Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth.

Leia C Shuhaibar,Thibault Horville,Tracy F Uliasz,Jeremy R Egbert,Emilie Dambroise,Nabil Kaci,Richard E Honkanen,Mark R Swingle,Martin Biosse Duplan,Laurence Legeai-Mallet,Laurinda A Jaffe,Léa Loisay,Giulia Vigone

doi:10.1172/jci.insight.141426

Abstract

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP in chondrocytes and severe short stature, causing achondroplasia (ACH) and acromesomelic dysplasia, type Maroteaux, respectively. Previously, we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+). Here, because FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein, we tested whether a phosphatase inhibitor (LB-100) could enhance BMN-111–stimulated bone growth in ACH. Measurements of cGMP production in chondrocytes of living tibias, and of NPR2 phosphorylation in primary chondrocytes, showed that LB-100 counteracted FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, the combination of BMN-111 and LB-100 increased bone length and cartilage area, restored chondrocyte terminal differentiation, and increased the proliferative growth plate area, more than BMN-111 alone. The combination treatment also reduced the abnormal elevation of MAP kinase activity in the growth plate of Fgfr3Y367C/+ mice and improved the skull base anomalies. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.

Highlights

Achondroplasia (ACH), the most common form of dwarfism, is due to a gain-of-function mutation in the fibroblast growth factor receptor type 3 (FGFR3) gene [1, 2]
Perfusion of A-type natriuretic peptide (ANP), which activates the NPR1 guanylyl cyclase, or perfusion of a nitric oxide donor (DEA/NO), which activates soluble guanylyl cyclases, did not increase cGMP (Supplemental Figure 2), showing that — among the several mammalian guanylyl cyclases — only natriuretic peptide receptor 2 (NPR2) is active in the chondrocytes of the mouse growth plate
The findings described here identify the PPP-family phosphatase inhibitor LB-100 as a stimulator of bone growth when used in combination with this CNP analog to stimulate production of cGMP by NPR2

Summary

Introduction

Achondroplasia (ACH), the most common form of dwarfism, is due to a gain-of-function mutation in the fibroblast growth factor receptor type 3 (FGFR3) gene [1, 2]. FGFR3 is expressed in growth plate cartilage and bone, which explains the bone anomalies observed in patients with ACH. In the absence of Fgfr, the most prominent phenotype of the mice is overgrowth, indicating that FGFR3 is a negative regulator of bone growth [4, 5]. Mice expressing a Fgfr3-activating mutation develop dwarfism and have reduced linear growth and impaired endochondral ossification, with reduced chondrocyte proliferation and reduced hypertrophic differentiation [6,7,8,9,10]. A complex intracellular network of signals, including FGFR3, mediates this skeletal phenotype. Activating mutations in FGFR3 lead to upregulated FGFR3 protein [11] and to increased activity of several downstream intracellular signaling pathways, including MAPK, PI3K/AKT, PLCγ, and STATs [12]

Methods

Results

Conclusion