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The expression of the immediate early gene c-fos was studied at the mRNA and the protein level in cells of the pituitary tumour cell line GH₃B₆. The induction of c-fos mRNA as detected by Northern blot analysis was stimulated by TRH and by depolarization with KCl, both leading to a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i), and also by epidermal growth factor (EGF). To assess the role of the changes in [Ca²⁺]_i in the induction of c-fos, Ca²⁺ was chelated in the extracellular medium with EGTA to prohibit Ca²⁺ influx during stimulation, or intracellular Ca²⁺ stores were emptied by prolonged exposure to EGTA, a treatment which abolished all [Ca²⁺]_i changes. In the latter case, the effect of TRH on c-fos mRNA expression was almost completely abolished, whereas EGF still caused substantial c-fos induction. Full induction of c-fos mRNA by TRH required a prolonged phase of stimulated Ca²⁺ influx. c-fos mRNA induction by TRH and KCl was markedly inhibited by two blockers of Ca²⁺/calmodulin-dependent protein kinase (CaM kinase), KN-62 and calmidazolium. In contrast, KCl induction of c-fos and the effects of KN-62 on TRH induction of c-fos were not observed in a closely related pituitary line GH₄C₁ in which TRH exerts its effects on immediate early genes predominantly via the protein kinase C pathway. In GH₃B₆ cells stimulated with TRH or KCl, enhanced FOS protein levels were detected by immunofluorescence and localized in the nucleus with confocal microscopy. Analysis by immunoblotting showed that TRH induced two protein species with apparent molecular masses of 52 and 57 kDa. In GH₃B₆ cells stimulated with KCl or TRH, the 52 kDa species was mainly found whereas, in the GH₄C₁ cells, TRH predominantly stimulated the 57 kDa species. These data show that distinct signalling pathways (CaM kinase and protein kinase C) involve Ca²⁺ influx to induce the transcription of the early gene c-fos, and that the resulting FOS protein species may depend on the pathways involved.

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