Mechanical unloading negatively affects the skeletal homeostasis. Endothelial cells (ECs) are vital bone regulators and sensitive to mechanical stimuli. We hypothesised that ECs are implicated in the control of bone metabolism during unloading. We treated mouse primary osteoblasts with Conditioned Medium from ECs subjected to MicroGravity (MG-EC-CM), demonstrating its ability to increase osteoblast proliferation (+1.5-fold; p=0.003), and decrease Alkaline Phosphatase (ALP) activity (−53%; p<0.001) and matrix nodule formation (−66%; p=0.010) compared with unit gravity EC-CM. MG-EC-CM increased expression of osteoblast RANKL and osteoclastogenesis in osteoblast-bone marrow mononuclear cell co-cultures (+56%; p<0.001). Furthermore, it induced the expression of the osteoblast de-differentiating factor, Lipocalin 2 (Lcn2) (+48-fold; p<0.001), whose silencing recovered osteoblast ALP activity (+52%; p=0.024), decreased RANKL expression and reduced osteoclast formation (−38%; p=0.004), with no effect on osteoblast proliferation. MG-EC-CM enhanced osteoblast NO-Synthase 2 (NOS2) and CycloOXygenase 2 (COX2) expression. Inhibition of NOS2 or NO signalling reduced osteoblast proliferation and rescued ALP activity (+38%; p=0.004). Nuclear translocation of the Lcn2/NOS2 transcription factor, NF-kB, was observed in MG-EC-CM-treated osteoblasts and MG-ECs, alongside a high expression of the NF-kB activator, IL-1β (+96-fold; p=0.002), with NF-κB inhibition reducing osteoblast proliferation and rescuing ALP. Lcn2 (+22-fold) and NOS2 (+12-fold) were also incremented in ex-vivo calvaria cultured with MG-EC-CM, and in-vivo tibias and calvaria (p=0.021) injected with MG-EC-CM. Furthermore, the tibias of in-vivo models of mechanical unloading, including tail-suspended mice and mice treated with botulin A toxin to induce transient muscle paralysis, which featured decreased bone mass, showed increased expression of IL-1β (+5.8-fold; p=0.004), Lcn2 (+10-fold; p=0.002) and NOS2 (+23-fold; p=0.001), suggesting their involvement in the in-vivo EC-osteoblast crosstalk. We conclude that, through IL-1β, MG-EC-CM induces NSO2 and Lcn2 in osteoblasts. By means of its downstream NO/COX2 pathway, NOS2 increases osteoblast proliferation, while Lcn2 impairs differentiation and enhances RANKL expression and osteoclastogenesis. Targeting this EC-osteoblast-osteoclast regulatory loop could help improve the bone phenotype in unloading conditions.
Disclosure: The authors declared no competing interests.