SUN-543 Myostatin Inhibits IGF1-Dependent Citrate Production during Osteogenesis in a Mouse Mesenchymal Stem Cell Line

Bone formation and osteoblast activation require glucose. Citrate is a key intermediate in the tricarboxylic acid (TCA) cycle that is the major energy-yielding metabolic pathway in cells. Citrate homeostasis, therefore, is critically important for glucose metabolism and energy production. In addition, citrate is an indispensable component of bone. The vast amount of citrate with its carboxylate groups provides a huge capacity for calcium binding on apatite that stabilizes the size of nanocrystal of bone. Reduced citrate levels in bone and serum are reported in elderly compared to young subjects, as well as in osteoporotic patients. IGF1, a growth factor that elicits downstream signaling events similar to insulin and also controls TCA activity, is essential in bone matrix mineralization. However, the source of citrate in bone and what roles that IGF1 plays in regulating citrate metabolism and deposition during osteogenesis are largely unknown. Myostatin (Mstn), a muscle cell-secreted cytokine, is involved in regulating energy metabolism, however, its effect on osteogenesis and bone formation are not well delineated. In this study, the interactive effect of Mstn and IGF1 on citrate production during osteogenesis was examined in C3H10T1/2 mouse mesenchymal stem cells (MSC). MSCs cultured in osteogenic medium induced high levels of citrate secretion with the peak (5.6-fold) was observed at a later stage (14-21d) of osteogenic differentiation. The levels of IGF1 mRNA and protein were also upregulated with a similar pattern, but of reduced magnitude than that of citrate secretion. In contrast, cells treated with Mstn showed a significant inhibition in both citrate secretion and IGF1 expression during the same period of osteogenesis. Alizarin Red staining (ARs) that was used to confirm the biological effect of Mstn showed a significant decrease in ARs in Mstn-treated cells compared to that of control cells. In addition, alkaline phosphatase (ALP) staining revealed a dramatical decrease in ALP expression and activity. These results suggest that inhibited citrate levels by Mstn led to defects in osteogenic differentiation and mineralization. To determine whether IGF1 plays role in these processes, an anti-IGF1 neutralizing antibody that inhibits IGF1 expression was employed. MSCs treated with IGF1 antibody showed a 35% reduction in citrate secretion compared to that of control cells. Moreover, the addition of exogenous IGF1 partially reversed the inhibitory effect of Mstn on citrate secretion, indicating that increased levels of citrate during osteogenesis is partially IGF1-dependent and that Mstn-inhibited citrate production is, at least in part, mediated by reduced IGF1 levels. These results imply a novel effect of Mstn on modulating osteogenesis of bone marrow MSCs by preventing IGF1-dependent citrate production that leads to defects in osteogenic differentiation and mineralization.