Aging negatively impacts the ability of megakaryocytes to stimulate osteoblast proliferation and bone mass

Abstract

Osteoblast number and activity decreases with aging, contributing to the age-associated decline of bone mass, but the mechanisms underlying changes in osteoblast activity are not well understood. Here, we show that the age-associated bone loss critically depends on impairment of the ability of megakaryocytes (MKs) to support osteoblast proliferation. Co-culture of osteoblast precursors with young MKs is known to increase osteoblast proliferation and bone formation. However, co-culture of osteoblast precursors with aged MKs resulted in significantly fewer osteoblasts compared to co-culture with young MKs, and this was associated with the downregulation of transforming growth factor beta. In addition, the ability of MKs to increase bone mass was attenuated during aging as transplantation of GATA1^low/low hematopoietic donor cells (which have elevated MKs/MK precursors) from young mice resulted in an increase in bone mass of recipient mice compared to transplantation of young wild-type donor cells, whereas transplantation of GATA1^low/low donor cells from old mice failed to enhance bone mass in recipient mice compared to transplantation of old wild-type donor cells. These findings suggest that the preservation or restoration of the MK-mediated induction of osteoblast proliferation during aging may hold the potential to prevent age-associated bone loss and resulting fractures.

Keywords: Aging; Bone loss; Bone mass; Megakaryocytes; Osteoblasts.

Figure 1.. The effect of aging on megakaryocyte formation.

Young (3-4 mo), middle-aged (11-14 mo), and old-aged (22-24 mo) C57BL/6 mice were characterized for MK phenotype. (A) Frequency of total bone marrow cells that stain positive for the MK marker CD41 by FACS analysis (n = 3 mice per age cohort). (B) Circulating platelet counts from whole blood (n = 21-31 mice per age cohort). (C) Total number of MKs generated per spleen from young or old spleens cultured in the presence of TPO and isolated by BSA gradient (n = 4 independent experiments). Data are expressed as means ± S.E.M. Kruskal-Wallis test with Dunn’s post hoc analysis (A), 1-Way ANOVA with Holm-Sidak post hoc analysis (B), and Unpaired t-test (C). * p < 0.05. MK = megakaryocyte.

Figure 2.. Megakaryocytes from aged mice are ineffective at stimulating bone cell proliferation.

(A) MKs derived from young (3 mo) or old-aged (24 mo) mice were co-cultured (1:2 ratio) for 5 days with calvarial cells isolated from newborn pups by collagenase digestion. Results are from 6 separate experiments and normalized to within experiment CC only cell counts. (B) mRNA was isolated from lysates of MKs derived from young or old mice, reverse transcribed, and analyzed by qPCR. Gapdh was used as a normalization control. (n = 4-6) (C) Long bone cells were isolated by collagenase digestion from young or old-aged mice and co-cultured (10,000 cells per well) for 5 days with fetal-liver derived MKs (5,000 cells per well). (n = 3 mice per age group). Data are expressed as means ± S.E.M. Kruskal-Wallis with Dunn’s post hoc analysis (A), Student’s T-test (B), or 2-Way ANOVA with Holm-Sidak post hoc analysis (C). ** p < 0.01; *** p < 0.001. CC = calvarial cell. MK = megakaryocyte.

Figure 3.. Adoptive transfer of young, but not old megakaryocyte skewed hematopoietic cells, increased bone mass in young wild-type recipient mice.

Young C57BL/6 male mice were lethally irradiated and transplanted with young (3 mo) or old (18 mo) wild-type or GATA1^low/low spleen cells by retro-orbital injection. Adoptively transferred (recipient) mice were euthanized and analyzed for femoral bone phenotype by μCT after 8 weeks. (A) Representative 3D renderings of distal trabecular bone from each group. (B) μCT analyses of bone volume fraction (BV/TV) of distal trabecular region. (C) Representative 3D renderings of midshaft cortical bone with trabecular bone highlighted in white. (D) μCT analyses of BV/TV of midshaft trabecular bone region. (E) Comparisons of spleen mass at sacrifice. (F) Platelet counts taken from whole blood at sacrifice. (B,D) Each data point represents a unique animal. Black bars indicate average of population. (E,F) Bars are averages with S.E.M. Two-way ANOVA with Flolm-Sidak post-hoc analyses. * p < 0.05,*** p < 0.001. Tb = trabecular.