Mitogenic G(i) protein-MAP kinase signaling cascade in MC3T3-E1 osteogenic cells: activation by C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)] and attenuation of activation by cAMP.

In osteogenic and other cells the mitogen-activated protein (MAP) kinases have a key role in regulating proliferation and differentiated functions. The osteogenic growth peptide (OGP) is a 14 mer mitogen of osteogenic and fibroblastic cells that regulates bone turnover, fracture healing, and hematopoiesis, including the engraftment of bone marrow transplants. It is present in the serum and extracellular fluid either free or complexed to OGP-binding proteins (OGPBPs). The free immunoreactive OGP consists of the full length peptide and its C-terminal pentapeptide OGP(10-14). In the present study, designed to probe the signaling pathways triggered by OGP, we demonstrate in osteogenic MC3T3 E1 cells that mitogenic doses of OGP(10-14), but not OGP, enhance MAP kinase activity in a time-dependent manner. The OGP(10-14)-induced stimulation of both MAP kinase activity and DNA synthesis were abrogated by pertusis toxin, a G(i) protein inhibitor. These data offer direct evidence for the occurrence in osteogenic cells of a peptide-activated, mitogenic Gi protein-MAP kinase-signaling cascade. Forskolin and dBu(2)-cAMP abrogated the OGP(10-14)-stimulated proliferation, but induced only 50% inhibition of the OGP(10-14)-mediated MAP kinase activation, suggesting additional MAP kinase-dependent, OGP(10-14)-regulated, cellular functions. Finally, it is demonstrated that OGP(10-14) is the active form of OGP, apparently generated proteolytically in the extracellular milieu upon dissociation of OGP-OGPBP complexes.

Structure-bioactivity of C-terminal pentapeptide of osteogenic growth peptide [OGP(10-14)].

The amino acid sequence of osteogenic growth peptide (OGP) consists of 14 residues identical to the C-terminal tail of histone H4. Native and synthetic OGP are mitogenic to osteoblastic and fibroblastic cells and enhance osteogenesis and hematopoiesis in vivo. The C-terminal truncated pentapeptide of OGP, H-Tyr-Gly-Phe-Gly-Gly-OH [OGP(10-14)], is a naturally occurring osteoblastic mitogen, equipotent to OGP. The present study assesses the role of individual amino acid residues and side chains in the OGP(10-14) mitogenic activity which showed a very high correlation between osteoblastic and fibroblastic cell cultures. Truncation of either Tyr10 or its replacement by Ala or D-Ala resulted in substantial, but not complete, loss of activity. Nevertheless, only a small loss of activity was observed following removal of the Tyr10 amino group. No further loss occurred consequent to the monoiodination of desaminoTyr10 on meta-position. However, a marked decrease in proliferative activity followed removal of the Tyr10 phenolic or the Phe12 aromatic group. Loss of activity of a similar magnitude also occurred subsequent to replacing Gly11 with L- or D-Ala. Approximately 50% loss of mitogenic activity occurred subsequent to truncation of Gly14 or blocking the C-terminal group as the methyl ester. All other modifications of the C-terminus and L- or D-Ala substitution of Gly13 resulted in 70-97% decrease in activity. Collectively, these data suggest that the integrity of the pharmacophores presented by Tyr and Phe side chains, as well as the Gly residues at the C-terminus, are important for optimal bioactivity of OGP(10-14).

Isolation of mitogenically active C-terminal truncated pentapeptide of osteogenic growth peptide from human plasma and culture medium of murine osteoblastic cells.

The osteogenic growth peptide (OGP) is a 14-amino acid stromal cell mitogen that stimulates in vivo osteogenesis and hematopoiesis. In the blood circulation and cell culture conditioned medium immunoreactive OGP (irOGP), identified using antibodies raised against the OGP C-terminal region, presents free and bound forms. The bound form consists entirely of the full length peptide. The present study was designed to investigate the identity of free irOGP under nondenaturing conditions. Fresh human serum and culture medium conditioned with murine osteoblastic MC3T3 E1 cells were fractionated using ultrafiltration (3000 molecular weight cut-off). Hydrophobic chromatography of the ultrafiltrate, immunoscreening of chromatographic fractions with antibodies directed against the OGP C-terminal region and amino acid sequencing of immunoreactive peaks demonstrated the presence of two mitogens, the full length OGP and a C-terminal truncated form, OGP(10-14). The OGP(10-14) derived from both serum and conditioned medium, as well as the synthetic pentapeptide [sOGP(10-14)], shared the in vitro OGP proliferative activity. However, in a competitive binding assay, devised to assess the OGP-OGP binding protein (OGPBP) complex formation, sOGP(10-14) failed to compete out radiolabeled OGP from the complex. It is concluded that OGP(10-14) is a naturally occurring human and murine mitogen. In addition, the data suggests that the OGP(10-14) is generated from OGP by proteolytic cleavage upon dissociation of the OGP-OGPBP complexes.

Human alpha 2-macroglobulin is an osteogenic growth peptide-binding protein.

The osteogenic growth peptide (OGP) is a 14mer mitogen of osteoblastic and fibroblastic cells. Physiologically, OGP is present in high abundance in human and other mammalian sera. Most of the serum OGP is complexed noncovalently to heat sensitive, high molecular weight OGP-binding proteins (OGPBPs). Changes in serum OGP levels that follow bone marrow ablation and the low doses of exogenous OGP required for the stimulation of bone formation suggest a regulatory role for the OGPBPs. In the present work, the OGP binding activity was monitored by competitive binding to [3-125I(Tyr10)]-sOGP and the corresponding complexes were demonstrated on nondenaturing cathodic polyacrylamide gel electrophoresis. We show that OGP binds to both native and activated human plasma alpha 2-macroglobulin (alpha 2M). alpha 2M was also immunoidentified in reduced and nonreduced SDS-polyacrylamide gel electrophoresis of OGP-affinity purified plasma-derived proteins. Immunoreactive OGP was detected in commercial preparations of both forms of alpha 2M; OGP was purified to homogeneity from the commercial preparation of activated alpha 2M. In MC3T3 E1 cells, native alpha 2M, at concentrations < 50 ng/mL, had a substantially increased mitogenic effect in the presence of synthetic, native-like, OGP (sOGP). Similar amounts of activated alpha 2M inhibited the sOGP proliferative effect. These results suggest that the native alpha 2M enhances the immediate availability of OGP to its target cells. Activated alpha 2M may participate in the removal of OGP from the system.

Osteogenic growth peptide increases blood and bone marrow cellularity and enhances engraftment of bone marrow transplants in mice.

The osteogenic growth peptide (OGP) was characterized recently in regenerating bone marrow (BM) and normal serum. In vitro, the OGP regulates stromal-cell proliferation and differentiated functions. In vivo, an increase in serum OGP accompanies the osteogenic phase of postablation BM regeneration. The present results in normal mice show that OGP induces a balanced increase in WBC counts and overall BM cellularity. In mice receiving myeloablative irradiation and syngeneic or semiallogeneic BM transplants, OGP stimulates hematopoietic reconstruction and doubles the survival rate; these effects are dependent on initiating the OGP administration before irradiation. Chimerism measurements in semiallogeneic graft recipients suggest no preferential effect of OGP on residual host cells. The data implicate OGP in the acceleration of hematopoiesis secondary to expansion of the stromal microenvironment and/or enhancement of stroma-derived signals to stem cells. The low-dose effectiveness of OGP is explained by the demonstration of an autocrine positive feedback loop that together with the OGP-binding protein sustains high serum levels of the peptide. A potential OGP-based treatment in combination with chemoradiotherapy is attractive because of the OGP-induced balanced multi-lineage enhancement of hematopoiesis and possible replacement of expensive recombinant cytokines by a readily synthesized peptide.

Structural and functional characterization of osteogenic growth peptide from human serum: identity with rat and mouse homologs.

The osteogenic growth peptide (OGP) was recently characterized in regenerating bone marrow. In experimental animals, OGP increases osteogenesis. Immunoreactive OGP (irOGP) in high abundance was demonstrated in normal animal serum mainly as an OGP-OGP-binding protein (OGPBP) complex. Here we show the presence of an OGP-OGPBP system in normal human serum. The total irOGP content, of which the bound peptide comprises at least 80-90%, ranged from 480-4460 mumol/L, several orders of magnitude higher than that of other regulatory polypeptides. The steady state/total irOGP ratio declined between 23 and 49 yr of age. The bound irOGP, purified by boiling, ultrafiltration, and hydrophobic high pressure liquid chromatography, was identical to OGP obtained previously from rat regenerating marrow and mouse stromal cell cultures in terms of its amino acid sequence, immunoreactivity, and mitogenicity. These data demonstrate the usefulness of our immunoassay to measure circulating OGP. More importantly, the identity of the human OGP with that of other species indicates the peptide's evolutionary conservation and, thus, its biological importance. The natural occurrence of OGP in man signifies its potential role in the prevention of bone loss and rescue of bone mass, especially in osteoporosis.