download report - Istituto Pasteur

P a r t i c i p a n t s :
Maurizio Alimandi, Deborah French, Patrizia Mancini,
Vincenzo Visco, professors; Francesca Belleudi, researcher;
Salvatore Raffa, post-doc fellow; Giulia Bolasco, Valerio
Nobili, Paola Pisano, Danilo Ranieri, PhD students; Antonio
Sabatucci, technician.
C o l l a b o r a t i o n s :
Laboratorio di Virologia, Istituto Regina Elena IFO-IRE, Roma
(Dr. Aldo Venuti).
Report of activity
The high-risk human papillomaviruses (HPVs) play
key roles in the pathogenesis of cervical cancers. The
E5 protein encoded by HPV type 16 is an oncoprotein
which contributes to skin carcinogenesis, since its
expression in vivo induces epidermal hyperplasia,
aberrant differentiation and skin tumors. The 16E5
protein transforms epithelial cells by deregulating
cell growth, survival and differentiation through the
modulation of growth factor receptors, such as the
epidermal growth factor receptor (EGFR). Among
the epithelial growth factors, the keratinocyte growth
factor (KGF/FGF7) and the fibroblast growth factor
10 (FGF10/KGF2) are major paracrine mediators of
proliferation, differentiation, survival and migration
of epithelial cells. Both KGF and FGF10 bind to and
activate exclusively the keratinocyte growth factor
receptor (KGFR/FGFR2b). The KGFR, in contrast
to most of the growth factor receptors, appears to
play an unique and unusual role in epithelial tissues,
exerting a tumor suppressive function in vitro and in
vivo. Interestingly, the KGFR/FGFR2b null-mice
phenotype closely reminds that shown by the 16E5
transgenic mice, characterized by a similar behaviour
in skin carcinogenic model. Thus, KGFR and 16E5
might be inversely correlated in their expression and
might exert opposite and interplaying roles in skin
homeostasis and tumorigenesis. With the aim to bet-
Pathogenetic mechanisms of microbially associated diseases - AREA 2
Role of the keratinocyte growth factor receptor on the molecular
and cellular alterations induced by the expression
of HPV16 E5 oncoprotein
Principal investigator: Maria Rosaria Torrisi
Professor of General Pathology
Dipartimento di Medicina Sperimentale
Tel: (+39) 06 4468450
mara.torrisi@uniroma1.it
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ter elucidate the molecular events involved in the
pathological effects induced by HPV infection and
UVB exposure of human epidermis, our research
project will attempt: a) to establish in vitro the correlation
between the expression levels of 16E5 and
those of KGFR and other epithelial RTKs and the
keratinocyte growth, differentiation, survival, and
transformation; b) to identify the mechanisms and
signaling pathways controlling the possible effects of
16E5 expression in modulating the ligand-dependent
KGFR activation and endocytosis and the cellular
response to UVB.
During the first year of the project, to investigate if
16E5 expression would be able to modulate KGFR
in vitro, we used human keratinocytes, grown at different
cell densities and expressing a modulated
number of KGFRs following transfection or differentiation,
as a model to study the 16E5 effects on the
receptor activation and modulation and on the related
epithelial proliferation/differentiation. First, we
utilized the human immortalized keratinocytes
HaCaT: stable transfectants of HaCaT cells
expressing 16E5, HaCaT E5 (pMSG) and HaCaT
E5 (RXR), were kindly provided by Dr. Venuti
(Regina Elena Cancer Institute of Roma). E5 and
KGFR transcript levels were analyzed by RT-PCR
and we found that the induced expression of E5
under the control of dexamethasone-inducible
pMSG promoter was able to down-modulate KGFR
mRNA. According to this KGFR modulation, by
quantitative immunofluorescence we observed that,
while treatment with KGF of control cells could
trigger, as expected from our previous studies,
HaCaT cell differentiation, testified by an increase of
the expression of the early differentiation marker
K1 and by keratinocyte stratification, the induction
of E5 expression appeared to abolish the KGF differentiative
effects. In addition, although HaCaT E5
cells appeared to grow more rapidly than HaCaT
control cells transfected with the empty vectors, the
proliferative response to KGF, assessed by Ki67 pos-