ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase

Abstract

ORMDL proteins are believed to be negative regulators of serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single-nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and total SL levels. Ormdl3-knockout (Ormdl3) and transgenic (Ormdl3) mice were generated to study the effect of ORMDL3 on total SL levels in plasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormdl3 mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Furthermore, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population-based cohort of 971 individuals. Total C-long chain bases were not significantly altered in the plasma and tissues of Ormdl3 mice, whereas C-sphinganine showed a small and significant increase in plasma, lung, and liver tissues. Mouse embryonic fibroblast cells from Ormdl3 mice did not show an altered SPT activity compared with Ormdl3 and Ormdl3 mice. Overexpression or knockdown of ORMDL3 in HEK293 cells did not alter SPT activity; however, parallel knockdown of all 3 ORMDL isoforms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long-chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seem not to be directly associated with changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.-Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.

Decreased sphingolipid synthesis in children with 17q21 asthma–risk genotypes

“…Whether this difference asthma-risk alleles are associated with decreased whole-blood sphingolipids. Of note, these results cannot be compared with a study involving a Swiss adult population, in which no associations were found between hydrolytic products of all plasma sphingolipids and rs7216389 genotypes (27). Those analyses cannot distinguish long-chain bases originating from de novo synthesis and were not performed in whole blood.…”

Sphingosine-1-Phosphate Signaling and Metabolism Gene Signature in Pediatric Inflammatory Bowel Disease: A Matched-case Control Pilot Study

“…38 ORMDL3 has been proposed as a negative regulator of serine palmitoyltransferase (SPT) enzymes that catalyze the initiation of de novo sphingolipid synthesis, 39 although direct in vivo evidence for ORMDL3 interaction with SPT enzymes has been recently been questioned. 40 Expression levels of ORMDL3 showed no significant changes between controls and IBD patients, regardless of disease severity or disease subtype (UC or CD) (Fig. 1).…”

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice

“…ORMDL3 knockout (Ormdl3 2/2 ) mice and ORMDL3 transgenic (Ormdl3 Tg/wt ) mice were previously described. 25 All mice were backcrossed for at least 10 crosses onto a C57BL/6 background. 1-DER mice express an MHC class II-restricted T-cell receptor specific for Der p 1 on their CD4 T cells and were generated, as previously described.…”

“…Ormdl3-LacZ reporter mice were generated by inserting a targeting construct into the first intron of Ormdl3 to be able to better address ORMDL3 expression ( Fig 1, A). 25 Although these mice still express some basal Ormdl3 (Fig 1, B), b-galactosidase activity can be used as a readout for Ormdl3 promoter activation.…”

“…Mammalian SPT activity seems to be affected only when all ORMDL paralogues are overexpressed or downregulated simultaneously, 17,[24][25][26] making it unlikely that SNPs in only ORMDL3 influence asthma through SPT inhibition. As an ER-resident protein, ORMDL3 has also been described to affect calcium metabolism and the unfolded protein response (UPR), influencing cytokine secretion by structural or immune cells.…”