Stimulation of astrocytes with epidermal growth factor (EGF) induced proliferation and triggered the biosynthesis of the transcription factor Egr-1, involving the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway. No differences in the proliferation rate of astrocytes prepared from wild-type or Egr-1-deficient mice were detected. However, expression of a dominant-negative mutant of Egr-1 that interfered with DNA-binding of all Egr proteins prevented EGF-induced proliferation of astrocytes. Site-directed mutagenesis of two crucial cysteine residues within the zinc finger DNA-binding domain revealed that DNA-binding of the Egr-1 mutant was essential to inhibit proliferation of EGF-stimulated astrocytes. Expression of NAB2 (a negative co-regulator of Egr-1, Egr-2 and Egr-3) or a dominant-negative mutant of Elk-1 (a key regulator of Egr-1 biosynthesis) abolished EGF-induced proliferation of astrocytes. Chromatin immunoprecipitation experiments showed that Egr-1, Egr-2 and Egr-3 bound to the gene expressing basic fibroblast growth factor (bFGF) in EGF-stimulated astrocytes. Egr-2 and Egr-3 also interacted with the bFGF gene in EGF-stimulated astrocytes prepared from Egr-1-deficient mice, indicating that loss of Egr-1 is compensated by other Egr proteins. Together, these data show that Egr transcription factors are essential for conversion of the mitogenic signal of EGF into a proliferative response.

The neurosteroid pregnenolone sulfate acts on the nervous system by modifying neurotransmission and receptor functions, thus influencing synaptic strength, neuronal survival, and neurogenesis. Here we show that pregnenolone sulfate induces a signaling cascade in insulinoma cells leading to enhanced expression of the zinc finger transcription factor Egr-1 and Egr-1-responsive target genes. Pharmacological and genetic experiments revealed that influx of Ca²⁺ ions via transient receptor potential M3 and voltage-gated Ca²⁺ channels, elevation of the cytosolic Ca²⁺ level, and activation of ERK are essential for connecting pregnenolone sulfate stimulation with enhanced Egr-1 biosynthesis. Expression of a dominant-negative mutant of Elk-1, a key regulator of gene transcription driven by a serum response element, attenuated Egr-1 expression following stimulation, indicating that Elk-1 or related ternary complex factors connect the transcription of the Egr-1 gene with the pregnenolone sulfate-induced intracellular signaling cascade elicited by the initial influx of Ca²⁺. The newly synthesized Egr-1 was biologically active and bound under physiological conditions to the regulatory regions of the Pdx-1, Synapsin I, and Chromogranin B genes. Pdx-1 is a major regulator of insulin gene transcription. Accordingly, elevated insulin promoter activity and increased mRNA levels of insulin could be detected in pregnenolone sulfate-stimulated insulinoma cells. Likewise, the biosynthesis of synapsin I, a synaptic vesicle protein that is found at secretory granules in insulinoma cells, was stimulated in pregnenolone sulfate-treated INS-1 cells. Together, these data show that pregnenolone sulfate induces a signaling cascade in insulinoma cells that is very similar to the signaling cascade induced by glucose in β-cells.

Ca²⁺ ions function as second messengers regulating many intracellular events, including neurotransmitter release, exocytosis, muscle contraction, metabolism and gene transcription. Cells of a multicellular organism express a variety of cell-surface receptors and channels that trigger an increase of the intracellular Ca²⁺ concentration upon stimulation. The elevated Ca²⁺ concentration is not uniformly distributed within the cytoplasm but is organized in subcellular microdomains with high and low concentrations of Ca²⁺ at different locations in the cell. Ca²⁺ ions are stored and released by intracellular organelles that change the concentration and distribution of Ca²⁺ ions. A major function of the rise in intracellular Ca²⁺ is the change of the genetic expression pattern of the cell via the activation of Ca²⁺-responsive transcription factors. It has been proposed that Ca²⁺-responsive transcription factors are differently affected by a rise in cytoplasmic versus nuclear Ca²⁺. Moreover, it has been suggested that the mode of entry determines whether an influx of Ca²⁺ leads to the stimulation of gene transcription. A rise in cytoplasmic Ca²⁺ induces an intracellular signaling cascade, involving the activation of the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin and various protein kinases (protein kinase C, extracellular signal-regulated protein kinase, Ca²⁺/calmodulin-dependent protein kinases). In this review article, we discuss the concept of gene regulation via elevated Ca²⁺ concentration in the cytoplasm and the nucleus, the role of Ca²⁺ entry and the role of enzymes as signal transducers. We give particular emphasis to the regulation of gene transcription by calcineurin, linking protein dephosphorylation with Ca²⁺ signaling and gene expression.