Insulin-like growth factor-1 rescues the mutated FGF receptor 3 (G380R) expressing ATDC5 cells from apoptosis through phosphatidylinositol 3-kinase and MAPK.
An activated mutation in the FGFR3 gene causes ACH. To examine the effects of IGF-1, which is an important mediator of GH, on apoptosis, we analyzed a chondrogenic cell line expressing the FGFR3 mutants. Our findings that IGF-1 prevented the apoptosis through PI3K and MAPK pathways may explain how GH treatment improves the disturbed bone growth in ACH.


BACKGROUND
Achondroplasia (ACH), which is caused by a point mutation of the fibroblast growth factor receptor 3 (FGFR3) gene in the transmembrane domain (G380R), is one of the most common genetic forms of dwarfism. Recently, using a chondrogenic cell line, ATDC5, we have showed that the constitutively active FGFR3 mutants induced an apoptosis of chondrocytes. We have also reported that growth hormone (GH) treatment increased the growth rate in achondroplasia in parallel with the increment of serum levels of insulin-like growth factor (IGF)-1, suggesting an important role of IGF-1 in skeletal development. In this study, to clarify the mechanism by which GH treatment improved the phenotype of ACH patients, we examined the possible effects of IGF-1 on an apoptosis induced by FGFR3 mutant in ATDC5.


METHODS
Using adenovirus vector, wildtype or mutant FGFR3 (G380R) was introduced into ATDC5. Analysis of apoptosis was estimated by TUNEL assay. Expression levels of apoptosis-related genes and activation of signaling molecules were analyzed by immunoblot.


RESULTS
MTT assay showed that the cell number was reduced in ATDC5 cells expressing the mutant FGFR3 (G380R; ATDC5-mtR3 cells), suggesting that ATDC5-mtR3 cells might fall into apoptosis. IGF-1, which is an important mediator of GH, restored cell proliferation and reduced apoptosis in ATDC5-mtR3 cells. IGF-1 also decreased the ratio of Bax/Bcl-2 in the cells. To investigate which signaling cascade is responsible for antiapoptotic effects of IGF-1, we examined the role of phosphatidylinositol 3-kinase (PI3K) and MAPK in ATDC5-mtR3 cells. Specific inhibitors of PI3K or MAPK blocked the antiapoptotic effects of IGF-1 in ATDC5-mtR3 cells.


CONCLUSIONS
Our findings, showing IGF-1 prevents the apoptosis induced by FGFR3 mutation through the PI3K pathway and MAPK pathway, explain the mechanisms by which GH treatment improves the disturbed bone growth in ACH.
Koike
Mio
M
Department of Pediatrics, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Yamanaka
Yoshitaka
Y
Inoue
Masaru
M
Tanaka
Hiroyuki
H
Nishimura
Riko
R
Seino
Yoshiki
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001973:Bromodeoxyuridine; D002455:Cell Division; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics* / Q000378:metabolism*
D017468:Receptors, Fibroblast Growth Factor; D007334:Insulin-Like Growth Factor I; D019869:Phosphatidylinositol 3-Kinases; C496365:FGFR3 protein, human; C496366:Fgfr3 protein, mouse; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3; D020928:Mitogen-Activated Protein Kinases; D001973:Bromodeoxyuridine