Runt-related transcription factor 2 (RUNX2) and RUNX2-related osteogenic genes are down-regulated throughout osteogenesis in type 1 diabetes mellitus

Abstract

Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.

Figure 1

Bone formation is reduced in T1DM. Radiographic (A) and histological (B) assessments of bone formation in control mice, STZ-induced diabetic mice, and insulin-treated diabetic mice are shown. Analyses were performed as described in Materials and Methods. P values for specific between-group comparisons are shown. C, Representative histology sections from distraction gaps of a control mouse, STZ-induced diabetic mouse, and insulin-treated diabetic mouse are presented. New bone formation is outlined in black. D, The effects of diabetes and insulin treatment on bone architecture were assessed by μCT.

Figure 2

Bone marrow adipocyte content is increased in T1DM, and nu cells are decreased. The percentage of nonfat nucleated cells (i.e. nu cells) (A) and adipocytes (B) in bone marrow adjacent to the DO gap was assessed in control animals, STZ-induced diabetic animals, and insulin-treated diabetic animals as described in Materials and Methods. C, Representative micrographs of each condition are shown. P values for specific between-group comparisons are shown.

Figure 3

Gene expression profiles in regenerate bone from control, diabetic, and insulin-treated diabetic mice. Each graph demonstrates the expression level for a gene of interest as assessed by qRT-PCR (described in Materials and Methods) in control, diabetic, and insulin-treated diabetic animals. A, RUNX; B, MMP-1; C, MMP-9; D, Akp2; E, Ibsp; F, Dmp1; G, Colla2; H, Spp1; I, Phex; J, Vdr; K, osteocalcin; L, PPARγ. P values for specific between-group comparisons are shown. PPAR, Peroxisome proliferator-activated receptor.

Figure 4

Genes involved in osteoblast commitment and differentiation: expression in regenerate bone from control, diabetic, and insulin-treated diabetic mice. Each graph demonstrates the expression level for a gene of interest as assessed by qRT-PCR (described in Materials and Methods) in control, diabetic, and insulin-treated diabetic animals. A, BMP-2; B, TAZ; C, TWIST2; D, MSX1; E, osterix; F, DLX5. P values for specific between-group comparisons are shown.