Stimulation of Elk1 Transcriptional Activity by Mitogen-activated Protein Kinases Is Negatively Regulated by Protein Phosphatase 2B (Calcineurin)

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 ␤-cells.Steroids synthesized in the central and peripheral nervous system that are, at least in part, independent of steroidogenic gland secretion are termed neurosteroids. They include progesterone, pregnenolone, pregnenolone sulfate, and dehydroepiandrosterone. Pregnenolone sulfate directly acts in the nervous system by modifying neurotransmission, receptor functions, and the strength of synaptic transmission (1). Stimulation with pregnenolone sulfate has been shown to exert modulatory effects on several types of receptors and ion channels including the N-methyl-D-aspartate receptor, the ␥-amino butyric acid-A receptor (1-6), voltage-gated Ca 2ϩ channels, and Kir2.3 K ϩ channels (5, 7-9). Intracerebral infusions of pregnenolone sulfate was shown to influence cognitive processes, neuronal survival, and neurogenesis (10, 11). Interestingly, the molecular cell biology of ␤-cells shows remarkable similarity to that of neurons. Neuronal genes are not only expressed in neurons, but also in endocrine cells. Pancreatic ␤-cells express synaptic vesicle proteins such as synapsin I, synaptophysin or synaptotagmin, neurotransmitters, and neurotransmitter-synthesizing enzymes. In line with this, it has recently been reported that the n...

Section: Discussionsupporting

confidence: 71%

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells

Mayer¹,

Muller²,

Mannebach

et al. 2011

“…Although c-fos expression was maintained at an elevated level through a mechanism independent of increased MAPK activity, constitutive SRE activity could be either because of deficient antagonistic phosphatase function (59,60) or by stimuli-independent phosphorylation by other kinases (61). Alternatively, SRE activity could be also regulated by a pathway involving the Rho family GTPases (62).…”

Section: Discussionmentioning

confidence: 99%

Loss of Net as Repressor Leads to Constitutive Increased c-fos Transcription in Cervical Cancer Cells

Riggelen¹,

Buchwalter²,

Soto³

et al. 2005

We have investigated the expression of c-fos in cervical carcinoma cells and in somatic cell hybrids derived therefrom. In malignant cells, c-fos was constitutively expressed even after serum starvation. Dissection of the c-fos promoter showed that expression was mainly controlled by the SRE motif, which was active in malignant cells, but repressed in their non-malignant counterparts. Constitutive SRE activity was not mediated by sustained mitogen-activated protein kinase activity but because of inefficient expression of the ternary complex factor Net, which was either very low or even barely discernible. Chromatin immunoprecipitation assays re-

“…We postulate several mechanisms underlying these two opposing effects. CaMKII and calcineurin may have different activation kinetics (44). For example, upon Ca 2ϩ influx, after opening VR1, calcineurin activates first and dephosphorylates the channels, leading to channel desensitization.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Vanilloid Receptor 1 by Ca2+/Calmodulin-dependent Kinase II Regulates Its Vanilloid Binding

Jung

Shin

Lee

et al. 2004

244

227

Vanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca 2؉ -calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [ 3 H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/ desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli.