Gene Expression—Total cel-lular RNA was isolated from the uterus, ovaries, liver, andkidneys from each of four SSAT-transgenic (ST) and four non-transgenic (NT) control mice. Equal amounts of RNA from eachtissue of ST or NT groups were pooled for all subsequent RNAanalyses (ST, �lanes; NT, �lanes). Prior to ddRT-PCR, thelevels of SSAT mRNAs were examined by Northern blot toconfirm their differential expression in tissues of ST and NTlittermates, using porcine SSAT cDNA previously cloned inthese laboratories (17) as hybridization probe. Two expectedRNA transcripts, a major species of �1.3 kb and a minorspecies of �3.5 kb, were abundantly expressed in all four tis-sues of ST mice, whereas these were barely or non-detectable incorresponding tissues of the NT mice (Fig. 1). The sizes of thesetwo transcripts are identical to those previously reported forthe SSAT gene of other mammalian species (17).

A representative portion of a typical differential display gel,which illustrates how candidate SSAT-regulated cDNAs/mRNAswere identified, is shown in Fig. 2. All combinations of twoarbitrary primers and seven anchored primers, the sum total ofwhich theoretically covers �7% of the total mRNA population(Technical Bulletin, GENOMYX Corp.), were used to examinegene expression changes in the four tissues of ST and NT mice.Visual inspection of resultant autoradiograms revealed bands

that were of similar intensity between a particular tissue typeof ST and NT mice as well as a smaller percentage of bandswhose intensities differed between the mouse groups. Interest-ingly, the majority of these putative differentially expressedtranscripts were confined to uterus and ovary, tissues withmarked observable phenotypes due to the presence of thetransgene (15).

Confirmation of Differentially Expressed Genes and Determi-nation of Identities—Twenty-five of the cDNA fragments notedabove were excised from gels and subjected to a second round ofPCR, using the original combination of primers for each. Of thetwenty-five bands identified on the original gels, 19 were suc-cessfully re-amplified by PCR and subcloned. All 19 were sub-jected to Northern blot analysis to confirm differential mRNAexpression. Fourteen of these exhibited changes in gene expres-sion that confirmed the original differential-display patterns.Of these, one pair and one set of three had identical mRNAexpression patterns and the same transcript sizes (data notshown), suggesting that those within a group were derivedfrom the same mRNA(s), despite having distinct migrationpositions on the original differential display gels. This wasconfirmed when differential display products were sequencedin their entirety and subjected to computer analysis. The iden-tities of the final resultant 11 different cDNAs are summarizedin Table I, and their corresponding Northern blots presented inFigs. 3 and 4, respectively. Many of these RNAs appear to benovel with respect to function. One (O 1-4-5) has 100% identityto mouse lipoprotein lipase (LPL). Another (L 1-2-3) has 99%similarity to a mouse EST reported in GenBankTM. This cDNAfragment has a complete open reading frame encoding a pro-tein with strongest sequence relatedness (although not identi-cal) to the calcium binding, signal transducing protein, calmod-ulin, and is designated here as CALM-Rel (Fig. 5) (accessionnumber AY061807). Another ddRT-PCR product (U 2-6-5) isthe mouse homolog of the mRNA/cDNA encoding human sub-unit 5 (�, 47 kDa) of eukaryotic translation initiation factor-3(eIF-3) (Fig. 6) (accession number AY061808). The U 1-3-7 andU 1-3-10 ddRT-PCR products had strong similarity but werenot identical to each other and to the endogenous murineleukemia provirus (MLV) genome/transcripts and are here des-ignated as MLV-Rel1 (accession number AY061810) and MLV-Rel2 (accession number AY061809). Sequence analysis of MLV-Rel2 indicated the interesting possibility that this defective

FIG. 1. Northern analysis of non-transgenic (�) and SSATtransgenic (�) mouse tissues for SSAT mRNAs. The upper panel isthe autoradiogram of the Northern blot whereas the lower panel is theethidium bromide-stained gel prior to blotting. Sizes of mRNAs areindicated to the right.

FIG. 2. Representative mRNA differential display results foruterus, ovary, liver, and kidney of non-transgenic (�) and SSATtransgenic (�) mice. An equal amount of RNA was pooled withintissues among groups of mice, used in ddRT-PCR (arbitrary primer 1and anchored primer 4 results shown), and the products run on a 4.5%polyacrylamide gel. Arrows indicate candidate SSAT-induced or -sup-pressed bands in a portion of the gel.

SSAT-modulated Genes in Female Reproductive Tissues 3649

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

retroviral transcript encodes amino-terminal and carboxyl-ter-minal truncated viral envelope (Env) proteins in normal mousetissues (Fig. 7). The U 1-1-5 sequence is an identical matchwith the mouse hepatitis virus receptor which is also found inGenBankTM under the designation of biliary glycoprotein. U1-4-4 appears to be a new member of the mouse retrovirus-related sequence (MuRRS) transcript family (accession numberAY061811).

Differentially Expressed Genes across Tissues—Of the clonedddRT-PCR products, a few of the corresponding mRNAs wereup-regulated and many more were down-regulated in the rel-evant tissues of ST mice, although some of these changesclearly were tissue-specific. To account for possible variationsin RNA loading and gel transfer during Northern analysis,blots were hybridized with glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) cDNA probe since this is conventionallyused as the loading control. Surprisingly, the GAPDH probeitself yielded a substantially higher mRNA signal in all SSAT-

transgenic relative to control, tissues (Fig. 3), althoughethidium bromide staining of 28 and 18 S rRNAs showed com-parable amounts of rRNA in each lane and subsequent hybrid-ization with other candidate gene probes, in some tissues,indicated no changes between mouse groups (see later results).As a result, all hybridization results were corrected for changesin the intensities of ethidium bromide-stained 28 S and 18 SrRNA bands.

The LPL mRNA, a single transcript of �3.8 kb, was barelydetectable in the uterus and ovaries, but was abundantly ex-pressed in the kidneys of NT mice. Levels of this transcriptwere up-regulated in uterus (�2.3-fold) and in ovaries (�1.9-fold) of ST mice, with no corresponding changes observed inthose of the kidney (Fig. 3). This transcript, however, was notexpressed at detectable levels in the liver of ST or NT mice. Thecalmodulin-related (CALM-Rel) transcript also was induced ina tissue-dependent manner upon overexpression of SSAT. This�1.3-kb transcript, while undetectable in all four tissues of NTmice, was readily apparent in liver and kidneys of ST mice. Incontrast, no detectable levels of CALM-Rel mRNA were ob-served for uterus and ovaries of the ST mice.

Expression of eIF-3 s5 mRNA, a single transcript of �1.5 kb,was high in all four tissues of NT mice. However, this level ofexpression was dramatically reduced in uterus and ovaries ofST mice, with a similar downward trend, albeit of lesser mag-nitude, observed for liver and kidney. Murine leukemia provi-rus-related (MLV-Rel1) transcripts exhibit three distinctivesizes, one major species of 5.3 kb and two minor species of �8.2and 3.5 kb, respectively (Fig. 4). The highly related MLV-Rel2sequence showed a similar pattern of three transcripts (7.2, 5.4,and 3.5 kb), with the 5.4-kb transcript being the major speciesand the minor species being only weakly discernible (Fig. 4 anddata not shown). MLV-Rel1 and -Rel2 transcripts were abun-dantly expressed in all four tissues of NT mice, with highestexpression in ovaries. However, expression of these transcriptswas reduced to nearly undetectable levels in all four corre-sponding tissues of ST mice. Expression of yet another retro-virus-related RNA sequence, a new member of the MuRRSfamily, also was similarly altered in tissues of ST mice. At leastfive distinct MuRRS transcripts were detected, four minor spe-cies of �13, 7.7, 3.9, and 2.6 kb, and one major species of 5.1 kb.The major MuRRS transcript was most abundantly expressedin ovary � uterus � kidney � liver, of NT mice and expressionof this transcript was suppressed to undetectable levels in the

TABLE ISummary of cDNA clones identified by differential display analysis of

uterus, ovary, liver, and kidney of non-transgenic andSSAT-transgenic mice

ddRT-PCRproduct

Accessionnumber of

closest match

Size (bp) ofcDNA

fragment

Identity (perfect match)or similarity (similar but

not identical to)a

U 1–1-5 X67279 629 Mouse hepatitis viralreceptor/biliaryglycoprotein

U 1–1-11 AA277455 722 Unknown functionL 1–2-3 AI18547 868 CALM-RelU 1–3-7 K03230 640 MLV-Rel2a

U 1–3-10 M17326 708 MLV-Rel1a

U 1–3-15 BF020640 489 Unknown functionO 1–4-5 NM008509 605 Lipoprotein lipaseU 1–4-4 X02487 650 MuRRSa

U 2–3-0 AI527208 674 Unknown functionU 2–7-1 AI604532 697 Unknown functionU 2–6-5 AW741978 654 eIF-3s5

a Similar but not identical.

FIG. 3. Northern analysis of “candidate” differentially ex-pressed mRNAs in tissues of non-transgenic (�) and transgenic(�) mice. All panels are autoradiograms of the Northern blots with theexception of the lowermost panel which is the ethidium bromide-stainedgel prior to blotting. The size(s) of the major transcript(s) for each geneis indicated to the right.

FIG. 4. Northern blots of endogenous retrovirus-related tran-scripts in tissues of non-transgenic (�) and transgenic (�) mice.All panels are autoradiograms of the Northern blots with the exceptionof the lowermost panel which is the ethidium bromide-stained gel priorto blotting. The size(s) of the major transcript(s) for each gene is indi-cated to the right.

SSAT-modulated Genes in Female Reproductive Tissues3650

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

corresponding tissues of ST mice. The four minor transcriptsalso appeared to behave in a similar manner. In contrast to theabove, the murine hepatitis viral receptor cDNA clone has twotranscript sizes of �4.2 and 3.8 kb, respectively. The majortranscript in kidney is 3.8 kb whereas the major transcript inliver and uterus is the 4.2-kb variant. Both transcripts wereup-regulated in the uterus of ST mice, whereas the reverse wastrue for liver. There were no alterations in the expression levelsof these transcripts in ovary and kidney between the two mousegroups.

Abundance of two other transcript classes (U 1-3-15 and U1-1-11) was altered in a tissue-selective manner upon SSAToverexpression (Fig. 3). U 1-3-15 transcripts (2 and 1.1 kb) werepresent in the uterus and ovaries of NT mice, but only thesmaller transcript was detected in the liver and kidney. Ex-pression of the larger transcript was nearly undetectable inuterus and ovaries of ST mice, while that of the smaller tran-script was not significantly altered in these same tissues. Inliver and kidney, however, the expression of the 1.1-kb tran-script was increased in ST relative to NT littermates. The

expression of the 1.4-kb U 1-1-11 transcript was down-regu-lated in uterus and ovaries, but was up-regulated in liver andkidney of ST relative to NT, mice. The U 2-3-0 and U 2-7-1RNAs also exhibited tissue-specific increases or decreases inthe ST versus NT mice (Fig. 3).

Expression of IGFs and Their Binding Proteins (IGFBPs)—In conjunction with the arbitrary ddRT-PCR approach, theexpression of IGFs and their binding proteins also was exam-ined in the NT and ST tissues, since corresponding proteins forthese genes have been shown to play important role(s) in cellgrowth and differentiation of multiple tissues, including thoseof the female reproductive tract (18). Northern blot analysis forIGF-I mRNA revealed three transcripts, two major species of7.5 and 0.9 kb and one minor species of 1.3 kb, respectively, inovary, uterus, and liver, but not kidney, of NT mice (Fig. 8). Nochanges in the levels of any of these transcripts were observedbetween corresponding tissues of ST and NT mice. Similarly,the expression levels of IGF-II mRNA were not altered intissues of ST mice, compared with those of NT mice (data notshown). By contrast, the expression of the IGFBP-2 and -3 were

FIG. 5. DNA sequence and openreading frame of ddRT-PCR product(panel A), and corresponding proteinrelatedness to murine calmodulins(panel B), for the novel calmodulin-related (CALM-Rel) gene transcriptinduced in liver and kidney of SSAT-transgenic mice. Shown for comparisonare the sequences of the protein productsof the mouse calmodulin-1, -2, and -3genes.

SSAT-modulated Genes in Female Reproductive Tissues 3651

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

dramatically altered in a tissue-specific manner with SSAToverexpression (Fig. 8). The levels of IGFBP-2 mRNA (a singletranscript of 1.4 kb) were undetectable in uterus and ovary ofNT mice, but were robustly induced in corresponding tissues ofST mice (Fig. 8, Table II). Conversely, expression of thismRNA, which was already low in liver and kidney of NT mice,was further reduced in corresponding ST mouse tissues. Theexpression of IGFBP-3 mRNA (2.4 kb) was most abundant inthe uterus of normal mice; however, this was significantlyreduced with SSAT overexpression (Fig. 8, Table II). In theother tissues examined, the levels of IGFBP-3 mRNA were notcorrespondingly altered in ST mice. The dramatic changes inIGFBP-2 and -3 mRNA levels in relevant tissue(s) were ob-served at the level of their respective proteins (Fig. 9). IGFBP-3protein levels were diminished (130%) in the uterus, whilethose of IGFBP-2 were induced (96%) in the uterus and to alesser extent (58%) in ovary, by SSAT overexpression. Uterineand ovarian expression of the IGFBP-3 acid-labile subunitmRNA was undetectable in NT and ST mice. In contrast, thismRNA was observed in liver � kidney, although this wasunaffected by SSAT overexpression.

Direct Effects of SSAT and Polyamines on Uterine IGFBP-2Expression—In the absence of any continuous mouse uterinecell line, we utilized a human uterine cell line (Hec-1-A) toexamine whether the in vivo effects of SSAT on IGFBP-2 geneexpression observed above were direct and/or involved poly-amines. Indeed, exogenous spermidine and spermine at 400 �M

concentration elevated in parallel, the SSAT and IGFBP-2mRNA abundance in Hec-1-A uterine cells (Fig. 10, A and B).

Putrescine, at this same concentration, increased IGFBP-2 butnot SSAT mRNA abundance. In addition, putrescine as wellas co-transfected SSAT mammalian expression vector (senseversus antisense; Fig. 10, C and D), stimulated IGFBP-2 genepromoter activity in transfected Hec-1-A cells.

IGFBP-2 Gene Cognate Transcription Factors in SSAT-Transgenic Mice—In view of the marked induction of the IG-FBP-2 mRNA in uterus and ovaries of SSAT-transgenic mice,we performed Northern analysis of the normal and transgenicmouse tissues with probes representing three functionally andstructurally related KLF family transcription factors (BTEB1,BTEB2, and Sp1) that are implicated in IGFBP-2 gene tran-scriptional activity in other cell systems. The rationale for thislast set of studies was to examine the possibility that alteredintracellular polyamine levels affects expression of one or moreof these transcription factors with subsequent alterations inIGFBP-2 gene expression. Our results (Fig. 11, Table II) indi-cated that Sp1 mRNAs were significantly induced by SSAToverexpression in uterus, liver, and kidney, whereas BTEB1mRNA was induced only in kidney. BTEB2 mRNA levels wereunaltered by SSAT overexpression in all tissues examined.

DISCUSSION

Previous studies of SSAT-transgenic mice have shown thatoverexpression of SSAT leads to changes in intracellular poly-amine pools, including an accumulation of putrescine (15).Since these alterations are accompanied by a distinctive phe-notype including hair loss, reduced subcutaneous fat, hypopla-sia of the uterus (both endometrial (stroma and glands) and

FIG. 6. DNA sequence and openreading frame of ddRT-PCR product(panel A), and corresponding proteinrelatedness to the carboxyl terminusof human translation initiation fac-tor 3, subunit 5 (panel B), for the RNAtranscript suppressed in uterus,ovary, liver, and kidney of SSAT-transgenic mice. This cDNA/mRNArepresents the mouse homolog of the hu-man eIF-3 s5 protein. The upper line isthe deduced mouse amino acid sequence;the corresponding human sequence iscompared (third line) to this, with con-served residues indicated on the secondline. The 5� end of the mouse cDNA clonecorresponds to amino acid 210 of the hu-man protein.

SSAT-modulated Genes in Female Reproductive Tissues3652

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

myometrial compartments are affected) and ovarian dysfunc-tion (reduced folliculogenesis and absence of corpora lutea)(15), it is reasonable to speculate that some or all of theseeffects may reflect changes in gene expression profiles as aconsequence of chronically altered or aberrant intracellularpolyamine levels and/or pool composition. In this initial at-tempt to identify genes whose expression, at the level of mRNA,are altered in response to changed polyamine homeostasis, weused the differential display technique as well as the candidategene approach to compare mRNA populations from uterus,ovaries, liver, and kidneys of ST mice with those of NT litter-mates. Uterus and ovary were selected based upon their pro-nounced morphological and functional changes in the trans-genic line. The present results clearly demonstrate thatdysregulation of polyamine pools (i.e. increased putrescine, N1-acetylspermine and N1-acetylspermidine in vivo, 15) via long-term overexpression of SSAT leads to marked changes in geneexpression profiles in these and other tissues.

Of the differentially expressed genes evaluated here, two(LPL and GAPDH) are involved in energy metabolism and wereinduced in the ST transgenic mice, albeit in a tissue-dependent

manner. Although LPL and GAPDH represent but a smallfraction of genes involved in energy metabolism, their identifi-cation as potentially polyamine-regulated suggests an impor-tant role for these polycations in the regulation of energy me-tabolism. Indeed, this supposition is consistent with an earlierstudy of rat adipocytes (19), where exogenous polyamines(spermine, spermidine, and putrescine) significantly inhibitedadenosine deaminase-stimulated lipolysis. The physiologicalimplications of these observed changes remain unclear at pres-ent, but such alterations may be partly responsible for thephenotypic changes (i.e. reduced adiposity) associated withSSAT overexpression. LPL is a key regulatory enzyme respon-sible for hydrolysis of triglycerides in plasma lipoprotein, gen-erating free fatty acids (FFA) and cholesterol. The intracellularmetabolism of FFA differs in various tissues subsequent tocellular uptake. In adipose tissue, FFA is re-esterified anddeposited as lipid droplets for storage. In contrast, FFAs aremainly utilized for �-oxidation and energy production in non-adipose tissues such as muscle. The marked up-regulation ofLPL mRNA expression in uterus and ovaries of transgenicmice, therefore, suggests increased FFA and cholesterol uptake

FIG. 7. MLV-Rel 2 cDNA sequenceand corresponding open readingframes (�2 and �3) encoding puta-tive (truncated) amino-terminal andcarboxyl-terminal MLV Env pep-tides. Asterisks indicate presumptive ter-mination codons.

SSAT-modulated Genes in Female Reproductive Tissues 3653

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

by both tissues in response to increased SSAT. However, in-creased levels of LPL expression are not always beneficial sincesuch changes inevitably lead to increased �-oxidation rate,which may in turn lead to cell death. Recent studies withtransgenic mouse lines have shown that muscle-specific over-expression of LPL causes a severe myopathy (20, 21). Up-regulation of LPL gene expression may be directly responsiblein part, for the morphological and functional changes observedin the uterus (i.e. myopathy of the myometrium) and ovaries ofST mice, possibly occurring via similar mechanisms.

The up-regulation of GAPDH mRNA levels in tissues of STmice is an interesting and novel observation, especially sinceoxidative stress has been shown to increase levels of GAPDH ina rodent cell line (22). Thus, increased GAPDH mRNA levels inthe present study may be indicative of increased oxidativestress in tissues of SSAT-transgenics, a linkage that has beenpreviously documented in unrelated studies with a humannon-small cell line (23) and human breast cancer cell lines (24),wherein polyamine analogue-induced programmed cell deathwas shown to be a consequence in part, of the oxidative stressresulting from generation of H2O2. Induction of GAPDH ex-pression has been suggested to constitute a defense mechanismfor protection of cells against environmental stresses, includingoxidative stress (24, 25), however, increased levels of GAPDHalso induced apoptosis in a number of cell types, particularlyneuronal cells (26). In COS-7 cells, overexpression of GAPDH

induced apoptosis (27), while its suppression by antisense tech-nology led to a subsequent attenuation of apoptosis in cerebel-lar granule cells (28, 29). Therefore, any significant increase inGAPDH gene expression may have a detrimental effect on cellviability. Surprisingly, morphologic and functional alterationsin the present study were only apparent in uterus and ovary,but not in liver and kidney, despite the greater increase inexpression of this gene occurring in the latter tissues withSSAT overexpression. Whatever the cause and consequence (ifany) of GAPDH induction in the current animal model, theseresults differ from those obtained with an in vitro model ofacute SSAT induction in MCF-7 cells (30), where no change inGAPDH mRNA expression was observed upon altered SSAT orpolyamines.

Another novel finding was that chronically altered poly-amine pools in SSAT transgenic mice were correlated withmassive reductions in the mRNA levels of endogenous virus-related genes (MLV-Rel1, Rel2, and MuRRS), all of which werehighly expressed in the corresponding tissues of normal mice.Interestingly, this marked suppression was most apparent foruterus and ovary, which exhibited higher basal expression ofthese transcripts than kidney and liver. The abundant expres-sion of these transcripts in reproductive tissues suggests theirintegration into or near uterine/ovarian genetic loci, althoughtheir specific functions in reproductive and other processes aretotally unknown. A similar pattern of tissue MLV proviral-related RNA (related to but not identical in sequence to theMLV-Re1 and -Rel2 transcripts reported here) expression waspreviously reported in another study with the C57BL/6 mousestrain, where a single transcript of 5.2 kb was predominantlyexpressed in the reproductive tissues of both sexes (31). It iswell recognized that a number of retroviruses, including thosehighly related to the transcripts identified in the present study,are capable of transforming normal cells into neoplastic typesvia activation of proto-oncogenes (32). The involvement of ret-rovirus expression in oncogenesis is best illustrated by theMMTV induction of mouse mammary tumors (33), wherebyretrovirus integration into preferred sites of the genome alterstranscriptional mechanisms in cis and leads to the activation ofan adjacent proto-oncogene (e.g. wnt). Although the majority ofthe retrovirus-related sequences in the mouse germ line aredefective and therefore, incapable of producing viral particles,one of the multiple MLV-Rel transcripts (8.3 kb) detected in thepresent study appears to be the non-defective, infectious MLVproviral RNA, based on its size (31). The expression of thistranscript is very low compared with those of the defectiveMLV transcripts, nevertheless, the presence of a full-lengthtranscript suggests the capability of synthesizing viral protein.Moreover, the MLV-Rel2 transcript described in the presentstudy has two open reading frames potentially encoding trun-

FIG. 8. Northern blots of IGF-I and IGFBP gene transcripts intissues of non-transgenic (�) and transgenic (�) mice. The panelsare the autoradiograms of the Northern blots except for the lowermostpanel which is the ethidium bromide-stained gel prior to blotting. Thesizes of the corresponding transcripts are indicated to the right.

FIG. 9. Autoradiogram of a ligand-blot containing microsomalmembrane fractions (30 �g protein) of tissues from non-trans-genic (�) and transgenic (�) mice probed with radioiodinatedhuman IGF-II. Protein identity was based upon molecular weights asestimated from electrophoretic mobility.

TABLE IIRelative changes in gene expression in transgenic versus

normal mice (%)

Gene Uterus Ovary Liver Kidney

LPL �230% �190 ND NCGAPDH �100% NC �130% �80%CALM-Rel NC NC �230% �340%eIF-3S5 �67% �69% �15% �28%MLV-Rel1 �65% �75% �60% NCMLV-Rel2 �63% �64% �46% NCMuRRS �64% �85% �55% �77%Mouse hepatitis viral receptor �35% N/A NC NCU 1–1-11 �34% �46% �50% �66%U 1–3-15 �100% NC �120% �265%U 2–7-1 NC �56% �56% �89%U 2–3-0 �53% �60% �19% �25%IGFBP-2 �700% �843% NC �45%IGFBP-3 �70% NC NC NCAcid labile subunit NC NC NC NCSp1 �80% NC �110% �122%BTEB1 NC NC NC �67%BTEB2 NC NC NC NC

SSAT-modulated Genes in Female Reproductive Tissues3654

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

FIG. 10. A and B, effects of polyamines on SSAT and IGFBP-2 mRNA abundance in Hec-1-A cells. Cells were incubated in serum-free medium(CONT, control) containing 400 �M polyamine (PUT, putrescine; SPD, spermidine; SPM, spermine) for 24 h. Total cellular RNA was isolated andanalyzed (25 �g/lane) by Northern hybridization, using labeled cDNA probes. B, Northern blot band intensities were obtained from twoindependent experiments (two or three replicate cell/RNA preparations per treatment per experiment as in A) and statistically analyzed aftercorrection for 18 S ribosomal RNA intensity. C, activity of transfected IGFBP-2 gene promoter (5 �g/well) is increased by 100 �M putrescine inserum-free medium (CONT). D, activity of transfected IGFBP-2 gene promoter (5 �g/well) is positively associated with SSAT expression. Shownis luciferase activity after co-transfection with SSAT sense (S) or antisense (As) expression vectors (1 �g of DNA/well) in the absence of serum inthe medium. Panels C and D each represent results of three to four independent experiments; data in B–D are expressed as LSM � S.E.M., withasterisks indicating statistical differences (p 0.05).

SSAT-modulated Genes in Female Reproductive Tissues 3655

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

cated Env peptides; it will be interesting to examine whetherthese peptides are actually expressed in tissues of normal miceand if so, whether polyamines and/or SSAT can alter theirsteady-state levels. Given this situation, the observation thatoverexpression of SSAT leads to suppression of this and othervirus-related transcripts may have important and as yet unex-plored ramifications with respect to polyamine involvementwith viral super infection; expression, splicing, and polyadenyl-ation of adjacent cellular genes; and retroviral etiologies (34–37). The present findings also raise the very interesting possi-bility that the anti-mitogenic actions demonstrated previouslyfor polyamine analogues may be associated, in part, with alter-ations of endogenous retroviral gene expression, although themechanisms underlying this regulation remain unknown atthe present time. Whatever the molecular mechanism(s) thatultimately apply, the present results provide strong evidencethat SSAT and/or polyamines may have a role in the physio-logic regulation of endogenous retroviral gene expression andsubsequently, in their activities and/or functions which havebeen previously implicated in cell proliferation (38–40).

The relative expression of the genes encoding IGFs and twoof their binding proteins was also investigated in the presentstudy, since the IGF system plays an important role in cellproliferation and differentiation within the female reproduc-tive tract and has been previously temporally linked to uterineSSAT gene expression during early pregnancy by our labora-tories (17). The current results indicated that SSAT overex-pression was associated with induced IGFBP-2 gene expressionat the levels of mRNA and protein for the uterus and ovary, butdiminished IGFBP-3 synthesis in the uterus. Furthermore,these alterations occurred without accompanying changes inthe expression of IGF-I and IGF-II genes. IGFBP-3 and IG-FBP-2 constitute the major IGF-binding proteins in reproduc-tive tissues, hence, changes in their concentrations are likely tohave pronounced effects on the bioavailability and/or bio-activ-ity of IGF-I and IGF-II. Several studies have shown that IG-FBP-2 inhibits IGF-stimulated growth via its competition withIGF receptors (41, 42). These in vitro results are consistentwith those of a recent in vivo study of transgenic mice in whichchronic overexpression of IGFBP-2 reduced postnatal body gain(43). In contrast, IGFBP-3 can sometimes enhance the mito-genic effects of IGF-I and -II by increasing their correspondinghalf-lives (44), although this protein can also inhibit cell pro-liferation and DNA synthesis under conditions of acute admin-istration (45). The former is well demonstrated by a recent

study, in which a complex of IGFBP-3 and IGF-I enhancedprotein synthesis under conditions of semistarvation (46). Thiswas not observed when free IGF-I alone was administered.Therefore, the combination of increased expression of IGFBP-2and the simultaneous decreased IGFBP-3 expression is pre-dicted to lead to a substantial overall decrease in the availabil-ity/delivery of IGF-I and IGF-II to target tissues, possibly re-sulting in the observed uterine hypoplasia and ovarian hypo-function in ST mice. Interestingly, these phenotypes aresomewhat mimicked by IGF-I null mutant female mice (47),further suggesting a possible overlap in reproductive functionsof SSAT and the IGF system. We capitalized on the observedpositive relationship of SSAT and IGFBP-2 in mouse uterus toexamine whether SSAT and polyamines directly affect IG-FBP-2 gene expression or alternatively, whether the observedin vivo phenomenon might be a secondary effect of alteredSSAT expression. In the absence of any continuous uterine celllines of murine origin, we used the well-characterized humanHec-1-A uterine cell line to attempt to link the polyaminesand/or SSAT with altered IGFBP-2 gene activity. This wasbased on the previous observation that uteri and ovaries of theSSAT transgenics did not differ from non-transgenics withrespect to spermidine and spermine contents, but did havemarkedly higher intracellular levels of putrescine (15). Theobserved parallel induction of SSAT and IGFBP-2 mRNA abun-dance in this cell line by spermidine and spermine, coupledwith the rapid effect of either SSAT or putrescine to stimulateexogenous IGFBP-2 gene promoter activity and IGFBP-2mRNA abundance, are supportive of a proposed direct linkageof increased SSAT with increased intracellular polyamine con-tent(s) and stimulation of IGFBP-2 gene expression.

The expression of a novel calmodulin-related gene was dra-matically induced from non-detectable levels, in liver and kid-ney, but not in the ovary and uterus, of female mice upon SSAToverexpression. The significance of this highly pronounced tis-sue-specific induction is totally unclear at the present time.However, the similarity of its deduced amino acid sequence tocalmodulins of multiple species and the presence of an appar-ent homolog in the human genome suggests possible roles incalcium signaling pathways, which might stimulate or inhibitcell and tissue growth. In this regard, a different CALM-relatedhuman gene has recently been suggested to be a tumor sup-pressor in breast epithelium (48). Further studies, includingthe biochemical characterization of the gene product, are un-derway to clarify these possibilities.

Chronic overexpression of SSAT was accompanied by de-ceased eIF3 subunit 5 (�, 47 kDa) mRNA abundance, an effectmost apparent for the uterus and ovaries. Eukaryotic transla-tion initiation factor 3 is a large, structurally complex, 10-subunit complex that has a central role in the initiation oftranslation. This complex binds to 40 S ribosomal subunits inthe absence of other initiation factors and helps to maintain 40and 80 S ribosomal subunits in a dissociated state. The eIF3complex also stabilizes initiator methionyl-tRNA binding to 40S subunits and is absolutely required for mRNA binding (49).Several of the other eIF3 protein subunits have been previouslyimplicated in normal and abnormal cellular growth (50–53),although subunit 5 has evidently not been previously implicat-ed/examined with respect to such linkages. Subunit 5 (eIF3-p47) of this complex is a member of the Mov-34 family ofeukaryotic proteins (54). As inferred from the studies of othersubunits, an observed down-regulation of the eIF-3 s5 genewould lead to an inhibition of protein synthesis and cell growth.Similarly, other initiation factors have been tied to cell death.eIF-5 has been implicated as a major trigger in the apoptosis ofa hepatoma cell line DH23A upon induced accumulation of

FIG. 11. Sp1 and BTEB1 but not BTEB2 gene transcripts areinduced in some tissues of SSAT-transgenic mice. Shown are theautoradiograms of the Northern blots. The sizes of the correspondingtranscripts are indicated to the right.

SSAT-modulated Genes in Female Reproductive Tissues3656

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

putrescine (55). A similar role has been proposed for eIF4GII(56) and eIF-2� (57). Although a role for eIF3 has not beendirectly examined, the dramatic down-regulation of expressionof this gene in the uterus and ovary, with accompanying alter-ations in phenotype or function, suggest a physiological linkageof eIF-3 with altered SSAT levels, cell proliferation, and/orapoptosis.

Chronic overexpression of SSAT resulted in altered expres-sion levels of other genes whose identities are currently un-known. Interestingly, the transcripts for these genes were, forthe most part, down-regulated in ST mice, in a tissue-selectivemanner. These observations may represent a general phenom-enon for tissues overexpressing SSAT, although this could alsosimply reflect preferential amplification of certain cDNA frag-ments due to the primer sets utilized. However, if indeed thereis a general inhibition of gene expression associated with SSAToverexpression, this differs from a recent study in which treat-ment of Rat-2 cells with difluromethyl ornithine, a specificinhibitor of polyamine biosynthesis, caused the induction of 26of 35 differentially expressed mRNAs, including that forGAPDH (58). Nevertheless, in the same report, kidneys oftransgenic mice overexpressing ODC and S-adenosylmethi-onine decarboxylase had diminished rather than enhanced lev-els of certain transcripts (58).

In summary, the present study has demonstrated that thechronic overexpression of SSAT leads to marked changes ingene expression in reproductive and non-reproductive tissuesof female mice. Of the differentially expressed genes examinedhere, the number was found to be disproportionately higher inthe uterus and ovary than in kidney and liver, consistent withthe greater phenotypic and functional changes observed in theformer tissues with SSAT overexpression. Although furtherstudies are required to define the functionality of many of thegenes identified on the SSAT phenotypes observed, our find-ings suggest that functional changes associated with SSAToverexpression are mediated at least in part, via the long-termeffects of polyamines (e.g. putrescine, spermidine, and/orspermine) on expression of genes encoding metabolic enzymes,endogenous retroviral transcripts, IGF-binding proteins, andcertain members of the KLF transcription factor family.

Acknowledgments—We thank Frank Michel and Ge Zhao for excel-lent technical support and other members of our laboratories for helpfuldiscussions and assistance.

REFERENCES

1. Tabor, C. W., and Tabor, H. (1984) Annu. Rev. Biochem. 53, 749–7902. Pegg, A. E. (1986) Biochem. J. 234, 249–2623. Asotra, S., Mladenov P. V., and Burke, R. D. (1988) Comp. Biochem. Physiol.

90, 885–8904. Takigawa, M., Enomoto, M., Nishida Y., Pan, H. O., Kinoshita, A., and Suzuki,

F. (1990) Cancer Res. 50, 4131–41385. He, Y., Suzuki, T., Kashiwagi, K., and Igarashi, K. (1994) Biochem. Biophys.

Res. Commun. 203, 608–6146. Alhonen, L., Halmekyto, M., Kosma, V. M., Wahlfors, J., Kauppinen, R., and

Janne, J. (1995) Int. J. Cancer 63, 402–4047. Alhonen, L., Heikkinen, S., Sinervirta, R., Halmekyto, M., Alakuijala P., and

Janne, J. (1996) Biochem. J. 314, 405–4088. Heljasvaara, R., Veress, I., Halmekyto, M., Alhonen, L., Alakuijala P., Janne,

J., Laajala, P., and Pajunen, A. (1997) Biochem. J. 323, 457–4629. Shappell, N. W., Fogel-Petrovic, M. E., and Porter, C. W. (1993) FEBS Lett.

321, 179–18310. Porter, C. W., Ganis, B., Libby, P. R., and Bergeron, R. J. (1991) Cancer Res.

51, 3715–372011. Casero, R. A., Mank, A. R., Xiao, L., Smith, J., Bergeron, R. J., and Celano, P.

(1992) Cancer Res. 52, 5359–536312. Alhonen, L., Karppinen, A., Uusi-Oukari, M., Vujcic, S., Korhonen, V. P.,

Halmekyto, M., Kramer, D. L., Hines, R., Janne, J., and Porter, C. W. (1998)J. Biol. Chem. 273, 1964–1969

13. Byers, T. L., Bitonti, A. J., and McCann, P. P. (1990) Biochem. J. 269, 35–40

14. Karahalios, P., Mamos, P., Vynios, D. H., Papaioannou, D., and Kalpaxis, D. L.(1998) Eur. J. Biochem. 251, 998–1004

15. Pietila, M., Alhonen, L., Halmekyto, M., Kanter, P., Janne, J., and Porter,C. W. (1997) J. Biol. Chem. 272, 18746–18751

16. Ko, Y., Simmen, R. C. M., Lee, C. Y., and Simmen, F. A. (1994) Endocrine 2,495–504

17. Green, M. L., Blaeser, L. L., Simmen, F. A., and Simmen, R. C. M. (1996)Endocrinology 137, 5447–5455

18. Cerro, J. A., Grewal, A., Wood, T. L., and Pintar, J. E. (1993) Regul. Pept. 48,189–198

19. Richelsen, B., Pedersen, S. B., and Hougaard, D. M. (1989) Biochem. J. 261,661–665

20. Levak-Frank, S., Radner, H., Walsh, A., Stollberger, R., Knipping, G., Hoefler,G., Sattler, W., Weinstock, P. H., Breslow, J. L., and Zechner, R. (1995)J. Clin. Invest. 96, 976–986

21. Hoefler, G., Noehammer, C., Levak-Frank, S., el-Shabrawi, Y., Schauer, S.,Zechner, R., and Radner, H. (1997) Biochimie (Paris) 79, 163–168

22. Ito, Y., Pagano, P. J., Tornheim, K., Brecher, P., and Cohen, R. A. (1996) Am. J.Physiol. 270, H81–87

23. Ha, H. C., Woster, P. M., Yager, J. D., and Casero, R. A. (1997) Proc. Natl.Acad. Sci. U. S. A. 94, 11557–11562

24. Chopra, S., and Wallace, H. M. (1998) Biochem. Pharmacol. 55, 1119–112325. Yan, Q., Briehl, M., Crowley, C. L., Payne, C. M., Bernstein, H., and Bernstein,

C. (1999) Biochem. Biophys. Res. Commun. 255, 133–13626. Sirover, M. A. (1999) Biochim. Biophys. Acta 1432, 159–18427. Tajima, H., Tsuchiya, K., Yamada, M., Kondo, K., Katsube, N., and Ishitani, R.

(1999) Neuroreport 10, 2029–203328. Ishitani, R., and Chuang, D. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,

9937–994129. Chen, R. W., Saunders, P. A., Wei, H., Li, Z., Seth, P., and Chuang, D. M.

(1999) J. Neurosci. 19, 9654–966230. Vujcic, S., Halmekyto, M., Diegelman, P., Gan, G., Kramer, D. L., Janne, J.,

and Porter, C. W. (2000) J. Biol. Chem. 275, 38319–3832831. Cornwall, G. A., Orgebin-Crist, M.-C., and Hann, S. R. (1992) Biol. Reprod. 47,

665–67532. Patarca, R. (1998) Crit. Rev. Oncog. 9, 151–19833. Dickson, C. (1990) Int. J. Cancer Suppl 5, 51–5434. Nihrane, A., Lebedeva, I., Lyu, M. S., Fujita, K., and Silver, J. (1997) J. Gen.

Virol. 78, 785–79335. Kowalski, P. E., Freeman, J. D., and Mager, D. L. (1999) Genomics 57, 371–37936. Baust, C., Seifarth, W., Germaier, H., Hehlmann, R., Leib-Mosch, C. (2000)

Genomics 66, 98–10337. Medstrand, P., Landry, J. R., and Mager, D. L. (2001) J. Biol. Chem. 276,

1896–190338. Dragani, T. A., Manenti, G., Della-Porta, G., and Weinstein, I. B. (1987) Cancer

Res. 47, 795–79839. Lin, L., Xu, B., and Rote, N. S. (2000) Placenta 21, 73–7840. Boese, A., Sauter, M., Galli, U., Best, B., Herbst, H., Mayer, J., Kremmer, E.,

Roemer, K., and Mueller-Lantzsch, N. (2000) Oncogene 19, 4328–433641. Wetterau, L. A., Moore, M. G., Lee, K. W., Shim, M. L., and Cohen, P. (1999)

Mol. Genet. Metab. 68, 161–18142. Duan, C., Ding, J., Li, Q., Tsai, W., and Pozios, K. (1999) Proc. Natl. Acad. Sci.

U. S. A. 96, 15274–1527943. Hoeflich, A., Wu, M., Mohan, S., Foll, J., Wanke, R., Froehlich, T., Arnold,

G. J., Lahm, H., Kolb, H. J., and Wolf, E. (1999) Endocrinology 140,5488–5495

44. Hodgson, D. R. (2000) Regul. Pept. 90, 33–3745. Matsumoto, T., Tsurumoto, T., Goldring, M. B., and Shindo, H. (2000) J.

Endocrinol. 166, 29–3346. Svanberg, E., Ohlsson, C., Kimball, S. R., and Lundholm, K. (2000) Eur.

J. Clin. Invest. 30, 438–44647. Baker, J., Hardy, M. P., Zhou, J., Bondy, C., Lupu, F., Bellve, A. R., and

Efstratiadis, A. (1996) Mol. Endocrinol. 10, 903–91848. Rogers, M. S., Foley, M. A., Crotty, T. B., Hartmann, L. C., Ingle, J. N., Roche,

P. C., and Strehler, E. E. (1999) Neoplasia 1, 220–22549. Chaudhuri, J., Chowdhury, D., and Maitra, U. (1999) J. Biol. Chem. 274,

17975–1798050. Asano, K., Merrick, W. C., and Hershey, J. W. B. (1997) J. Biol. Chem. 272,

23477–2348051. Verlhac, M. H., Chen, R. H., Hanachi, P., Hershey, J. W., and Derynck, R.

(1997) EMBO J. 16, 6812–682252. Nupponen, N. N., Isola, J., and Visakorpi, T. (2000) Genes Chromosomes &

Cancer 28, 203–21053. Guo, J., Hui, D. J., Merrick, W. C., and Sen, G. C. (2000) EMBO J. 19,

6891–689954. Asano, K., Vornlocher, H.-P., Richter-Cook, N. J., Merrick, W. C., Hinnebusch,

A. G., and Hershey, J. W. B. (1997) J. Biol. Chem. 272, 27042–2705255. Tome, M. E., Fiser, S. M., Payne, C. M., and Gerner, E. W. (1997) Biochem. J.

328, 847–85456. Svitkin, Y. V., Gradi, A., Imataka, H., Morino, S., and Sonenberg, N. (1999)

J. Virol. 73, 3467–347257. Satoh, S., Hijikata, M., Handa, H., and Shimotohno, K. (1999) Biochem. J. 342,

65–7058. Veress, I., Haghighi, S., Pulkka, A., and Pajunen, A. (2000) Biochem. J. 346,

185–191

SSAT-modulated Genes in Female Reproductive Tissues 3657

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Porter, Juhani Jänne and Frank A. SimmenSeok Hong Min, Rosalia C. M. Simmen, Leena Alhonen, Maria Halmekytö, Carl W.

-Acetyltransferase (SSAT) 1 NOther Tissues of Female Mice Overexpressing Spermidine/Spermine

Altered Levels of Growth-related and Novel Gene Transcripts in Reproductive and

doi: 10.1074/jbc.M100751200 originally published online November 14, 20012002, 277:3647-3657.J. Biol. Chem. 

  10.1074/jbc.M100751200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/277/5/3647.full.html#ref-list-1

This article cites 58 references, 27 of which can be accessed free at

by guest on May 19, 2020

http://ww

w.jbc.org/

Dow

nloaded from