Page 68 - 77_01

FIBROBLAST
GROWTH
FACTOR
RECEPTOR
3….

1. INTRODUCTION

Achondroplasia
is
the
most
common
form
of
dwarfism
in
humans.
This

disorder
is
characterized
by
a
mutation
in
the
gene
that
encodes
for
the
FGFR3

receptor,
being
the
mutation
in
97%
of
the
patients
a
Glycine
to
Arginine

substitution
at
position
380.

FGFR3
is
a
regulator
of
endochondral
bone
growth
and
its
mutation
causes

gain
of
function
disturbing
chondrocyte
proliferation
and
differentiation
during

endochondral
ossification
process.
As
a
result,
the
rate
of
cartilage
template

formation
and
turnover
required
for
bone
elongation
are
dramatically
reduced
(1).

At
the
molecular
level,
sustained
activation
of
mutant
FGFR3
stimulates

several
intracellular
signalling
pathways,
including
extracellular
signal-­-regulated

kinases
1
and
2
(ERK1/2)
cascade
and
signal
transducer
and
activators
of

transcription
(STAT-­-1)
pathway,
that
account
for
the
critical
changes
in

chondrocyte
proliferation
(2,
3),
differentiation
(4)
and
extracellular
matrix

homeostasis
(5).

Potential
therapeutic
approaches
have
been
proposed
for
achondroplasia

treatment.
The
main
strategies
are
based
on
blocking
FGFR3
activation
(6,
7)
or

interfering
with
the
pathways
that
modulate
the
downstream
propagation
of

FGFR3
signals
(5,
8).
However,
despite
the
efforts,
achondroplasia
remains
as
an

orphan
pathology
with
no
pharmacological
treatment
so
far.

RNA
interference
is
a
posttranscriptional
process
triggered
by
the

introduction
of
double-­-stranded
RNA,
which
leads
to
gene
silencing
in
a
sequence-­-
specific
manner.
This
is
one
of
the
most
exciting
new
findings
in
functional

genomics
of
the
past
decade
and
its
potential
for
experimental
and
therapeutic

purposes
is
currently
under
investigation
(9).

In
this
work,
we
transfected
immortalized
human
chondrocytes
carrying

the
heterozygous
achondroplasia
mutation
(G380R)
with
three
different
small

interfering
RNAs
(siRNAs)
and
analyzed
their
effect
on
FGFR3
expression
and

prolonged
signalling
to
evaluate
the
therapeutical
potential
of
siRNAs
in

achondroplasia
treatment.

2. MATERIALS AND METHODS

2.1.
Cells

Immortalized
human
chondrocytes
carrying
the
heterozygous

achondroplasia
mutation
(G380R)
were
generated
and
characterized
by
Dr.

Laurence
Legeai-­-Mallet
(10).
Cells
were
cultured
in
Dulbecco´s
modified
Eagle´s

medium
(Invitrogen,
Paisley,
UK)
supplemented
with
10%
fetal
calf
serum,
1%

penicillin/streptomicyn
and
500
ng/ml
geneticin
(all
obtained
from
Invitrogen)

and
were
incubated
at
37
ºC
with
5%
CO2.

2.2.
Small
interfering
RNA
design
and
Transfection

Using
siRNA
design
software,
three
siRNA
duplexes
targeting
FGFR3
were

obtained
from
Ambion
(Applied
Biosystems,
Foster
City,
CA,
USA).
The
three

sequences
used
are
shown
in
Table
1.
A
scrambled
siRNA
without
sequence

homology
to
any
known
human
gene
was
also
obtained
from
Ambion
and
used
as
a

5