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M. R. Torrisi - Role of the keratinocyte growth factor receptor on the molecular and cellular alterations
itivity to identify cycling cells, was much less pronounced
in HaCaT cells expressing E5 compared to
the control cells. These results indicate that E5
expression down-modulates KGFR transcription
and the consequent KGF-induced proliferation and
differentation of human keratinocytes.
Since 16E5 is known to inhibit the EGFR endocytic
degradative pathway and to enhance receptor recycling,
we performed also a series of experiments to
investigate if the E5 protein would be able to interfere
with the two alternative KGFR endocytic pathways.
In fact, we have demonstrated that
FGF10/KGF2 induces receptor recycling, whereas
KGF/FGF7 stimulates receptor degradation
(Belleudi et al., 2007): since the recycling endocytic
pathway followed by KGFR upon FGF10 stimulation
correlates with the higher mitogenic activity
exerted by this ligand on epithelial cells compared to
KGF, suggesting that the two ligands may play different
functional roles through the regulation of the
receptor endocytic transport, our hypothesis was
that 16E5 would play a similar role in targeting
KGFR to the recycling route. To this aim, first we
performed transient transfection of HaCaT cells
with a pCIneo-HA E5 expression vector (kindly provided
by Dr. Venuti) and, 48 hours post-transfection,
we immunostained E5 protein with anti-HA antibodies.
Immunfluorescence and confocal analysis showed
that the E5 signal colocalized with the endoplasmic
reticulum marker calreticulin, as expected, while
only a minor colocalization was observed with the
marker of the Golgi complex giantin and no colocalization
was detected with the mitochondrial marker
MitoTracker. Next, to analyze if E5 expression
could affect KGFR endocytosis, we studied the intracellular
endocytic traffic followed by KGFR during
internalization induced by the two ligands KGF and
FGF10 in HaCaT E5/KGFR cotrasfected cells. In
particular, we focused our attention on the late steps
of KGFR internalization to search for a possible differential
sorting of receptors destined to recycling
or degradation in the presence of E5. The KGFR
endocytic intracellular transport was analyzed in
detail at immunofluorescence and confocal
microscopy and the intracellular endocytic structures
and compartments involved in the KGFR inter-
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nalization process induced by the ligands were identified
using the following markers: EEA1 for early
endosomes, LysoTracker for lysosomes and internalized
Transferrin for the recycling compartment.
HaCaT KGFR/E5 cotrasfected and HaCaT KGFR
transfected cells were treated with the ligands and
with the anti-KGFR antibody at 4°C and warmed for
1h at 37°C. Cells were also incubated in the presence
of LysoTracker to identify lysosomes or in the presence
of Tf-Tx to identify the juxtanuclear recycling
compartment. The results showed that, upon KGF
treatment of cells expressing E5, most of KGFRs
did not reach the lysosomes, as demonstrated by the
very low percentage of colocalization with
LysoTracker. In contrast, in agreement with our previous
results (Belleudi et al., 2007), the receptor
extensively colocalized with LysoTracker in cells
overexpressing KGFR alone. After FGF10 treatment,
the KGFR signal colocalized with internalized
Tf, but not with LysoTracker in both cotransfeted or
singly transfected cells. suggesting that E5 expression
does not inhibit receptor recycling to the plasma
membrane. Thus, E5 appears to interfere with the
KGF-induced KGFR transport to the degradative
pathway, but not with the FGF10-induced receptor
traffic through the recycling compartment (Belleudi
et al. in preparation).
Selected Publications
Belleudi F, Leone L, Nobili V, Raffa S,
Francescangeli F, Maggio M, Morrone S, Marchese
C, Torrisi MR. Keratinocyte growth factor receptor
ligands target the receptor to different intracellular
pathways. Traffic 2007, 8:1854-72.
Lotti LV, Rotolo S, Francescangeli F, Frati L,
Torrisi MR, Marchese C. AKT and MAPK signaling
in KGF-treated and UVB-exposed human epidermal
cells. J Cell Physiol. 2007, 212:633-42.
Cardinali G, Bolasco G, Aspite N, Lucania G, Lotti
LV, Torrisi MR, Picardo M. Melanosome transfer
promoted by keratinocyte growth factor in light and
dark skin derived keratinocytes. J Invest Dermatol.
2008, 128:558-67.