Hanna Gavish

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.

Highly efficient synchronous embryogenesis was induced in suspension cultures of sour orange ( Citrus aurantium L.) by a change in the carbon source of the growth medium from sucrose to glycerol. In liquid culture the embryos developed into globular structures during a three week period. Embryo development showed an absolute requirement for the continued presence of glycerol. The embryo cell cultures turned green in the light, but light did not affect the course of development. The profiles of soluble cellular protein extracts of embryo and proembryogenic (PEM) cells were very similar as judged by two‐dimensional polyacrylamide gel electrophoresis. However, major differences were detected in the profiles of extracellular proteins. PEM cells accumulated extracellular glycoproteins of 53–57 kDa mass. Upon subculture in glycerol containing medium, the accumulation of these proteins ceased within two days. Developing embryos accumulated at least 4 new extracellular polypeptides of 41–42 kDa mass. In addition to these polypeptides, stage specific peroxidases and proteases were found. The relatively extended duration and synchrony in which these early developmental events take place make Citrus cultures an especially useful system for the study of early events in plant embryogenesis.