Endoplasmic Reticulum Stress Increases the Expression of Methylenetetrahydrofolate Reductase through the IRE1 Transducer

Rima Rozen,Daniel Leclerc

doi:10.1074/jbc.m708598200

Abstract

Methylenetetrahydrofolate reductase (MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels. Mild deficiency of MTHFR is common in many populations and modifies risk for several complex traits including vascular disease, birth defects, and cancer. We recently demonstrated that MTHFR can be up-regulated by NF-kappaB, an important mediator of cell survival that is activated by endoplasmic reticulum (ER) stress. This observation, coupled with the reports that homocysteine can induce ER stress, prompted us to examine the possible regulation of MTHFR by ER stress. We found that several well characterized stress inducers (tunicamycin, thapsigargin, and A23187) as well as homocysteine could increase Mthfr mRNA and protein in Neuro-2a cells. The induction of MTHFR was also observed after overexpression of inositol-requiring enzyme-1 (IRE1) and was inhibited by a dominant-negative mutant of IRE1. Because IRE1 triggers c-Jun signaling, we examined the possible involvement of c-Jun in up-regulation of MTHFR. Transfection of c-Jun and two activators of c-Jun (LiCl and sodium valproate) increased MTHFR expression, whereas a reported inhibitor of c-Jun (SP600125) and a dominant-negative derivative of c-Jun N-terminal kinase-1 reduced MTHFR activation. We conclude that ER stress increases MTHFR expression and that IRE1 and c-Jun mediate this activation. These findings provide a novel mechanism by which the ER can regulate homeostasis and allude to an important role for MTHFR in cell survival.

Highlights

(MTHFR), an enzyme in folate and homocysteine metabolism, influences many cellular processes including methionine and nucleotide synthesis, methylation reactions, and maintenance of homocysteine at nontoxic levels
GRP78 is a chaperone protein that is a standard marker of the endoplasmic reticulum (ER) stress response, whereas Nos2 activation can be attributed to activation of NF-κB signaling, since it is abolished when BAY11-7082 (Fig. 1C) or PDTC are administered with tunicamycin
Bay11-7082 did not inhibit the upregulation of Grp78 and Mthfr mRNAs by tunicamycin (Figs. 1A & B), indicating that these 2 genes are activated by ER stress independently of NF-κB

Summary

Experimental Procedures

Cell culture conditions and transfection__ Neuro-2a neuroblastoma cells and RAW264.7 macrophages were maintained in high glucose Dulbecco’s modified Eagle’s medium containing 10% serum at 37 ̊C. Overexpression of DNA constructs harboring the short or long isoforms of MTHFR was performed in E. coli, as previously described [15]. RNA purification and real time RTPCR__ Total RNA extraction from cultured cells and real-time RT-PCR were performed as previously described [14]. Cell lysis was performed in a lysis buffer (1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) in the presence of Complete Mini (protease inhibitor cocktail from Roche Applied Science, Laval, Quebec). 50 μg protein from each lysate were diluted with loading buffer, boiled and loaded onto SDS-polyacrylamide gels. To ensure reproducibility of observations, all Western blot experiments were performed at least twice, using extracts from independent experiments. Antibodies against GRP78, FLAG and ß-actin were from Sigma (Oakville, Ontario). Polyclonal antibody against MTHFR was previously described [15]. Activities were measured in duplicate and less than 6% variation was observed between repeats

RESULTS

DISCUSSION

FIGURE LEGENDS