to the GenBankTMIEMBL Data Bank with accession number(s) U04204. $ This work was ...... Klagsbmn, M., and Baird, A. (1991) Cell 67,229-231. Burgess, W. H. .... Bohren, K. M., Page, J. L., Shankar, R., Henry, S. P., and Gabbay, K. H. (1991).
THEJOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 11, Issue of March 18, pp. 8604-8609, 1994 Printed in U.S.A.
A Delayed-early Gene Activatedby Fibroblast Growth Factor-1 Encodes a Protein Related to Aldose Reductase* (Received for publication, October 21, 1993)
Patrick J. DonohueS, GregoryF. Alberts, Brian S. Hampton, and Jeffrey A. Winkles9 From the Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockuille, Maryland 20855
The addition of polypeptide mitogensto quiescent cell though it is known that FGF-1 binding initiates numerous lines induces the expression of various gene products, biochemical changes, including the activation of specific genes some of which arelikely to perform functionscritical for (1). Genes that are known to be FGF-1-regulated in murine cell cycle progression, DNA synthesis, and mitosis. We NIH 3T3 fibroblasts include those encodingc-fos, c j u n , c-myc, have used a differential display approach to identify early growth response gene-1, thrombospondin-1, ornithine defibroblast growth factor (FGF)-1-inducible genes in NIH- carboxylase,proliferin,glyceraldehyde-3-phosphatedehydro3T3 cells. Oneof these genes, termedFGF-regulated genase, fatty acid synthase, phosphofructokinase, and sarco(FR)-l, encodesa316-aminoacidproteinwith -82% (endo)plasmic reticulum Ca2+-ATPase (5-7L2 It is likelythat at amino acid sequence identity to an abundant protein least some of these proteins perform functions essential for expressed in mouse vas deferens and -70% identity to FGF-1 mitogenic activity on thiscell line. human aldose reductase.The function of the vas deferTo gain further insight into the FGF-1 signaling mechanism, ens protein is unknown; however, aldose reductase is an we have used a reverse transcription-PCR differential display NADPH-dependentmonomericoxidoreductaseimpliapproach to identify FGF-1-inducible genes in NIH3T3 fibrocated in the pathogenesis of diabeticcomplications. FGF-1 induction ofFR-1mRNA expression is first de- blasts (7). Briefly, RNA isolated from serum-starved or FGF-1cDNA, and PCR assays are is dependent stimulated cells is converted to tectable at 4 h after mitogen addition and on de novo RNA and proteinsynthesis. FGF-2 or phorbol performedwithvariousdegenerateoligonucleotideprimers. ester treatment can also increase FR-1 mRNA levels; in Amplification products are displayed using agarose gel electrocontrast, whole blood serum or individual growth fac- phoresis and ethidium bromide staining. Those DNAfragments are isolated, tors present in serum have only minimal effects on FR-1 representingdifferentiallyexpressedmRNAs mRNA expression. FR-1 mRNAis detectable in a numbercloned, and characterized. In this report, we present data inof mouse tissues but is most abundant in newborn liver dicating that FGF-1 can inducethe expression of a transcript and in adult intestine, ovary, and testis. These results encoding a member of the aldo-keto reductase superfamily(8raise the possibility that aldose reductase-related pro- 10). Specifically, thepredictedpolypeptideexhibits -82% teins may play a role in FGF-1- and FGF-2-stimulated amino acid sequence identity with a major protein expressed in mitogenesis. epithelial cells of the mouse vas deferens(11)a n d -70% identity with the enzyme aldose reductase (alditol:NAD(P)+ l-oxidoreductase;EC 1.1.1.21), whichcatalyzestheNADPH-dependent reduction of various carbonyl compounds (12). Aldose The fibroblast growth factor (FGF)’ family presently consists reductase has abroad substratespecificity but is best knownas of nine structurally related polypeptides (1, 2 ) . FGF-1, also referred toas acidic FGF, isa multifunctional protein that can the first enzyme in thepoly01 pathway, and consequently, as a stimulate cell proliferation, migration, differentiation, and sur-potential mediatorof diabetic complications (13). To our knowlfirst report indicatingthat the expressionof a n of edge, this is the vival (1).These effects are mediated through the interaction FGF-1 with a family of receptor tyrosine kinases (3) and with aldose reductase-related protein is regulated by polypeptide of growth factors. heparan sulfate proteoglycans (4) present on the surface responsive cells. The subsequent molecular events associated MATERIALSANDMETHODS with FGF-1 signal transduction are not well understood, alCell Culture-Murine NIH 3T3 cells (American QpeCulture Collection) were grown at 37 “C in Dulbecco’s modified Eagle’s medium (Me* This study was supported in part by National Institutes of Health diatech) supplemented with 10% (v/v) heat-inactivated bovine calf seGrant HL-39727 (to J. A.W.) and a grant-in-aid from the American rum (HyClone Laboratories) and a 1:lOO dilution of apenicilli4 Heart Association (to J. A. W.). The costs of publication of this article streptomycidfungizone solution (JRH Biosciences). The cellswere were defrayedin part by the payment of page charges. This article must expanded by trypsin-EDTA (JRH Biosciences) treatment and subcultherefore be hereby marked “advertisement” in accordance with 18 turing at a split ratio of 1:7every 2-3 days. To induce a relatively U.S.C. Section 1734 solely to indicate this fact. quiescent cell population, subconfluent cells were incubated for -72 h The nucleotide sequencefs) reported inthis paper has been submitted to the GenBankTMIEMBL Data Bank with accession number(s)U04204. in the above medium containing a reduced serum concentration (0.5%). Cells were then either left untreated or treated for various times with $ This work was performed in partial fulfillment of the requirements for the degree of Doctor of Philosophyfrom the Graduate Genetics either 10% calf serum or 0.5% calf serum supplemented with 10 ng/ml recombinant human FGF-1 (gift ofW. Burgess, American Red Cross) Program, George Washington University, Washington, D. C. 5 To whom correspondence and reprint requests should be addressed: and 5 unitdm1 heparin (Upjohn), 10%calf serum, 10 ng/ml FGF-2 Dept. of Molecular Biology, Holland Laboratory, American Red Cross, (Bachem Inc.), 10 ng/mlPDGF-BB (Genzyme), 20ng/mlEGF(Genzyme), 2 ng/ml TGF-Pl (R & D Systems), 20 ng/ml IGF-1 (Bachem),or 15601 Crabbs Branch Way, Rockville, MD 20855.Tel.:301-738-0655 30ng/mlPMA (Sigma). In some experiments, cells were either left Fax: 301-738-0465. The abbreviations used are: FGF, fibroblast growth factor; FR, FGF- untreated or treated with FGF-1 and/or 10pg/mlcycloheximide regulated; EGF, epidermal growth factor; IGF, insulin-like growth fac- (Sigma). tor; MVDP, mouse vas deferens protein; PDGF, platelet-derived growth P. J. Donohue, D. K. W. Hsu, and J. A. Winkles, unpublished obserfactor; PCR, polymerase chain reaction; PMA,phorbol myristate acvations. etate; TGF, transforming growth factor. ~
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FGF-1 Regulation Aldo-keto of an Reductase
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RNA Isolation-Cells were harvested by trypsin-EDTA treatment degenerate C2H2 zinc finger and protein tyrosine kinase doand total RNA isolated using RNazol B (Tel-Test) or RNA Stat-60 (Tel- main oligonucleotide primers. Amplification products were disTest) according to the manufacturer's instructions. Tissues from new- played using agarose gel electrophoresisand ethidiumbromide born (1-5 days old) or adultFVB/N mice (Taconic Farms) were homogstaining. The patternof amplified cDNAs obtained from quiesenized in RNA Stat-60 (3 ml of reagentf500 pg of tissue) using a cent and FGF-1-stimulated cellular RNA were, for the most Tissumizer (Tekmar). RNA concentrations were calculated from the part, similar(Fig. lA).However, a DNAfragment of -700 base absorbance at 260 m.All RNA samples contained intact rRNA and were free of contaminating genomic DNA as evaluated by electrophoresis in pairs in size was amplified to a greater degree when cDNA agarose/formaldehyde gels. representing the RNA isolated from cells treated with FGF-1 Differential Display-RNA (1 pg) isolated from serum-starved or for 12 h was used as template. InitialRNA gel blot hybridizaFGF-1-stimulated cells was converted to cDNA as described (14) using tion experiments indicated that thisDNA fragment hybridized random hexamer primers (Boehringer Mannheim). PCR reactions were performed in 10 m~ Tris-HC1 (pH 8.31, 1.5 m~ MgC12, 50 m~ KCl, 0.2 to a transcript of -1.5 kilobases in size. Therefore, this fragmM each deoxyribonucleotide triphosphate and included 2.5 units of Taq ment, termed FR-1, was radiolabeled and used to screen a DNA polymerase (Boehringer Mannheim), 0.25 pgof a zinc finger do- Balb/c 3T3 cell hgtl0 cDNA library in order to isolate larger main sense primer, and0.25 pg of a tyrosine kinase domain antisense cDNA clones. Two positive phage were isolated and theircDNA primer. The degenerate zinc finger domain primer was designed by inserts subcloned. Both inserts were -1.5 kilobases in size; one aligning various mouse C,H, zinc finger cDNA sequences (15-18) and establishing a consensus sequence. It is 5' GGNGAGAARCCCTWYG- cDNA clone was used for the subsequent experiments. Regulation of FR-1 mRNA Expression in NIH 3T3 CellsARTG 3'. The degenerate protein tyrosine kinase domain primer was described previouslyby Wilks (19)and is 5'GGAATTCCAWAGGACCA- RNA gel blot hybridization analysis was performed to confirm SACRTC 3'. Degenerate bases in the primers listed above are abbrevi- the differential display results indicating that FR-1 mRNA levated as recommended by apreviousnomenclaturecommittee(20). els were elevated in FGF-1-stimulated NIH3T3 cells. A single Samples were subjected to 32cycles of PCR amplification using a PerFR-1 transcript wasdetected in both serum-starved and FGFkin-Elmer 9600 thermocycler. Stage1included eight cycles with dena1-stimulated cells. However, FR-1 mRNA expression was elturation at 94 "C for 30 s, annealing at 46 "C for 30 s, and primer addition; increasedFR-l extension a t 68 "C for 30 s. Stage 2 included 24cycles with denaturation evated significantly afterFGF-1 mRNA levels were apparent at 4 h, and maximal levels were at 94 "C for 30 s, annealing at 58 "C for 30 s, and primer extension at 72 "C for 30 s. An aliquot of each amplification mixture was subjected t o detected at 12 and 18 h (Fig. lI3). FR-1 mRNA induction by electrophoresis i n a 1.8% agarose (Life Technologies, Inc.) gel. 0X174/ FGF-1 is due, at least in part, to transcriptional activation, HaeIII restriction fragments (Clontech Laboratories) were used as size since it does not occur if RNA synthesis is inhibited (datanot standards. DNA was visualized by ethidium bromide staining. The appropriate DNAfragment, named FR-1, was excised, recovered using the shown). Since the FR-1mRNA expression kinetics were typical freeze-squeeze method (211, reamplified using the Stage 2 conditions of other FGF-1-inducible delayed-early genes, we determined described above, and ligated into thecloning vector pCRlOOO (Invitro- whether FGF-1induction of FR-1 mRNAlevels was dependent gen Corp.). on de novo protein synthesis. The protein synthesis inhibitor cDNA Library Screening-A mouse Balb/c 3T3 cell A g t l O cDNA li- cycloheximide repressed FGF-1induction of FR-1 mRNA levels brary (gift of L. Lau, University of Illinois College of Medicine) was (Fig. 1C). Taken together, these results indicate that the FR-1 screened withthe subcloned PCR-derived FR-1 DNAfragment to obtain gene is an FGF-1-inducible delayed-early gene in NIH3T3 a larger cDNA clone. Briefly, the DNA fragmentwaslabeledwith [32PldCTP (3000 CVmmol, DuPont NEN) using a random primer label- fibroblasts. ing kit (Boehringer Mannheim). Approximately 180,000 phage were Whole blood serum contains numerouspolypeptide mitogens plated a t a density of 20,000 plaque-forming units/l50-mm dish using for NIH 3T3 cells but does not contain significant amounts of Escherichia coli C600 Hfl a s host. Duplicate plaque lifts (ColonyiPlaque FGF-1 (23). Therefore, we investigated whether serum stimuScreen, Du Pont) were hybridized and washed as described (22).Posilation of NIH 3T3 cells would also induceFR-1 geneexpression. tive signals were purifiedby two additional rounds of screening. PuriRNA wasisolated from serum-starved or serum-stimulated fied A clones were amplifiedon E. coli C600 Hfl and A DNA isolated by polyethylene glycoVNaC1 precipitation. The cDNAinserts were releasedcells and equivalent amountsanalyzed by RNA gelblot hybridfrom the A g t l O vector by EcoRI digestion andsubcloned into pGEM3Zf+ ization. FR-1 mRNA expression was notinduced by serum (Promega Corp.). treatment; in fact, the expression level actually decreased after cDNA Sequence Analysis-Plasmid DNA was purified using aMagic 8 h of treatment (Fig. 2 A ) . It should be noted that the level of Miniprep Kit (Promega Corp.) and both strands ofthe cDNAinsert were FR-1 mRNA expression in serum-starved cells shown here apsequenced by the dideoxynucleotide chain termination method either pears significantly higher than that shown in the preceding automatically usinga n Applied Biosystems model 373A DNA sequencer or manually using a Sequenase 2.0 kit (U. S. Biochemical Corp.). The figure. This is because this autoradiogram wasexposed 12-fold deduced protein sequence was compared with sequences present in the longer in order t o detect the inhibitory effect of serum treatPIR(release 36) andSwiss-Prot(release25)databases on a Sun ment on FR-1 mRNA expression. Spark-10 server using a Genetics Computer Group software package. We next determinedwhetherthe FGF-1-relatedmitogen RNA Gel Blot Hybridization-Ten pg of each RNA sample was denaFGF-2 or individual growth factors present in whole serum tured and subjected t o electrophoresis in 1.2% agarose gels containing could regulate FR-1 mRNA levels in serum-starved NIH 3T3 2.2 M formaldehyde. The gels were stained with ethidium bromide to cells. Cells were also treated with the phorbol ester PMA to verify that each lane contained similar amountsof undegraded rRNA. RNA was electroblotted onto Zetabind nylon membranes (Cuno Inc.) investigate whether protein kinase C activation altered FR-1 and cross-linked by U V irradiation using a Stratalinker (Stratagene). gene expression. In this particular experiment, FR-1 mRNA The cDNA insert was radiolabeledas described above for cDNA library levels were increased -11.5-fold after 8 h of FGF-1 treatment screening. Hybridization and membrane washing conditions were as (Fig. 2 B ) . FGF-2 and the phorbol ester PMA increased FR-1 described(22).Hybridizationsignals and rRNA ethidiumbromide staining intensities were quantitated by densitometry usingVisage 4.6 mRNA levels -7- and -9.8-fold, respectively. Incontrast, software (BioImage Products). The FR-1mRNA signal was normalized PDGF-BB, TGF-P1, EGF, or IGF-1 treatment had relatively t o the 28 S rRNA signal to correctfor slight differences in the amount small effects on FR-1 mRNA expression (-2.6-, -1.1-, -1.8-, of RNA per gel lane. and -0.9-fold induction, respectively). Thus, although serum
itself decreased FR-1 mRNA levels, some of the major growth factors present in serum either had no effect on or slightly Identification of a n FGF-1-inducible Gene by Differential increased FR-1 gene expression. Display-RNA isolated from serum-starved or FGF-1-stimuFR-1 cDNA Sequence Analysis-The nucleotide sequence of a lated NIH 3T3cells was converted to cDNA using reverse tran- nearly full-length FR-1 cDNA is shown in Fig. 3. It contains a scriptase and random primers. PCR was then performed using long open reading frame beginning at nucleotide 68 that enRESULTS
-
FGF-1 Regulation of an Aldo-keto Reductase Gene
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tained a 289 nucleotide 3‘-untranslated region with a typical polyadenylation signal (25) 23-28 basesupstream of the poly(A)tail. Comparison of the PCR primer sequences with the FR-1 cDNA sequence identified the two regions that flanked the 700-base pair cDNA clone originally identified by differential display. The protein tyrosine kinase antisense oligonucleFIG.1. Identification ofan FGF-1-inducible mRNAin M H 3T3 otide actually acted as a sense primer. It had -61% nucleotide fibroblasts by differential display. A, serum-starved cells were either left untreated or treated with FGF-1 for 2 or 12 h. RNA was sequence identity, primarily at its3’ end, to a sequence in the isolated, cDNA was synthesized, and the PCR performed using CzHz 5‘-untranslated region of the FR-1 cDNA. The C2H2zinc finger zinc finger and protein tyrosine kinaseoligonucleotide primers. Ampli- sense oligonucleotide actually acted as an antisenseprimer. It fication products were separated by agarose gel electrophoresis and had -85% nucleotide sequence identity to a sequence within visualized by ethidium bromide staining. TheDNA size markers(M,in base pairs) are0X174IHaeIII restriction fragments. Thearrow denotes the FR-1 cDNA coding region. Therefore, based on these findB, ings, it seemed unlikely that the protein encoded by FR-1 a cDNA fragment representing a differentially expressed transcript. serum-starved cells were either left untreated or treated withFGF-1 for mRNA would contain tyrosine kinase or zinc finger structural the indicated timeperiods. RNAwas isolated and equivalent amounts of motifs. each sample analyzed by RNA gel blot hybridization. In this and the FR-1 Amino Acid Sequence Comparisons-A computer subsequent RNA gel blot hybridization figures, the blots were probed search of protein data bases revealed that the deduced FR-1 with radiolabeled FR-1cDNA, and only the region of the autoradiogram amino acid sequence was homologous to MVDP and aldose that contained a hybridization signal is shown. Also, to demonstrate equivalent loading of samples, the gels were stained with ethidium reductase, two members of the NAPDH-dependent aldo-keto bromide; the bottom panel of each figure is a photograph of the 28 S reductase superfamily (8-10). The amino acid sequence idenrRNA band. C, serum-starved cells were either left untreated or treated with FGF-1, FGF-1 and cycloheximide (CHX), or cycloheximide alone tity between FR-1 and MVDP ( l l ) , human aldose reductase (lo),rabbit aldose reductase (26), rataldose reductase (27,28), for 8 h. RNA was isolated and equivalent amounts of each sample analyzed by RNA gel blot hybridization. and bovine aldose reductase (29, 30) is 82.3, 70.2, 69.3, 69.0, and 68.7%, respectively. Lower but significant levels of amino acid sequence identity are also observed with several other codes a protein of 316 amino acids with a predicted molecular members of the aldo-keto reductase superfamily: human aldemass of 36,120 daltons. The presumed initiating AUG is the hyde reductase (52%), human chloredecone reductase (47.1%), first methionine codon and is also flanked by a consensus se- frog p-crystallin (46.8%), rat 3a-hydroxysteroid dehydrogenase quence for translation initiation (24). The cDNA clone con- (46.8%), and bovine prostaglandin F synthetase (45.6%). An
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alignment of FR-1, MVDP, and the human and rat aldose reductases is shown in Fig. 4. FR-1 mRNA Expression Levels in Mouse Tissues-We used RNA gel blot hybridization analysis to examine the tissue distribution of FR-1 mRNA. Six different tissues were obtained from newborn animals and 12 different tissues were obtained from adult animals. In thenewborn animals, FR-1 transcripts were undetectable or expressed at a low level in heart, intestine, kidney, lung, and skin, whereasa relatively high level of expression was found in the liver (Fig. 5A). In the adult animals, FR-1mRNA was undetectableor expressed at a low level in brain, heart, kidney, liver, lung,salivarygland,skeletal muscle, skin, and spleen but was relatively abundant in intestine, ovary and testis (Fig. 5B).These results indicate that FR-1 gene expression is regulated in vivo in both a developmental stage- andtissue-specific manner.
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DISCUSSION
Murine NIH 3T3 fibroblasts express FGF cell surface receptors and proliferate in response to FGF-1 treatment (5). It is probable that at least some of the proteins importantfor FGF1-stimulated NIH 3T3 cell growth are encoded by FGF-l-inducible immediate-early or delayed-early genes. Therefore, we have used a reverse transcription-PCR differential display approach to isolate cDNA clones representing FGF-1-inducible mRNAs (7). In this report, we describe the identification and characterization of the FR-1 cDNA clone. The original FR-1 cDNA was identified using degenerateoligonucleotide primers designed to recognize sequences encoding zinc finger or protein tyrosine kinase structural domains. However, under the PCR conditions used, the oligonucleotides were able to anneal to regions of minimal sequence homology, one of which was actu-
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FIG.4. Homology between the predicted F R - 1 amino acid sequence, MVDP, and aldose reductase. The aligned sequences are FR-1, MVDP, human aldose reductase(HAR1, and rat aldose reductase( R A R ). Numbers at therzght side refer t o the last amino acids on the lines.Amino acids that are identical in all four polypeptides a t a given position are boxed.
FIG.5. F R - 1 mRNA expression levels in various mouse tissues. RNA was isolated from the indicatedtissuesand equivalentamounts of eachsampleanalyzed byRNA gel blot hybridization. A, newborn mouse tissues; B, adult mouse tissues.
A) FR-1
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ally in the 5'-untranslatedregion of the FR-1 cDNA template. cated that lysine 262 is involved in NADPH binding and cataThus, as discussed previously (7), the differential display ap- lytic activity (31, 32), and cysteine 298 is important for the regulation of catalytic activity and inhibitor sensitivity (33). proach we have used will identifyvariousFGF-1-regulated genes, only some of which may actually encode proteins with These 2 amino acid residues are conserved between FR-1 and aldose reductase. Furthermore, all 18 of the human aldose the targeted structural domains. acid residuesreported to interact with The FR-1gene has properties consistent with previously reductaseamino identified mitogen-inducible delayed-early genes. First, FGF-1 NADPH on the basis of x-ray crystallography data (34) are stimulation of serum-starved cells increases FR-1 mRNA levels conserved in mouse FR-1. FR-1 and MVDP exhibit the highest degree of amino acid with relatively late kinetics (peak expression a t 12-18 h). Second, in contrast to the immediate-earlyclass of mitogen-induc- sequence identity F"2%).They each exhibit a similar amount ible genes, FR-1 mRNA accumulation requires de nouo protein of sequence identity (-70%) to the human, rat, rabbit, and synthesis. This latter result indicates FR-1 that gene activation bovine aldose reductases. It ispresently unclear whether FR-1 and/or FR-1 mRNA stabilization is dependent on immediate- or MVDP should be considered the mouse form of aldose reductase, since -85% amino acid sequence conservation is apparent early proteins. In general, many of the previously identified mitogen-regu- when the aldose reductases from other species are compared. lated genes are activated to a similar extent by numerous Furthermore, analysis of various rat (35) or human (36, 37) growth-promoting agents, including whole serumand indi- tissues by RNA gel blot hybridization or immunoreactivity asvidual polypeptide growth factors. These findings imply that says, respectively, have indicated that the kidney medulla is a there may be considerable overlap in the signal transduction major site of aldose reductase synthesis. Neither MVDP mRNA pathways activated by distinct mitogens. An interesting prop- nor protein expression has been detected in the mouse kidney erty of FR-1 geneinduction in NIH 3T3 cells is that FGF-1 and (38, 39). FR-1 mRNA is expressed in a variety of tissues, inFGF-2 are significantly more potent than whole serum or vari- cluding kidney, but is most abundant in intestine, ovary, and contain aldose ous purified serum mitogens. However, in regard to the effect of testis. It isknown that these latter three tissues PDGF-BB, TGF-P1, EGF, or IGF-1, we should note that our reductase protein (36, 37, 4042). We also observed that the FR-1 mRNA expression level in some tissues is regulated deassay tested one concentration at a single time point. The deduced FR-1 amino acid sequence is structurally re- velopmentally. For example, FR-1mRNA is expressed at a low level in newborn intestine anda t a relatively high level in adult lated to members of the aldo-keto reductase superfamily (810). The amino acid sequence identity betweenFR-1 and intestine. Also, FR-1 mRNAexpressionis high innewborn liver MVDP, human aldose reductase, human aldehyde reductase, but not adult liver. We should emphasize that it is presently unknown whether thelevel of FR-1 mRNA expression is a good human chloredecone reductase, frog pcrystallin, rat 3a-hyindicator of FR-1 protein abundance. This issue will be addroxysteroid dehydrogenase, and bovine prostaglandin F synthetase is 82.3, 70.2, 52.0, 47.1, 46.8, 46.8, and 45.6%, respec- dressed when FR-1-specific antibodies become available. tively. Although it is generally assumed that all members of Thereare relatively few reports on factors or conditions this family are NADPH-dependent monomeric oxidoreducta- which can regulate the expression level of aldo-keto reductase ses, theenzymatic nature of MVDP and pcrystallin hasnot yet superfamily genes. It hasbeen shown that MVDP gene expresbeen demonstrated directly. Carper et al. (9) have reported, sion is increased following testosterone injection (11,38,43). In addition, aldose reductase gene expression is elevated in varihowever, that pcrystallin can bind NADPH. At the present time we do not know whether the FR-1 protein has NADPH- ous cell types subjected to hypertonic stress (44-48). TheFR-1 dependent reductase activity; however, the degree of homology gene is induced by FGF-1, FGF-2, PMA, and toa lesser extent, to aldose reductase implies that itmay. For example, previous by PDGF-BB and EGF. This is, to our knowledge, the first aldose reductase site-directed mutagenesis studies have indi- demonstration that an aldose reductase-related gene can be
FGF-1 RegulationAldo-keto of an Reductase regulated by phorbol ester or polypeptide growth factors. At this time it is difficult to propose a physiological role for FR-1 in FGF-stimulated cell division. However, if FR-1is an enzyme with aldosereductase-like substrate specificity,its elevated expression during cell cycleprogression could function to enhance synthesis. the rateof glycolysis and/or membrane phospholipid Acknowledgments-We thank Dr. L. Lau for the cDNAlibrary, Dr. W. Burgess for the FGF-1, Dr. R. Friesel for the tyrosine kinase oligonucleotide, S. Appleby forperforming the automated DNA sequence analysis, and Dr. C. Bieberich forthe mouse tissue samples. We are also grateful to Dr. D. Hsu and Dr. W. Burgess for critical review of the manuscript, Dr. D. Carper for helpful discussions, and K. Wawzinski for excellent secretarial assistance. REFERENCES 1. Burgess, W. H., and Winkles, J.A. (1994) in Regulation of the Proliferation of Neoplastic Cells (Pusztai, L., Lewis, C. E., and Yap, E., eds) Oxford University Press, Oxford, in press 2. Miyamoto, M., NaruO, K., Seko, C., Matsumoto, S., Kondo, T., and Kurokawa, T. (1993) Mol. Cell. Bid. 13, 42514259 3. Johnson, D. E., and Williams, L. T. (1993) Adu. Cancer Res. 80, 1-40 4. Klagsbmn, M., and Baird, A. (1991) Cell 67,229-231 5. Burgess, W. H., Shaheen, A. M., Ravera, M., Jaye, M., Donohue, P. J., and Winkles, J. A. (1990) J . Cell Bid. 111,2129-2138 6. Burgess, W. H., Shaheen, A. M., Hampton, B., Donohue, P. J., andWinkles, J. A. (1991) J. Cell. Biochem. 45, 131-138 7. Hsu, D. K. W.,Donohue, P. J.,Alberts, G. F., and Winkles, J. A. (1993)Biochem. BioDhvs. Res. Commun. 197. 1483-1491 8. W e r m k k B. (1985) in Enzymology of Carbonyl Metabolism 2: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase (Flynn, T. G., and Weiner, H., eds)pp. 209-230, Alan R. LISS, Inc., New York 9. Carper, D. A,, Wistow, G., Nishimura, C., Graham, C., Watanabe, K., Fujii, Y., Hayashi, H., and Hayaishi,0. (1989) Exp. Eye Res. 49,377-388 10. Bohren, K. M., Bullock, B., Wermuth, B., and Gabbay, K. H. (1989) J. Biol. Chem. 264,9547-9551 11. Pailhoux, E.A,, Martinez, A., Veyssiere, G. M., and Jean,C. G. (1990) J . Bid. Chem. 266,19932-19936 12. Bhatnagar, A., and Srivastava, S. K. (1992)Biochem. Med. Metabolic Bid. 48, 91-121 13. Tomlinson, D. R., Willars, G. B., and Camngton,A. L. (1992)Pharmacol. Ther. 64, 151-194 14. Winkles, J. A., Friesel, R., Alberts, G. F., Janat, M. F., and Liau, G. (1993)Am. J . Pathol. 143, 518-527 15. Chowdhury, K., Deutsch, U., and Gruss, P. (1987) Cell 48, 771-778 16. Chavrier, P., Zerial, M., Lemaire, P., Almendral, J., Bravo, R., and Charnay,P (1988) EMBO J . 7, 29-35 17. Nelki, D.. Dudlev, K., Cunnineham, P.. and Akhavan.M. (1990)Nucleic Acids Res. 18, 365518. Lemaire, P., Revelant, O., Bravo, R., and Charnay,P. (1988) Proc. Natl. Acad.
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