Epidermal Growth Factor Up-regulates the Transcription ofMouse Lon Homology ATP-Dependent Protease Through

Extracellular Signal-Regulated Protein Kinase- andPhosphatidylinositol-3-Kinase-dependent Pathways

Yunfeng Zhu,* Mei Wang,* Hong Lin,* Chuanshu Huang,† Xianglin Shi,‡ and Jia Luo*,1

*Department of Microbiology, Immunology, and Cell Biology, West Virginia University School of Medicine, Morgantown,West Virginia 26506; †New York University Medical Center, Nelson Institute of Environmental Medicine, Tuxedo, New York 10987; and

Epidermal growth factor (EGF) induces tumorigenictransformation of mouse epidermal cells (JB6 P�). Wecloned a full-length EGF-responsive cDNA in JB6P�

cells; EGF up-regulated mRNA expression of this gene5- to 6-fold. The deduced amino acid sequence of thiscDNA exhibited 84 and 96% homology with human andrat Lon homology ATP-dependent protease, respec-tively, and all conserved domains of Lon, such asATPase/protease domains, are present in the mousegene, indicating that this gene is mouse Lon. EGF in-creased the transcriptional rate without affecting themRNA stability of m-Lon. The level of m-Lon in irre-versibly transformed mouse epidermal cells (JB7) was3.4-fold higher than that in parental JB6 P� cells. Sim-ilarly, human mammary epithelial cells overexpress-ing the proto-oncogene ErbB2 expressed significantlyhigher levels of Lon than normal mammary epithelialcells. EGF failed to regulate Lon expression in ERK-deficient JB6 P� cells or cells that expressed the dom-inant-negative p85 P13-K regulatory unit. Further-more, selective chemical blockers for MEK1 and P13-K(PD98059 and LY294002) inhibited EGF-mediated in-duction. Mitochondria-localized Lon protease plays acritical role in the regulation of mitochondrial geneexpression and genome integrity. Disruption of mito-chondrial homeostasis is a general characteristic oftumorigenic transformation. Thus, the role of Lon intumor promotion warrants further study. © 2002 Elsevier

Science (USA)

Key Words: mitochondria; subtractive hybridization;transformation; tumor promotion.

1 To whom reprint requests should be addressed at Department ofMicrobiology, Immunology, and Cell Biology, West Virginia Univer-sity School of Medicine, Robert C. Byrd Health Science Center,Morgantown, WV 26506-9177. Fax: 304-293-7823. E-mail: jluo@

hsc.wvu.edu.

97

INTRODUCTION

Chemical carcinogenesis is a complex process thatcan be divided experimentally into three stages,namely, initiation, promotion, and progression. Initia-tion is associated with irreversible, carcinogen-medi-ated DNA mutation. In contrast, promotion is a revers-ible process in which there are increases in the rate ofcell replication and/or alterations in gene expression.Progression represents the final genetic changes asso-ciated with the conversion of benign tumors into fullymalignant cells. The molecular mechanisms of carcino-genesis remain to be elucidated. JB6 mouse epidermalcells offer an excellent model system to investigate themolecular events that are associated with tumor pro-motion. The JB6 cell system includes transformation-sensitive (P�) and transformation-resistant (P�) cells.Promotion-sensitive JB6 P�, originally derived fromprimary mouse epidermal cells, undergoes a responseanalogous to second stage tumor promotion in mouseskin when treated with tumor promoters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or epidermalgrowth factor (EGF) [1]. Exposure of JB6 P� cells toeither TPA or EGF induces anchorage-independentgrowth and tumorigenic transformation. In contrast,P� cells are extracellular signal-regulated protein ki-nase (ERK)-deficient cells and resistant to tumor pro-moter induction of tumorgenicity [2, 3].

Gene expression is dramatically altered during tu-mor promotion. Identification of genes potentially re-sponsible for tumor promotion is critical for under-standing the mechanisms of carcinogenesis. In thepresent study, we used suppression subtractive hybrid-ization to screen genes that are inducible by EGF inJB6 P� cells. The suppression subtractive hybridiza-tion offers two major advantages, i.e., the inclusion ofall of the sequences in the mouse cDNA pool and the

‡Pathology and Physiology Research Branch, National Institute fo

ccupational Safety and Health, Morgantown, West Virginia 26505 r O

Experimental Cell Research 280, 97–106 (2002)doi:10.1006/excr.2002.5621

equalization of rare and abundant messages following0014-4827/02 $35.00

© 2002 Elsevier Science (USA)All rights reserved.

the initial hybridization [4]. With suppression subtrac-tive hybridization, we herein demonstrate that thegene encoding mouse Lon homology (m-Lon) ATP-de-pendent protease is EGF-responsive and the activationof ERK and PI3-K is required for EGF-mediated regu-lation.

MATERIALS AND METHODS

Cell Culture and Treatment

The JB6 P� mouse epidermal cell line (Cl 41), the JB6 P� cell line(Cl 30.7b), JB6�p85, and transformed JB7 cells were grown in Eagle’sMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine,and 25 �g/ml of gentamicin at 37°C with 5% CO2. JB6�p85 cells are aJB6 P� line with stable transfection of a dominant-negative p85regulatory unit of PI3-K. The JB7 cell line is an irreversibly trans-formed clone selected from EGF-treated Cl 41 cells. The humanmammary epithelial cell line (HB2) and ErbB2 overexpressing HB2(HB2ErbB2) cells were obtained from Dr. Jianping Ye (Louisiana StateUniversity). These cells were grown in the same medium as JB6cells, except that 5 �g/ml of hydrocortisone and 10 �g/ml of bovineinsulin were added. Cells were treated with either P13-kinase inhib-itor LY294002 (10 �M, Sigma Chemical Co., St. Louis, MO) or MAPKkinase 1 inhibitor PD98059 (50 �M, Calbiochem, La Jolla, CA), JNKinhibitor D-JNKI1 (1 �M, Alexis Biochemicals, San Diego, CA), andp38 MAPK inhibitor SB202190 (10 �M, Calbiochem) 30 min prior toEGF exposure. EGF was purchased from Sigma Chemical Co., basicfibroblast growth factor (bFGF) was from Upstate Biotech. Inc. (LakePlacid, NY), and heregulin �1 (HRG) was from NeoMarkers, Inc.(Fremont, CA). Cells were exposed to growth factors (30 ng/ml) forspecified periods.

Subtractive Hybridization Cloning

JB6 P� cells were treated with EGF (30 ng/ml) for 3 days. The totalRNA was isolated using a TRI REAGENT kit (Molecular ResearchCenter, Inc., Cincinnati, OH). Messenger RNA was isolated using aPoly A tract (Promega, Madison, WI). Subtractive hybridization wasperformed using a PCR-Select cDNA Subtraction Kit (Clontech Lab-oratories, Palo Alto, CA) [5]. The library of subtracted fragments wasligated into the TA vector pCR4-TOPO (Invitrogen, Carlsbad, CA)and then transfected into TOP10 cells (Invitrogen). The subtractivefragments were confirmed further by reverse dot blot and Northernblot, and their sequences were determined (Research Genetics,Huntsville, AL).

Reverse Dot Blot Hybridization

Amplification of subtractive fragment library. The transformedclones were randomly selected and grown in 100 �l of LB with 50�g/ml of ampicillin at 37°C for 6 h in 96-well plates. Cloned DNAfragments from the subtracted library were amplified by PCR. ThePCR mixtures (10 �l) contained templates (transformed clones), 50�dNTP (10 mM ATP, dGTP, dTTP, and dCTP), Taq DNA polymerase(5 U/�l), and primers (the same as subtractive hybridization PCRprimers). Amplification was performed at 95°C for 4 min for dena-turing, followed by 30 cycles of additional reaction (94°C for 30 s,55°C for 30 s, and 72°C for 1 min). The PCR product was denaturedin a solution containing 10 �l of 0.6 M NaOH and 10 �l of 0.5%bromophenol blue. Equal amounts of DNA fragments were trans-ferred to Nytran membranes by a Minifold I dot blotting apparatus(Schleicher & Schuell, Keene, NH).

Preparation of cDNA library probes and hybridization. [32P]-cDNA library probes were prepared from the mRNA of both controland EGF-treated (30 ng/ml, 6 h) samples using a method similar to

that previously described [6, 7]. Briefly, reverse transcription andisotope labeling were carried out in a 35-�l reaction mixture thatcontained 2 �g of mRNA, 1 �l of oligo(dT) (10 �M), 1 �l of 50� dNTP(10 mM ATP, 10 mM dGTP, 10 mM dTTP, and 100 �M dCTP), 3 �lof reverse transcriptase (M-MLV, 200 U/�l, BRL), 16 �l of [32P]dCTP(3000 Ci/mM, ICN), and 7 �l of 5 � reverse transcription buffer. Thereaction was carried out at 42°C for 90 min, followed by incubation at75°C for 10 min. After reverse transcription, the mixture was treatedwith 1 �l of RNase A (20 ng/�l) and 1 �l of RNase H (1U/�l) at 37°Cfor 45 min. Unincorporated nucleotides were removed using a G-50Sephadex spin column and the specific activity was detected by ascintillation counter. Nytran membranes were prehybridized with ahybridization buffer (ULTRAhyb Buffer, Austin, TX) at 42°C for 30min. Then, filters were hybridized with cDNA probes (1 � 106 cpm/ml) in the same buffer at 42°C overnight. The membranes werewashed three times (5 min each) at 42°C with 2� SSC, 0.1% SDSsolution followed by three additional washes (15 min each) at 42°C in0.1� SSC, 0.1% SDS solution. The signals were processed with aPhosphorImager and analyzed with Image QuaNT software (Molec-ular Dynamics, Sunnyvale, CA).

5�-Rapid Amplification of cDNA Ends (5�-RACE)

The extension of isolated cDNA fragments was performed using acommercial kit (Rapid Amplification of cDNA Ends, version 2.0, LifeTechnologies, Grand Island, NY). Three gene-specific primers (GSP)were designed based on the target gene sequence. In brief, the cDNAwas prepared by reverse transcription with GSP1 and tailed withpoly(dC) by terminal deoxynucleotidyl transferase. Amplificationwas performed by PCR with a bridged anchor primer (provided bythe kit) and GSP2. The extended fragment was inserted into the TAcloning vector pCR4-TOPO and transfected to TOP10 cells. Thepositive clones were screened by PCR using a bridged universalamplification primer and GSP3.

Northern Blot Hybridization

Probe preparation. The mouse cDNA probe for m-Lon was de-rived from subcloned DNA. The probe was labeled with [32P]dCTP byPCR. The reaction mixture contained the following components:[32P]dCTP (3000 Ci/mM), templates (plasmids harbored subtractiveDNA fragments, 20 ng/ml), 50� dNTP (10 mM ATP, dGTP, 10 mMdTTP, and 100 �M dCTP), Taq DNA polymerase (5 U/�l), and theprimers (the same as the subtractive hybridization PCR primers).Amplification was performed at 95°C for 3 min for denaturing fol-lowed by 30 cycles of additional reaction (94°C for 30 s, 55°C for 30 s,and 72°C for 2 min). Labeled probe was then purified by G-50 column(Promega, Madison, WI). Since mouse and human Lon share highhomology, the same m-Lon cDNA probe was used for the analysis ofhuman Lon (h-Lon) expression in human breast epithelial cells. ThecDNA for mouse cytochrome c oxidase subunit I (COX-I) was pre-pared by RT–PCR using a JB6 P� cDNA library and specific primers(5�-CACGATACATACTATGTAGTA-3� and 5�-GAATGTGTGATAT-GGTGGA-3�). The PCR products were sequenced and labeled with32P using a PCR procedure described above.

Blotting and hybridization. The procedure for blotting and hy-bridization was the same as that previously described [8]. A house-keeping gene, cyclophilin, was used as an internal control becauseEGF did not affect its expression. The signals were processed with aPhosphorImager and analyzed with Image QuaNT software.

mRNA Stability

The stability of the mRNA was determined by a previously de-scribed method [8]. Briefly, total RNA was extracted from JB6 P�

cells before and at various times (30 min–6 h) after treatment withactinomycin D (5 �g/ml). RNA samples were then subjected to North-

98 ZHU ET AL.

ern blot analysis as described above. The signals were visualizedwith a PhosphorImager and analyzed with Image QuaNT software.

Nuclear Runoff Assay

The assay was described previously in detail [8]. In brief, nuclearRNA was isolated and labeled with [32P]-ATP. The RNA transcriptswere partially hydrolyzed with 200 mM NaOH at 0°C for 10 min andthen neutralized with Hepes acid. Nytran filters (Schleicher &Schuell) were coated with 1 �g of denatured cDNAs of m-Lon orcyclophilin by slot blotting. Then the filters were UV cross-linked andprehybridized with a hybridization buffer (50% formamide, 250 mMsodium phosphate, pH 7.2, 1 mM EDTA, 250 mM NaCl, 100 �g/ml ofssDNA, 40 �g/ml of tRNA, and 7.0% SDS) at 55°C for 4 h. Hybrid-ization was performed in the same solution containing 2�106 cpm ofthe 32P-RNA at 55°C for 48 h. Filters were washed four times (20 mineach) in 2� SSC, followed by a 15-min wash in 2� SSC containing 5�g/ml of RNase A and 100 �g/ml of RNase T1 at 65°C. Finally, thefilters were rinsed in 0.3� SSC containing 1.0% SDS for 1 h. Thehybridization signals were visualized with a PhosphorImager andanalyzed with Image QuaNT software.

Statistical Analysis

Differences among treatment groups were tested using a one-wayanalysis of variance (ANOVA). Differences in which P � 0.05 wereconsidered statistically significant. In cases where significant differ-ences were detected, specific post-hoc comparisons between treat-ment groups were examined with Student–Newman–Keuls tests.

RESULTS

Identification of EGF-Responsive Genes

Approximately 1000 clones derived from suppressionsubtractive hybridization were subjected to reverse dotblot analysis. A representative reverse dot blot imageis shown in Fig. 1. With reverse dot blot hybridization,eight differentially expressed cDNA fragments wereverified. One of them (clone 4F1, 605 bp) displayed the

most drastic alteration upon EGF treatment. TheEGF-induced fragment was sequenced and comparedfor homology to any entry in an existing nuclear aciddatabase available through the National Center forBiotechnology Information (GenBank/EMBL). Clone4F1 insert exhibited 100% homology with a clone of aMus musculus adult male lung cDNA RIKEN full-length enriched library (Clone 1200017E13, AccessionNo. AK004820). To confirm that this fragment is Clone1200017E13, the full length of this gene was preparedby 5�-RACE, and an additional 2-kb sequence was ob-tained. Sequencing analysis showed that this gene has100% homology with Clone 1200017E13 and 93% ho-mology with Rattus norvegicus Lon homology ATP-dependent protease (r-Lon; Accession No. AB064323).

Alignment of the deduced amino acid of this mousegene with h-Lon and r-Lon exhibited 84 and 96% ho-mology, respectively. Furthermore, all conservative do-mains of h-Lon and r-Lon, such as the ATPase andprotease domains are present in this mouse gene (Fig.2). Therefore, we concluded that Clone 4F1 representsmouse Lon homology ATP-dependent protease.

Expression of m-Lon mRNA

Up-regulation of m-Lon mRNA by EGF in JB6 P�

cells was further confirmed by Northern blot analysis.The detected band was about 2.9 kb (Fig. 3), a sizeconsistent with the putative length of m-Lon aminoacids. Time sequence analysis showed that up-regula-tion of the m-Lon level was evident at 6 h and peakedat around 72 h following EGF exposure (Fig. 4). EGFsignificantly increased the transcriptional rate (Fig. 5),with little effect on mRNA stability (Fig. 6). Therefore,EGF-mediated alteration resulted from enhanced tran-scription of m-Lon. It is reported that Lon-like proteasein yeast increased the stability of mRNA of COX-I [9].To determine whether there is a correlation betweenup-regulation of m-Lon transcription and increasedCOX-I mRNA level, we examined the expression ofCOX-I by Northern analysis. As shown in Figs. 3 and 4,EGF significantly increased the steady-state expres-sion of COX-I mRNA, and the profile of EGF-inducedCOX-I mRNA alteration was similar to that of m-Lon.Furthermore, as shown in Fig. 6, EGF treatment sig-nificantly increased the half-life of COX-I mRNA. Todetermine whether EGF regulation of Lon expressionin JB6 P� cells is specific, we further investigated theeffects of serum, bFGF, and heregulin �1 on thesteady-state expression of Lon mRNA, and the resultsindicated that serum and other growth factors hadlittle effect on Lon expression (Fig. 7).

Since EGF induces transformation of JB6 P� cellsand up-regulates Lon transcription, we sought to de-termine whether transformed cells express higher lev-els of Lon. JB7 cells are an irreversibly transformed

FIG. 1. Determination of EGF-responsive fragments with re-verse dot blot hybridization. The subtractive fragments were ampli-fied by PCR, and equal amounts were transferred to Nytran mem-branes by dot blotting. The membrane was hybridized with[32P]cDNA library probes. The probes were prepared by reverse tran-scription of mRNA from both control and EGF-treated (30 ng/ml, 6 h)samples (see Materials and Methods). A representative membrane ispresented. Clone 4F1 (m-Lon) is indicated by arrows.

99EGF REGULATES MOUSE LON HOMOLOGY ATP-DEPENDENT PROTEASE

FIG. 2. Alignment of the predicted amino acid sequence of mouse Lon (m-Lon) with human and rat Lon (h-Lon and r-Lon). An arrowheadindicates a serine residue at the putative active site [37]. The proposed ATP-binding sites, which have Walker-type consensus motif, areunderlined [38]. The identity of an amino acid in different species is indicated with a dot.

100

clone selected from EGF-treated JB6 P� cells. Asshown in Fig. 8, the mRNA level for m-Lon was signif-icantly higher in transformed JB7 cells than in JB6 P�

cells. ErbB2 is a proto-oncogene belonging to the familyof tyrosine kinase receptors [10]. Overexpression ofErbB2 is found in some breast carcinomas and is asso-ciated with tumor aggressiveness and poor prognosis[11, 12]. Our results showed that the level of Lon

mRNA in a human breast epithelial cell line overex-pressing ErbB2 (HB2ErbB2) was significantly higherthan that in its parental cells (HB2) (Fig. 8).

EGF-Mediated m-Lon Expression Requires theActivation of ERK and PI3K

It is known that EGF activates ERK and PI3-K inJB6 P� cells and these signal pathways are essentialfor EGF-induced transformation of JB6 P� cells [3, 13].JB6 P� cells are ERK-deficient and resistant to EGFinduction of tumorgenicity [2, 3], and JB6�p85 cells are aJB6 P� line with stable transfection of a dominant-negative p85 regulatory unit of PI3-K. As shown inFigs. 9A and B, EGF failed to regulate Lon expressionin JB6 P� and JB6�p85 cells, indicating that these signalpathways were required for EGF regulation of Lon. Toverify that the ERK and the PI3-K were involved inEGF-induced up-regulation, selective chemical inhibi-tors were employed to block either the MAPK or thePI3-K pathway. As shown in Fig. 9C, LY294002 com-pletely abolished EGF-mediated up-regulation of m-Lon mRNA, while PD98059 only partially inhibitedEGF-mediated alteration. SB202190 and D-JNKI1 didnot affect EGF regulation of Lon expression, suggest-ing that p38 MAPK and JNK were not involved.

DISCUSSION

Epidermal growth factor, a tumor promoter of mouseepidermal JB6 P� cells, drastically alters the pattern ofgene expression. Herein, we demonstrate that mouseLon homology ATP-dependent protease in JB6 P� cellsis one of the genes that undergo dramatic up-regula-tion following EGF treatment. EGF-induced expres-sion of m-Lon is evident at 6 h following EGF treat-

FIG. 3. Northern blot analysis of m-Lon gene expression. (A) Total RNA (10 �g) prepared from JB6 P� cells was subjected to Northernblot using 32P-labeled cDNA of m-Lon. Molecular weight markers are shown on the left. (B) JB6 P� cells were treated with EGF (30 ng/ml)for 3 days. Ten micrograms of total RNA isolated from control and EGF-treated cells was loaded into each lane. The expressions of m-Lon,COX-I, and cyclophilin were examined with Northern blot hybridization. Cyclophilin was used as an internal standard. Triplicate experi-ments were performed independently.

FIG. 4. Time sequence for EGF-mediated m-Lon expression. JB6P� cells were treated with EGF (30 ng/ml) for various times (6 h–7days). The expression of m-Lon and COX-I was examined by North-ern blot hybridization. The relative amounts of m-Lon and COX-Iexpression were determined with Image QuaNT. The same blot wasstripped of m-Lon cDNA and rehybridized with a probe of the house-keeping gene cyclophilin. The expression of m-Lon and COX-I wasnormalized with the level of cyclophilin. The result is the mean �SEM of triplicates.

101EGF REGULATES MOUSE LON HOMOLOGY ATP-DEPENDENT PROTEASE

ment and persists for at least 7 days. EGF significantlyincreases the transcription rate of m-Lon, but has littleeffect on RNA stability.

It appears that the effect of EGF on Lon expression isspecific, since serum, bFGF, and heregulin �1 do notsignificantly induce Lon up-regulation. Both ERK andPI3-K are essential for EGF-mediated transformationand up-regulation of m-Lon, i.e., blocking either path-way inhibits EGF-mediated induction. However, theextent and temporal profile of inhibition are different.Blockage of the PI3-K pathway results in a rapid andcomplete inhibition of m-Lon expression, whereasblockage of ERK produces a partial and slower atten-uation. The result may reflect cross-talk between thetwo pathways, and it suggests that PI3-K is a direct or

major signal pathway that mediates m-Lon transcrip-tion. Considerable convergence as well as cross-talkhas been shown between MAPK and PI3-K pathways[14–16]. For example, the activation of ERK requiresthe participation of PI3-K [15, 17, 18].

The involvement of ERK and PI3-K in Lon regula-tion is further supported by the finding that ErbB2overexpression up-regulates Lon expression. Proto-on-cogene ErbB2 belongs to the family of tyrosine kinasereceptors which include EGF receptors ErbB1, ErbB3,and ErbB4 [10]. Although no specific ligand for ErbB2has been identified, ErbB2 forms heterodimers withErbB1, ErbB3, or ErbB4 and is believed to be an es-sential component of high-affinity receptors of EGFand heregulin (ligand for ErbB3/4) [19]. Overexpres-

FIG. 5. Analysis of the transcription rate of m-Lon in JB6 P� cells. Cells were treated with EGF (30 ng/ml) for 24 h. (A) Nuclei wereisolated and subject to nuclear runoff assay as described under Materials and Methods. A constant concentration (1 �g) of m-Lon andcyclophilin cDNA was coated in each lane. (B) The relative transcription rate was determined with Image QuaNT and expressed as apercentage of the control. The result is the mean � SEM of triplicates. *P � 0.05 versus control values.

FIG. 6. Stability of m-Lon and COX-I mRNA. JB6 P� cells were treated with EGF (30 ng/ml) for 24 h, and total RNA was extracted beforeand at various times (30 min–8 h) after treatment with actinomysin D (5 �g). RNA samples were then subjected to Northern blot analysis.The signals were visualized with a PhosphorImager and expressed as a percentage of the amount measured prior to application ofactinomycin D. The result is the mean � SEM of triplicates.

102 ZHU ET AL.

sion of ErbB2 results in constitutive activation of itsintrinsic kinase as well as ERK and PI3-K [20, 21].Therefore, ErbB2-mediated Lon up-regulation couldresult from the activation of ERK and PI3-K.

ATP-dependent proteases are the major proteases inorganisms, and the Lon protease encoded by the longene of Escherichia coli was one of the first to beidentified and remains the best studied so far [22–24].In bacterial cells, Lon protease plays an important rolein radiation resistance, cell division, filamentation,production of capsular polysaccharide, lysogeny of cer-tain bacteriophages, and proteolytic degradation ofseveral classes of regulatory (cell cycle-related) andabnormal proteins [24, 25]. Lon protease is also knownas a heat shock protein that is induced under stressfulconditions [26–28].

Lon-like proteases are present in eukaryotic cellsand organelles (chloroplasts and mitochondria) [24].Eukaryotic Lon protease is encoded in the nucleus and

localized in mitochondria. Lon protease representsubiquitin-independent protein degradation pathway,i.e., Lon-mediated protein degradation does not requireubiquitination [24]. Human Lon homology ATP-depen-dent protease has been cloned and is ubiquitously ex-pressed in all human tissues [29]. However, the phys-iological role of eukaryotic Lon-like proteases is poorlyunderstood. A major known function of eukaryotic Lonprotease is to regulate the biogenesis and homeostasisof mitochondria [9, 30–32]. For example, ATP-depen-dent PIM1 protease, a Lon-like protease of yeast, playsmultiple and essential roles in the regulation of mito-chondrial gene expression and genome integrity [9].PIM1 protease enhances the stability of COX-I mRNAin the mitochondria of yeast [9]. Our results show acorrelation between increased Lon expression and en-hanced COX-I mRNA stability in JB6 P� cells, suggest-ing that mammalian Lon is also involved in the regu-lation of mitochondrial gene expression. Moreover,

FIG. 7. Effects of serum and growth factors on m-Lon expression. (A) JB6 P� cells were serum starved for 48 h and then exposed to amedium containing either 10% FBS or growth factors (30 ng/ml of EGF, bFGF, or heregulin �1) for 24 h. The expressions of m-Lon andcyclophilin were examined with Northern blot hybridization. (B) The relative amounts of m-Lon expression were determined with ImageQuaNT and normalized to the cyclophilin level. The result is the mean � SEM of triplicates. *P � 0.05 versus control values.

FIG. 8. Expression of m-Lon in various cell lines. (A) Total RNA was isolated from JB6 P�, JB7, HB2, and HB2ErbB2 cells. Ten microgramsof total RNA was loaded into each lane. The expression of Lon and cyclophilin was examined with Northern blot. (B) The relative amountsof m-Lon expression were determined with Image QuaNT and normalized to the cyclophilin level. The result is the mean � SEM of triplicates.*P � 0.05 versus control values.

103EGF REGULATES MOUSE LON HOMOLOGY ATP-DEPENDENT PROTEASE

mitochodrial Lon may serve a chaperone-like functionin mediating both the assembly of protein complexesand the disposal of substrates that assemble incor-rectly [31].

The role of Lon in tumorigenic transformation re-mains to be established. Here, we demonstrate thatmammalian Lon is EGF inducible and that trans-formed cells overexpress Lon. Human mammary epi-thelial cells that overexpress ErbB2 also exhibit signif-icantly higher expression of Lon. Overexpression ofErbB2 is believed to be associated with tumor aggres-siveness and a poor prognosis [11, 12]. Lon is alsooverexpressed in hepatoma cells when compared tonormal liver cells, and overexpression is associatedwith enhanced mitochondrial biogenesis [32]. Themechanisms for up-regulation of Lon during tumori-

genic transformation are not clear. It is likely to be aresponse to an elevated level of mitochondrial biogen-esis or alternatively to the accumulation of abnormalprotein complexes during the process of tumorigenictransformation. In bacterial cells, the presence of ab-normal proteins significantly stimulates lon transcrip-tion [33]. Regardless of the causes for Lon up-regula-tion, abnormal expression of mitochondrial Lon maycause disruption of mitochondrial genome integrityand homeostasis. Mitochondria play a critical role inmalignancy, and mitochondrial genomic aberration is ageneral characteristic of tumor cells [34–36]. Thus, thedisturbance of Lon regulation may underlie carcino-genesis-related deregulation of mitochondrial ho-meostasis.

FIG. 9. Requirement of MAPK and PI3-K for m-Lon expression. (A) JB6 P�, JB6 P�, and JB6�p85 cells were treated with EGF (0 or 30ng/ml) for 3 days. Ten micrograms of total RNA from control and EGF-treated samples was loaded into each lane. The expressions of m-Lonand cyclophilin were examined with Northern blot hybridization. (B) The relative amounts of m-Lon expression were determined with ImageQuaNT and normalized to cyclophilin level. The result is the mean � SEM of triplicates. (C) JB6 P� cells were treated with EGF (30 ng/ml)in the presence/absence of selective blockers for MAPK or PI3-K for the indicated times (6–24 h). The expression of m-Lon was examined withNorthern blot hybridization. The relative m-Lon level was determined with a PhosphorImager and normalized to the cyclophilin level. Theinhibitory effect of each selective blocker on Lon expression was expressed as a percentage of inhibition. The result is the mean � SEM oftriplicates. *P � 0.05 versus control values.

104 ZHU ET AL.

We thank Dr. Daniel C. Flynn for his critical reading of themanuscript. This research was supported by Grants AA12968 andCA90385 from the National Institutes of Health.

REFERENCES

1. Colburn, N. H., Former, B. F., Nelson, K. A., and Yuspa, S. H.(1979). Tumour promoter induces anchorage independence ir-reversibly. Nature 281, 589–591.

2. Bernstein, L. R., and Colburn, N. H. (1989). AP1/jun function isdifferentially induced in promotion-sensitive and resistant JB6cells. Science 244, 566–569.

3. Huang, C., Ma, W. Y., Young, M. R., Colburn, N., and Dong, Z.(1998). Shortage of mitogen-activated protein kinase is respon-sible for resistance to AP-1 transactivation and transformationin mouse JB6 cells. Proc. Natl. Acad. Sci. USA 95, 156–161.

4. Duguid, J. R., and Dinauer, M. C. (1990). Library subtraction ofin vitro cDNA libraries to identify differentially expressedgenes in scrapie infection. Nucleic Acids Res. 18, 2786–2792.

5. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Mo-qadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya,N., Sverdlov, E. D., and Siebert, P. D. (1996). Suppressionsubtractive hybridization: A method for generating differen-tially regulated or tissue-specific cDNA probes and libraries.Proc. Natl Acad. Sci. USA 93, 6025–6030.

6. Kim, M. G., Chen, C., Flomerfelt, F. A., Germain, R. N., andSchwartz, R. H. (1998). A subtractive PCR-based cDNA librarymade from fetal thymic stromal cells. J. Immunol. Methods 213,169–182.

7. Stubbs, A. P., Abel, P. D., Golding, M., Bhangal, G., Wang, Q.,Waxman, J., Stamp, G. W., and Lalani, E. N. (1999). Differen-tially expressed genes in hormone refractory prostate cancer:association with chromosomal regions involved with geneticaberrations. Am. J. Pathol. 154, 1335–1343.

8. Zhu, Y., Lin, H., Wang, M., Li, Z., Wiggins, R. C., and Luo, J.(2001). Up-regulation of transcription of smooth muscle myosinalkali light chain by ethanol in human breast cancer cells. Int.J. Oncol. 18, 1299–1305.

9. Van Dyck, L., Neupert, W., and Langer, T. (1998). The ATP-dependent PIM1 protease is required for the expression of in-tron-containing genes in mitochondria. Genes Dev. 12, 1515–1524.

10. Ullrich, A., and Schlessinger, J. (1990). Signal transduction byreceptors with tyrosine kinase activity. Cell 61, 203–212.

11. King, C. R., Kraus, M. H., and Aaronson, S. A. (1985). Ampli-fication of a novel v-erbB-related gene in a human mammarycarcinoma. Science 229, 974–978.

12. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J., Ulrich,A., and McGuire, W. L. (1987). Human breast cancer: correla-tion of relapse and survival with amplification of the HER-2/neuoncogene. Nature 366, 177–182.

13. Huang, C., Ma, W. Y., and Dong, Z. (1996). Requirement forphosphatidylinositol 3-kinase in epidermal growth factor-in-duced AP-1 transactivation and transformation in JB6 P� cells.Mol. Cell. Biol. 16, 6427–6435.

14. Marshall, M. S. (1995). Ras target proteins in eukaryotic cells.FASEB J. 9, 1311–1318.

15. Kitamura, T., Kimura, K., Jung, B. D., Makondo, K., Okamoto,S., Canas, X., Sakane, N., Yoshida, T., and Saito, M. (2001).Proinsulin C-peptide rapidly stimulates mitogen-activated pro-tein kinases in Swiss 3T3 fibroblasts: Requirement of proteinkinase C, phosphoinositide 3-kinase and pertussis toxin-sensi-tive G-protein. Biochem. J. 355, 123–129.

16. Yart, A., Laffargue, M., Mayeux, P., Chretien, S., Peres, C.,Tonks, N., Roche, S., Payrastre, B., Chap, H., and Raynal, P.(2001). A critical role for phosphoinositide 3-kinase upstream ofGab1 and SHP2 in the activation of ras and mitogen-activatedprotein kinases by epidermal growth factor. J. Biol. Chem. 276,8856–8864.

17. Lopez-Ilasaca, M. (1998). Signaling from G-protein-coupled re-ceptors to mitogen-activated protein (MAP)-kinase cascades.Biochem. Pharmacol. 56, 269–277.

18. Reyes-Reyes, M., Mora, N., Zentella, A., and Rosales, C. (2001).Phosphatidylinositol 3-kinase mediates integrin-dependentNF-kappaB and MAPK activation through separate signalingpathways. J. Cell Sci. 114, 1579–1589.

19. Karunagaran, D., Tzahar, E., Beerli, R. R., Chen, X., Graus-Porta, D., Ratzkin, B. J., Seger, R., Hynes, N. E., and Yarden, Y.(1996). ErbB-2 is a common auxiliary subunit of NDF and EGFreceptors: Implications for breast cancer. EMBO J. 15, 254–264.

20. Ben-Levy, R., Paterson, H. F., Marshall, C. J., and Yarden, Y.(1994). A single autophosphorylation site confers oncogenicityto the Neu/ErbB2 receptor and enables coupling to the MAPkinase pathway. EMBO J. 13, 3302–3311.

21. Ignatoski, K. M., Maehama, T., Markwart, S. M., Dixon, J. E.,Livant, D. L., and Ethier, S. P. (2000). ERBB-2 overexpressionconfers PI 3� kinase-dependent invasion capacity on humanmammary epithelial cells. Br. J. Cancer 82, 666–674.

22. Goldberg, A. L., and St. John, A. C. (1976). Intracellular proteindegradation in mammalian and bacterial cells: Part 2. Annu.Rev. Biochem. 45, 747–803.

23. Hershko, A., and Ciechanover, A. (1982). Mechanisms ofintracellular protein breakdown. Annu. Rev. Biochem. 51,335–364.

24. Goldberg, A. L. (1992). The mechanism and functions of ATP-dependent proteases in bacterial and animal cells. Eur. J. Bio-chem. 203, 9–23.

25. Goldberg, A. L., Moerschell, R. P., Chung, C. H., and Maurizi,M. R. (1994). ATP-dependent protease La (lon) from Esche-richia coli. Methods Enzymol. 244, 350–375.

26. Goff, S. A., Casson, L. P., and Goldberg, A. L. (1984). Heat shockregulatory gene htpR influences rates of protein degradationand expression of the lon gene in Escherichia coli. Proc. Natl.Acad. Sci. USA 81, 6647–6651.

27. Phillips, T. A., VanBogelen, R. A., and Neidhardt, F. C. (1984).Lon gene product of Escherichia coli is a heat-shock protein. J.Bacteriol. 159, 283–287.

28. Baker, T. A., Grossman, A. D., and Gross, C. A. (1984). A generegulating the heat shock response in Escherichia coli alsoaffects proteolysis. Proc. Natl. Acad. Sci. USA 81, 6779 –6783.

29. Wang, N., Gottesman, S., Willingham, M. C., Gottesman,M. M., and Maurizi, M. R. (1993). A human mitochondrialATP-dependent protease that is highly homologous to bacterialLon protease. Proc. Natl. Acad. Sci. USA 90, 11247–11251.

30. Rep, M., Van Dijl, J. M., Suda, K., Schatz, G., Grivell, L. A., andSuzuki, C. K. (1996). Promotion of mitochondrial membranecomplex assembly by a proteolytically inactive yeast Lon. Sci-ence 274, 103–105.

31. Suzuki, C. K., Suda, K., Wang, N., and Schatz, G. (1994). Require-ment for the yeast gene LON in intramitochondrial proteolysisand maintenance of respiration. Science 264, 273–276.

32. Luciakova, K., Sokolikova, B., Chloupkova, M., and Nelson,B. D. (1999). Enhanced mitochondrial biogenesis is associatedwith increased expression of mitochondrial ATP-dependent Lonprotease. FEBS Lett. 444, 186–188.

105EGF REGULATES MOUSE LON HOMOLOGY ATP-DEPENDENT PROTEASE

33. Goff, S. A., and Goldberg, A. L. (1985). Production of abnormalproteins in E. coli stimulates transcription of lon and other heatshock genes. Cell 41, 587–595.

34. Dorward, A., Sweet, S., Moorehead, R., and Singh, G. (1997).Mitochondrial contributions to cancer cell physiology: Redoxbalance, cell cycle, and drug resistance. J. Bioenerg. Biomembr.29, 385–392.

35. Cavalli, L. R., and Liang, B. C. (1998). Mutagenesis, tumorige-nicity, and apoptosis: Are the mitochondria involved? Mutat.Res. 398, 19–26.

36. Penta, J. S., Johnson, F. M., Wachsman, J. T., and Copeland,W. C. (2001). Mitochondrial DNA in human malignancy. Mutat.Res 488, 119–133.

37. Amerik, A. Yu, Antonov, V. K., Gorbalenya, A. E., Kotova, S. A.,Rotanova, T. V., Shimbarevich, E. V. (1991). Site-directed mu-tagenesis of La protease. A catalytically active serine residue.FEBS Lett. 287, 211–214.

38. Walker, J. E., Saraste, M., Runswick, M. J., Gay, N. J. (1982).Distantly related sequences in the alpha- and beta-subunits ofATP synthase, myosin, kinases and other ATP-requiring en-zymes and a common nucleotide binding fold. EMBO J. 1,945–951.

Received February 11, 2002Revised version received July 10, 2002Published online September 5, 2002

106 ZHU ET AL.