Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells

Abstract

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...

Signal transduction via TRPM3 channels in pancreatic β-cells

“…The connection between an elevated Ca 2C concentration and the activation of the ERK signaling pathway is accomplished by protein kinase C (PKC), most likely PKCa and PKCbII (Benes et al 1999) and the protein kinase Raf (Kolch et al 1993, Corbit et al 2003, Duan & Cobb 2010. Given the similarities between glucose and pregnenolone sulfateinduced signaling, it is not surprising that Raf and ERK are activated in pregnenolone sulfate-stimulated insulinoma cells (Mayer et al 2011 (Mayer et al 2011), suggesting that calcineurin may be part of a second negative feedback loop in the pancreatic b-cells (Fig. 3).…”

“…Recently, the screening of a compound library revealed that citrus fruit flavanones such as naringenin and hesperetin and fabacea secondary metabolites selectively inhibit TRPM3 channel activation (Straub et al 2013). The neurosteroid pregnenolone sulfate, the sulfated form of pregnenolone, has been found to activate TRPM3 channels in insulinoma cells and pancreatic b-cells (Wagner et al 2008, Klose et al 2011, Mayer et al 2011 as well as in other cell types. Stimulation with pregnenolone sulfate also activates TRPM3 channels in HEK293 cells, vascular smooth muscle cells, and synovial fibroblasts (Ciurtin et al 2010, Klose et al 2011, confirming that TRPM3 functions as a Ca 2C -permeable non-selective cation channel.…”

“…Pregnenolone sulfate is not a TRPM3-specific ligand, as this steroid also exerts modulatory effects on several types of receptors and ion channels including the N-methyl-D-aspartate (NMDA) receptor, the gamma amino butyric acid-A receptor, voltage-gated Ca 2C channels, and Kir2.3 K C channels (Horak et al 2004, Chen & Sokabe 2005, Hige et al 2006, Kobayashi et al 2009, Chen et al 2010, Kostakis et al 2011, Mayer et al 2011, Wang 2011. The analysis of chimeric NMDA receptors composed of NR2B and NR2D domains demonstrated that a 'steroid modulatory domain' (SMD1) on the NMDA receptor subunit NR2B is crucial for the stimulation of NR2B-containing NMDA receptors by pregnenolone sulfate.…”

Activation of gene transcription via CIM0216, a synthetic ligand of transient receptor potential melastatin-3 (TRPM3) channels

“…We discovered that TRPM3 stimulation induces a intracellular signaling cascade that results in the activation of the stimulus-responsive transcription factors AP-1, c-Fos, cJun, CREB, Egr-1, and Elk-1. [1][2][3][4][5] The natural ligands for TRPM3 channels are still unknown, but several compounds have been suggested to function as TRPM3-specific agonists. In most of these studies, the influx of Ca 2C ions into the cells and the subsequent rise in the intracellular Ca 2C concentration was used as an indication of activated TRPM3 channels.…”

“…The lentiviral transfer vectors pLL.TRPM3, encoding a TRPM3-specific shRNA, has been described. 1,4 The viral particles were produced as previously described 13,14 by triple transfection of 293T/17 cells with the gag-pol-rev packaging plasmid, the env plasmid encoding VSV glycoprotein, and the transfer vector.…”