Insulin Receptor deletion in S100a4-lineage cells accelerates age-related bone loss

Abstract

Type I and Type II Diabetes dramatically impair skeletal health. Altered Insulin Receptor (IR) signaling is a common feature of both diseases, and insulin has potent bone anabolic functions. Several previous studies have demonstrated that loss of IR in bone cells results in disrupted bone homeostasis during early post-natal growth. Here we have deleted IR in S100a4-lineage cells (IRcKO^S100a4) and assessed the effects on bone homeostasis in both young (15 weeks) and older adult (48 weeks) mice. S100a4-Cre has previously been shown to target the perichondrium during bone development, and here we show that S100a4 is expressed by adult trabecular and cortical bone cells, and that S100a4-Cre effectively targets adult bone, resulting in efficient deletion of IRβ. Deletion of IRβ in S100a4-lineage cells does not affect initial bone acquisition or homeostasis with no changes in cortical, trabecular or mechanical properties at 15-weeks of age, relative to wild type (WT) littermates. However, by 48-weeks of age, IRcKO^S100a4 mice display substantial declines in trabecular bone volume, bone volume fraction and torsional rigidity, relative to age-matched WT controls. This work establishes the utility of using S100a4-cre to target bone and demonstrates that IRβ in S100a4-lineage cells is required for maintenance of bone homeostasis in adult mice.

Keywords: Aging; Bone; Bone homeostasis; Insulin receptor; S100a4.

Fig. 1

S100a4-lineage cells are located in cortical and trabecular bone. (A) To determine the spatial localization of S1004-lineage cells in 15- and 48-week old femurs S100a4-Cre; Rosa-Ai9 (S100a4^Ai9) mice were used. At 15- and 48-weeks of age S100a4-lineage cells were located in the cortical and trabecular bone, and the bone marrow. Scale bars represent either 50 or 100 μm as noted on each image. White arrows indicate trabecular osteoblasts that are S100a4^Ai9+. Yellow arrows identify S100a4^Ai9+ cells lining the endosteum. (B) To determine the proportion of S100a4^Ai9+ cells in cortical and trabecular bone, S100a4^Ai9+ cells/DAPI⁺ cells were quantified in each compartment. (*) indicates p < 0.05 between 15- and 48-week old animals. (C & D) Co-immunofluorescence for Red Fluorescent Protein (S100a4^Ai9+) and (C) Osterix (green) or (D) Osteocalcin (green). White arrows indicate co-expression of S100a4^Ai9 and Osterix or Osteocalcin. Orange arrows in (D) identify Osteocalcin⁺ cells that are not derived from the S100a4-lineage. Sections were also stained with DAPI to identify nuclei. Scale bars represent 20 μm. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 2

Cells actively expressing S100a4 are located in the trabecular bone, and cortical bone of aged 48-week but not 15-week old mice. To determine the spatial localization of cells actively expressing S100a4 at 15 and 48-weeks of age S100a4GFP^promoter were used. At 15 weeks of age several S100a4⁺ cells were located in trabecular bone and the bone marrow, with very few S100a4^GFP+ cells in the cortical bone. At 48-weeks S100a4⁺ cells were observed in cortical and trabecular bone, as well as the bone marrow. Scale bars represent 50 μm. White arrows indicate S100a4^GFP+ cells. (B) S100a4^GFP+ cells were quantified in cortical and trabecular bone at 15- and 48-weeks of age. S100a4^GFP+ cells were significantly increased in cortical bone at 48-weeks, relative to 15-weeks. No change in the proportion of S100a4^GFP+ cells was observed in trabecular bone between 15 and 48 weeks of age. (***) indicates p = 0.0002.

Fig. 3

Deletion of IR in S100a4-lineage cells decreases IR expression in bone. At 48 weeks of age total mRNA and protein was isolated from the femurs of WT and IRcKO^S100a4 mice. (A) qPCR analysis demonstrates a significant 64% reduction in IRβ mRNA expression in IRcKO^S100a4 relative to WT. (B) Western blotting for IRβ demonstrates a substantial decrease in IRβ protein expression in IRcKO^S100a4 relative to WT. Expression was normalized to βactin.

Fig. 4

IRcKO^S100a4 does not alter cortical bone parameters during aging. (A & B) 3D micro CT reconstructions of the cortical bone of the femurs from (A) 15-week old, and (B) 48-week-old male mice. (C–F) Micro CT analysis of (C) Cortical Bone Volume, (D) Cortical Bone Volume/Total Volume, (E) Cortical Thickness, and (F) Cortical Mineral Density from WT and IRcKO^S100a4 femurs at 15 and 48 weeks of age. (*) indicates p < 0.05.

Fig. 5

IRcKO^S100a4 enhances trabecular bone volume loss during aging. (A & B) 3D micro CT reconstructions of femur trabecular bone from (A) 15- and 48-week-old male mice. (B–F) Micro CT analysis of (B) Trabecular Bone Volume, (C) Trabecular Bone Volume/Total Volume, (D) Trabecular Number, (G) Trabecular Spacing, (F) Trabecular Thickness from WT and IRcKO^S100a4 at 15 and 48 weeks of age. (*) indicates p < 0.05.

Fig. 6

IRcKO^S100a4 decreases femur torsional rigidity at 48-weeks of age. To determine the effects of IRcKO^S100a4 on the torsional mechanical properties, femurs from WT and IRcKO^S100a4 littermates underwent torsion testing at 15- and 48-weeks of age to assess A) Ultimate Torque at Failure, and B) Torsional Rigidity. (*) indicates p < 0.05.