Ganglioside GD1a enhances osteogenesis by activating ERK1/2 in mesenchymal stem cells of Lmna mutant mice

Abstract

Mutations in Lmna usually cause a series of human disorders, such as premature aging syndrome (progeria) involving the skeletal system. Gangliosides are known to be involved in cell surface differentiation and proliferation of stem cells. However, the role of gangliosides in Lmna dysfunctional mesenchymal stem cells (MSCs) is unclear. Therefore, Ganglioside's role in osteogenesis of Lmna dysfunctional MSCs analyzed. As a result of the analysis, it was confirmed that the expression of ganglioside GD1a was significantly reduced in MSCs derived from Lmna^Dhe/+ mice and in MSCs subjected to Lamin A/C knockdown using siRNA. Osteogenesis-related bone morphogenetic protein-2 and Osteocalcin protein, and gene expression were significantly decreased due to Lmna dysfunction. A result of treating MSCs with Lmna dysfunction with ganglioside GD1a (3 μg/ml), significantly increased bone differentiation in ganglioside GD1a treatment to Lmna-mutated MSCs. In addition, the level of pERK1/2, related to bone differentiation mechanisms was significantly increased. Ganglioside GD1a was treated to Congenital progeria Lmna^Dhe/+ mice. As a result, femur bone volume in ganglioside GD1a-treated Lmna^Dhe/+ mice was more significantly increased than in the Lmna^Dhe/+ mice. Therefore, it was confirmed that the ganglioside GD1a plays an important role in enhancing osteogenic differentiation in MSC was a dysfunction of Lmna.

Keywords: GD1a; Lmna; aging; mesenchymal stem cells; osteogenesis.

Figure 1

Micro-array analysis of genes expression in Lmna^Dhe/+ mutant MSCs compared to normal MSCs. Gene expression was analyzed by a microarray using an antibody. (A) Antibody microarray assay (Ray L1000 Antibody Array) can analyze 1000 genes and has 15 domains such as inflammatory response, neurogenesis, RNA splicing, secretion, extracellular matrix, angiogenesis, cell cycle, apoptotic processes, cell differentiation, cell death, cell migration, cell proliferation, DNA repair, aging, and immune response. (B) We analyzed the differences in gene expression in Lmna^Dhe/+ mutant MSC compared to normal MSC. (C) Decreases of osteogenesis-related genes expression in Lmna^Dhe/+ mutant MSCs compared to normal MSCs.

Figure 2

Osteogenic analysis in Lmna dysfunction MSCs compared to normal MSCs. (A) Alizarin Red staining. (B) Expression of osteogenic genes in Lmna dysfunction MSCs compared with normal MSCs. (C) Expression of osteogenic proteins in Lmna dysfunction MSCs compared with normal MSCs. (D) Lamin A/C knock down by siRNA. and (E) Alizarin Red staining. (F) Expression of osteogenic proteins in Lmna dysfunction MSCs compared with normal MSCs. (G) Expression of osteogenic genes in Lmna dysfunction MSCs compared with normal MSCs. ^***p < 0.001 indicates a significant difference from the normal MSCs.

Figure 3

Ganglioside expression patterns in Lmna-dysfunction MSCs. (A) Ganglioside expression patterns in primary Lmna^Dhe/+ mutation MSCs. (B) Lamin A/C-knockdown MSCs by siRNA. ^***p < 0.001 indicates a significant difference from the normal MSCs.

Figure 4

Increases of osteogenesis and ERK1/2 activation in GD1a was-treated Lmna dysfunction MSCs. Cell viability (A) and osteogenesis (B) in Lmna^Dhe/+ mutation MSCs were treated with GD1a. (C) Alizarin Red staining in GD1a was-treated Lmna dysfunction MSCs. (D) Increases of osteogenic proteins and (E) osteogenic genes in GD1a was-treated Lmna dysfunction MSCs compared with Lmna dysfunction MSCs. (F) Primary Lmna^Dhe/+ mutation MSCs were isolated from Lmna^Dhe/+ mutation mouse. (G) Lamin A/C was knocked down in mouse MSCs using siRNA. (H) Phosphorylation of ERK1/2 in Lmna dysfunction MSCs was treated with GD1a. (I) Alizarin Red staining in U0126-treated MSCs. (J) Increases of osteogenic proteins in GD1a-treated MSCs compared with pERK1/2-inhibited MSCs by U0126. Phosphorylation of ERK1/2 was determined by western blotting with anti-p-ERK1/2. ERK1/2 was used as a loading control. Values represent mean ± SD; ^&&&p < 0.001 indicates a significant difference from the normal MSCs; ^***p < 0.001 indicates a significant difference from the Lmna^Dhe/+ mutant MSCs; ^###p < 0.001 indicates a significant difference from the Lamin A/C KD MSCs. ^$$$p < 0.001 indicates a significant difference from the U0126-treated MSCs.

Figure 5

Micro-array analysis of genes expression in Lmna^Dhe/+ mutant MSCs was treated with GD1a compared to Lmna^Dhe/+ mutant MSCs. Gene expression was analyzed by a microarray using an antibody. (A) Antibody microarray assay (Ray L1000 Antibody Array) can analyze 1000 genes and has 15 domains such as inflammatory response, neurogenesis, RNA splicing, secretion, extracellular matrix, angiogenesis, cell cycle, apoptotic processes, cell differentiation, cell death, cell migration, cell proliferation, DNA repair, aging and immune response. (B) We analyzed the differences in gene expression in Lmna^Dhe/+ mutant MSCs was treated with GD1a compared to Lmna^Dhe/+ mutant MSCs. (C) Decreases of osteogenesis-related genes expression in Lmna^Dhe/+ mutant MSCs was treated with GD1a compared to Lmna^Dhe/+ mutant MSCs.

Figure 6

Analysis of trabecular and cortical bone of the femur. Lmna^Dhe/+ mutant mice were intraperitoneally injected with ganglioside GD1a at 5, 10, and 30 mg/kg/2-day for 7 weeks. (A) The mice were weighed once a week for 7 weeks, and (B) Hematoxylin and eosin staining of the femur nearby the metaphysic area. (C) Micro-CT analysis of trabecular and cortical bone of femur. (D) Representative parameters of trabecular and cortical bone, respectively. ^*p < 0.05 indicates a significant difference the normal; ^**p < 0.01 indicates a significant difference the Normal; ^#p < 0.05 indicates a significant difference the Lmna^Dhe/+ mutant mouse.