EGF-mediated phosphorylation of extracellular signal-regulated kinases in osteoblastic cells

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal-regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time-dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF-stimulated cells is not due to an increase in ERK protein, since EGF-treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (approximately 45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1-selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12-myristate 13-acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lasser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF-stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down-regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF-mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback.