RESEARCH Open Access
E ec s o human papilloma i us (HPV) ype 16
oncop o eins on he exp ession o in oluc in in
human ke a inocy es
Esz e Gyöngyösi, Ani a Szalmás, Annamá ia Fe enczi, Józse Kónya, Lajos Ge gely and Gyö gy Ve ess
*
Abs ac
Backg ound: The human papilloma i us (HPV) li e cycle is closely linked o ke a inocy e di e en ia ion. Oncogenic
HPV in ec ion has been shown o hampe he no mal di e en ia ion o ke a inocy es; howe e , he unde lying
mechanisms esponsible o his phenomenon a e ye o be cla i ied. He e, we aimed o s udy he e ec s o HPV16
E6 and E7 oncogenes on he exp ession o in oluc in (IVL), an es ablished ma ke o ke a inocy e di e en ia ion, in
human o eskin ke a inocy e (HFK) cells.
Resul s: The di e en ia ion o HFK cells by se um and high calcium signi ican ly inc eased bo h he mRNA and he
p o ein le els o IVL. The E6 and E7 oncop o eins o HPV16 oge he caused s ong down- egula ion o IVL mRNA
and p o ein bo h in p oli e a ing and in di e en ia ing HFK cells. To s udy he e ec s o HPV oncogenes on he IVL
p omo e , we made ansien ans ec ion assays and luci e ase es s and ound ha HPV 16 E6 bu no E7
ep essed IVL p omo e ac i i y in p oli e a ing HFK cells. The inhibi o y e ec o HPV 16 E6 on he human IVL
p omo e could be localised o he p oximal egula o y egion (PRR) o he gene.
Conclusions: These esul s sugges ha he down- egula ion o IVL p omo e ac i i y by HPV 16 E6 signi ican ly
con ibu e o he inhibi ion o endogenous IVL exp ession by he HPV 16 oncop o eins. In con as , he down-
egula ion o endogenous IVL exp ession by HPV16 E7 is p obably no caused by a di ec and speci ic e ec o E7
on he IVL p omo e .
Keywo ds: HPV 16, Oncogenes, Ke a inocy e di e en ia ion, In oluc in
Backg ound
Papilloma i uses a e small DNA i uses, wi h a ci cula
double-s anded DNA genome o abou 8 kbp leng h
[1]. O e 100 human papilloma i us (HPV) ypes ha e
been iden i ied un il now, o which abou 40 is able o
in ec he geni al mucosa [2]. Low- isk HPV ypes (HPV
6, 11, 42) a e mainly ound in benign geni al lesions
(condyloma acumina um) o low g ade ce ical dyspla-
sias, while high- isk o oncogenic geni al ypes (HPV 16,
18, and o he s) a e causally linked o he de elopmen
o ce ical cance [3].
The E6 and E7 oncop o eins o high- isk HPVs a e
esponsible o he ans o ming ac i i y o he i us [4].
High- isk HPV E6 induces he deg ada ion o he p53
umou supp esso p o ein h ough he ubiqui in-p o-
eosome pa hway [5]. In addi ion, HPV E6 is able o
bind se e al o he cellula p o eins, some o which can
media e ans o ming ac i i y independen ly om he
p53 pa hway [5]. High- isk HPV E7 is able o bind o
he pRB ( e inoblas oma) umou supp esso p o ein,
esul ing in he unc ional inac i a ion and deg ada ion
o pRB [6]. By binding o pRB/E2F complex and, by
eleasing ee E2F ansc ip ion ac o s, HPV E7 induces
he p og ession o he cell cycle [6].
The li e cycle o human papilloma i uses is closely
linked o ke a inocy e di e en ia ion. HPVs ini ially
in ec p oli e a ing basal cells o he squamous epi he-
lium, while i us p oduc ion is associa ed wi h e minally
di e en ia ed laye s [7]. The cellula DNA eplica ion
machine y is eac i a ed by he E7 oncogene in di e en-
ia ing ke a inocy es o p o ide a cellula en i onmen
* Co espondence: [email p o ec ed].hu
Depa men o Medical Mic obiology, Medical and Heal h Science Cen e,
Uni e si y o Deb ecen, 4032 Deb ecen, Nagye dei k . 98, Hunga y
Gyöngyösi e al.Vi ology Jou nal 2012, 9:36
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© 2012 Gyöngyösi e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
ha is pe missi e o he eplica ion o he i al genome
[7,8]. This ac i i y o HPV 16 E7 was shown o delay he
induc ion o he ke a inocy e di e en ia ion ma ke s
in oluc in and ke a in 10 [9].
Du ing he mul i-s ep p ocess o ke a inocy e di e en-
ia ion, he exp ession o genes in ol ed in he p ocess
(such as ke a ins, ansglu aminase 1, in oluc in, e c.) is
igh ly egula ed. The p esence o HPV 16 E6 oncogene
was shown o hampe he no mal di e en ia ion o ke -
a inocy es induced by se um and calcium o by no mal
s a i ica ion in o gano ypic cell cul u e [10-12]. How-
e e , he unde lying mechanisms esponsible o he
pe u bed di e en ia ion o ke a inocy es by he HPV
oncogenes a e only pa ially elucida ed.
I is easonable o assume ha he E6 and/o E7 onco-
genes may ha e an e ec on he ansc ip ion o key cel-
lula genes in ol ed in he di e en ia ion o squamous
epi helium. Indeed, HPV 16 E6 was shown o modula e
he exp ession o se e al di e en ia ion-associa ed genes
in human o eskin ke a inocy es [13]. Using a xenog a
model, Leh and co-wo ke s showed ha in ec ion o
human ke a inocy es by ce ain HPVs ( ype 11 and 59)
causes al e ed exp ession o ce ain CCE (co ni ied cell
en elope) p o eins, such as lo ic in and small p oline
ich p o eins (SPRR), bo h on he mRNA and on he
p o ein le el [14,15]. Howe e , i is no known whe he
he HPV oncop o eins ha e e ec s on he p omo e s o
di e en ia ion- egula ed genes o exe hei e ec s
pos - ansc ip ionally.
The aim o his s udy was o in es iga e he e ec s o
HPV 16 E6 and E7 oncogenes on he exp ession o he
in oluc in (IVL) and ansglu aminase 1 (TG1)genes,
which a e es ablished ma ke s o di e en ia ion o squa-
mous epi helium. IVL p o ein is a 68 kDa, od-shaped
molecule con aining se e al glu amine esidues. I is
ound in he cy oplasm and c oss-linked o memb ane
p o eins by ke a inocy e ansglu aminases in di e en-
ia ing ke a inocy es [16].
He e, we ound ha he HPV oncogenes down- egu-
la ed bo h IVL mRNA and p o ein le els in human o e-
skin ke a inocy e (HFK) cells. In o de o s udy he
molecula mechanisms ha a e esponsible o he gene
exp ession al e a ions by he HPV oncop o eins, expe i-
men s we e pe o med using HPV 16 E6 and/o E7
exp ession plasmids along wi h luci e ase epo e con-
s uc s con aining pa s o he egula o y egion o he
human IVL gene.
Resul s
Gene a ion and cha ac e iza ion o human o eskin
ke a inocy e (HFK) cells exp essing HPV 16 oncogenes
To s udy he e ec s o HPV oncogenes on he exp es-
sion o cellula genes, human o eskin ke a inocy e
(HFK) cells we e ansduced by ecombinan
e o i uses ca ying ei he he con ol ec o (LXSN)
o ec o s encoding HPV16 E6 o E7 o bo h onco-
genes. The p esence o HPV E6 and/o E7 mRNA in he
ansduced cells was demons a ed by RT-PCR (Figu e
1A).The2PCRbandsshownonFigu e1A(uppe
panel) ep esen he ull leng h E6 mRNA and he E6*I
spliced a ian , in acco dance wi h p e ious esul s [17].
Thep esenceo unc ionallyac i eHPVoncop o eins
was con i med by demons a ing hei e ec on he le el
o he cellula p53 oncop o ein (Figu e 1B). As expec ed,
ansduc ion o HPV16 E6 esul ed in a dec eased le el
o p53 p o ein (abou 10% o ha seen in con ol ans-
duced cells, as indica ed by densi ome y), while exp es-
sion o HPV 16 E7 led o he s abiliza ion and hus
highe le el (1.6- old inc ease) o p53 p o ein [5,6].
The e ec s o he HPV oncogenes on he endogenous
mRNA le els o di e en cellula genes we e s udied using
eal- ime RT-PCR assays. In hese expe imen s, cell lines
we e used wi hin 5 o 6 passages a e ansduc ion, as we
wan ed o see he acu e e ec s o he HPV oncogenes on
he exp ession o he cellula genes. The ansduced cells
main ained he p oli e a ing, non-di e en ia ing pheno-
ype when le in se um- ee medium, while di e en ia ion
was induced by changing he cul u e medium o DMEM
(wi h se um and high calcium) o 24 h, in acco dance
wi h he esul s o p e ious s udies [9,11]. An es ablished
unc ion o he HPV 16 E6 oncogene is he ansc ip ional
ans-ac i a ion o he human TERT ( elome ase e e se
ansc ip ase) p omo e [18,19]. Acco dingly, he p esence
o E6 led o a conside able (abou 300- old) inc ease in he
le el o TERT mRNA (p< 0.001), while E7 had a weake
bu s illsigni ican (p< 0.05) e ec (Figu e 2A). As
expec ed, induc ion o di e en ia ion had no signi ican
e ec on he le el o TERT mRNA in any o he ans-
duced cells.
E ec s o HPV 16 oncogenes on he exp ession o
selec ed cellula genes in ol ed in ke a inocy e
di e en ia ion
Real- ime RT-PCR assays we e used o examine he
e ec s o he HPV oncogenes on he exp ession o he
squamous di e en ia ion ma ke ansglu aminase 1
(TG1) and one o i s majo subs a e in oluc in (IVL)in
HPV oncogene ansduced cells. As expec ed, induc ion
o di e en ia ion o HFK cells by se um and inc eased
calcium esul ed in highly inc eased le els o bo h IVL
and TG1 mRNA (p< 0.005) (Figu e 2B and 2C). In p o-
li e a ing cells, bo h E6 and E7 had signi ican inhibi ing
e ec (p< 0.01) on IVL mRNA le els (Figu e 2B). We
ound a e y s ong down- egula ion o IVL mRNA in
cells exp essing bo h HPV oncop o eins (HFK-16E6E7)
compa ed o ec o ansduced (LXSN) cells (p< 0.001).
Bo h E6 and E7 caused signi ican (p< 0.001) down- eg-
ula ion o IVL mRNA in di e en ia ing HFK cells.
Gyöngyösi e al.Vi ology Jou nal 2012, 9:36
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Again, he s onges e ec was seen in cells exp essing
E6 and E7 oge he (p< 0.001). In p oli e a ing cells,
HPV16E6andE7oncogenes oge he hadonly
mode a e inhibi ing e ec on he endogenous mRNA
le el o TG1 (p< 0.05), while in di e en ia ing cells, he
HPV oncogenes had no signi ican e ec on TG1
Figu e 1 The exp ession o HPV oncogenes in ansduced human o eskin ke a inocy e (HFK) cells.(A) Cells we e ansduced by
ecombinan e o i uses ca ying ei he he con ol ec o (LXSN) o ec o s encoding HPV16 E6, E7, o E6/E7. The cells we e ei he le in
se um- ee medium (p oli e a ing cells) o induced o di e en ia e in DMEM (wi h se um and high calcium). RT-PCR analysis was used o con i m
he exp ession o he app op ia e HPV oncogenes in he di e en cell lines (GAPDH was used as an endogenous con ol). (B) The e ec o
HPV16 E6 and E7 on he le el o p53 p o ein. P o ein ex ac s om ansduced cells we e subjec ed o Wes e n blo assay o p53 o ac in ( he
la e as loading con ol). Pa s o ep esen a i e X- ay ilms a e shown.
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Figu e 2 The e ec o HPV oncogenes on he mRNA le els o selec ed genes in HFK cells. Gene a ion o cell lines and induc ion o
di e en ia ion was pe o med as desc ibed in he legend o Figu e 1. Rela i e Quan i ica ion (RQ) alues we e ob ained by eal- ime RT-PCR
analysis o see he e ec s o HPV 16 oncogenes on he le el o TERT (A), IVL (B), o TG1 (C) mRNA in HFK cells. The RQ alue o p oli e a ing
cells ansduced by LXSN was se o 1, and o he alues a e shown ela i e o his. Each eac ion was pe o med in iplica e a leas h ee imes.
Rep esen a i e g aphs a e shown, wi h e o ba s indica ing s anda d de ia ion.
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mRNA exp ession (Figu e 2C). These esul s sugges
ha he e ec o he HPV oncogenes on IVL exp ession
is ei he mo e di ec o mo e speci ic han ha on TG1
exp ession.
To see he e ec s o he HPV oncogenes on he le el
o he IVL p o ein, we pe o med Wes e n blo analysis
using monoclonal an ibody o IVL (Figu e 3). Induc ion
o di e en ia ion by se um and high calcium esul ed in
inc eased IVL p o ein le els, as compa ed o ha seen
in p oli e a ing cells. In p oli e a ing cells, HPV 16 E6
o E7 alone had li le e ec on endogenous IVL p o ein,
while he wo oncogenes oge he caused s ong down-
Figu e 3 The e ec o HPV oncogenes on he le el o IVL p o ein in HFK cells. Gene a ion o cell lines and induc ion o di e en ia ion was
pe o med as desc ibed in he legend o Figu e 1. Ten μg o p o ein ex ac s p epa ed om he indica ed cell lines we e subjec ed o Wes e n
blo analysis o IVL o ac in p o ein ( he la e as loading con ol). (A) Pa s o ep esen a i e X- ay ilms a e shown. (B) The amoun o IVL
p o ein was quan i a i ely analyzed by densi ome y and s anda dized o ac in p o ein le el. The s anda dized densi y alue o cycling cells
ansduced by LXSN was se o 1, and o he alues a e shown ela i e o his. Values show he means om ou independen expe imen s wi h
s anda d e o s shown as e o ba s.
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egula ion o IVL p o ein le el as compa ed o ha
ound in ec o ansduced (LXSN) cells (p< 0.001).
This down- egula ing e ec o E6/E7 on IVL p o ein
le el was smalle bu s ill signi ican in di e en ia ing
cells (p= 0.032).
E ec s o HPV 16 oncogenes on he ansc ip ional
ac i i y o he human IVL p omo e
Nex , we aimed o de e mine whe he he down- egula ion
o IVL exp ession by he HPV oncop o eins was caused by
inhibi ing he ansc ip ional ac i i y o he IVL p omo e .
To his end, low-passage HFK cells we e ansien ly ans-
ec ed by ei he ec o con ol o HPV 16 E6 and/o E7
exp ession ec o s along wi h luci e ase epo e con-
s uc s con aining he egula o y egion o he human IVL
gene. In pilo expe imen s, we used an in e nal con ol
ec o (a Renilla luci e ase epo e ec o ) o s anda dize
o ans ec ion e iciency. Howe e , we ound ha HPV
16 E6 exp ession had an e ec on he ac i i y o he in e -
nal con ol ec o . The e o e, he e we show luci e ase
esul s ob ained by s anda diza ion o p o ein concen a-
ion. The exp ession o unc ionally ac i e HPV 16 E6 and
E7 p o eins in he ans ec ed HFK cells was con i med by
co- ans ec ion o epo e cons uc s con aining ei he
binding si es o he p53 p o ein o he adeno i us E2
(AdE2) p omo e ca ying binding si e o he E2F an-
sc ip ion ac o . As expec ed, he exp ession o HPV 16 E6
caused he down- egula ion o he p53 epo e cons uc
in p oli e a ing HFK cells (Figu e 4A), while HPV 16 E7
inc eased he ac i i y o he adeno i us E2 p omo e (Fig-
u e 4B).
In p oli e a ing HFK cells, HPV 16 E6 caused signi ican
down- egula ion o a epo e cons uc (pGL3-IVL) con-
aining he ull-leng h egula o y egion o he human IVL
gene (p< 0.001). On he con a y, E7 alone had no signi i-
can e ec (p= 0.485) on he IVL epo e cons uc in
p oli e a ing cells (Figu e 4C). Induc ion o di e en ia ion
by se um and high calcium esul ed in highly (15- old)
inc eased ac i i y o he pGL3-IVL cons uc in he p e-
sence o he emp y exp ession ec o pcDNA. In di e en-
ia ing HFK cells, bo h HPV 16 E6 (p= 0.124) and E7 (p=
0.253) had a non-signi ican endency o inhibi IVL p o-
mo e ac i i y, while HPV 16 E6 and E7 oge he caused
signi ican down- egula ion o he IVL p omo e (p=
0.014) (Figu e 4C). Taken oge he , hese esul s indica e
ha he down- egula ion o IVL p omo e ac i i y by HPV
16 E6 signi ican ly con ibu e o he inhibi ion o endogen-
ous IVL exp ession by he HPV 16 oncop o eins.
Localisa ion o he e ec s o HPV 16 oncogenes on he
human IVL p omo e
In o de o localise he e ec s o HPV 16 oncogenes on
he human IVL p omo e , a se ies o luci e ase epo e
ec o s was cons uc ed ca ying di e en agmen s o
he p omo e all ex ending o n +41 ela i e o he
ansc ip ional s a si e (Figu e 5A). Fi s , we checked
in ansien ans ec ion expe imen s whe he hese con-
s uc s esponded o di e en ia ion s imuli in HFK cells.
As shown on Figu e 5B, he ac i i y o each epo e
cons uc was induced by di e en ia ion o HFK cells.
Induc ion by di e en ia ion was highes (15- old) o he
longes cons uc (IVL-2418), while i was lowe (abou
4- old) o he sho e IVL p omo e cons uc s. Nex ,
we s udied he e ec s o HPV 16 E6 and E7 on he an-
sc ip ional ac i i y o he IVL epo e cons uc s in
HFK cells. In p oli e a ing HFK cells, HPV 16 E6 was
able o down- egula e ansc ip ion om each epo e
cons uc . When compa ed o ec o con ol ans ec ed
cells, E6 educed he ac i i y o he ull-leng h epo e
cons uc IVL-2418 (p= 0.026) and also ha o he
sho es epo e cons uc IVL-272 (p= 0.011) con ain-
ing only he PRR (p oximal egula o y egion) o he
IVL p omo e (Figu e 5C). E6 caused signi ican down-
egula ion o all he es ed IVL epo e cons uc s also
in di e en ia ing HFK cells (Figu e 5D). These esul s
indica e ha mos o he down- egula ing ac i i y o
HPV 16 E6 on he human IVL p omo e can be mapped
o he p oximal egula o y egion o he IVL gene ( o
he p omo e egion -231 o +41 ela i e o he an-
sc ip ional s a si e).
HPV 16 E7 had no signi ican e ec on any IVL epo -
e cons uc in p oli e a ing cells (Figu e 5C). In di e -
en ia ing cells, E7 had a mode a e bu signi ican
inhibi ing e ec (p=0.020)on he ull-leng hIVL
epo e cons uc (IVL-2418), while i had no signi i-
can e ec on he sho e IVL epo e cons uc s.
Discussion
In his s udy, we ound ha he HPV 16 E6 and E7 onco-
p o eins caused a syne gis ic down- egula ion o endo-
genous IVL mRNA and p o ein le els in HFK cells,
which a e na u al hos cells o he i us. Ou inding is in
acco dance wi h p e ious s udies pe o ming mic oa ay
analysis o genes in ol ed in ce ical ca cinogenesis. IVL
and/o o he ke a inocy e di e en ia ion associa ed genes
(such as ce ain ke a ins and small p olin- ich p o eins)
a e down- egula ed in ce ical cance specimens com-
pa ed o no mal ce ical samples [20,21]. Acco dingly,
se e al s udies using cul u ed human ke a inocy es as in
i o models o ce ical ca cinogenesis ound ha he
exp ession o HPV oncogenes causes a down- egula ion
o exp ession o IVL and/o o he genes in ol ed in
epi helial di e en ia ion [13,22,23]. Howe e , mic oa ay
analysis does no p o ide in o ma ion on he mechanism
o changes in gene exp ession. The e o e, ou app oach
was o analyse in HFK cells gene exp ession al e a ions o
a ew selec ed genes in ol ed in ke a inocy e di e en ia-
ion using eliable eal- ime RT-PCR assays and o
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Figu e 4 The e ec o HPV oncogenes on he ansc ip ional ac i i y o di e en p omo e s in HFK cells.(A)HFKcellswe eco-
ans ec ed wi h 0.5 μg o he p53 epo e cons uc p53-Luc, along wi h 0.25 μg o ei he con ol ec o (pcDNA) o 0.25 μg o exp ession
cons uc s encoding HPV16 E6 o E7 o bo h exp ession ec o s (0.125-0.125 μg) as indica ed. (B) HFK cells we e co- ans ec ed wi h 0.5 μgo
he adeno i us E2 epo e cons uc pAdE2Luc, along wi h 0.25 μg o ei he con ol ec o (pcDNA) o 0.25 μg o exp ession cons uc s
encoding HPV16 E6 o E7 o bo h as indica ed. (C) HFK cells we e co- ans ec ed wi h 0.5 μg o he IVL epo e cons uc pGL3-IVL, along wi h
0.25 μg o ei he con ol ec o (pcDNA) o 0.25 μg o exp ession cons uc s encoding HPV16 E6 o E7 o bo h as indica ed. A e ans ec ion,
HFK cells we e ei he le in se um- ee medium o induced o di e en ia e in DMEM (wi h se um and high calcium). The luci e ase ac i i ies a e
shown ela i e o he ac i i y o cells co- ans ec ed wi h epo e cons uc s and he emp y exp ession ec o (pcDNA). Da a a e om a leas
h ee independen expe imen s wi h s anda d e o s shown as e o ba s.
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explo e he molecula mechanisms behind gene exp es-
sion al e a ions using luci e ase epo e assays. We also
ound i impo an o s udy he e ec s o he HPV onco-
genes on he exp ession o di e en ia ion- egula ed
genes bo h in p oli e a ing and in di e en ia ing HFK
cells as we hough ha di e en ia ing cells a he han
p oli e a ing cells e lec be e he cellula en i onmen
equi ed o he p oduc i e li e cycle o HPV.
As expec ed, induc ion o di e en ia ion o ke a ino-
cy es highly inc eased he endogenous mRNA le els o
bo h IVL and TG1 in HFK cells (Figu e 2). In e es ingly,
he HPV 16 E6 and E7 oncogenes oge he had a e y
s ong down- egula ing e ec on IVL mRNA bu only a
mode a e e ec on TG1 mRNA.Thissugges s ha he
HPV oncogenes may ha e di e en e ec s on he exp es-
sion o di e en genes in ol ed in he di e en ia ion o
Figu e 5 Localisa ion o he e ec s o HPV 16 oncogenes on he human IVL p omo e .(A) Schema ic ep esen a ion o epo e cons uc s
con aining di e en pa s o he human IVL p omo e . The IVL p omo e agmen s we e cloned in he epo e ec o pGL3 in on o he
luci e ase gene. Nucleo ide posi ions a e gi en ela i e o he ansc ip ion s a si e. Selec ed ansc ip ion ac o ecogni ion si es a e indica ed
(C/EBP, CCAAT enhance binding p o ein). (B) The e ec o di e en ia ion on he ansc ip ional ac i i y o cons uc s con aining di e en
agmen s o he IVL p omo e in human o eskin ke a inocy es. HFK cells we e co- ans ec ed wi h pGL3 luci e ase epo e cons uc s con aining
di e en agmen s o he human IVL p omo e (IVL-2418, IVL-1809, IVL-744, IVL-272) along wi h pcDNA con ol ec o . Cells we e le in se um-
ee medium o induced o di e en ia e in DMEM wi h se um and high calcium. (C) and (D) The e ec o HPV 16 E6 o E7 oncogenes on he
ansc ip ional ac i i y o cons uc s con aining di e en agmen s o IVL p omo e in p oli e a ing (C) o in di e en ia ing (D) HFK cells. HFK cells
we e co- ans ec ed wi h pGL3 luci e ase epo e cons uc s along wi h ei he con ol ec o (pcDNA) o exp ession cons uc s encoding HPV16
E6 o E7. The luci e ase ac i i ies a e shown ela i e o he ac i i y o cells co- ans ec ed wi h IVL-2418 epo e cons uc along wi h he con ol
ec o . Da a a e om h ee independen expe imen s wi h s anda d e o s shown as e o ba s.
Gyöngyösi e al.Vi ology Jou nal 2012, 9:36
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squamous epi helial cells. Wes e n blo analysis showed
ha he join e ec o HPV 16 E6 and E7 on ansc ip-
ional down- egula ion esul ed in excessi e dec ease o
IVL p o ein le els as well, bo h in p oli e a ing and in di -
e en ia ing cells (Figu e 3). In a p e ious s udy, he
exp ession o HPV 6 o HPV 16 E7 was shown o esul
in a dec ease o IVL p o ein le els in HFK cells [24]. We
can conclude ha he HPV 16 E6 and E7 oncogenes
oge he seem o down- egula e basal IVL exp ession and
also dec ease he di e en ia ion-induced exp ession o
he IVL gene in HFK cells.
The exp ession o genes in ol ed in ke a inocy e di -
e en ia ion (including IVL) a e gene ally egula ed on
he le el o ansc ip ion [25]. The e o e, i seemed ea-
sonable o in es iga e he e ec s o HPV 16 oncop o-
eins on IVL p omo e ac i i y. This app oach included
ans ec ing HFK cells by HPV 16 E6 and/o E7 exp es-
sion ec o s along wi h luci e ase epo e cons uc s
con aining he whole ups eam- egula o y egion (URR)
o he human IVL gene. In ag eemen wi h p e ious
esul s [26], di e en ia ion o HFK cells led o a signi i-
can inc ease in he ansc ip ional ac i i y o he IVL
p omo e . In p oli e a ing HFK cells, HPV 16 E6, bu
no E7 caused a signi ican down- egula ion o IVL p o-
mo e ac i i y. The HPV 16 E6 and E7 oncop o eins
oge he caused a down- egula ion o IVL p omo e
ac i i y in di e en ia ing HFK cells (Figu e 4). Taken
oge he , hese esul s sugges ha he down- egula ion
o endogenous IVL mRNA and p o ein le els in HFK
cells by he HPV 16 E6 oncop o ein is caused by inhibi-
ion o IVL p omo e ac i i y. Howe e , i can no be
uled ou ha HPV 16 E6 down- egula es he exp ession
o IVL o o he di e en ia ion-associa ed genes also by
o he mechanisms. Fo example, HPV 16 E6 was shown
o down- egula e he exp ession o No ch1, which was
sugges ed o ha e a ole in he supp ession o ke a ino-
cy e di e en ia ion by E6 [27].
In o de o localise he e ec o he HPV oncogenes
wi hin he IVL p omo e , we made luci e ase epo e
cons uc s con aining di e en pa s o he URR o he
human IVL gene. The URR o he human IVL gene con-
ains a dis al egula o y egion (DRR, -2473/-1953 om
ansc ip ion s a si e) and a p oximal egula o y egion
(PRR, -241/-7 om he ansc ip ion s a si e) [26].
F om he 5 possible AP1 (ac i a o p o ein 1) binding
si es in he URR, AP1-5 (in DRR) and AP1-1 (in PRR) a e
essen ial o op imal p omo e ac i i y [28]. AP1 ac o s
(c- os, osB, F a-1, F a-2, c-jun, junB and junD) a e
exp essed a speci ic epide mal laye s and he exp ession
pa e n o hese ac o s is hough o ha e a ole in di e -
en ia ion- egula ed gene exp ession in ke a inocy es
[25,29,30]. F a-1, junB and junD in e ac wi h AP1 si es
wi hin he human IVL p omo e and media e pho bol
es e esponsi eness [28]. In ou expe imen s, he le el o
inhibi ion by HPV 16 E6 was he highes o he con-
s uc con aining he whole URR o he IVL gene, bu an
IVL epo e cons uc ca ying only he PRR was s ill
signi ican ly inhibi ed by he HPV 16 E6 p o ein, bo h in
p oli e a ing and in di e en ia ing HFK cells (Figu e 5).
This sugges s ha he PRR o IVL gene con ains binding
si es o ansc ip ion ac o s ha a e egula ed by HPV
16 E6.
HPV 16 E7 had a signi ican inhibi o y e ec only on
he cons uc con aining he ull-leng h IVL p omo e
(IVL 2418), and his e ec was seen only in di e en ia -
ing cells (Figu e 5). This may sugges ha he e ec o E7
on he IVL p omo e is less di ec and/o less speci ic
han ha o E6. We ind i concei able ha he e ec s o
E7 seen on IVL exp ession (syne gis ic down- egula ing
e ec wi h E6) and on IVL p omo e (sligh down- egula-
ion only in di e en ia ing cells) a e caused no by a
di ec and speci ic in e ac ion wi h he IVL p omo e , bu
a he by ecen ly desc ibed o he mechanisms. Fo
ins ance, he DEK p o ein was ound o be ansc ip ion-
ally up- egula ed by HPV 16 E7, and his was shown o
be impo an in he induc ion o cell p oli e a ion and
inhibi ion o he epi helial di e en ia ion p og am
[31,32]. Fu he mo e, nucleophosmin (NPM) was
epo ed o be up- egula ed by HPV 16 E7 a he pos -
ansc ip ional le el, and his up- egula ion was sug-
ges ed o ha e a ole in he inhibi ion o di e en ia ion in
ke a inocy es [33].
Bo h he DRR and he PRR o he human IVL gene
con ains binding si es o AP1 ansc ip ion ac o s
(PRR). The p omo e s o di e en ia ion-associa ed ke a i-
nocy e genes usually con ain binding si es o he AP1
ac o s, and hese a e hough o be impo an in he eg-
ula ion o gene exp ession by di e en ia ion s imuli
[25,29]. I is also in e es ing o no e ha he HPV 16 E7
p o ein was shown o bind o AP1 ansc ip ion ac o s,
including c-jun, junB, junD and c- os [34]. The e o e, we
suppose ha he AP1 mo i s in he p omo e o he
human IVL genemayha ea olein he egula iono
gene exp ession by HPV oncop o eins. In o de o p o e
his hypo hesis, u he esea ch will be equi ed using
p omo e mu agenesis and ch oma in immunop ecipi a-
ion (ChIP) assays. I would be also in e es ing o s udy
he e ec s o he HPV oncogenes on he exp ession o
o he genes (such as ke a ins, small p olin- ich p o eins,
S100 calcium binding p o eins) in ol ed in ke a inocy e
di e en ia ion.
The dec eased exp ession o IVL and o he di e en ia-
ion- egula ed genes by he HPV oncop o eins may ha e
an impo an ole in he p oduc i e li e cycle o he
i us. HPV eplica ion akes place in di e en ia ing
epi helial cells, which would exi he cell cycle in he
absence o i al in ec ion. The E7 oncogene is able o
induce he p og ession o he cell cycle in di e en ia ing
Gyöngyösi e al.Vi ology Jou nal 2012, 9:36
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