Elsevier

Bone

Volume 41, Issue 1, July 2007, Pages 13-18
Bone

PTH has the potential to rescue disturbed bone growth in achondroplasia

https://doi.org/10.1016/j.bone.2007.02.028Get rights and content

Abstract

Introduction

Achondroplasia (Ach), the most common form of short-limb short stature, and related disorders are caused by constitutively active point mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. Recent studies have provided a large body of evidence for the role of the proliferation and differentiation of chondrocytes in these disorders. Furthermore, a G380R mutation in FGFR3 (FGFR3Ach), which results in achondroplasia, induces apoptosis in the chondrogenic cell line ATDC5. This is associated with a decrease in the expression of PTHrP, which shares the same receptor with PTH, and it is significant that PTHrP rescues these cells from apoptosis.

Methods

Fetuses derived from transgenic mice expressing FGFR3Ach under the control of the type II collagen promoter (AchTG) or from wild-type mice were obtained on the 15th day of pregnancy. The femurs were collected from these specimens and cultured for 4 days with PTH. The effects of PTH treatment were then determined by morphometric and histological analyses, in situ hybridization of type X collagen mRNA, and the TUNEL assay.

Results

AchTG femurs showed suppressed growth compared with wild type (0.29 ± 0.10 mm vs. 0.46 ± 0.06 mm, respectively; p < 0.05), particularly in cartilage. PTH treatments improved the growth velocity in the femurs of the AchTG (0.50 ± 0.06 mm; p < 0.01 vs. control). This was associated with the inhibition of both differentiation and apoptosis in chondrocytes.

Conclusions

Our data suggest that PTH inhibits differentiation and apoptosis in chondrocytes and improves bone growth. These effects thus counterbalance the effects of FGFR3 mutations. PTH therefore is a potential therapeutic agent for achondroplasia.

Introduction

Fibroblast growth factor receptor 3 (FGFR3) has been identified as a critical regulator of enchondral bone growth [8]. Mutations in the coding sequence of the FGFR3 gene have also been identified as the cause of achondroplasia (Ach) [17], [20], thanatophoric dysplasia (TD) [23], [24], and related disorders [4], [5], [22], [25]. Ach is the most common form of genetic short stature and exhibits characteristic phenotypes of rhizomeric short limbs, relative macrocephaly, and exaggerated lumbar lordosis. TD, a common but more severe and lethal skeletal dysplasia during the neonatal period, consists of two phenotypes distinguishable by radiological and other clinical criteria and referred to as TD types I and II (TDI and TDII) [24].

FGFR3-deficient mice exhibit skeletal overgrowth, suggesting that FGFR3 is a negative regulator of long bone growth during enchondral formation [7], [9]. Several in vitro studies have also demonstrated that mutations in FGFR3, which are responsible for Ach and TD, cause ligand-independent activation of the receptor [27], [28]. The effects of activated FGFR3 signals on bone growth in Ach have been studied recently in vivo using transgenic [14] and cDNA knock-in [6], [26] models. Mutations in FGFR3 were found to cause short stature and to result in features that mimicked human Ach. These experiments also demonstrated that a gain of FGFR3 function causes Ach, leading to the inhibition of chondrocyte proliferation.

In the growth plate, parathyroid hormone (PTH)-related peptide (PTHrP) is expressed in the perichondrium and in proliferating chondrocytes, whereas its receptor is found primarily in the proximal prehypertrophic layer [12]. Mice in which the pthrp gene is disrupted die immediately after birth and show skeletal dysplasia and short limbs due to accelerated chondrocyte differentiation [10], [13]. However, although transgenic mice that overexpress pthrp in proliferative chondrocytes also have shorter and thicker limbs, this is a result of decelerated terminal chondrocyte differentiation and delayed mineralization [2], [29].

In a recent study, we showed that both Ach- and TDII-type mutant FGFR3 induce apoptotic changes in ATDC5 cells, a mouse chondrogenic cell line [11]. We also found that the expression of pthrp was markedly suppressed in ATDC5 cells expressing Ach or TDII mutant FGFR3, and that treatment with PTHrP, which functions in chondrocyte differentiation through the PTH/PTHrP receptor, blocks the apoptotic response in these cells [30]. These findings indicate that the acceleration of FGFR3 signaling, caused by these ACH- or TDII-inducing mutations, induces apoptosis in chondrocytes via the suppression of PTHrP [31].

In the present study, we evaluate the effects of PTH, which shares the same receptor with PTHrP and has been available for clinical usage, upon bone growth suppression caused by mutant FGFR3 in mouse organ cultures.

Section snippets

Organ cultures

The generation of the Achondroplasia mouse model (AchTG), which expresses activated FGFR3 in the growth plate under the control of col2a1 promoter and enhancer sequences, has been described previously [14]. To obtain fetuses from AchTG and wild-type mice (WT), WT female mice were mated with AchTG male mice. On day 15 of pregnancy, the bilateral femurs were dissected aseptically from the fetuses. Fetuses whose femurs were significantly different from the average expected size were discarded. The

Morphometric analysis

The total longitudinal length of each mouse femur was found to increase time-dependently during the 4-day organ culture. AchTG femurs had a significantly reduced longitudinal bone growth compared with WT (0.29 ± 0.10 mm and 0.46 ± 0.06 mm, respectively; p < 0.05). rhPTH treatment, however, restored the bone growth of AchTG femurs to levels comparable to those of WT control (0.50 ± 0.06 mm; p < 0.01 vs. control), whereas in WT there was no appreciable change (0.55 ± 0.09 mm; n.s.) (Fig. 1B). AchTG femurs

Discussion

Achondroplasia is the most common genetic form of rhizomeric short stature, and many patients have been diagnosed with this condition throughout history. Some surgical treatments are available for complications such as spinal stenosis, hydrocephalus, and short stature; recently we have used growth hormone therapy to treat achondroplastic patients [18], [19], [21]. However, these individuals never reach the normal height range, which indicates the limitations of such therapies. Hence, it is

Acknowledgments

We thank Dr. David M. Ornitz for permission to use ColII-FGFR3Ach transgenic mice, and Eli Lilly & Company for sharing with us recombinant human PTH(1–34).

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