Abstract
Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.
Highlights
Received 23.8.12; revised 22.12.12; accepted 17.1.13; Edited by A Verkhratsky transcobalamin alone.[6,7] The most discernible consequences found in TO cells include reduced proliferation, accelerated differentiation and augmented apoptosis.[7,8] Reduced proliferation has been observed in other vitamin B12-deficient non-neuronal cell models, including that created by the downregulation of transcobalamin receptor TCblR/CD320.9 These B12 deficiency-relevant changes are among the signs known to associate with early aging and death, and they bear certain resemblances to those observed in cells under stress
As the high phosphorylation of eukaryotic initiation factor 2a (P-eIF2a) level in TO cells could be reversed upon the addition of vitamin B12 and SAM, we suggest this stress response to be vitamin B12and methylation-dependent (Figure 1a and Supplementary Figure 1)
The large increase in P-eIF2a level observed in TO cells was concluded as being mainly related to endoplasmic reticulum (ER) stress, considering the increased expression of PKR-like ER kinase (PERK) (Figure 1a) and the unchanged expression of the other eIF2a kinases, namely double-stranded RNA-activated protein kinase (PKR), general control non-repressed 2 (GCN2) and heme-regulated eIF2a kinase (HRI) (Supplementary Figure 2)
Summary
Received 23.8.12; revised 22.12.12; accepted 17.1.13; Edited by A Verkhratsky transcobalamin alone.[6,7] The most discernible consequences found in TO cells include reduced proliferation, accelerated differentiation and augmented apoptosis.[7,8] Reduced proliferation has been observed in other vitamin B12-deficient non-neuronal cell models, including that created by the downregulation of transcobalamin receptor TCblR/CD320.9 These B12 deficiency-relevant changes are among the signs known to associate with early aging and death, and they bear certain resemblances to those observed in cells under stress. Whereas SIRT deacetylation renders p53 less active in inducing apoptosis and senescence, the deacetylation of FOXO3 by SIRT1 potentiates its transcriptional activity, leading to the upregulation of genes for cell cycle arrest and DNA damage repair. Another key player of the SIRT1dependent stress response is heat-shock factor 1 (HSF1), whose activation increases the transcription of molecular chaperones such as HSP70 and HSP90. Cell Death and Disease impaired fatty acid oxidation with myocardium hypertrophy in methyl donor-deficient young rodents.[25] Taken these data together, we asked whether vitamin B12 deficiency induces cellular stress through an altered expression of SIRT1. This article reports that cellular vitamin B12 availability, by controlling SIRT1expression, modulates the severity of ER stress by influencing the extent of HSF1 expression and acetylation in N1E115 dopaminergic cells
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