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The role of cytosolic free Ca²⁺ ([Ca²⁺]_i) in the induction of the immediate early gene c-fos by TRH or by phorbol 12-myristate 13-acetate (PMA) was studied in the clonal pituitary cell line GH₄C₁. It was found that c-fos mRNA levels were rapidly and transiently increased by TRH at physiological concentrations (1–100 nM). The effect of TRH was dependent on a rise in [Ca²⁺]_i, and TRH stimulation of Ca²⁺ influx was essential for c-fos induction. Cell depolarization with K⁺, which produces a [Ca²⁺]_i rise by soliciting Ca²⁺ influx via voltage-gated Ca²⁺ channels, was insufficient to induce c-fos. Blockade or downregulation of protein kinase C (PKC) strongly attenuated TRH stimulation of c-fos expression. Direct stimulation of PKC by PMA raised c-fos mRNA levels, but only under conditions permitting Ca²⁺ influx. We conclude that TRH induces c-fos mRNA by a mechanism dependent on PKC activation and on Ca²⁺ influx. The essential role of Ca²⁺ influx for PMA stimulation of c-fos mRNA suggests a novel pathway linking PKC stimulation to early gene expression.

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