PEMF and ceramides

This journey starts with the notion that 50 Hz from powerlines contributes to leukemia via ceramides in lipid membranes. It does so via interaction of PEMF with voltage gated Ca²⁺ channels. Signaling kinases related to changes in intracellular Ca²⁺ were examined. Another kinase, Akt, was found to be the signaling kinase and intracellular store operated Ca²⁺ channel the source of endogenous Ca²⁺release. Far from leukemia, this pathway is important in muscle stretch.

The basics

A kinase is an enzyme that transfers a phosphate group from adenosine triphosphate (ATP) to a serine, threonine, or tyrosine amino acid side chain of a protein. Phosphorylation is a common way of turning enzymes on. Phosphorylation, transfer of a phosphate group, may also occur in lipids as is shown in the featured image for sphingosine kinase.

Lipid rafts are rigid regions in the cell membrane enriched in cholesterol and “sphingolipids. ” Rafts tend to be hot zones of “signal transduction” of growth hormone receptors on the outside of the cell to intracellular kinases and other signaling enzymes that tell the nucleus to respond to changes in the environment.

Ceramides and 50 Hz

This 2009 study study by Qiu and coauthors tested the hypothesis that the very low electric grid 50 Hz PEMF could be associated with childhood leukemia. The ceramide connection came from previous studies demonstrating that 50-Hz, 0.4 mT PEMF exposure resulted in epidermal growth factor (EGF) receptor clustering that other studies demonstrated to be influenced by ceramide.

Anionic fluid FL cells were chosen for study.

For ceramide metabolism experiments, FL cells were exposed to MF at 0.4 mT for 60 min with or without pretreatment of imipramine (50 lM, 4 h). Lipid extraction and quantitation were followed the protocol described above.

Imipramine is a tricyclic antidepressants which block block histamine H₁ receptors, α₁-adrenergic receptors and muscarinic receptors as well as inhibit the reuptake of norepinephrine and serotonin. Use of imipramine as a sphingomyelinase inhibitor is well documented in the PubMed peer reviewed literature. Acid sphingumyelinase catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine.

The ceramide family

The first focus was which ceramides were are influenced by PEMF.

These data were adapted from bar graphs in the Qiu 2016 publication. Sham PEMF stayed constant at 100% over the 60 minute exposure. By 60 minutes, all ceramide species were statistically significantly increased. The 24 acyl chain species was over doubled making it likely to be functionally significantly increased.

The C16 carbon acyl chain increased more with less time whereas the C24 chain took longer exposure to really increase. The `10 minutes exposure was chosen for further experiments.

treatment	C16	C18	C24
sham SM	100	100	100
MF SM	75	75	65
MF +Imip	80	100	100
sham Cer	100	100	100
MF Cer	125	120	120
MF + Imip Cer	160	80	100

A table of approximations of Qiu 2016 Figure 3 bar graphs. It makes sense that if PEMF is increasing sphingomyelinase activity, SM levels would decrease and ceramide (CER) levels would increase. Perhaps imipramine sort of resembles the product phosphoryl choline. It makes sense that imipramine would keep C18 sphingomyelin levels at 100% in PEMF. The system breaks down with the large increase (significant only over the sham) of C16 ceramide. promote C

Qiu 2016 hypothesized that PEMF was doing something to promote synthesis of C16 ceramide, perhaps by addition of a C16 acyl group to sphingosine.

Something else is going on

Difference first author, Xiaobo Yang, same Wenjun Sun Lab, same PEMF treatment, and the same amniotic fluid FL cells…. The goal of this 2019 was to determine the signal transduction pathway and how ceramides were being increased. The previous study, Qiu 2016, failed to provide convincing evidence that sphingomyelinase alone could explain the PEMF increase in ceramides.

kinase inhibitors used

Two new inhibitors were added to the testing:

• Go6976 an inhibitor of protein kinase C alpha, PKC
• Nifedipine (NIF), an L-type Ca²⁺ channel inhibitor.
• U0126 in an ERK inhibitor. Extracellular signal-Regulated Kinases transfer phosphate groups to serine or threonine amino acid side chains upon activated (phosphorylated) when growth factors bind extracellular receptors.
• SKI II is a sphingosine kinase inhibitor that reduces levels of sphingosine -1-phosphate.

It should be noted that PKCα is activated by increases in Ca²⁺. The additional twist is that enzyme activity of sphingosine kinase (SK1), PKCα, and PKCα’s target ERK kinase were traditionally assumed to be turned on by phosphorylation. Antibodies against kinase specific phosphorylation sites were used to determine if these kinases had been turned on.

PEMF increases intracellular ca²⁺

Further downstream, ERK kinase phosphorylation was increased by the standard 10 min 50 Hz PEMF by about 10%. Both Nif (L-type Ca²⁺ channel inhibitor) and U0126 ( ERK inhibitor) prevented the increase in SK1 phosphorylation (activation.) Additionally, Nif prevented the tiny 10-20% increase in ERK phosphorylation. The PKCα’ did not result in a significant decrease in the small increase in SK1 phosphorylation (activation). The spingosine kinase 1 inhibitor SK1 II reduced both endogenous and PEMF phosphorylation of ERK and PKCα phosphorylation levels.

L-type Ca²⁺ channels about face

Ye 2022, Same everything except remove the extracellular Ca²⁺. A 50-Hz MF exposure did not affect the activation of ERK and PKCα. This time the Sun Lab was still interested in the concept that 50 Hz from powerlines could contribute to leukemia. Akt was activated by 50 Hz PEMF except when intracellular Ca²⁺ was chelated with BAPTA,. L-type Ca²⁺channel inhibitor Nif was washout effect under Ca²⁺ free conditions . Treatment of FL cells with LY294002, the inhibitor of Akt, could delete the 50 Hz-induced SK1 activation under the condition of calcium-free medium. Store activated endogenous Ca²⁺ release was involved in 50-Hz PEMF-induced cell proliferation via Akt-SK1 signal cascade.PK1 had also been reported to be opened by lowering of Ca²⁺ in the sarcoplasmic reticulum.

rcsb.org Crystal structure of sphingosine kinase 3VZB

Instead let us look at the structure of sphingosine kinase. Is there something that this structure published by Wang and others at Amgen can tell us? This is a quote from the PubMed abstract

PubMed Abstract: 

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.

Some images of the crystal structure of sphingosine kinase 1. Note the alternating hydrophobic and hydrophilic domains.

The ligands are (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol (Erythrosphingosine), ethandiol, and sulfate. Can mechanical pushing of this enzyme influence its activity? Can movement of charges over the surface influence activity? Yang 2019 demonstrated that 50 Hz PEMF could, but also hinted at other pathways such as Akt.

References

• Formigli L, Sassoli C, Squecco R, Bini F, Martinesi M, Chellini F, Luciani G, Sbrana F, Zecchi-Orlandini S, Francini F, Meacci E. (2009) Regulation of transient receptor potential canonical channel 1 (TRPC1) by sphingosine 1-phosphate in C2C12 myoblasts and its relevance for a role of mechanotransduction in skeletal muscle differentiation. J Cell Sci. 2009 May 1;122(Pt 9):1322-33. sci-hub free paper
• Ke XQ, Sun WJ, Lu DQ, Fu YT, Chiang H. 50-Hz magnetic field induces EGF-receptor clustering and activates RAS. Int J Radiat Biol. 2008 May;84(5):413-20. Sci-Hub free paper
• Qiu L, Feng B, Ni Z, Wu X, Sun W. (2016) Exposure to a 50-Hz magnetic field induced ceramide generation in cultured cells. Int J Radiat Biol. 2016;92(4):215-21. Sci-Hub free paper
• Yang X, Ye A, Chen L, Xia Y, Jiang W, Sun W. (2019) Involvement of calcium in 50-Hz magnetic field-induced activation of sphingosine kinase 1 signaling pathway. Bioelectromagnetics. 2019 Apr;40(3):180-187. Sci-hub free paper
• Ye AF, Liu XC, Chen LJ, Xia YP, Yang XB, Sun WJ. Endogenous Ca²⁺ release was involved in 50-Hz MF-induced proliferation via Akt-SK1 signal cascade in human amniotic epithelial cells. Electromagn Biol Med. 2022 Apr 3;41(2):142-151. PubMed