Selective Runx2-II deficiency leads to low-turnover osteopenia in adult mice

Abstract

Runx2 transcribes Runx2-II and Runx2-I isoforms with distinct N-termini. Deletion of both isoforms results in complete arrest of bone development, whereas selective loss of Runx2-II is sufficient to form a grossly intact skeleton with impaired endochondral bone development. To elucidate the role of Runx2-II in osteoblast function in adult mice, we examined heterozygous Runx2-II (Runx2-II(+/-)) and homozygous Runx2-II (Runx2-II(-/-))-deficient mice, which, respectively, lack one or both copies of Runx2-II but intact Runx2-I expression. Compared to wild-type mice, 6-week-old Runx2-II(+/-) had reduced trabecular bone volume (BV/TV%), cortical thickness (Ct.Th), and bone mineral density (BMD), decreased osteoblastic and osteoclastic markers, lower bone formation rates, impaired osteoblast maturation of BMSCs in vitro, and significant reductions in mechanical properties. Homozygous Runx2-II(-/-) mice had a more severe reduction in BMD, BV/TV%, and Ct.Th, and greater suppression of osteoblastic and osteoclastic markers than Runx2-II(+/-) mice. Non-selective Runx2(+/-) mice, which have an equivalent reduction in Runx2 expression due to the lack one copy of Runx2-I and II, however, had an intermediate reduction in BMD. Thus, selective Runx2-II mutation causes diminished osteoblastic function in an adult mouse leading to low-turnover osteopenia and suggest that Runx2-I and II have distinct functions imparted by their different N-termini.

Fig. 1

Diagrams of Runx2 alleles and gene products in non-selective Runx2 and selective Runx2-II-deficient mice. (A) Heterozygous Runx2^+/− mice have one copy of wild-type Runx2 alleles predicted to generate 1/2 of the normal Runx2-I and Runx2-II gene products. (B) Homozygous Runx2^−/− mice have neither wild-type Runx2 alleles nor Runx2-I and Runx2-II gene products. (C) Heterozygous Runx2-II^+/− mice have one wild-type Runx2 allele and one Runx2-II mutant allele. These mice should express normal amounts of Runx2-I and 1/2 the amount of Runx2-II gene products. (D) Homozygous Runx2-II^−/− mice have two mutant Runx2-II alleles predicted to produce normal amounts of Runx2-I but no Runx2-II gene products. P1 or P2 designates the promoter regions for Runx2-II or Runx2-I, respectively.

Fig. 2

Effects of genotype and age on bone mineral density (BMD). (A, B) BMD of femur and spine in selective Runx2-II-deficient mice. (C, D) BMD of femur and spine in non-selective Runx2-deficient mice. BMD analysis of the femoral and lumbar spine was assessed by the PIXImus^™ mouse densitometer at 6-, 12-, and 24-week-old in Runx2-II^+/+, selective Runx2-II^+/−, Runx2-II^−/−, and non-selective Runx2^+/+, Runx2^+/−, Runx2^−/− mice. Reductions in both femoral and lumbar spine BMD were greater in 6-week-old Runx2-II null mice compared to heterozygous selective Runx2-II^+/− and non-selective Runx2^+/− mice. Wild-type mice from non-selective Runx2-deficient strain have a lower BMD compared to wild-type mice used in the proceeding analysis, likely due to the fact that that they are derived from a different genetic background. Data represent the mean ± SEM. *Difference from wild-type Runx2-II^+/+ mice; ^#difference from heterozygous Runx2-II^+/− mice at P values specified below the bar graft at each time point. The time-dependent increments in BMD at 6, 12, and 24 weeks were also significantly different (as indicated by brackets above the bar graphs).

Fig. 3

3D images of femurs in 6- and 24-week wild-type and mutant mice. 3D images of metaphyseal bone (upper panel) and cortical bone (lower panel) generated from μCT analysis. There was an age-dependent reduction in trabecular bone volume and an increase in cortical thickness in wild-type mice. The trabecular bone volume and cortical thickness was reduced in Runx2-II^+/− and Runx2-II^−/− mice proportionate to the reduction in gene dosage. Runx2-II^+/− mice displayed an age-dependent decrease in trabecular bone and increase in cortical bone similar to wild-type mice.

Fig. 4

Nondecalcified histological sections of tibia bone in 6-week-old Runx2-II wild-type and mutant mice. (A, B) Goldner-stained sections of the epiphyseal bone (×200 magnification) and growth plate and metaphyseal bone (×100 magnification) of 6-week-old Runx2-II^+/+, Runx2-II^+/−, and Runx2-II^−/− mice. Mineralized trabecular bone (Tb) is blue and unmineralized osteoid is reddish brown in color. (C, D) Villanueva-stained sections of trabecular (×200 magnification) and cortical bone (×400 magnification) viewed under fluorescent light in 6-week-old Runx2-II^+/+, Runx2-II^+/−, and Runx2-II^−/− mice prelabeled with tetracycline followed by calcein (double label). Heterozygous Runx2-II^+/− mice have reduced trabecular bone volume, normal appearing growth plate, and reduced bone formation rates. Homozygous Runx2-II^−/− mice show a marked reduction of trabecular bone volume, a widened growth plate consisting of increased zone of hypertrophic chondrocytes, and severe impairment of bone formation as evidenced by a diffuse single label or unlabeled trabecular bone surfaces. In contrast, mineral apposition rates on the periosteal surfaces of cortical bone were preserved in both heterozygous Runx2-II^+/− and homozygous Runx2-II^−/− mice. Tb.B., trabecular bone, Ct.B., cortical bone, B.M., bone marrow, G.P., growth plate, H.Z., hypertrophic zone, D.L., double label, S.L., single label.

Fig. 5

Assessment of osteoblastic differentiation in BMSCs cultures from 6- and 24-week wild-type and mutant mice. (A) Histochemical staining of mineralization nodules. BMSCs from 6- and 24-week mice were cultured for 12 days in differentiation media and stained with Alizarin Red-S. There is an age-dependent decrease in mineralized nodule formation and the heterozygous BMSC cultures showed less abundant mineralized nodules. (B) Quantification of mineralization. Alizarin Red-S was extracted with 10% cetylpyridinium chloride and quantified as described in Materials and methods. The heterozygous BMSCs had significantly lower Alizarin Red-S accumulation at day 12 of culture compared with age-matched wild type. (C) Alkaline phosphatase (ALP) activity. The heterozygous BMSCs had significantly lower ALP activity at day 8 of culture compared with age-matched wild-type, indicating reduced osteoblast function in Runx2-II mutant mice. Data represent the mean ± SEM. *Difference from wild-type Runx2-II^+/+ mice at P values specified below the bar graft at each time point. The time-dependent decrements in mineralization but not ALP activity at 6 and 24 weeks were also significantly different (as indicated by brackets above the bar graphs).