Meclozine Facilitates Proliferation and Differentiation of Chondrocytes by Attenuating Abnormally Activated FGFR3 Signaling in Achondroplasia

Masaki Matsushita,Hiroshi Kaneko,Hiroshi Kitoh,Akio Masuda,Naoki Ishiguro,Kenichi Mishima,Mikako Ito,Bisei Ohkawara,Kinji Ohno,Oreste Gualillo

doi:10.1371/journal.pone.0081569

Abstract

Achondroplasia (ACH) is one of the most common skeletal dysplasias with short stature caused by gain-of-function mutations in FGFR3 encoding the fibroblast growth factor receptor 3. We used the drug repositioning strategy to identify an FDA-approved drug that suppresses abnormally activated FGFR3 signaling in ACH. We found that meclozine, an anti-histamine drug that has long been used for motion sickness, facilitates chondrocyte proliferation and mitigates loss of extracellular matrix in FGF2-treated rat chondrosarcoma (RCS) cells. Meclozine also ameliorated abnormally suppressed proliferation of human chondrosarcoma (HCS-2/8) cells that were infected with lentivirus expressing constitutively active mutants of FGFR3-K650E causing thanatophoric dysplasia, FGFR3-K650M causing SADDAN, and FGFR3-G380R causing ACH. Similarly, meclozine alleviated abnormally suppressed differentiation of ATDC5 chondrogenic cells expressing FGFR3-K650E and -G380R in micromass culture. We also confirmed that meclozine alleviates FGF2-mediated longitudinal growth inhibition of embryonic tibia in bone explant culture. Interestingly, meclozine enhanced growth of embryonic tibia in explant culture even in the absence of FGF2 treatment. Analyses of intracellular FGFR3 signaling disclosed that meclozine downregulates phosphorylation of ERK but not of MEK in FGF2-treated RCS cells. Similarly, meclozine enhanced proliferation of RCS cells expressing constitutively active mutants of MEK and RAF but not of ERK, which suggests that meclozine downregulates the FGFR3 signaling by possibly attenuating ERK phosphorylation. We used the C-natriuretic peptide (CNP) as a potent inhibitor of the FGFR3 signaling throughout our experiments, and found that meclozine was as efficient as CNP in attenuating the abnormal FGFR3 signaling. We propose that meclozine is a potential therapeutic agent for treating ACH and other FGFR3-related skeletal dysplasias.

Highlights

Achondroplasia (ACH) is one of the most common skeletal dysplasias with an incidence of one in 16,000 to 26,000 live births [1]
As rat chondrosarcoma (RCS) chondrocytic cells express high levels of fibroblast growth factor receptor 3 (FGFR3), exogenous administration of FGF2 readily recapitulates cellular processes occurring in FGFR3-related skeletal dysplasias [22]
Among the 1,186 FDA-approved drugs that have favorable or validated pharmacokinetic and toxicological profiles, we identified meclozine as a novel inhibitor of the FGFR3 signaling, which can potentially be applied to clinical practice for short stature in FGFR3-related skeletal dysplasias

Summary

Introduction

Achondroplasia (ACH) is one of the most common skeletal dysplasias with an incidence of one in 16,000 to 26,000 live births [1]. Gain-of-function mutations of FGFR3 cause several short-limbed skeletal dysplasias such as hypochondroplasia (HCH) [8], severe ACH with developmental delay and acanthosis nigricans (SADDAN) [9], and thanatophoric dysplasia (TD) types I and II [10]. Loss of function mutations in FGFR3 lead to the CATSHL syndrome in humans, which is characterized by overgrowth of the skeleton including camptodactyly, tall stature, scoliosis, and hearing loss [11], as well as spider lamb syndrome in sheep [12]. These findings indicate that the FGFR3 signaling functions as a negative regulator of endochondral bone growth

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