Immature and mature megakaryocytes enhance osteoblast proliferation and inhibit osteoclast formation

Abstract

Recent data suggest that megakaryocytes (MKs) play a role in skeletal homeostasis. In vitro and in vivo data show that MKs stimulate osteoblast (OB) proliferation and inhibit osteoclast (OC) formation, thus favoring net bone deposition. There are several mouse models with dysregulated megakaryopoiesis and resultant high bone mass phenotypes. One such model that our group has extensively studied is GATA-1 deficient mice. GATA-1 is a transcription factor required for normal megakaryopoiesis, and mice deficient in GATA-1 have increases in immature MK number and a striking increase in bone mass. While the increased bone mass could simply be a result of increased MK number, here we take a more in depth look at the MKs of these mice to see if there is a unique factor inherent to GATA-1 deficient MKs that favors increased bone deposition. We show that increased MK number does correspond with increased OB proliferation and decreased OC formation that stage of maturation does not alter the effect of MKs on bone cell lineages beyond the megakaryoblast stage, and that GATA-1 deficient MKs survive longer than wild-type controls. So while increased MK number in GATA-1 deficient mice likely contributes to the high bone mass phenotype, we propose that the increased longevity of this lineage also plays a role. Since GATA-1 deficient MKs live longer they are able to exert both more proliferative influence on OBs and more inhibitory influence on OCs.

Figure 1

Affect of MK number on OB proliferation. Increasing numbers of GATA-1 deficient and C57BL/6 MKs similarly enhanced OB proliferation. A significant, greater than 90% increase in OB proliferation was observed when OBs were cultured with either 2500 C57BL/6 or GATA-1 deficient MKs. Culturing OBs with 5000 MKs further enhanced OB proliferation. * Denotes a significant difference in proliferation compared to the OB alone control group (p<0.05).

Figure 2

Affect of MK CM concentration on osteoclastogenesis. Increasing concentrations of GATA-1 deficient and C57BL/6 MK CM similarly inhibited OC development. A significant, greater than 65% reduction in the number of OCs formed was observed with just 3% CM. With 30% CM from either C57BL/6 or GATA-1 deficient MKs, complete inhibition in OC formation was seen. * Denotes a significant difference (p<0.05) in OC number compared to control groups cultured without CM (0%).

Figure 3

Affect of MK maturation stage on OB proliferation. 2500 BSA separated C57BL/6 MKs or flow cytometry separated immature and mature MKs all similarly enhanced OB proliferation, while 2500 megarkaryoblasts did not alter OB proliferation. * Denotes a significant difference in proliferation compared to the OB control group (p<0.05).

Figure 4

Affect of MK maturation stage on OC inhibition. 3% C57BL/6 MK CM from BSA separated or flow cytometry separated immature and mature MKs similarly inhibited OC development, while 3% CM from megakaryoblasts did not inhibit OC development. * Denotes a significant difference in OC number compared to OC control group (p<0.05).

Figure 5

Viable MK number over time. C57BL/6 and GATA-1 deficient MKs were cultured alone, with TPO, with OBs, or with TPO and OBs, and viable cell number recorded. The combination of OBs and TPO increased MK survival. GATA-1 deficient MKs survived for longer than did C57BL/6 MKs.