Effects of human papillomavirus (HPV) type 16 oncoproteins on the expression of involucrin in human keratinocytes

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 h p://www. i ologyj.com/con en /9/1/36 © 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 h p://www. i ologyj.com/con en /9/1/36 Page 2 o 12 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. Gyöngyösi e al.Vi ology Jou nal 2012, 9:36 h p://www. i ologyj.com/con en /9/1/36 Page 3 o 12 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. Gyöngyösi e al.Vi ology Jou nal 2012, 9:36 h p://www. i ologyj.com/con en /9/1/36 Page 4 o 12 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. Gyöngyösi e al.Vi ology Jou nal 2012, 9:36 h p://www. i ologyj.com/con en /9/1/36 Page 5 o 12 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 Gyöngyösi e al.Vi ology Jou nal 2012, 9:36 h p://www. i ologyj.com/con en /9/1/36 Page 6 o 12 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. Gyöngyösi e al.Vi ology Jou nal 2012, 9:36 h p://www. i ologyj.com/con en /9/1/36 Page 7 o 12 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 h p://www. i ologyj.com/con en /9/1/36 Page 8 o 12 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 h p://www. i ologyj.com/con en /9/1/36 Page 9 o 12