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. 2022 Oct;12(10):1717-1728.
doi: 10.1002/2211-5463.13483. Epub 2022 Sep 14.

Mutation of the galectin-3 glycan-binding domain (Lgals3-R200S) enhances cortical bone expansion in male mice and trabecular bone mass in female mice

Kevin A Maupin  1 , Cassandra R Diegel  1 , Payton D Stevens  1 , Daniel Dick  1 ; VAI Vivarium and Transgenic Core  1 ; Bart O Williams  1
Affiliations

Mutation of the galectin-3 glycan-binding domain (Lgals3-R200S) enhances cortical bone expansion in male mice and trabecular bone mass in female mice

Kevin A Maupin et al. FEBS Open Bio. 2022 Oct.

Abstract

We previously observed that genomic loss of galectin-3 (Gal-3; encoded by Lgals3) in mice has a significant protective effect on age-related bone loss. Gal-3 has both intracellular and extracellular functionality, and we wanted to assess whether the affect we observed in the Lgals3 knockout (KO) mice could be attributed to the ability of Gal-3 to bind glycoproteins. Mutation of a highly conserved arginine to a serine in human Gal-3 (LGALS3-R186S) blocks glycan binding and secretion. We generated mice with the equivalent mutation (Lgals3-R200S) and observed a subsequent reduction in Gal-3 secretion from mouse embryonic fibroblasts and in circulating blood. When examining bone structure in aged mice, we noticed some similarities to the Lgals3-KO mice and some differences. First, we observed greater bone mass in Lgals3-R200S mutant mice, as was previously observed in Lgals3-KO mice. Like Lgals3-KO mice, significantly increased trabecular bone mass was only observed in female Lgals3-R200S mice. These results suggest that the greater bone mass observed is driven by the loss of extracellular Gal-3 functionality. However, the results from our cortical bone expansion data showed a sex-dependent difference, with only male Lgals3-KO mice having an increased response, contrasting with our earlier study. These notable sex differences suggest a potential role for sex hormones, most likely androgen signaling, being involved. In summary, our results suggest that targeting extracellular Gal-3 function may be a suitable treatment for age-related loss of bone mass.

Keywords: CRISPR/Cas9; bone μCT; galectin; genetic animal models; sexual dimorphism.

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Conflict of interest statement

BOW has received a sponsored research award from Janssen Pharmaceuticals and is a member of the scientific advisory board and a shareholder of Surrozen. Neither of these are directly related to the work in this manuscript.

Figures

Fig. 1
Fig. 1
Generation of Lgals3‐R200S knock‐in allele by CRISPR/Cas9. (A) Comparison of human and mouse Gal‐3 amino acid sequence and identification of an essential arginine to mutate to generate the glycan‐binding deficient Gal‐3. (B) The target sequence of the Lgals3 gene showing PAM and sgRNA binding site. Red letters indicate nucleotides to be mutated following homologous recombination of the single‐stranded oligonucleotide (ssODN) template. Sanger sequencing confirmation of incorporation of mutations. (C) PCR mediated identification of mice with wild‐type and Lgals3‐R200S alleles. M, molecular weight ladder; MUT, mutant; WT, wild‐type. Expected amplified product sizes are 483 bp for wild‐type and 131 and 586 bp for the R200S knock‐in allele. (D) Plasma serum levels of Gal‐3 in 36‐week‐old WT (+/+) female n = 10 and male n = 12, HET (KI/+) female n = 15 and male n = 11, and MUT (KI/KI) female n = 15 and male n = 13 animals. The samples were run in duplicate and the assay was run once. Values are expressed as mean ± SEM (n = 9–15). Dunnett's post hoc analysis adjusted P‐values compared to wild‐type; bold values highlight *P < 0.05, **P < 0.01, ***P < 0.001. (E) Immunoblot of Gal‐3 and vinculin on the cell surface of mouse embryonic cells isolated from WT (+/+), HET (KI/+), and MUT (KI/KI) embryos. Two biological replicates were evaluated per genotype and the assay was run once. (F) Gal‐3 immunocytochemistry of permeabilized MEFs from Lgals3‐R200SKI/KI and WT mice. Gal‐3 is present throughout the cytosol and nucleus in both conditions. Gal‐3 protein levels are overall reduced by approximately 30%. Scale bar = 100 μm. One biological sample per genotype and five technical slide replicates per genotype were evaluated, with a total of n = 31 WT MEF cells and n = 40 Lgals3‐R200S mutant MEF cells. Quantification of Gal‐3 staining intensity is indicated on the right, data is mean ± SEM. Asterisks indicate a significant difference, *P < 0.05.
Fig. 2
Fig. 2
Enhanced trabecular bone mass at 36 weeks in Lgals3‐R200S females. (A) Volume of interest for L3 trabecular bone measurements, as assessed by μCT. (B) Representative trabecular bone volumes of mice in each of the treatment groups. Images were selected based upon median bone volume fraction within each sex:genotype. Scatter plots for bone volume fraction (BV/TV; C), trabecular (Tb.) thickness (D), Tb. Number (E), and Tb. Spacing (F). The following number of samples were analyzed: Wildtype (+/+) female n = 9 and male n = 9, Het (KI/+) female n = 15 and male n = 11, and Mut (KI/KI) female n = 14 and male n = 13. Statistical significance determined after Holm‐Sidak post‐hoc analyses of 2‐way ANOVAs. Asterisks indicate a significant difference, *P < 0.05.
Fig. 3
Fig. 3
Enhanced cortical bone mass at 36 weeks in Lgals3‐R200S males. (A) Region of interest for cortical bone measurements, as assessed by μCT. (B) Representative cortical bone volumes of mice in each of the treatment groups. Images were selected based upon median Total Area measurements within each sex:genotype (C) Diagram showing areas represented by total area, bone area, and marrow area. Scatter plots from μCT data for total area (D), bone area (E), marrow area (F), and mean polar moment of inertia (MMI Polar; G). Scatterplots from derived tissue mechanical properties were measured by 4‐point bending, including max stress (H) and elastic modulus (I). The following number of samples were analyzed: Wildtype (+/+) female n = 9 and male n = 9, Het (KI/+) female n = 15 and male n = 11, and Mut (KI/KI) female n = 14 and male n = 13. Statistical significance determined after Holm‐Sidak post hoc analyses of 2‐way ANOVAs. Asterisks indicate a significant difference, *P < 0.05.
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