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Trans-2-enoyl-CoA reductase limits Ca2+ accumulation in the endoplasmic reticulum by inhibiting SERCA2b Ca2+ pump

ABSTRACT: The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). How FA metabolic enzymes cooperate with other ER functions, such as calcium (Ca2+) accumulation and its regulated release to the cytosol, is elusive. Trans-2-enoyl-CoA reductase (TER) is a FA reductase present in the ER membrane, and catalyzes the last step of FA elongation cycle and sphingosine degradation pathway. Here we show that TER directly binds to an ER Ca2+ pump, sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), and regulates its activity. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes. Depletion of TER increases the ER Ca2+ storage and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.

INSTRUMENT(S): LCMS-IT-TOF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Toshiaki Sakisaka

LAB HEAD: Toshiaki Sakisaka

PROVIDER: PXD020792 | Pride | 2021-01-13

REPOSITORIES: Pride

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Project description:Altered Ca2+ handling has both immediate physiological effects and long-term genomic effects on vascular smooth muscle function. Previously we have shown that elevation of cytoplasmic Ca2+ through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element binding protein (CREB) in cerebral arteries. Here we demonstrate that stimulation of these different Ca2+ influx pathways results in transcriptional activation of a distinct, yet overlapping set of genes, and that the induction of selected CRE-regulated genes is prevented by the addition of corresponding Ca2+ channel blockers. Using oligonucleotide array analysis, changes in mRNA levels were quantified following membrane depolarization with K+ or depletion of intracellular Ca2+ stores with thapsigargin in human cerebral vascular smooth muscle cells. Array results for differentially regulated genes containing a CRE were confirmed by quantitative RT-PCR, and corresponding changes in protein expression were shown by Western blot analysis and immunofluorescence. Membrane depolarization induced a transient increase in c-fos mRNA and a sustained increase in early growth response-1 (Egr-1) mRNA and protein that were inhibited by application of the VDCC blocker, nimodipine, and the SOCC inhibitor, 2-aminoethoxydiphenylborate (2-APB). Thapsigargin induced a sustained increase in c-fos mRNA and MAP kinase phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB but not by nimodipine. Our findings thus indicate that Ca2+ entry through VDCCs and SOCCs can differentially regulate CRE-containing genes in vascular smooth muscle and imply that signals involved in growth modulation are both temporally and spatially regulated by Ca2+. Experiment Overall Design: each of three experiments cell cultures were split three ways; one of the resulting samples was left untreated (C), another was treated with thapsigargin (TG), and the third was treated with elevated K+ (K). The resulting nine samples were used for triplicate estimates of the response of each gene to TG and K treatments. Experiment Overall Design: We tested the prospective hypothesis that genes having a CRE are differentially expressed after TG or K treatment using a permutation test: each of the 22,283 probe sets on the Affymetrix GeneChip was categorized two ways based on 1) whether or not it contains a CRE or not . Independence of CRE and threshold differential expression was rejected by Fisherâs exact test for both TG treatment ( ) and K treatment ( ). Target genes (c-fos, egr-1, and mkp-1 ) were identified based on ranking.

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Trans-2-enoyl-CoA reductase limits Ca2+ accumulation in the endoplasmic reticulum by inhibiting SERCA2b Ca2+ pump

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