Figure 12.

Signaling pathway leading to Egr-1 biosynthesis in thrombin or carbachol-stimulated 39M1-81 cells. Stimulation of protease-activated and M₁ muscarinic acetylcholine receptors leads to the activation of phospholipase C (PLC), the generation of IP₃ and the release of Ca²⁺-ions into the cytosol via stimulation of ionotropic IP₃ receptors of the endoplasmic reticulum. The increase of the intracellular Ca²⁺-concentration is prevented by pretreating the cells with BAPTA-AM. Elevation of the intracellular Ca²⁺-concentration may induce a transactivation of the EGF receptor or an activation of PKC. Protein kinase C regulates directly or indirectly Raf activity. While preincubation of the cells with AG1478 was used to inhibit the tyrosine kinase activity of the EGF receptor, PKC activity was either blocked by incubation with the bisindolylmaleimide GF109203X or by prolonged treatment with TPA. Both transactivation of the EGF receptor or activation of PKC stimulated the ERK signaling pathway. The compound PD98059 was used to inhibit the phosphorylation of MAP kinase kinase by Raf, thus blocking the stimulus-induced phosphorylation and activation of ERK. Major nuclear substrates for ERK are ternary complex factors such as Elk-1, essential components of the serum response element ternary complex. The Egr-1 promoter contains five SREs that mediate signal-induced activation of Egr-1 gene transcription. Stimulus-induced Egr-1 biosynthesis was blocked by inhibiting ternary complex factor activity via expressing of a dominant-negative mutant (REST/Elk-1ΔC). Likewise, forced dephosphorylation of ERK by overexpression of MKP-1 blocked the signaling cascade leading to enhanced Egr-1 biosynthesis.