Distinct Pathways of Extracellular Signal-regulated Kinase Activation by Growth Factors, Fibronectin and Parathyroid Hormone 1-34

Growth factors, hormones, and matrix proteins regulate osteoblast proliferation and differentiation, acting through cognate receptors. Since each of the receptors are coupled to a variety of distinct signal transduction pathways, in this report we evaluated whether there is a common convergent intermediate step that allows cross-talk among the various pathways. Since extracellular signal-regulated kinases 1 and 2 (Erk1/2) play a role in mitogenesis and differentiation processes, we evaluated the effects of various osteotrophic factors on Erk1/2 phosphorylation in osteoblasts. Osteoblasts isolated from the metaphyseal marrow (MM) and diaphyseal marrow (DM) of 4-6 week old male rat longitudinal bones were grown to confluency and Erk1/2-phosphorylation was evaluated using antibodies that recognized either the total or the phosphorylated form of the kinase. There was very little Erk1/2 phosphorylation in cells kept in suspension. Both MM and DM cells attached to fibronectin (FN), demonstrated Erk1/2 phosphorylation that persisted for at least up to 8h. Platelet-derived growth factor AB (PDGF-AB) induced a transient and robust Erk1/2 phosphorylation that was attenuated by 2h. Studies with specific inhibitors indicated that the effects of these factors were mediated by protein kinase C, by receptor tyrosine kinase, as well as by protein phosphatases. Parathyroid hormone (PTH 1-34), a bone anabolic agent however, caused a down-regulation of FN stimulated Erk1/2 phosphorylation in MM derived cells. The inhibitory effect of PTH was mediated through cAMP-dependent protein kinase A (PKA) activation. The data collectively suggest that a combination of diverse extracellular stimuli regulates Erk1/2 phosphorylation that may ultimately influence osteoblast proliferation and/or differentiation.