L-Me hionine A ailabili y Regula es Exp ession o he Me hionine
Adenosyl ans e ase 2A Gene in Human Hepa oca cinoma Cells
ROLE OF S-ADENOSYLMETHIONINE*
Recei ed o publica ion, No embe 13, 2002, and in e ised o m, Ma ch 20, 2003
Published, JBC Pape s in P ess, Ma ch 26, 2003, DOI 10.1074/jbc.M211554200
Ma ia L. Ma ı´nez-Chan a ‡§¶, M. Ujue La asa‡§储, Ma a Va ela-Rey‡¶, Shelly C. Lu**,
Elena R. Ga cı´a-T e ijano‡, Jose´ M. Ma o‡ ‡‡, and Ma ı´as A. A ila‡ ‡‡§§
F om he ‡Labo a o io de P o eo´mica, Geno´mica y Bioin o ma´ ica, and Di isio´n de Hepa ologı´a y Te apia Ge´nica,
Uni e sidad de Na a a, Facul ad de Medicina, 31008 Pamplona, Spain and he **Di ision o Gas oen e ology
and Li e Diseases, USC Resea ch Cen e o Li e Diseases, USC-UCLA Resea ch Cen e o Alcoholic Li e and
Panc ea ic Diseases, Keck School o Medicine, Uni e si y o Sou he n Cali o nia, Los Angeles, Cali o nia 90033
In mammals, me hionine adenosyl ans e ase (MAT),
he enzyme esponsible o S-adenosylme hionine
(AdoMe ) syn hesis, is encoded by wo genes, MAT1A
and MAT2A. In li e , MAT1A exp ession is associa ed
wi h high AdoMe le els and a di e en ia ed pheno ype,
whe eas MAT2A exp ession is associa ed wi h lowe
AdoMe le els and a dedi e en ia ed pheno ype. In he
cu en s udy, we examined egula ion o MAT2A gene
exp ession by L-me hionine a ailabili y using HepG2
cells. In L-me hionine-de icien cells, MAT2A gene ex-
p ession is apidly induced, and me hionine adenosyl-
ans e ase ac i i y is inc eased. Res o a ion o L-me hi-
onine apidly down- egula es MAT2A mRNA le els; o
his e ec , L-me hionine needs o be con e ed in o
AdoMe . This no el ac ion o AdoMe is no media ed
h ough a me hyl ans e eac ion. MAT2A gene exp es-
sion was also egula ed by 5ⴕ-me hyl hioadenosine, bu
his was dependen on 5ⴕ-me hyl hioadenosine con e -
sion o me hionine h ough he sal age pa hway. The
ansc ip ion a e o he MAT2A gene emained un-
changed du ing L-me hionine s a a ion; howe e , i s
mRNA hal -li e was signi ican ly inc eased ( om 100
min o mo e han 3 h). The e ec o L-me hionine wi h-
d awal on MAT2A mRNA s abiliza ion equi es bo h
gene ansc ip ion and p o ein syn hesis. We conclude
ha MAT2A gene exp ession is modula ed as an adap -
i e esponse o he cell o L-me hionine a ailabili y
h ough i s con e sion o AdoMe .
Regula ion o cellula unc ions by nu ien s, including he
con ol o gene exp ession, is well documen ed in p oka yo es
and lowe euka yo es. In mammals, hese esponses ha e also
been obse ed, and lipids and ca bohyd a es, among o he di-
e a y cons i uen s, ha e been shown o be impo an egula o s
o gene exp ession (1–3). Mammalian cells can also adap o
die a y o pa hological luc ua ions in amino acid a ailabili y
(4, 5). Amino acid limi a ion elici s a se ies o cellula e-
sponses, among which he up- egula ion o amino acid ans-
po sys ems was one o he i s o be ecognized ( e iewed in
Re s. 6 and 7). Subsequen ly, a numbe o genes ha e been
shown o speci ically change hei exp ession le els ollowing
amino acid dep i a ion, al hough he unde lying mechanisms
a e no comple ely known (4, 5). Unde such limi ing condi-
ions, amino acids play a signaling ole in he cell, con eying
egula o y messages o he ansc ip ional machine y and a -
ec ing he u no e o speci ic mRNAs and p o eins (5, 8).
These p ocesses a e beginning o be unde s ood wi h wo k
ca ied ou mainly in cul u ed cells, gi en he complexi y o he
esponse o amino acid dep i a ion in in ac animals, which
includes nu ien -induced changes in ho mone le els.
L-Me hionine is an essen ial amino acid equi ed o p o ein
syn hesis and pa icipa es, oge he wi h ATP, in he o ma ion
o S-adenosylme hionine (AdoMe ),
1
he p incipal biological
me hyl dono (9–11). Upon ans e o i s ac i a ed me hyl
g oup o many di e en accep o molecules, AdoMe is con-
e ed in o S-adenosylhomocys eine (AdoHcy), a molecule ha
can be u he hyd olyzed o adenosine and homocys eine in a
e e sible eac ion (10). Homocys eine in u n may be con-
e ed in o cys a hionine o eme hyla ed back o L-me hio-
nine, depending on he cellula needs o his amino acid (10).
Mammalian cells ha e de eloped a se ies o adap i e mecha-
nisms ha espond o luc ua ions in L-me hionine a ailabili y,
which ha e been bes cha ac e ized in he li e and in ol e he
modula ion o enzyma ic ac i i ies implica ed in me hionine
me abolism (10, 12). Some o hese mechanisms, including he
capaci y o eme hyla e homocys eine, a e impai ed in he
ans o med cell, gi ing ise o he condi ion o “me hionine
dependence” in cance ( e iewed in Re . 13).
The i s s ep in L-me hionine me abolism, he syn hesis o
* This wo k was suppo ed by Na ional Cen e o Complemen a y
and Al e na i e Medicine, Na ional Ins i u es o Heal h (NIH), G an
R01 AT-1576 ( o S. C. L., J. M. M., and M. A. A.); NIAA, NIH, G an s
R01 AA-12677 and R01 AA013847 ( o S. C. L., J. M. M., and M. A. A.),
Plan Nacional de I⫹D G an 99/0038 ( o J. M. M.); Minis e io de
Sanidad y Consumo G an FIS 01/0712 ( o M. A. A.); a g an om he
Fundacio´n In˜igo Al a ez de Toledo ( o M. A. A.); Gobie no de Na a a
G an s 681/2000 and 394/2001 ( o J. M. M. and M. A. A., espec i ely);
NIH G an R01 DK-51719 ( o S. C. L.); Ins i u o de Salud Ca los III
G an s G03/015 Red Tema´ ica de In es igacio´n Coope a i a and C03/
C02 Red Nacional de In es igacio´n en Hepa ologı´a y Gas oen e ologı´a
( o J. M. M. and M. A. A.); and G an PI0220369 om he Minis e iode
Sanidad y Consumo ( o E. R. G. T.). The cos s o publica ion o his
a icle we e de ayed in pa by he paymen o page cha ges. This
a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance
wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac .
§ Bo h au ho s con ibu ed equally o his wo k.
¶Fellow o he Uni e sidad de Na a a.
储Fellow o he Spanish Minis e io de Ciencia y Tecnologı´a.
‡‡ Bo h au ho s sha e senio au ho ship.
§§ To whom co espondence should be add essed: Labo a o io de
P o eo´mica, Geno´mica y Bioin o ma´ ica, Di isio´n de Hepa ologı´a y
Te apia Ge´nica, Facul ad de Medicina, Uni e sidad de Na a a, 31008
Pamplona, Spain. Tel.: 34-948-425678; Fax: 34-948-425677; E-mail:
[email p o ec ed].
1
The abb e ia ions used a e: AdoMe , S-adenosylme hionine; Ac D,
ac inomycin D; AdoHcy, S-adenosylhomocys eine; C
3
-Ado, 3-deazaade-
nosine; C
3
-AdoHcy, S-3-deaza-adenosylhomocys eine; GAPDH, glyce -
aldehyde-3-phospha e dehyd ogenase; KRB, K ebs-Ringe bica bona e;
MAT, me hionine adenosyl ans e ase; MEM, minimum Eagle’s me-
dium; MTA, 5⬘-me hyl hioadenosine; MTAP, 5⬘-me hyl hioadenosine
phospho ylase.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 22, Issue o May 30, pp. 19885–19890, 2003
© 2003 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A.
This pape is a ailable on line a h p://www.jbc.o g 19885
This is an Open Access a icle unde he CC BY license.
AdoMe , is ca alyzed by he enzyme me hionine adenosyl ans-
e ase (MAT). In mammals, he e a e h ee isoenzymes o
MAT (MAT I, MAT II, and MAT III); MAT I and MAT III a e
wo oligome ic o ms (a e ame and dime , espec i ely) o
he p o ein coded by MAT1A gene, whe eas MAT II is he
p oduc o he MAT2A gene (11, 14, 15). The exp ession o bo h
genes is di e en ially egula ed; MAT1A is exp essed in he
adul hepa ocy e, whe eas MAT2A is exp essed in all o he
cells o he o ganism and in he e al hepa ocy e (11, 14).
In e es ingly, MAT2A exp ession is induced when he adul
hepa ocy e p oli e a es (as occu s du ing li e egene a ion)
and upon neoplas ic ans o ma ion o he li e , whe e MAT1A
exp ession is silenced (11, 16–18). Al hough bo h enzymes ca -
alyze he same eac ion, hey display dis inc ca aly ic and
egula o y p ope ies ha impac on he in acellula concen-
a ions o AdoMe (11). We ha e ecen ly shown ha in li e ,
AdoMe plays a pi o al ole in he egula ion o essen ial cel-
lula unc ions, such as he con ol o cellula p oli e a ion,
di e en ia ion, and apop osis (18–24); consequen ly, he le els
o his me aboli e mus be igh ly con olled in his o gan. In
he p esen wo k, we desc ibe how exp ession o he MAT2A
gene is acu ely egula ed by he a ailabili y o L-me hionine
and in es iga e he mechanisms behind his esponse. Ou
obse a ions indica e ha luc ua ions in L-me hionine a ail-
abili y egula e MAT2A exp ession mainly a he le el o
mRNA u no e ; addi ionally, we demons a e an essen ial
ole o AdoMe in his esponse, u he suppo ing he hy-
po hesis o AdoMe as a signaling molecule in he li e (25).
EXPERIMENTAL PROCEDURES
Chemicals—AdoMe , in he s able o m o sul a e-p- oluenesul ona e
sal p oduced by Knoll (Milan, I aly) was om Eu opha ma (Mad id,
Spain). 5⬘-Me hyl hioadenosine (MTA) was gene ously p o ided by
Knoll (Milan, I aly). All o he eagen s we e o analy ical g ade, and
unless o he wise s a ed, hey we e pu chased o m Sigma.
Cell Cul u e and T ea men Condi ions—Human HepG2 hepa oma
cells we e ob ained om he Ame ican Type Cul u e Collec ion (ATCC)
(Manassas, VA), and HuH7 human hepa oma cells we e om ou issue
cul u e acili y a he Uni e si y o Na a a. Cells we e main ained in
minimum Eagle’s medium (MEM) supplemen ed wi h 10% e al cal
se um, 2 mMglu amine, 100 uni s/ml penicillin, and 100
␮
g/ml s ep-
omycin (all om In i ogen) a 37 °C in a humid a mosphe e o 5% CO
2
in ai . To es MAT2A exp ession, cells we e cul u ed in L-me hionine-
de icien MEM supplemen ed wi h 5% e al cal se um and a ying
concen a ions o L-me hionine (Sigma). I has been es ima ed ha
L-me hionine concen a ion in bo ine se um is a ound 15
␮
M,so he
inal concen a ion o L-me hionine in ou expe imen s would be a ound
0.75
␮
M; in his wo k, his is e e ed o as L-me hionine es ic ion. In
some expe imen s, cells we e kep in K ebs-Ringe bica bona e (KRB)
and a ying concen a ions o L-me hionine. No signi ican di e ences
in cell iabili y, as de e mined by ypan blue exclusion es , we e
obse ed unde he di e en ea men s.
De e mina ion o MAT Ac i i y and AdoMe Le els—Cells (3 ⫻10
6
)
we e lysed by eeze hawing in 300
␮
lo 10mMT is-HCl (pH 7.5)
con aining 0.3 mMsuc ose, 0.1%
␤
-me cap oe hanol, 1 mMbenzami-
dine, and 0.1 mMphenylme hylsul onyl luo ide. The homogena e was
cen i uged o 30 min a 10,000 ⫻g, and MAT ac i i y was assayed in
he supe na an as desc ibed p e iously (27). Fo he de e mina ion o
AdoMe concen a ions, cells we e lysed and dep o einized in 0.4 M
pe chlo ic acid and cen i uged a 12,000 ⫻g o 15 min a 4 °C.
Supe na an s we e il e ed and analyzed by high pe o mance liquid
ch oma og aphy as desc ibed p e iously (27).
RNA Isola ion and No he n Blo Analysis—To al RNA was isola ed
by he guanidinium hiocyana e me hod as desc ibed (28, 29). RNA
concen a ion was de e mined spec opho ome ically be o e use, and
he in eg i y was checked by elec opho esis wi h subsequen e hidium
b omide s aining. Elec opho esis o RNA and gel blo ing we e ca ied
ou as desc ibed (27, 29). No he n hyb idiza ion analysis was pe -
o med on o al RNA using s anda d p ocedu es as desc ibed (27, 29).
All p obes (human MAT2A and glyce aldehyde-3-phospha e dehyd o-
genase (GAPDH) cDNAs) we e labeled wi h [
32
P]dCTP using a andom
p ime ki (RediP ime DNA Labeling Sys em; Ame sham Bisociences).
To ensu e equal loading o RNA samples, memb anes we e also hyb id-
ized wi h
32
P-labeled 18 S RNA DNA p obe. Quan i a ion was pe -
o med by scanning densi ome y o he x- ay ilms, using he Molecula
Analys so wa e (Bio-Rad), and alues we e no malized o 18 S RNA.
Nuclea Run-on T ansc ip ion Assay—Nuclei om 3 ⫻10
7
HepG2
cells kep in MEM wi hou L-me hionine o di e en du a ions we e
isola ed as desc ibed p e iously (27). T ansc ip ion eac ions we e pe -
o med o 30 min a 37 °C wi h 60
␮
l o he isola ed nuclei in a inal
olume o 200
␮
l in he p esence o 100
␮
Ci o [
␣
-
32
P]u idine iphos-
pha e (800 Ci/mmol; Ame sham Biosciences) and 0.5 mMunlabeled
nucleo ides essen ially as desc ibed (27). Elonga ed RNA ansc ip s
we e pu i ied by phenol ex ac ion as desc ibed abo e, dena u ed, and
hyb idized (a equi alen amoun s o adioac i i y (i.e. 1–2⫻10
6
cpm))
o 5
␮
g o he co esponding cDNAs and immobilized on Ny an il e s
in hyb idiza ion bu e (0.2 MNaPO
4
(pH 7.2), 1 mMEDTA, 7% SDS,
45% o mamide, and 250 mg/ml Esche ichia coli ans e RNA as ca -
ie ) o 48 h a 42 °C. The DNAs used we e he human MAT2A,
GAPDH, MTAP, S-adenosylhomocys eine hyd olase, and 18 S cDNAs;
pUC18 was used o backg ound con ol. The il e s we e washed and
exposed o x- ay ilms. Quan i a ion was pe o med by scanning densi-
ome y o he x- ay ilms, using he Molecula Analys so wa e (Bio-
Rad). Da a we e no malized o he ansc ip ion o he GAPDH gene,
he s eady-s a e mRNA le els o which we e no a ec ed by sho e m
L-me hionine deple ion.
S a is ics—Unless o he wise s a ed, he da a a e means ⫾S.E. o a
leas h ee independen expe imen s pe o med in duplica e. S a is ical
signi icance was es ima ed wi h S uden ’s es . A p alue o ⬍0.05 was
conside ed signi ican .
RESULTS
L-Me hionine Res ic ion Induces MAT2A mRNA Le els and
MAT Ac i i y in HepG2 Cells—To e alua e he e ec o L-
me hionine le els on MAT2A gene exp ession, HepG2 cells
we e cul u ed o di e en pe iods o ime in L-me hionine-
de icien MEM wi h 5% e al cal se um, and MAT2A exp es-
sion was assessed by No he n blo ing. As shown in Fig. 1A,
MAT2A mRNA le els we e d ama ically induced by L-me hio-
nine es ic ion in HepG2 cells in a ime-dependen ashion.
The in acellula le els o AdoMe in HepG2 cells be o e L-
me hionine es ic ion ( ⫽0) we e 1.2 ⫾0.10 nmol/mg o
p o ein and we e educed o 0.28 ⫾0.02 nmol/mg o p o ein
a e 4ho incuba ion in L-me hionine-de icien medium. The
magni ude o MAT2A mRNA accumula ion was also dependen
FIG.1. L-Me hionine deple ion induces MAT2A mRNA le els
and MAT ac i i y in HepG2 cells. A, HepG2 cells we e incuba ed in
MEM wi hou L-me hionine o di e en pe iods o ime, and MAT2A
mRNA le els we e analyzed by No he n blo ing. The exp ession o
GAPDH and he 18 S RNA genes was also examined. B, HepG2 cells
we e cul u ed o 24 h in MEM wi h inc easing concen a ions o L-
me hionine, and he exp ession o MAT2A, GAPDH, and 18 S RNA
genes was assessed by No he n blo ing. Rep esen a i e blo s o h ee
di e en expe imen s pe o med in duplica e a e shown. C, speci ic
MAT II ac i i y in HepG2 cells cul u ed o 24 h in MEM supplemen ed
wi h L-me hionine (100
␮
M)(⫹) o wi hou L-me hionine (⫺). Da a a e
means ⫾S.E. o h ee expe imen s pe o med in iplica es (as e isk
indica es p⬍0.05).
S-Adenosylme hionine Regula es MAT2A mRNA S abili y19886
on he le els o L-me hionine in he cul u e medium, as ob-
se ed when HepG2 cells we e cul u ed o 24 h in MEM
supplemen ed wi h inc easing concen a ions o his amino
acid (Fig. 1B). The in acellula le els o AdoMe in cells cul-
u ed du ing 24 h in MEM supplemen ed wi h 0, 100, o 1000
␮
ML-me hionine we e 0.16 ⫾0.02, 1.04 ⫾0.06, and 1.52 ⫾0.01
nmol/mg o p o ein, espec i ely. The le els o GAPDH mRNA
and 18 S ibosomal RNA we e also measu ed, and no signi i-
can changes we e obse ed (Fig. 1, Aand B). The induc ion o
MAT2A gene exp ession co ela ed wi h he up- egula ion o
MAT II ac i i y when measu ed 24 h a e cul u e in L-me hi-
onine- es ic ed MEM (Fig. 1C). The same obse a ions we e
made in HuH7 cells (da a no shown).
Speci ici y o MAT2A Response o L-Me hionine Deple-
ion—To de e mine whe he he obse ed up- egula ion o
MAT2A mRNA le els was limi ed o L-me hionine es ic ion in
cul u e medium, HepG2 cells we e g own in comple e MEM
(100
␮
ML-me hionine) un il 70% con luence and subsequen ly
cul u ed in KRB bu e o 2 h in he p esence o 0 o 100
␮
M
L-me hionine. As shown in Fig. 2A,MAT2A gene exp ession
was induced in me hionine-de icien KRB medium, whe eas
he addi ion o L-me hionine p e en ed his esponse. The ex-
p ession o o he genes ela ed o AdoMe me abolic pa hways,
such as S-adenosylhomocys eine hyd olase and 5⬘-me hyl hio-
adenosine phospho ylase, was also es ed in HepG2 cells unde
he abo e men ioned condi ions, and no signi ican changes
we e obse ed (Fig. 2A).
We nex examined he mechanisms h ough which L-me hi-
onine could media e his acu e e ec on MAT2A gene exp es-
sion. As p e iously s a ed, besides i s in ol emen in p o ein
syn hesis, in acellula L-me hionine is me abolized in o
AdoMe by he ac ion o MAT (Fig. 3 summa izes AdoMe
me abolic pa hways). We es ed whe he AdoMe could mimic
he e ec o L-me hionine on MAT2A gene exp ession. Fo his
pu pose, HepG2 cells we e cul u ed in me hionine- es ic ed
MEM o 24 h in he p esence o 4 mMAdoMe , and MAT2A
mRNA le els we e subsequen ly analyzed by No he n blo -
ing. In acellula AdoMe concen a ions we e measu ed in
con ol and AdoMe - ea ed cells 8 h a e he addi ion o his
compound. These concen a ions we e 0.20 ⫾0.01 and 5.02 ⫾
0.2 nmol/mg o p o ein in con ol and AdoMe - ea ed cells,
espec i ely. As shown in Fig. 2B, he po en induc ion o
MAT2A gene exp ession ha occu s in he absence o L-me hi-
onine (lane 2) was no obse ed o such an ex en when AdoMe
was added o he cul u e medium (Fig. 2B, compa e lanes 2 and
3). Howe e , unde he same condi ions, he nonme abolizable
D-isome o me hionine (a 100
␮
M) had no e ec on MAT2A
gene exp ession (Fig. 2B,lane 4). In e es ingly, MTA (a 500
␮
M), a p oduc o AdoMe me abolism gene a ed in he poly-
amine biosyn he ic pa hway and he i s molecule in he me-
hionine sal age ou e (30–32) (Fig. 3), was e y e ec i e in
p e en ing he induc ion o MAT2A mRNA le els by me hio-
nine es ic ion (Fig. 2B,lane 5). In li e cells, homocys eine
can be eme hyla ed and con e ed back o me hionine h ough
wo al e na i e pa hways ha in ol e he enzyme me hionine
syn hase o he enzyme be aine-homocys eine me hyl ans-
e ase (10, 11) (Fig. 3). Me hionine syn hase is a cobalamin-de-
penden enzyme ha equi es 5-me hyl e ahyd o ola e o con-
e homocys eine in o me hionine (33). We incuba ed HepG2
cells wi h homocys eine (200
␮
M), olinic acid (100
␮
M, a p e-
cu so o 5-me hyl e ahyd o ola e), and cyanocobalamin (15
␮
M) o 24 h in he absence o L-me hionine. As shown in Fig.
2B,lane 6, his ea men had no e ec on MAT2A mRNA
le els ha we e s ongly induced a e 24 h in a me hionine-
de icien medium. Simila ly, we explo ed he pa hway ca a-
lyzed by be aine-homocys eine me hyl ans e ase o he con-
e sion o homocys eine in o me hionine by incuba ing cells
wi h be aine (2 mM) and homocys eine (200
␮
M) o 24 h in he
absence o he L-me hionine addi ion. This ea men was also
unable o modula e he induc ion o MAT2A mRNA in esponse
o L-me hionine es ic ion (Fig. 2B,lane 7).
The induc ion o MAT2A mRNA elici ed by me hionine e-
s ic ion could be due ei he o an inc ease in ansc ip ion o
he gene o o inc eased mRNA s abili y. Consequen ly, we
ha e measu ed he e ec o L-me hionine es ic ion on he
ansc ip ion a e o he MAT2A gene in HepG2 cells by pe -
o ming nuclea un-on s udies. We obse ed ha he an-
sc ip ion a e o he MAT2A gene emained cons an a di e -
en ime poin s a e L-me hionine wi hd awal o up o 4 h;
simila ly, he ansc ip ion a e o GAPDH was no a ec ed
unde his condi ion (Fig. 4A).
FIG.2. Speci ici y o MAT2A gene esponse o L-me hionine
deple ion. A, HepG2 cells we e g own on L-me hionine (100
␮
M)-
supplemen ed MEM un il 70% con luence, and hen medium was
changed o KRB bu e o 2hin hep esence (⫹) o absence (⫺)o
L-me hionine (100
␮
M). MAT2A, MTAP, S-adenosylhomocys eine hy-
d olase, GAPDH, and 18 S RNA gene exp ession was assessed by
No he n blo ing; blo s ep esen a i e o h ee expe imen s pe o med
in duplica e a e shown. B, HepG2 cells we e cul u ed o 24 h in he
p esence o 100
␮
ML-me hionine (lane 1); 0
␮
ML-me hionine (lane 2); 4
mMAdoMe (lane 3); 100
␮
MD-me hionine (lane 4); 500
␮
MMTA (lane
5); 200
␮
Mhomocys eine, 100
␮
M olinic acid, and 15
␮
Mcyanocobal-
amine (lane 6); o 2 mMbe aine and 200
␮
Mhomocys eine (lane 7).
MAT2A, GAPDH, and 18 S RNA gene exp ession we e examined by
No he n blo ing; blo s ep esen a i e o h ee expe imen s pe o med
in duplica e a e shown.
FIG.3.L-Me hionine and AdoMe me abolic pa hways. L-Me hi-
onine ecycling and sal age pa hways a e p esen ed.
S-Adenosylme hionine Regula es MAT2A mRNA S abili y 19887
We nex es ed whe he ongoing ansc ip ion o ansla ion
was necessa y o he obse ed induc ion o MAT2A mRNA
le els in esponse o L-me hionine es ic ion. Fo his pu pose,
cells we e g own in comple e MEM un il 70% con luence and
hen ans e ed o L-me hionine- es ic ed MEM in he p es-
ence o ei he ac inomycin D (Ac D) (5
␮
g/ml) o cycloheximide
(0.1 mM) o 3 h. As shown in Fig. 4, Band C, bo h ea men s
comple ely abolished he induc ion o MAT2A mRNA le els.
These obse a ions indica e ha bo h ansc ip ion and p o ein
syn hesis a e equi ed o he up- egula ion o MAT2A gene
exp ession du ing L-me hionine deple ion.
MAT2A mRNA Is S abilized in L-Me hionine-deple ed
Cells—Taken oge he , ou da a sugges ha L-me hionine e-
s ic ion inc eases MAT2A gene exp ession by s abilizing he
mRNA. To suppo his hypo hesis, we de e mined MAT2A
mRNA hal -li e in comple e and in L-me hionine-deple ed
MEM. HepG2 cells g owing in comple e MEM we e incuba ed
o di e en pe iods o ime in he p esence o absence o Ac D
(5
␮
g/ml), and MAT2A mRNA le els we e measu ed by No h-
e n blo ing. Unde his condi ion, we could es ima e ha
MAT2A mRNA decayed wi h a hal -li e o ⬃100 min in L-
me hionine-supplemen ed medium (Fig. 5A). In o de o de e -
mine MAT2A mRNA hal -li e in L-me hionine- es ic ed me-
dium, cells we e cul u ed in he absence o his amino acid o
4 h and hen ea ed wi h Ac D (5
␮
g/ml) o up o3hin he
same medium. As shown in Fig. 5B, he le el o MAT2A mRNA
emained cons an in L-me hionine-deple ed cells, sugges ing
an mRNA hal -li e much longe han 3 h.
We wan ed o u he explo e he mechanisms h ough
which L-me hionine could media e his acu e egula ion o
MAT2A mRNA le els. We i s de e mined whe he AdoMe
and MTA we e ac ing on MAT2A gene exp ession also a he
le el o mRNA s abili y. Fo his pu pose, HepG2 cells we e
incuba ed o 4hinL-me hionine-de icien MEM, and hen
AdoMe (4 mM) o MTA (500
␮
M) was added o he cul u es in
he absence o p esence o Ac D (5
␮
g/ml) and incuba ions
con inued up o 6 h mo e. Analysis o MAT2A gene exp ession
unde hese ea men s indica ed ha bo h AdoMe and MTA
sha ed wi h L-me hionine i s abili y o down- egula e MAT2A
mRNA in he absence o ongoing ansc ip ion (Fig. 6). In e -
es ingly, MTA needed o be me abolized h ough he me hio-
nine sal age pa hway o exe i s e ec on MAT2A mRNA
le els, since he concomi an addi ion o adenine (1 mM), an
inhibi o o MTAP, he i s and a e-limi ing enzyme in his
pa hway (33), comple ely blocked i s e ec (Fig. 6). O pa icu-
la ele ance is he obse a ion ha L-me hionine has o be
me abolized and con e ed in o AdoMe o media e i s e ec on
MAT2A mRNA le els, as e idenced when L-me hionine ea -
men was pe o med in he p esence o cycloleucine (20 mM), a
compe i i e inhibi o o MAT ac i i y (34) (Fig. 6). I unde
hese condi ions ( ea men wi h L-me hionine and cyclo-
leucine) exogenous AdoMe (4 mM) was added o he cells,
MAT2A mRNA le els we e down- egula ed (Fig. 6). The addi-
ion o ei he adenine o cycloleucine o L-me hionine-s a ed
cells had no e ec on MAT2A mRNA le els (da a no shown).
As p e iously men ioned, an essen ial ole o L-me hionine,
h ough i s con e sion in o AdoMe , is o p o ide he cell wi h
he necessa y ac i a ed me hyl g oups o a as numbe o
ansme hyla ion eac ions. Hence, i was impo an o know i
a ansme hyla ion eac ion(s) could be in ol ed in he egula-
ion o MAT2A mRNA le els. Fo his pu pose, L-me hionine-
dep i ed cells we e ea ed wi h L-me hionine in he p esence
o he adenosine analog 3-deazaadenosine (C
3
-Ado) (30
␮
M,30
min p io o L-me hionine addi ion) (37). C
3
-Ado is an inhibi o
o S-adenosylhomocys eine hyd olase and leads o a signi ican
inc ease in he in acellula le els o AdoHcy; addi ionally, i
can be con e ed in o he mo e s able 3-deaza-de i a i e o
AdoHcy (S-3-deaza-adenosylhomocys eine (C
3
-AdoHcy)) (37).
AdoHcy and C
3
-AdoHcy a e s ong inhibi o s o me hyla ion
eac ions (14, 37). We obse ed ha he e ec o L-me hionine
was p ese ed in he p esence o C
3
-Ado (Fig. 6), sugges ing
ha me hyla ion eac ions we e no in ol ed in he egula ion
o MAT2A gene exp ession by his amino acid.
FIG.4.E ec o L-me hionine le els on MAT2A gene ansc ip-
ion in HepG2 cells. A, nuclea un-on assay o MAT2A gene an-
sc ip ion pe o med in nuclei isola ed om HepG2 cells main ained o
ei he 2 o 4hin0o 100
␮
ML-me hionine. Labeled RNAs we e
syn hesized by isola ed nuclei and hyb idized agains MAT2A, GAPDH,
and 18 S RNA cDNAs on slo blo s (pUC18 plasmid DNA was used o
backg ound con ol). Expe imen s we e pe o med wo imes in dupli-
ca e, and a ep esen a i e blo is shown. B, No he n blo analysis o
MAT2A mRNA le els om HepG2 cells kep in MEM wi h 100
␮
M
L-me hionine (lane 1), 0
␮
ML-me hionine o 3h(lane 2), o 0
␮
M
L-me hionine plus 5
␮
g/ml Ac D o 3 h. C, No he n blo analysis o
MAT2A mRNA le els om HepG2 cells kep in MEM wi h 100
␮
M
L-me hionine (lane 1), 0
␮
ML-me hionine o 3h(lane 2), o 0
␮
M
L-me hionine plus 0.1 mMcycloheximide o 3 h. Analysis was pe -
o med also wi h an 18 S RNA cDNA p obe o loading con ol. Rep-
esen a i e blo s om h ee expe imen s pe o med in duplica e a e
shown.
FIG.5. E ec o L-me hionine deple ion on he hal -li e o
MAT2A mRNA in HepG2 cells. A, quan i a ion o MAT2A mRNA
le els in HepG2 cells kep in MEM supplemen ed wi h 100
␮
ML-
me hionine in he absence (closed ci cles) o p esence (closed iangles)
o 5
␮
g/ml Ac D o di e en pe iods o ime. B, quan i a ion o MAT2A
mRNA le els in HepG2 cells ha we e kep o 4hinL-me hionine-
deple ed MEM and hen ( ime 0) u he ea ed o up o3hin he
absence (closed ci cles) o p esence (closed iangles)o 5
␮
g/ml Ac D.
Da a a e means ⫹S.E. o h ee expe imen s pe o med in duplica e
(as e isk indica es p⬍0.05 wi h espec con ols wi hou Ac D). Values
we e no malized o 18 S RNA le els.
S-Adenosylme hionine Regula es MAT2A mRNA S abili y19888
DISCUSSION
In his wo k, we ha e p o ided e idence o he acu e egu-
la ion o MAT2A gene exp ession in esponse o he a ailabili y
o L-me hionine. Incuba ion o human hepa oma HepG2 cells
(and HuH7 cells; no shown) in medium es ic ed in his
amino acid esul ed in he apid induc ion o MAT2A mRNA
le els in a ime- and concen a ion-dependen manne . A pa -
allel inc ease in MAT ac i i y was obse ed in cells cul u ed
unde educed L-me hionine a ailabili y, sugges ing ha
ansla ion o MAT2A mRNA was aking place e en in he
p esence o limi ing amoun s o L-me hionine. This obse a ion
is in ag eemen wi h o he epo s showing he induc ion o
speci ic p o eins unde limi ed a ailabili y o L-me hionine (38,
39). MAT2A gene esponse seemed o be mainly due o changes
in L-me hionine le els and no o luc ua ions in o he amino
acids o nu ien s. This may be in e ed by ou expe imen s in
which HepG2 cells we e incuba ed in KRB bu e and induc ion
o MAT2A gene exp ession was modula ed by he sole addi ion
o L-me hionine.
As men ioned p e iously, besides i s ole in p o ein syn he-
sis, L-me hionine en e s a me abolic pa hway in which i is
con e ed, oge he wi h ATP, in o AdoMe , he main me hyl
dono compound in he cell. I was impo an o know i he
obse ed esponse o MAT2A gene o luc ua ions in L-me hio-
nine le els could be ela ed o his me abolic pa hway. We
obse ed ha AdoMe addi ion o L-me hionine-s a ed cul-
u es was e ec i e in down- egula ing he exp ession o
MAT2A gene. This obse a ion, oge he wi h he inding ha
he nonme abolizable D-isome o me hionine had no e ec on
MAT2A mRNA le els, p o ided he i s e idence suppo ing
he concep ha L-me hionine media es i s e ec on MAT2A
gene exp ession h ough i s con e sion in o AdoMe . We also
es ed he e ec o o he molecules ha a e subsequen me a-
bolic p oduc s o L-me hionine and AdoMe , such as homocys-
eine and MTA. Homocys eine ea men had no e ec on
MAT2A mRNA le els, sugges ing ha his me aboli e was no
in ol ed in he cellula signaling o L-me hionine limi a ion.
Homocys eine can se e as a p ecu so o L-me hionine in all
cells; howe e , ans o med cells, including hepa oma cells, a e
unable o g ow i homocys eine is used in place o me hionine in
cul u e medium (13, 40, 41). Ou p esen da a u he suppo
his no ion. Wi h ega d o MTA, a p oduc o AdoMe me ab-
olism in he polyamine biosyn he ic pa hway and a p ecu so o
L-me hionine in he so-called me hionine sal age pa hway, we
obse ed ha i was able o modula e MAT2A mRNA le els in
L-me hionine-s a ed cul u es. As we la e obse ed, MTA e -
ec was dependen on i s me abolism h ough he me hionine
sal age pa hway and he e o e p obably due o i s ul ima e
con e sion in o his amino acid and subsequen ly in o AdoMe .
Adenine, a p oduc o MTA me abolism and an inhibi o o
MTAP, he i s and a e-limi ing enzyme in he me hionine
sal age pa hway (30–32, 35), blocked MTA e ec . Me hionine
dependence o umo cells has been associa ed wi h de ec s in
MTAP exp ession leading o he supp ession o his sal age
pa hway (13, 41, 42); howe e , his is no he case o HepG2,
whe e we ound ha MTAP exp ession was no mal (da a no
shown). Addi ionally, hese da a a es o he impo ance o he
me hionine sal age pa hway in me hionine conse a ion unde
limi ed supply o his amino acid and u he suppo he
ele ance o his me abolic ou e as a a ge in an ineoplas ic
he apy.
We ha e p o ided e idence showing ha MAT2A gene an-
sc ip ion emained cons an du ing L-me hionine s a a ion a
leas up o 4 h a e he emo al o his amino acid om he
cul u e medium. These da a sugges ed ha he po en e ec o
L-me hionine on MAT2A gene exp ession migh ake place a
he pos - ansc ip ional le el. In he egula ion o gene exp es-
sion by amino acids, bo h ypes o mechanisms, namely he
egula ion o gene ansc ip ion and o mRNA s abili y, ha e
been desc ibed. Fo ins ance, ansc ip ion o he aspa agine
syn he ase gene and he s abili y o he high a ini y ca ionic
amino acid anspo e -1 mRNA a e known o be modula ed by
amino acid s a a ion (43, 44). In he case o ce ain genes,
such as ca ionic amino acid anspo e -1, changes in i s mRNA
u no e a e alone accoun o he d ama ic induc ion on he
exp ession o his gene in amino acid-deple ed medium (44, 46).
Because he MAT2A gene ansc ip ion a e was no signi -
ican ly al e ed by L-me hionine s a a ion, we measu ed
MAT2A mRNA hal -li e in L-me hionine- ed and -s a ed cul-
u es. We obse ed a signi ican inc ease in MAT2A mRNA
hal -li e in esponse o L-me hionine s a a ion. In e es ingly,
his e ec was comple ely dependen on de no o p o ein syn-
hesis. This obse a ion sugges s ha a p o ein ac o (s) may
be syn hesized ha binds and s abilizes MAT2A mRNA when
cellula L-me hionine le els a e educed. This kind o mecha-
nism has been epo ed o he egula ion o mRNAs encoding
p o eins such as ans e in ecep o (45), he ca ionic amino
acid anspo e -1 (46), and o he s (47).
Once we es ablished ha MAT2A gene exp ession was eg-
ula ed by L-me hionine a he le el o mRNA u no e , we
con i med ha he obse ed e ec s o AdoMe and MTA on
MAT2A s eady s a e mRNA le els we e also media ed a his
same le el. In hese expe imen s, i was also clea ly es ab-
lished ha L-me hionine pe se was no su icien o con ey he
cellula signal(s) ha modula ed MAT2A mRNA u no e .
Con e sion o L-me hionine in o AdoMe was absolu ely neces-
sa y, gi en he ac ha cycloleucine, a compe i i e inhibi o o
MAT II ac i i y (34), comple ely abolished L-me hionine ac ion.
In line wi h his is he obse a ion ha when exogenous
AdoMe was added, he blockade imposed by cycloleucine on
L-me hionine e ec was o e come. Taken oge he , hese ind-
ings p o ide an explana ion o ou p e ious obse a ions made
in MAT1A knockou mice. These animals display d ama ically
ele a ed le els o ci cula ing L-me hionine (a ound 8- old in-
c ease compa ed wi h wild ype mice), and a he same ime he
FIG.6. Mechanism o L-me hionine egula ion o MAT2A
mRNA s abili y. MAT2A mRNA le els we e de e mined by No he n
blo ing in HepG2 cells ha we e kep in L-me hionine-deple ed MEM
o 4 h and hen u he incuba ed o 3 o 6 h mo e in he absence o
p esence o 5
␮
g/ml Ac D and he indica ed ea men s: wi hou L-
me hionine, 100
␮
ML-me hionine, 4 mMAdoMe , 500
␮
MMTA, 500
␮
M
MTA plus 1 mMadenine, 100
␮
ML-me hionine plus 30
␮
MC
3
-Ado, 100
␮
ML-me hionine plus 20 mMcycloleucine, 100
␮
ML-me hionine, and 4
mMAdoMe plus 20 mMcycloleucine. Rep esen a i e blo s o h ee
expe imen s pe o med in duplica e a e shown.
S-Adenosylme hionine Regula es MAT2A mRNA S abili y 19889

exp ession o he MAT2A gene is induced in hei li e (21).
These seemingly con adic o y obse a ions may be explained
by he ac ha he hepa ic concen a ion o AdoMe in hese
knockou mice is educed o abou 25% o ha ound in wild
ype animals (21).
Ano he in e es ing inding was he obse a ion ha ea -
men wi h C
3
-Ado, a compound ha inc eases he in acellula
concen a ion o AdoHcy, and C
3
-AdoHcy, wo po en inhibi o s
o me hyla ion eac ions, did no in e e e wi h he e ec o
L-me hionine on MAT2A mRNA le els. This inding sugges s
ha a me hyla ion eac ion is no in ol ed in he e ec o
L-me hionine (and consequen ly o AdoMe ) on MAT2A gene
exp ession, al hough we canno exclude a me hyl ans e e-
ac ion ha would be esis an o AdoHcy o C
3
-AdoHcy inhibi-
ion. We ha e p e iously desc ibed ha AdoMe is able o
induce he ansc ip ion o he MAT1A gene in cul u ed a
hepa ocy es (24, 25) and ha in his e ec a me hyla ion eac-
ion is p obably in ol ed (24). Now we epo on a no el e ec
o AdoMe on gene exp ession ha occu s a he pos ansc ip-
ional le el and in which a di e en mechanism o ac ion is
seemingly in ol ed. An al e na i e mode o ac ion o AdoMe
could be i s di ec binding o a cellula a ge s uc u e. Such
in e ac ion o AdoMe is known o occu in euka yo ic cells, and
he bes cha ac e ized a ge is he enzyme cys a hionine
␤
-syn hase, which is allos e ically egula ed by AdoMe bind-
ing o a speci ic domain e med he cys a hionine
␤
-syn hase
domain (49). In e es ingly, his cys a hionine
␤
-syn hase do-
main is a widesp ead s uc u e ha was o iginally desc ibed in
a chaebac e ia and is well conse ed h oughou e olu ion (50).
Gi en he cen al ole played by AdoMe in cellula me ab-
olism, he sha p induc ion in MAT2A gene exp ession in e-
sponse o educed L-me hionine and AdoMe a ailabili y could
be ega ded as an adap i e mechanism o he cell aimed a
p ese ing he in acellula le els o AdoMe . As we ha e
shown, his is a inely uned esponse, and we can en ision
di e en po en ial explana ions o his o be so. In he i s
place, o each AdoMe molecule syn hesized, one ATP mole-
cule and one L-me hionine molecule a e consumed a he ex-
pense o he homeos asis o cellula ene ge ics and p o ein
syn hesis (10, 11). Addi ionally, ecen obse a ions sugges
ha AdoMe plays a ole in he cell ha anscends i s pu ely
me abolic unc ion as a me hyl dono and me abolic in e me-
dia e ( e iewed in Re . 26). Changes in AdoMe le els modula e
p oli e a ion, apop osis, and di e en ia ion, so i s syn hesis
needs o be ca e ully con olled. In his ega d, he inhibi o y
e ec o AdoMe on MAT II ac i i y has been well cha ac e ized
(12, 13, 48). Ou p esen obse a ions ep esen a no el ac ion
o AdoMe in he egula ion o cellula physiology, his ime
modula ing i s own p oduc ion also h ough he egula ion o
MAT2A mRNA s abili y.
Acknowledgmen —We g a e ully acknowledge Es e anı´a Fe na´ndez
o echnical suppo .
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S-Adenosylme hionine Regula es MAT2A mRNA S abili y19890