Signal transduction of pregnenolone sulfate in insulinoma cells: activation of Egr-1 expression involving TRPM3, voltage-gated calcium channels, ERK, and ternary complex factors.

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 Ca(2+) ions via transient receptor potential M3 and voltage-gated Ca(2+) channels, elevation of the cytosolic Ca(2+) 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 Ca(2+). 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 ß-cells.

Nicotinamide adenine dinucleotide-dependent binding of the neuronal Ca2+ sensor protein GCAP2 to photoreceptor synaptic ribbons.

Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approaches that GCAP2 specifically interacts with RIBEYE in photoreceptor synapses. We show that the flexible hinge 2 linker region of RIBEYE(B) domain that connects the nicotinamide adenine dinucleotide (NADH)-binding subdomain with the substrate-binding subdomain (SBD) binds to the C terminus of GCAP2. We demonstrate that the RIBEYE-GCAP2 interaction is induced by the binding of NADH to RIBEYE. RIBEYE-GCAP2 interaction is modulated by the SBD. GCAP2 is strongly expressed in synaptic terminals of light-adapted photoreceptors where GCAP2 is found close to synaptic ribbons as judged by confocal microscopy and proximity ligation assays. Virus-mediated overexpression of GCAP2 in photoreceptor synaptic terminals leads to a reduction in the number of synaptic ribbons. Therefore, GCAP2 is a prime candidate for mediating Ca2+-dependent dynamic changes of synaptic ribbons in photoreceptor synapses.

Epidermal-growth-factor-induced proliferation of astrocytes requires Egr transcription factors.

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.

Expression of the transcriptional repressor ATF3 in gonadotrophs is regulated by Egr-1, CREB, and ATF2 after gonadotropin-releasing hormone receptor stimulation.

Stimulation of GnRH receptors enhances expression of activating transcription factor (ATF) 3 in a pituitary gonadotroph cell line. The signaling pathway requires elevated cytosolic Ca2+ levels and activation of ERK and c-Jun N-terminal protein kinase. The signaling cascade was blocked by overexpression of either MAPK phosphatase (MKP)-1 or MAPK phosphatase-5 that dephosphorylate nuclear ERK and c-Jun N-terminal protein kinase. In addition, ATF3 biosynthesis was impaired after lentiviral-mediated expression of a constitutively active mutant of calcineurin A. Thus, MKP-1, MKP-5, and calcineurin may function as shut-off devices for GnRH receptor signaling. Expression of dominant-negative mutants of early growth response protein (Egr)-1, cAMP response element binding protein (CREB), and ATF2 blocked the biosynthesis of ATF3, indicating that these transcription factors connect the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the ATF3 gene. This view was corroborated by chromatin immunoprecipitation experiments revealing that Egr-1 and the phosphorylated forms of CREB and ATF2 bound to the 5'-upstream region of the ATF3 gene in buserelin-stimulated gonadotrophs. Together the data indicate that the ATF3 gene is a bona fide target gene of Egr-1, CREB, and ATF2 in gonadotrophs. Moreover, we show that in gonadotrophs ATF3 bound to its own promoter under physiological conditions. The analysis of a lentiviral-transmitted ATF3 promoter/luciferase reporter gene, embedded into the chromatin of the cells, revealed that ATF3 blocked the activity of its own promoter. We additionally identified the chromogranin B gene as bona fide target gene of ATF3 in gonadotrophs.

Elk-1, CREB, and MKP-1 regulate Egr-1 expression in gonadotropin-releasing hormone stimulated gonadotrophs.

Stimulation of gonadotropin-releasing hormone (GnRH) receptors with the GnRH analogue buserelin enhances expression of the zinc finger transcription factor Egr-1 in a pituitary gonadotroph cell line. The signaling cascade is blocked by overexpression of MAP kinase phosphatase-1 that dephosphorylates extracellular signal-regulated protein kinase in the nucleus. Chromatin immunoprecipitation experiments revealed that the phosphorylated form of Elk-1, a key regulator of gene transcription driven by serum response element (SRE), binds to the 5'-upstream region of the Egr-1 gene in buserelin-stimulated gonadotrophs. Expression of a dominant-negative mutant of Elk-1 completely blocked Egr-1 expression, indicating that Elk-1 connects the intracellular signaling cascade elicited by activation of GnRH receptors with transcription of the Egr-1 gene. GnRH receptor activation additionally induced the phosphorylation of CREB, which in its phosphorylated form bound to the Egr-1 gene. Expression of a dominant-negative mutant of CREB reduced GnRH receptor-induced upregulation of Egr-1 expression, indicating that CREB plays a role in the signaling pathway that regulates Egr-1 expression in gonadotrophs. We further identified the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, and transforming growth factor beta as bona fide target genes of Egr-1 in gonadotrophs. The analysis of gonadotroph cells that express--in addition to GnRH receptors--muscarinic M(3) acetylcholine receptors revealed that the nuclear events connecting GnRH receptors and muscarinic M(3) acetylcholine receptors with the Egr-1 gene are indistinguishable.

Egr-1-A Ca(2+)-regulated transcription factor.

The biosynthesis of the zinc finger transcription factor Egr-1 is stimulated by many extracellular signaling molecules including hormones, neurotransmitters, growth and differentiation factors. The Egr-1 gene represents a convergence point for many intracellular signaling cascades. An increase of the intracellular Ca(2+) concentration, by activating ionotropic or Galpha(q/11)-coupled receptors or voltage-gated L-type Ca(2+) channels, is often the prerequisite for enhanced Egr-1 gene transcription. This increase has been observed following stimulation with extracellular signaling molecules including ATP, glutamate, thrombin, carbachol, gonadotropin-releasing hormone, or glucose. Egr-1 is thus a Ca(2+) regulated transcription factor - similar to CREB, NFAT, NF-kappaB and others. This review also discusses the importance of the cytoplasmic and nuclear Ca(2+) concentration in transcriptional regulation of the Egr-1 gene.

Calcium influx into MIN6 insulinoma cells induces expression of Egr-1 involving extracellular signal-regulated protein kinase and the transcription factors Elk-1 and CREB.

Glucose induces many changes in the transcriptional pattern of beta-cells derived from the endocrine pancreas. The zinc finger protein Egr-1 belongs to the transcription factors that are activated in glucose-treated beta-cells. Egr-1 expression is additionally induced by treatment of MIN6 pancreatic beta-cells with tolbutamide, a compound that triggers a closure of ATP-dependent potassium channels, K(ATP), in the plasma membrane or by KCl that depolarizes the cell membrane. Stimulation with glucose, tolbutamide or KCl induces a Ca2+ influx into the beta-cells via L-type Ca2+ channels. Accordingly, incubation of the cells with the L-type Ca2+ channel blocker nifedipine or the acetoxymethylester of the cytosolic Ca2+ chelator BAPTA prevented Egr-1 expression. Moreover, diacylgycerol-dependent protein kinase C isoenzymes and activation of extracellular signal-regulated protein kinase (ERK) are required for glucose-, tolbutamide- and KCl-induced Egr-1 expression. The signaling cascade was blocked by MAP kinase phosphatase-1 (MKP-1) overexpression that dephosphorylated ERK in the nucleus. Stimulation of beta-cells by glucose, tolbutamide and KCl induced the phosphorylation of the transcription factors Elk-1 and CREB. ChIP experiments revealed that phosphorylated Elk-1 and CREB bound under physiological conditions to the Egr-1 gene. Lentiviral-mediated expression of dominant-negative mutants of Elk-1 or CREB interfered with glucose-, tolbutamide- and KCl-induced upregulation of Egr-1 biosynthesis. Together, these data indicate that stimulus-induced transcription of the Egr-1 gene in beta-cells requires combinatorial regulation by Elk-1 and CREB following activation of ERK. The newly synthesized Egr-1 is biologically active and binds under physiological conditions to the genes encoding basic fibroblast growth factor, tumor necrosis factor alpha, transforming growth factor beta and PTEN.