Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum

Abstract

Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. 1. The analysis of proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr .apprxeq. 81000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. 2. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (protein 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6'') while TSH specifically decreased the labeling of four polypeptides (protein 1''-4'') and increased the labeling of one polypeptide (protein 5'') whose synthesis was decreased by EGF and serum. 3. The specific effect of TSH on one protein labeling (protein 7; Mr .apprxeq. 39000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a corrlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. 4. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells. Our findings are discussed within the framework of the previously described coexistence of cyclic-AMP-dependent (stimulated by TSH) and cyclic-AMP-independent (stimulated by serum or EGF) regulatory pathways leading of the initiation to DNA synthesis.