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Phosphatase inhibition by LB-100 enhances BMN-111 stimulation of bone growth
Leia C. Shuhaibar, … , Laurinda A. Jaffe, Laurence Legeai-Mallet
Leia C. Shuhaibar, … , Laurinda A. Jaffe, Laurence Legeai-Mallet
Published May 10, 2021
Citation Information: JCI Insight. 2021;6(9):e141426. https://doi.org/10.1172/jci.insight.141426.
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Research Article Bone biology Therapeutics

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

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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.

Authors

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

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Figure 1

LB-100 counteracts the inactivation of NPR2 by FGF in growth plate chondrocytes of intact tibias from newborn mice.

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LB-100 counteracts the inactivation of NPR2 by FGF in growth plate chond...
(A) Confocal image of cGi500 fluorescence in chondrocytes in the columnar and prehypertrophic region of the tibia growth plate, as used for measurements of cGMP production. Scale bar: 100 μm. (B) cGMP increase in growth plate chondrocytes in response to perfusion of 0.1 μM CNP, and inhibition of the cGMP increase by pretreatment with 0.5 μg/mL FGF18 + 1 μg/mL heparin for 80 minutes. Control tibias were pretreated for 80 minutes with heparin only. The graph shows the mean ± SEM for 27 control tibias and 18 FGF-treated tibias. (C) Inhibitory effect of LB-100 on the activity of recombinant PPP1CA by LB-100 assayed using DiFMUP (diamonds) or [32P]-labeled histone (squares) as a substrate. Each point represents the mean ± SD (n = 3–4). IC50 values are provided in Table 1. (D) Confocal image of cGi500 fluorescence in growth plate chondrocytes after pretreatment with 10 μM LB-100 for 2 hours. No difference in morphology was seen compared with control tibias (A) without LB-100. Scale bar: 100 μm. (E and F) Effect of LB-100 (or cantharidin) preincubation on CNP-stimulated cGMP production in FGF-treated tibias. Tibias expressing cGi500 were preincubated with solutions with or without LB-100 for 60 minutes. FGF was then added, and 80 minutes later, tibias were placed in a perfusion slide for cGi500 imaging during CNP perfusion. (E) The CFP/YFP emission ratio as a function of time after CNP perfusion. (F) The CFP/YFP emission ratio at 15 minutes after CNP perfusion. Symbols indicate individual tibias (n = 5–27). For E and F, data are shown as mean ± SEM. Data were analyzed by 1-way ANOVA followed by the Holm-Sidak multiple comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

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