Download The involvement of endoplasmic reticulum stress in FGFR3

The Involvement of endoplasmic reticulum stress in FGFR3-related chondrodysplasias
+1Sato, R; 1Takahashi, M; 2Tsugawa, K; 2Miyake, M; 2Ito, T; 2Oyadomari, S; 1Yasui, N
+1Department of Orthopedics, The University of Tokushima, Tokushima, Japan
2
Department of division of molecular biology, Institute for genome research, The University of Tokushima, Tokushima, Japan
[email protected]
INTRODUCTION
Point mutations of the Fibroblast Growth Factor Receptor 3 Gene
are known to cause a variety of chondrodysplasias, ranging from
hypochondroplasia (mild dwarfism) to thanatophoric dysplasia (severe
dwarfism). Constitutive activation of the Jak/STAT-1 and MAPK
pathway by mutant FGFR3 has been shown, but the severity of
phenotype is not completely consistent with the activity of these
pathways.
Newly synthesized secretory and membrane proteins are folded
and matured in the lumen of the endoplasmic reticulum (ER). Various
physiological and pathological conditions perturb protein folding in the
ER lumen and lead to the accumulation of unfolded proteins, a cellular
condition referred to as ER stress. To deal with ER stress, cells activate
the unfolded protein response (UPR), involving the induction of
molecular chaperones, translational attenuation, and ER-associated
degradation(Fig. 1). When the UPR is perturbed or not sufficient to
restore normal ER function, apoptosis is induced, which is implicated in
the pathophysiology of several diseases. Accumulating evidence
indicates that the synthesis of mutant proteins such as collagen Ⅰ ,
collagenⅡ, collagenⅩ, COMP and matrilin3 can trigger ER stress.
In this study, we examined whether mutant FGFR3 proteins cause
ER stress.
Detection of induction of ER stress using luciferase assay
Human embryonic kidney (HEK) 293 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) contained 10% fetal
bovine serum (FBS). HEK293 cells were co-transfected with the ER
stress reporter plasmid and plasmids expressing mutant FGFR3 and
EGFP using polyethyleneimine by reverse-transfection method in 96well plate. Luciferase assay was performed 48 after transfection using
luciferase assay kit (ONE-Glo Promega) and determined by a plate
reader (Envision PerkinElmer).
Luciferase signal was normalized by EGFP fluorescence to correct for
transfection efficiency.
RESULT
Luciferase signals in cells transfected with R200C, R248C,
N540K, N540T, K650M, K650E, X807C and X807R mutant FGFR3
genes, increased compared to that of wild type FGFR3 (Fig. 3).
Fig 3. Induction of ER stress response by mutant FGFR3
Fig 1. Unfolded protein response
METHOD
Construction of FGFR3 expression plasmids and ER stress reporter
plasmid
The plasmid containing wild type FGFR3 gene was kindly gifted
from Tanaka (Okayama University). The full-length cDNAs of all
reported mutant FGFR3 genes were amplified with mutagenesis PCR
(Fig. 2). PCR fragments of mutant FGFR3 were subcloned into the
Hind Ⅲ site of pcDNA3 expression vector The ER stress reporter
contains three tandem copies of unfolded protein response elements
(UPRE), which had the binding sites of XBP1 induced by ER stress, was
subcloned into the NheⅠand Bgl Ⅱ sites of pGL-4.29 vector.
DISCUSSION
Membrane proteins, such as FGFR3, is necessary to be folded in
their proper tertiary structure in the ER to work functionally,
improper protein folding (misfolding) due to mutation can leads to the
accumulation of unfolded proteins in the ER and might trigger apoptosis.
It is generally accepted that FGFR3 is a negative regulator of
chondrogenesis and that constitutive activation of FGFR3 signaling is
the cause of chondrodysplasias. This study indicated that ER stress was
induced by FGFR3 mutants, might lead to chondrodysplasia inducing
an apoptosis of chondrocytes (Fig. 4). Further molecular elucidation of
role of ER stress in chondrodysplasia propose a new treatment approach
using “chemical chaperone”, that can stabilize various proteins against
misfolding.
Fig 4. Hypothesis of ER stress-induced apoptosis in FGFR3 –related
chondrodysplasias
Fig 2. Point mutations of FGFR3 gene resulting in a variety of
chondrodysplasias
SIGNIFICANCE
This study is the first report that provide the new insight into
molecular pathogenesis of chondrodysplasia due to mutant FGFR3genes
besides the hypothesis that activation of FGFR3 signaling contributes to
chondrodysplasias.
Poster No. 1717 • ORS 2012 Annual Meeting