Osteogenic Growth Peptide C-terminal Pentapeptide [OGP(10-14)] Acts On Rat Bone Marrow Mesenchymal Stem Cells To Promote Differentiation To Osteoblasts And To Inhibit Differentiation To Adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells(MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. A reciprocal relationship exists between bone marrow MSCs adipogenesis and osteogenesis. OGP increases bone formation and trabecular bone density and stimulates fracture healing. Transgenic mice overexpressing OGP have a markedly increased peak bone mass. In cultures of osteoblastic and other bone marrow stromal cells, OGP regulates proliferation, alkaline phosphatase activity, and matrix mineralization. OGP occurs physiologically in human and rodent serum as an inactive complex with a2-macroglobulin(a2M). Dissociating from the complex with a₂M, the peptide is proteolytically cleaved, thus generating the C-terminal pentapeptide OGP(10-14). OGP(10-14), but not OGP, is responsible for the binding to the OGP receptor and activates an intracellular Gi-protein-MAP kinase signaling pathway. OGP(10-14) is the minimal OGP-derived sequence that retains the full OGP-like biological activity. These observations prompted us to evaluate the possible mechanism of OGP(10-14) in prevention or treatment of osteoporosis. Whether OGP(10-14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. We hypothesized that OGP(10-14) may positively regulate bone formation by directly affecting differentiation of multipotential bone marrow MSCs.In the present study, OGP(10-14) was prepared according to the standard solid phase peptide synthesis methodology using Fmoc strategy or Boc strategy in a manual apparatus. The purity of the peptide established by analytical HPLC, was shown to exceed 95%. The structural integrity was confirmed by electrospray mass spectrometry. The activity of OGP(10-14) assessed in fibroblastic NIH 3T3 cell cultures, was showed a bell-shaped dose-response curve, and the maximum activity was found at 10^-10M. The MSCs were isolated from both femora in Wistar rats using all-bone marrow culture method. All cells used for the experiments were passage 3. The cells were cultured in osteogenic induction medium or adipogenic induction medium. By Oil Red O staining, alkaline phosphatase (ALP) staining, Von Kossa staining, contents of triglyceride (TG) and ALP activity measuring, semiquantitative RT-PCR assessing the expression of adipocyte-specific genes (PPARγ2, LPL, aP₂ and C/EBP) and osteogenic specific genes ( Cbfal, ALP and OC), the effects of OGP(10-14) on the shunting between adipocytic and osteoblastic lineages of MSCs wereevaluated.Our results showed that OGP(10-14) promoted osteogenic differentiation of the stemcells and concurrently inhibited adipocyte formation. OGP(10-14) increased ALP activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of Cbfa1. This osteogenesis promotion induced by OGP(10-14) was independent of the presence of Dex, but OGP(10-14) combined with Dex, could enhance osteogenesis further. In contrast, OGP(10-14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of PPARγ2. The expressions of ALP, OC, LPL, aP₂ and C/EBP were undetectable or expressed at very low levels in rMSCs treated with OGP (10-14) or vehicle.In conclusion, the MSCs could differentiate to osteoblasts and adipocytes when stimulated under appropriate conditions. OGP(10-14) directly regulates progenitor rat MSCs differentiating into more osteoblasts and less adipocytes. These effects appear to be at the level of commitment rather than at the level of maturation. The results of this study might be relevant for the pathogenesis and the treatment of osteoporosis.