Adenosine 3′,5′-Monophosphate Mediates Both the Mitogenic Effect of Thyrotropin and Its Ability to Amplify the Response to Insulin-Like Growth Factor I in FRTL5 Cells*

Publisher Website
Google Scholar
Abstract
In previous studies we have demonstrated that bovine TSH (bTSH) and insulin-like growth factor I (IGF-I) independently stimulated both the incorporation of [3H]thymidine into DNA and replication in quiescent FRTL5 cells. In the case of TSH, evidence was presented that these responses are cAMP mediated. In addition, responses of thymidine incorporation are greatly amplified when particular concentrations of two agents are added together, but this effect diminishes as the concentration of either bTSH or IGF-I is increased. The present experiments were undertaken to obtain further information concerning the mechanism of the independent mitogenic effects of bTSH and IGF-I and to explore the nature of the biphasic synergistic interaction with respect to thymidine incorporation that occurs when bTSH and IGF-I are added together. Verification that the increases in [3H]thymidine incorporation induced by bTSH and IGF-I, alone and together, are truly reflective of increases in DNA synthesis was obtained in experiments in which labeled nuclei were counted in cultures of cells grown in the presence of one or both mitogenic agents to which [3H]thymidine has been added. In these studies the number of cells with labeled nuclei was increased markedly by each of the two agents, and the response when the two mitogens were added together was far greater than the sum of their individual effects. Over a range of concentrations which included those that elicit a mitogenic response in FRTL5 cells, IGF-I, unlike bTSH, failed to increase cAMP generation when added alone. Moreover, IGF-I did not significantly enhance the cAMP response to varying concentrations of bTSH. A concentration-dependent increase in the incorporation of [3H]thymidine into DNA was induced by culturing cells in the presence of the cAMP analog (Bu)2cAMP (Bt2cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, and the stimulator of adenylate cyclase forskolin. When increasing concentrations of these agents were added together with IGF-I, a biphasic pattern of response of DNA synthesis, mimicking that produced by the combination of IGF-I and increasing concentrations of bTSH, was observed. Further evidence that cAMP mediates the mitogenic response to bTSH was the observation that adenosine inhibited the stimulation of both cAMP generation and DNA synthesis that bTSH produced. Although preincubation of quiescent FRTL5 cells for 24 h in the presence of bTSH resulted in only a small increase in DNA synthesis, measured during the last 3 h of a subsequent 24-h incubation carried out in the absence of btSH, it greatly amplified the response to IGF-I added alone during the second incubation. These effects of preincubating cells with bTSH were mimicked by preincubating them with Bt2cAMP. Although preincubation with IGF-I also increased DNA synthesis during a second incubation in the absence of any additive, it inhibited, rather than enhanced, the mitogenic responseto both bTSH and Bt2cAMP during the second incubation. These data indicate that the pathways by which bTSH and IGF-I stimulate growth in FRTL5 cells are different; the former is mediated by cAMP while the latter is not. The bTSH and IGF-I with respect to DNA synthesis in these cells is also cAMP dependent. This amplification results from a priming action of bTSH on the subsequent response to IGF-I; this action persists after bTSH is withdrawn and is also cAMP dependent.