PROTEIN-TYROSINE PHOSPHORYLATION-INDUCED BY EPIDERMAL GROWTH-FACTOR AND INSULIN-LIKE GROWTH-FACTOR-I IN A RAT CLONAL DENTAL PULP-CELL LINE

Both epidermal growth factor (EGF) and insulin-like growth factor-I (I GF-I) produce a dose-dependent stimulation in the rate of cell divisio n in a rat clonal dental pulp-cell line (RDP 4-1). To elucidate the in itial mitogen-induced cellular events that may mediate mitogenic actio n, the effects of EGF and IGF-I on cellular protein tyrosine phosphory lation were examined. In a dose-dependent manner, EGF (1-100 ng/ml) tr ansiently stimulated tyrosine phosphorylation in four major proteins w ith apparent molecular weights of 220, 180, 140 and 120 kDa, and in fi ve other more minor proteins (90, 80, 65, 55 and 44 kDa). IGF-I (1-100 ng/ml) dose-dependently stimulated the tyrosine phosphorylation of 16 0- and 140-kDa proteins, and had a smaller effect on the 80-, 65- and 44 kDa proteins. In contrast to the action of EGF, IGF-I-induced tyros ine phosphorylation was sustained for more than 60 min, particularly t hat of the 160-kDa phosphoprotein. From the results of specific immuno precipitation/Western-blot analyses, the 180-kDa EGF-sensitive protein could be identified as the EGF receptor (EGF-R). Among the IGF-I-sens itive pulp cell proteins, the 160-kDa protein was identified as insuli n-receptor substrate-1. Both mitogenic treatments stimulated the tyros ine phosphorylation of a weak, 44-kDa protein, which we have identifie d as the extracellular signal-regulated kinase-1. Despite the presence of phosphoproteins of the correct size, neither the IGF-I receptor (I GF-I-R) nor the phospholipase C gamma-isoform could be identified as t yrosine kinase substrates in either treatment. Pretreatment with the t yrosine kinase inhibitor genistein (20 mu g/ml) significantly inhibite d EGF- and IGF-I-induced tyrosine phosphorylation in permeabilized RDP 4-1 cells, and the tyrosine phosphatase inhibitor orthovanadate (1 mM ) significantly prolonged the duration of the mitogen-stimulated tyros ine phosphorylation in both intact or permeabilized cells. Phorbol 12- myristate 13-acetate (100 nM), which activates protein kinase C (PKC), inhibited the tyrosine phosphorylation induced by either growth facto r. This action was blocked by pretreatment with staurosporine (200 nM, 15 min), a selective PKC inhibitor. However, neither removing externa l Ca2+ with EGTA (1 mM) nor inducing Ca2+ influx with A23187 ionophore (2 mu M) significantly altered EGF- or IGF-I-induced phosphorylation. These findings strongly suggest that authentic EGF-R and IGF-I-R on R DP 4-1 cells are coupled to complex, tyrosine kinase-mediated, intrace llular signalling systems that are sensitive to a PKC-dependent mechan ism. EGF- and IGF-I-induced tyrosine phosphorylation cascades may have important roles in vivo in the regulation of dental pulp-cell prolife ration and ultimately may affect dentine formation.