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LGR4 deficiency results in delayed puberty through impaired Wnt/β-catenin signaling
Alessandra Mancini, … , Leonardo Guasti, Leo Dunkel
Alessandra Mancini, … , Leonardo Guasti, Leo Dunkel
Published June 4, 2020
Citation Information: JCI Insight. 2020;5(11):e133434. https://doi.org/10.1172/jci.insight.133434.
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Research Article Endocrinology Reproductive biology

LGR4 deficiency results in delayed puberty through impaired Wnt/β-catenin signaling

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Abstract

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

Authors

Alessandra Mancini, Sasha R. Howard, Federica Marelli, Claudia P. Cabrera, Michael R. Barnes, Michael J.E. Sternberg, Morgane Leprovots, Irene Hadjidemetriou, Elena Monti, Alessia David, Karoliina Wehkalampi, Roberto Oleari, Antonella Lettieri, Valeria Vezzoli, Gilbert Vassart, Anna Cariboni, Marco Bonomi, Marie Isabelle Garcia, Leonardo Guasti, Leo Dunkel

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Figure 3

LGR4 mutant receptors affect Wnt/β-catenin signaling owing to defects in protein production, trafficking, and protein turnover.

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LGR4 mutant receptors affect Wnt/β-catenin signaling owing to defects in...
(A) HEK293T were nontransfected (–) or transfected (+) with HA-hLGR4 plasmids (WT or mutants; 200 ng/well) and reporter vectors (TOP-Flash and Renilla [150 ng/well]). Signaling was activated with conditioned media treatment, and each transfection normalized by cotransfection with Renilla. HA-hLGR4 mutant receptors resulted in significant reduced luciferase activity. ****P < 0.0001, ***P = 0.004, *P = 0.0437; n = 4. (B) Western blot and densitometry analysis revealed reduced levels of LGR4 mutants. GAPDH was used as loading control. Molecular weight (KDa) of a protein standard is reported (left panel: WT vs. I96V **P = 0.0054; WT vs. G363C **P = 0.0039; WT vs. D844G **P = 0.0049); n = 3. (C) Representative plots and quantification of flow cytometry analysis of cell surface expression of WT and mutant LGR4 proteins expressed in HEK293T. Normalized median fluorescence intensity (nMFI) reveals reduced levels of mutant receptors at the plasma membrane compared with HA-hLGR4 WT (WT vs. p.G363C *P = 0.0228; WT vs. p.D844G *P = 0.0498). Control: HEK293T transfected with pcDNA3.1EGFP vector only. n = 3. (D) LGR4 mutants have a shorter half-life: transiently transfected HEK293T with HA-hLGR4 WT or mutant constructs treated with CHX (50 μg/mL) for different time periods (0, 3, 6, 9, and 12). Levels of LGR4 WT and mutant proteins were expressed relative to untreated LGR4 WT or mutant proteins (0 h); n = 4. Statistical analysis by 1-way ANOVA. Half-life was analyzed via a 1-phase decay equation, and degradation speed (K) compared between each mutant and WT protein using the extra sum-of-squares F test. Co, control medium; Rspo1, Rspondin-1.

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