PTHrP rescues ATDC5 cells from apoptosis induced by FGF receptor 3 mutation.
An activation mutation in the FGFR3 gene causes ACH. The effects of the FGFR3 mutants on apoptosis were analyzed in a chondrogenic cell line. ACH chondrocytes exhibited marked apoptotic with downregulation of PTHrP expression. Rescue of these cells by PTHrP replacement implies a potential therapy for this disorder.


BACKGROUND
Achondroplasia (ACH), the most common form of short-limb dwarfism, and its related disorders are caused by constitutively activated point-mutated FGFR3. Recent studies have provided a large body of evidence on chondrocyte proliferation and differentiation in these disorders. However, little is known about the possible effects of the FGFR3 mutants on apoptosis of chondrocytes.


METHODS
The mutant FGFR3 genes causing ACH and thanatophoric dysplasia (TD), which is a more severe neonatal lethal form, were introduced into a chondrogenic cell line, ATDC5. Analysis of apoptosis was estimated by TUNEL assay, DNA laddering, and fluorescent measurement of mitochondrial membrane potential. Expression levels of parathyroid hormone-related peptide (PTHrP) and apoptosis-related genes were analyzed by Northern blot or immunoblot.


RESULTS
The introduction of these mutated FGFR3s into ATDC5 cells downregulated PTHrP expression and induced apoptosis with reduction of Bcl-2 expression. Importantly, replacement of PTHrP prevented the apoptotic changes and reduction of Bcl-2 expression in ATDC5 cells expressing the ACH mutant. In parallel with the severity of disease and the activity of FGFR3, ATDC5 cells expressing TD-mutant FGFR3 showed less expression of PTHrP and Bcl-2 and induced more remarkable apoptotic changes compared with ACH-mutant expressing cells. Furthermore, overexpression of Bcl-2 inhibited apoptotic changes, suggesting that the mutant FGFR3 caused apoptosis, at least in part, through reduction of Bcl-2 expression, which seems to be downstream of PTHrP.


CONCLUSIONS
Our data suggest that excessive activation of signaling cascades mediated by the FGFR3 mutants inhibits the expression of PTHrP and Bcl-2, resulting in apoptosis of chondrocytes, possibly leading to short-limb dwarfism. Rescue of these cells by PTHrP replacement implies a potential therapy for this disorder.
Yamanaka
Yoshitaka
Y
Department of Pediatrics, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Tanaka
Hiroyuki
H
Koike
Mio
M
Nishimura
Riko
R
Seino
Yoshiki
Y
D000130:Achondroplasia / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis / Q000502:physiology*; D002455:Cell Division; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics; D004789:Enzyme Activation; D019254:Genes, bcl-2 / Q000235:genetics / Q000502:physiology; D006801:Humans; D020287:In Situ Nick-End Labeling; D009154:Mutation / Q000235:genetics*; D010012:Osteogenesis; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics* / Q000378:metabolism*
D004272:DNA, Mitochondrial; D044162:Parathyroid Hormone-Related Protein; D017468:Receptors, Fibroblast Growth Factor; C496365:FGFR3 protein, human; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3