Yamanaka Yoshitaka, Tanaka Hiroyuki, Koike Mio, Nishimura Riko, Seino Yoshiki
Department of Pediatrics, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
J Bone Miner Res. 2003 Aug;18(8):1395-403. doi: 10.1359/jbmr.2003.18.8.1395.
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.
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.
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.
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.
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.
FGFR3基因的激活突变导致软骨发育不全（ACH）。在一个软骨形成细胞系中分析了FGFR3突变体对细胞凋亡的影响。ACH软骨细胞表现出明显的凋亡，同时甲状旁腺激素相关蛋白（PTHrP）表达下调。用PTHrP替代物挽救这些细胞意味着对这种疾病有潜在的治疗方法。
软骨发育不全（ACH）是短肢侏儒症最常见的形式，其相关疾病由组成型激活的点突变FGFR3引起。最近的研究为这些疾病中软骨细胞的增殖和分化提供了大量证据。然而，关于FGFR3突变体对软骨细胞凋亡可能的影响知之甚少。
将导致ACH和致死性侏儒症（TD，一种更严重的新生儿致死形式）的突变FGFR3基因导入软骨形成细胞系ATDC5。通过TUNEL检测、DNA梯状条带分析和线粒体膜电位的荧光测量来评估细胞凋亡。通过Northern印迹或免疫印迹分析甲状旁腺激素相关肽（PTHrP）和凋亡相关基因的表达水平。
将这些突变的FGFR3导入ATDC5细胞会下调PTHrP表达并诱导细胞凋亡，同时Bcl-2表达减少。重要的是，PTHrP替代可防止表达ACH突变体的ATDC5细胞发生凋亡变化和Bcl-2表达减少。与疾病的严重程度和FGFR3的活性平行，与表达ACH突变体的细胞相比，表达TD突变体FGFR3的ATDC5细胞PTHrP和Bcl-2表达较少，并诱导更明显的凋亡变化。此外，Bcl-2的过表达抑制了凋亡变化，表明突变的FGFR3至少部分通过降低Bcl-2表达导致细胞凋亡，而Bcl-2表达降低似乎在PTHrP的下游。
我们的数据表明，由FGFR3突变体介导的信号级联过度激活会抑制PTHrP和Bcl-2的表达，导致软骨细胞凋亡，可能导致短肢侏儒症。用PTHrP替代物挽救这些细胞意味着对这种疾病有潜在的治疗方法。
