Possible Involvement of Nitric Oxide and Reactive Oxygen Species in Glucose Deprivation-Induced Activation of Transcription Factor Rst2

Glucose is one of the most important sources of cellular nutrition and glucose deprivation induces various cellular responses. In Schizosaccharomyces pombe, zinc finger protein Rst2 is activated upon glucose deprivation, and regulates gene expression via the STREP (stress response element of Schizosaccharomyces pombe) motif. However, the activation mechanism of Rst2 is not fully understood. We monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system. Hydrogen peroxide (H2O2) enhanced Rst2 transcriptional activity upon glucose deprivation and free radical scavenger inhibited Rst2 transcriptional activity upon glucose deprivation. In addition, deletion of the trx2 + gene encoding mitochondrial thioredoxin enhanced Rst2 transcriptional activity. Notably, nitric oxide (NO) generators enhanced Rst2 transcriptional activity upon glucose deprivation as well as under glucose-rich conditions. Furthermore, NO specific scavenger inhibited Rst2 transcriptional activity upon glucose deprivation. Altogether, our data suggest that NO and reactive oxygen species may be involved in the activation of transcription factor Rst2.


Introduction
Glucose is the main source of energy for most cells and glucose deprivation induces various cellular processes including gene expression, metabolic change, and oxidative stress [1][2][3]. The fission yeast Schizosaccharomyces pombe (S. pombe) is a good model system for studying mechanisms of glucose deprivation-induced gene expression in higher eukaryotes [4].
Zinc-finger protein Rst2 plays an important role in glucose deprivation-induced gene expression. Upon glucose deprivation, Rst2 induced expression of the fbp1 + gene, encoding a fructose-1,6-bis-phosphatase, via the STREP (stress response element of Schizosaccharomyces pombe) motif [5]. It has also been demonstrated that under glucose-rich conditions, cAMP-dependent kinase (PKA) directly phosphorylates and inhibits Rst2. Upon glucose deprivation PKA-independent activation of Rst2 is observed [5], however, the mechanism is not well understood.
In our previous study, we developed a method to monitor the transcriptional activity in living cells [6]. To identify the activation mechanisms of Rst2, we monitored Rst2 transcriptional activity. The results show that hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) generators enhanced Rst2 transcriptional activity. Free radical scavenger and NO specific scavenger inhibited glucose deprivation-induced activation of Rst2. These results highlight that reactive oxygen species (ROS) and NO may be involved in the activation of Rst2.

Strains, Media, and Genetic and Molecular Biology Methods
S. pombe strains used in this study are listed in Table 1. The normal minimal medium EMM (Edinburgh minimal medium), low glucose EMM and YES media have been described previously [7][8][9]. Standard genetic and recombinant-DNA methods [10] were used except where noted.

Disruption of the trx1 + Gene
To knockout the trx1 + gene, a PCR-based targeted gene deletion method was prepared by the Cre-loxP-mediated marker removal procedure as described previously [11]. The DNA fragments containing the disrupted trx1 + , were amplified by using the plasmid pKB6640 which contains the lys3 + marker as a template [11], and using the sense primer 5′-cgt taa atc gat ttt ttc ttt att tga gta tat att ttt aac tta att tcc cat ttc att tat ata caa cCC AAT AGG CCG AAA TCG GCA AAA TCC C-3′, and the antisense primer 5′-cat tta ttt ttg tta aat aaa aat att ttg tat tac aag ttc ata aca act aac tat cag att gcg taa aGG TGA TGG TTC ACG TAG TGG GCC-3′. The resulting products containing trx1::lys3 + disruption fragments were transformed into KP3157 (h -leu1-32 lys3::loxp) cells [11]. Stable integrants were selected on medium lacking lysine. The disruption of the gene was checked using PCR (data not shown).

Real-Time Monitoring Assay of Rst2-Mediated Transcriptional Activity
The multi-copy reporter plasmid (pKB8307) was transformed into fission yeast cells for reporter assays. The transformants were cultured at 27°C in normal EMM media overnight to midlog phase and recovered by centrifugation. Then the cells were resuspended in refresh EMM containing 2% glucose as glucose-rich medium (GR), or in low glucose EMM containing 0.1% glucose to induce glucose deprivation (GD). Coelenterazine was used as a substrate for Renilla luciferase and yielding luminescence was detected using a luminometer (AB-2350; ATTO Co., Tokyo, Japan) at 1-min intervals and reported as relative light units (RLU).

Real-Time Monitoring of Rst2 Transcriptional Activity in Living Cells
Transcriptional factor Rst2 regulates gene expression via the STREP motif [5]. We constructed reporter plasmid containing three tandem repeats of STREP fused to Renilla luciferase (3xSTREP::Renilla). In wild-type cells, glucose deprivation caused a marked increase in the transcription with a peak at about 80 min ( Figure 1A). In Δrst2 cells, glucose deprivationinduced transcription was completely abolished ( Figure 1B). These results indicate that the reporter assay reflects Rst2 transcriptional activity. Previous work indicated that Rst2 is activated by glucose deprivation [5]. To examine whether Rst2 is specifically activated by glucose deprivation, wild-type cells were subjected to oxidative stress (1 mM H 2 O 2 ), osmotic stress (300 mM KCl) or heavy metal stress (1 mM CdCl 2 ), respectively. The results clearly showed that 3xSTREP::Renilla responded to glucose deprivation, but not H 2 O 2 , KCl or CdCl 2 ( Figure 1C).

PKA Inhibited Rst2 Transcriptional Activity
S. pombe has a single gene encoding the catalytic subunit of PKA, pka1 + [12]. Previous work indicated that Rst2 is phosphorylated and inhibited by PKA under glucose-rich conditions [5]. We then monitored Rst2 transcriptional activity in Δpka1 cells. The Δpka1 cells showed high basal transcription activity with normal response to glucose deprivation (Figure 2A and B). We also monitored whether glucose deprivation-induced activation of Rst2 is repressed by adenosine-3′,5′-cyclic monophosphate (cAMP) addition. In wildtype cells, the addition of cAMP caused a dose-dependent decrease in glucose deprivation-induced activation of Rst2, whereas cAMP did not significantly inhibit Rst2 transcriptional activity in Δpka1 cells ( Figure 2C). The results indicate that cAMP inhibited glucose deprivation-induced activation of Rst2 through PKA. Altogether, these results suggest that PKA functions as a negative regulator of Rst2 and other mechanisms may be involved in the activation of Rst2.

Redox Change May Be Involved in Glucose Deprivation-Induced Transcriptional Activation of Rst2
Free radical ROS, such as H 2 O 2 and superoxide, cause oxidative stress and act as signal molecules [13]. Previous work indicated that glucose deprivation induces oxidative stress in S. pombe [14]. These results led us to investigate the relationship between free radical ROS and Rst2 transcriptional activity. Under glucose-rich conditions, 1 mM H 2 O 2 did not affect Rst2 transcriptional activity ( Figure 1C). In contrast, H 2 O 2 caused a dose-dependent increase in Rst2 transcription activity upon glucose deprivation ( Figure 3A and B). Free radical scavenger N-acetyl-L-cysteine (NAC; NACALAITESQUE, INC.) inhibits the oxidative stress-induced activation of the Sty1 MAPK pathway [6]. We next addressed whether NAC inhibits glucose deprivation-induced activation of Rst2. NAC caused a dose-dependent decrease in glucose deprivation-induced activation of Rst2 ( Figure 3C and D). These results suggest that free radical ROS may be involved in the Rst2 transcriptional activation induced by glucose deprivation.
We previously demonstrated that H 2 O 2 activates Sty1 and that NAC inhibits oxidative stress-induced activation of Sty1 [6]. In Δsty1 cells, H 2 O 2 increased Rst2 transcriptional activity upon glucose deprivation, and NAC inhibited Rst2 transcriptional activity upon glucose deprivation (data not shown). The results indicate that the effect of H 2 O 2 or NAC on Rst2 activity is independent on Sty1.

Deletion of the trx2 + Gene Enhanced Rst2 Transcriptional Activity
The free radical scavenger thioredoxin is conserved from prokaryote to eukaryote and plays a role in maintaining the cellular redox environment [15]. There are two thioredoxins, cytosolic thioredoxin Trx1 and mitochondrial thioredoxin Trx2 in S. pombe [16]. We looked at H 2 O 2 sensitivity of Δtrx1 and Δtrx2 cells. The results showed that on YES containing 3 mM H 2 O 2 the growth of Δtrx1 cells was completely inhibited, whereas that of Δtrx2 cells was partially inhibited ( Figure 4A). These results indicate that both cytosolic and mitochondrial thioredoxins are important in the detoxification of H 2 O 2 . It is demonstrated that the Δtrx1 cells required cysteine for growth [17,18]. Consistently, the Δtrx1 cells grew as well as wild-type cells on EMM supplemented with 500 mg/l cysteine whereas they failed to grow on EMM without cysteine ( Figure S1A).
The cytosolic thioredoxin peroxidase Tpx1 and the transcription factor Pap1 play a role in defense against oxidative stress in S. pombe [19,20]. Therefore, we monitored Rst2 transcriptional activity in Δtrx2, Δpap1, Δtpx1, and Δtrx1 cells. In Δtrx2 cells, Rst2 transcriptional activity was higher than that in wild-type cells under both glucose-rich and glucosedeprived conditions ( Figure 4B-D). In Δtpx1 and Δpap1 cells, Rst2 transcriptional activity was similar to that observed in wildtype cells ( Figure 4C and D). Unexpectedly, in Δtrx1 cells, Rst2 transcriptional activity was lower than that in wild-type cells under both conditions ( Figure S1B). These results suggest that intracellular redox state affects Rst2 transcriptional activity.

NO May Be Involved in the Transcriptional Activation of Rst2
Nitric oxide (NO) is also a free radical and acts as a signal molecule [21]. In mammalian cells, NO modulates various cellular processes including gene expression, metabolism, and mitochondrial function [21][22][23]. In S. pombe, NO may function as a signal molecule which induces transcriptional and physiological changes [24]. Here, we examined the effect of the NO generator S-Nitroso-N-acetylpenicillamine (SNAP; Wako) on Rst2 activation. Results showed that unlike H 2 O 2 , SNAP induced a dose-dependent increase in Rst2 transcriptional activity under both conditions ( Figure 5A-D). Similarly, other nitric oxide generators such as sodium nitroprusside dehydrate (SNP; Enzo) and diethylamine-NONOate (DEA-NONOate; Enzo) also increased Rst2 transcriptional activity under both conditions ( Figure 6A and B).
Next, we examined the effect of 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; Dojindo), a NO specific scavenger [25] on Rst2 transcriptional activity. The results showed that carboxy-PTIO inhibited glucose deprivation-induced activation of Rst2 in a dose-dependent manner ( Figure 6C and D). These results suggest that NO may be involved in the transcriptional activation of Rst2. In addition, we examined whether the effect of NO on Rst2 activity is dependent on PKA. In Δpka1 cells, SNAP increased Rst2 transcriptional activity under glucose-rich condition ( Figure S2). The result indicates that the effect of NO on Rst2 activity is independent on PKA.

Discussion
Here we show that free radicals, NO and ROS, caused a dose-dependent increase in Rst2 transcriptional activity upon glucose deprivation. NO specific scavenger carboxy-PTIO and free radical scavenger NAC caused a dose-dependent decrease in glucose deprivation-induced activation of Rst2. These results suggest that NO and/or ROS may be involved in glucose deprivation-induced activation of transcription factor Rst2. We also show that under glucose-rich conditions, NO, but not ROS, induced Rst2 transcriptional activation. Previous work demonstrated that NO and ROS affect cellular responses in part through reversible thiol modifications [23,[26][27][28]. Cross-talk between these reactive species might be common and have potentially important implications for normal and pathological cellular functions [29][30][31][32]. Altogether, these results indicate that H 2 O 2 and NO may act by different mechanisms.
S-nitrosylation, the covalent attachment of NO to cysteine thiol, regulates various cellular processes including gene expression and signal transduction [23,33]. We show that SNAP induced a markedly higher Rst2 transcriptional activity compared with DEA NONOate. Consistently, it is known that SNAP is a more potent reagent than DEA NONOate in inducing S-nitrosylation [34]. Therefore, we hypothesize that Snitrosylation level may affect Rst2 transcriptional activity.
Thioredoxin has been implicated in the regulation of the redox state of ROS-responsive signaling proteins [35,36]. Glucose deprivation induces mitochondrial ROS generation [2], and the mitochondrial thioredoxin modulates ROS emission from mitochondria [37]. Here, the mitochondrial thioredoxin Trx2 deletion cells showed higher Rst2 transcriptional activity than that in wild-type cells, whereas cytosolic antioxidant enzyme Trx1 or Tpx1, or oxidative stress response transcription factor Pap1 deletion cells did not enhance the activity. We hypothesize that mitochondrial ROS generation enhances Rst2 transcriptional activity. Also, multiple studies reported that thioredoxin may play an important role in protein denitrosylation [29,30]. In combination with our results, we hypothesize that in fission yeast, glucose deprivation induced the generation of NO and/or ROS in mitochondria that in turn resulted in the activation of Rst2.