Prx1 Regulates Thapsigargin-Mediated UPR Activation and Apoptosis

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) signaling via the accumulation of unfolded and misfolded proteins. ER stress leads to the production of reactive oxygen species (ROS), which are necessary to maintain redox homeostasis in the ER. Although peroxiredoxin 1 (Prx1) is an antioxidant enzyme that regulates intracellular ROS levels, the link between Prx1 and ER stress remains unclear. In this study, we investigated the role of Prx1 in X-box binding protein 1 (XBP-1) activation, the C/EBP homologous protein (CHOP) pathway, and apoptosis in response to ER stress. We observed that Prx1 overexpression inhibited the nuclear localization of XBP-1 and the expression of XBP-1 target genes and CHOP after thapsigargin (Tg) treatment to induce ER stress. In addition, Prx1 inhibited apoptosis and ROS production during ER stress. The ROS scavenger inhibited ER stress-induced apoptosis but did not affect XBP-1 activation and CHOP expression. Therefore, the biological role of Prx1 in ER stress may have important implications for ER stress-related diseases.


Introduction
Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes that reduce peroxides [1]. Prx1 is a member of the 2-Cys Prx family and is an abundant protein that is present mainly in cytoplasm. Under stress conditions, Prx1 undergoes a structural transition to function as a chaperone from a peroxidase [2]. In addition, Prx1 shows abnormal expression in several human cancers, including lung, breast, and prostate cancers, as well as modulates various ROS-mediated signaling pathways, such as phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST) [1,3,4]. Prx1 acts as a tumor suppressor that inhibits tumorigenesis and promotes tumor cell death by interacting with c-Myc and as an oncogene that inhibits tumor cell death by interacting with nuclear factor kappa B (NF-κB) and the androgen receptor (AR) [5][6][7].
The ER is an organelle involved in the folding and maturation of newly synthesized proteins to be secreted [8]. When unfolded proteins accumulate in the ER or when calcium is deficient, ER stress is induced, and cells activate an adaptive mechanism known as the UPR to overcome it [9]. The UPR activation depends on three ER stress sensors: inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like ER kinase (PERK) [10,11]. Under ER stress, IRE1α oligomerizes and autophosphorylates to activate its RNase domain [10]. Active IRE1α removes the intron of XBP-1 via an unconventional splicing reaction, resulting in the production of the functional transcription factor XBP-1s [10]. XBP-1s induces the expression of ER chaperones and ER-associated degradation (ERAD) genes to restore ER homeostasis [10]. Under ER stress, ATF6 is trafficked from the ER to the Golgi and cleaved by site-1 and site-2 proteases

Materials and Methods
Materials. The human full-length Prx1 gene sequence was amplified by PCR and ligated into a pCS4-3Myc-plasmid. Primary antibodies against c-Myc, XBP-1s, PARP1, and BAX were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The anti-β-actin antibody was purchased from Millipore (Billerica, MA, USA). The anti-Prx1 antibody was manufactured by AB Frontier (Seoul, South Korea). The ER stress inducer Tg was obtained from Sigma Aldrich (St. Louis, MO, USA).
Cell culture and transfection. HEK293 and HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (WelGENE, Daegu, South of Korea) containing 10% fetal bovine serum (FBS) (Capricorn, Germany) and 1% penicillin/streptomycin (WelGENE, Daegu, South of Korea). Cells were incubated at 37 • C in 5% CO 2 humidified air. Cells were seeded at a density of 1.25 × 10 5 cells on a 6 cm dish. After incubation for 16 h, the cells were transfected using the TransIT-X2 ® Transfection Kit (Mirus, MIR6000, Madison, WI, USA) as per the manufacturer's instructions. After incubation for 48 h, cells transfected with plasmid or siRNA were harvested. Prx1 was overexpressed using the plasmid pCS4-3Myc-Prx1. Prx1 levels were knocked down in HEK293 cells using siRNA. Both siPrx1 (sc-36177) and against a scrambled sequence (siCON) (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Tg was treated after 16 h of serum starvation.
Annexin V assay. HEK293 cells were induced with overexpressed Prx1 or treated with 300 nM Tg for 12 h. HEK293 cells were treated with trypsin and harvested. The harvested cells were washed thrice with PBS and centrifuged at 3000 rpm for 5 min. annexin V analysis was performed using the EzWay annexin V-FITC Apoptosis Detection Kit (Koma Biotech, Seoul, South Korea), as per the manufacturer's protocol. Flow cytometry was performed using BD FACSCalibur (BD Biosciences, Durham, NC, USA).
Immunofluorescence. HeLa cells were incubated at a density of 2 × 10 4 cells per 24 well on glass coverslips and transfected with overexpressed Prx1 using pCS4-3Myc-Prx1. Overexpressed Prx1 in HeLa cells were treated with 300 nM Tg for 12 h. Cells were fixed with 4% (v/v) formaldehyde in PBS for 15 min. Fixated cells were washed thrice with 0.1 M glycine in PBS to quench autofluorescence. Cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min. After washing with PBS, cells were incubated with 5% (w/v) bovine serum albumin (BSA; Bovogen Biologicals, Victoria, Australia) in PBS for 30 min. The slides were then incubated with an anti-Prx1 (1:100) antibody at 4 • C for 16 h. After washing thrice with PBS, anti-mouse IgG Alexa Fluor 488 antibody (1:500, A11001, Invitrogen, CA, USA) was added and the nuclei were stained using Vectashield mounting medium for fluorescence with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). Images were obtained using an LSM confocal microscope (Zeiss, Germany).
DCF-DA staining. HeLa cells were induced with overexpressed Prx1 or treated with 5 mM NAC and/or 300 nM Tg for 12 h. These cells were incubated with 20 µM of 2,7dichlorofluorescein diacetate (DCF-DA) (Invitrogen, CA, USA) in the dark at 37 • C for 30 min to measure intracellular ROS levels. The cells were washed with PBS once. Next, intracellular ROS levels were evaluated by calculating the relative fluorescence intensity (RFI) from each group at 488 nm using a confocal laser microscope.
Statistical analysis. All experiments were performed thrice, and data are presented as the mean ± standard deviation (SD). Comparisons between two groups were analyzed using Student's t-test. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

ER Stress Induces UPR Activation, CHOP Pathway, and Apoptosis in HEK293 Cells
The ER calcium pump is inhibited by the ER stress inducer Tg, which leads to ER malfunction [23]. We investigated XBP-1 splicing and nuclear translocation in HEK293 cells to test whether Tg induces UPR activation. The cells were treated with Tg in a timedependent manner, and RT-PCR was used to quantify the mRNA levels of unspliced XBP-1 (uXBP-1) and spliced XBP-1 (sXBP-1) (Figure 1a). The sXBP-1 mRNA levels were upregulated in response to Tg treatment ( Figure 1a). Additionally, we observed that the amount of XBP-1 in the nuclear fraction increased after Tg treatment in a time-dependent manner ( Figure 1b). sXBP-1 regulates several UPR target genes, including ER chaperones (BIP/GRP78, ERDJ4, ERDJ5, HEDJ, GRP58, and PDIP5) and ERAD components (EDEM, HERP, and p58IPK) [24]. The mRNA expression levels of ERDJ4 and HERP in response to Tg were measured using qRT-PCR. The mRNA expression levels of ERDJ4 and HERP increased by up to nine times after 12 h of Tg treatment (Figure 1b). for fluorescence with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). Images were obtained using an LSM confocal microscope (Zeiss, Germany). DCF-DA staining. HeLa cells were induced with overexpressed Prx1 or treated with 5 mM NAC and/or 300 nM Tg for 12 h. These cells were incubated with 20 μM of 2,7dichlorofluorescein diacetate (DCF-DA) (Invitrogen, CA, USA) in the dark at 37 °C for 30 min to measure intracellular ROS levels. The cells were washed with PBS once. Next, intracellular ROS levels were evaluated by calculating the relative fluorescence intensity (RFI) from each group at 488 nm using a confocal laser microscope.
Statistical analysis. All experiments were performed thrice, and data are presented as the mean ± standard deviation (SD). Comparisons between two groups were analyzed using Student's t-test. Differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

ER Stress Induces UPR Activation, CHOP Pathway, and Apoptosis in HEK293 Cells
The ER calcium pump is inhibited by the ER stress inducer Tg, which leads to ER malfunction [23]. We investigated XBP-1 splicing and nuclear translocation in HEK293 cells to test whether Tg induces UPR activation. The cells were treated with Tg in a timedependent manner, and RT-PCR was used to quantify the mRNA levels of unspliced XBP-1 (uXBP-1) and spliced XBP-1 (sXBP-1) ( Figure 1A). The sXBP-1 mRNA levels were upregulated in response to Tg treatment ( Figure 1A). Additionally, we observed that the amount of XBP-1 in the nuclear fraction increased after Tg treatment in a time-dependent manner ( Figure 1B). sXBP-1 regulates several UPR target genes, including ER chaperones (BIP/GRP78, ERDJ4, ERDJ5, HEDJ, GRP58, and PDIP5) and ERAD components (EDEM, HERP, and p58IPK) [24]. The mRNA expression levels of ERDJ4 and HERP in response to Tg were measured using qRT-PCR. The mRNA expression levels of ERDJ4 and HERP increased by up to nine times after 12 h of Tg treatment ( Figure 1B).  ER stress not only induces apoptosis through activation of the CHOP pathway but also directly induces apoptosis. Therefore, we examined whether Tg treatment activates such a pathway in HEK293 cells. The cells were treated with Tg for 6, 12, or 24 h, and CHOP mRNA levels were determined using qRT-PCR. CHOP mRNA levels were upregulated in response to Tg treatment ( Figure 1c). Apoptotic cells treated with Tg were measured by flow cytometry. After 24 h of Tg treatment, annexin V-PI-positive cells were increased approximately 3-fold compared to untreated cells (Figure 1d). In addition, Western blotting demonstrated that Tg raised the levels of PARP-1 cleavage and BAX in a time-dependent  (Figure 1d). These results indicate that the ER stress inducer, Tg, efficiently triggers UPR activation, the CHOP pathway, and apoptosis in HEK293 cells.

Prx1 Regulates ER Stress-Induced UPR Activation, CHOP Pathway, and Apoptosis
To determine the regulatory effect of Prx1 on ER stress-induced UPR signaling, we examined the effect of exogenous Prx1 overexpression in HEK293 cells. After transfecting cells with a control vector or Myc-Prx1, we measured the variation in XBP-1 splicing, the nuclear translocation of sXBP-1, and the expression levels of sXBP-1 target genes. In XBP-1 splicing, the mRNA levels of sXBP-1 in response to Tg treatment were similar to those of Prx1 overexpression and the vector (Figure 2a). Prx1 overexpression, however, inhibited nuclear translocation of sXBP-1 after receiving Tg treatment (Figure 2b). Similar to this finding, Prx1 overexpression inhibited the Tg-induced upregulation of ERDJ4 and HERP mRNA levels ( Figure 2c). Furthermore, we investigated whether Prx1 regulates the CHOP pathway and apoptosis during ER stress. After transfecting the cells with a control vector or Myc-Prx1, we treated them with Tg. As shown in Figure 2d, 12 h after Tg treatment, CHOP mRNA levels increased approximately 11 times. However, compared to control cells, Prx1-overexpressed cells had 26% lower CHOP mRNA levels.
ER stress not only induces apoptosis through activation of the CHOP pathway but also directly induces apoptosis. Therefore, we examined whether Tg treatment activates such a pathway in HEK293 cells. The cells were treated with Tg for 6, 12, or 24 h, and CHOP mRNA levels were determined using qRT-PCR. CHOP mRNA levels were upregulated in response to Tg treatment ( Figure 1C). Apoptotic cells treated with Tg were measured by flow cytometry. After 24 h of Tg treatment, annexin V-PI-positive cells were increased approximately 3-fold compared to untreated cells ( Figure 1D). In addition, Western blotting demonstrated that Tg raised the levels of PARP-1 cleavage and BAX in a timedependent manner ( Figure 1D). These results indicate that the ER stress inducer, Tg, efficiently triggers UPR activation, the CHOP pathway, and apoptosis in HEK293 cells.

Prx1 Regulates ER Stress-Induced UPR Activation, CHOP Pathway, and Apoptosis
To determine the regulatory effect of Prx1 on ER stress-induced UPR signaling, we examined the effect of exogenous Prx1 overexpression in HEK293 cells. After transfecting cells with a control vector or Myc-Prx1, we measured the variation in XBP-1 splicing, the nuclear translocation of sXBP-1, and the expression levels of sXBP-1 target genes. In XBP-1 splicing, the mRNA levels of sXBP-1 in response to Tg treatment were similar to those of Prx1 overexpression and the vector (Figure 2A). Prx1 overexpression, however, inhibited nuclear translocation of sXBP-1 after receiving Tg treatment ( Figure 2B). Similar to this finding, Prx1 overexpression inhibited the Tg-induced upregulation of ERDJ4 and HERP mRNA levels ( Figure 2C). Furthermore, we investigated whether Prx1 regulates the CHOP pathway and apoptosis during ER stress. After transfecting the cells with a control vector or Myc-Prx1, we treated them with Tg. As shown in Figure 2D, 12 h after Tg treatment, CHOP mRNA levels increased approximately 11 times. However, compared to control cells, Prx1-overexpressed cells had 26% lower CHOP mRNA levels. The intensity of sXBP-1 bands was normalized to Histone H3 levls and was quantified using ImageJ To examine the involvement of Prx1 in Tg-induced apoptosis, we measured the number of apoptotic cells using flow cytometry. When HEK293 cells were treated with Tg, the proportion of apoptotic cells increased by 2.6-fold. After 24 h of Tg treatment, the proportion of apoptotic cells was 23% lower in Prx1-overexpressed cells than that in control cells (2.02 ± 0.08 vs. 2.62 ± 0.12, p < 0.05) (Figure 2e). Similarly, Prx1 overexpression inhibited Tg-induced PARP-1 cleavage (Figure 2e). These results indicated that Prx1 overexpression inhibits UPR activation, CHOP expression, and apoptosis.
To confirm whether Prx1 mediated regulation of Tg-induced apoptosis via UPR signaling, we induced Prx1 knockdown using siRNA and analyzed apoptosis and mRNA levels of ERDJ4 and HERP. Prx1 knockdown increased Tg-induced upregulation of ERDJ4 and HERP mRNA levels (Figure 3a). In addition, CHOP mRNA expression levels were upregulated by Prx1 silencing after Tg treatment (Figure 3b). The annexin V/PI staining assay demonstrated that Tg-mediated apoptotic cell induction was significantly higher in Prx1-depleted cells (Figure 3c). Furthermore, we showed that depletion of Prx1 induced the cleavage of PARP-1 protein compared to Tg-treated control cells (Figure 3d). Consequently, Prx1 is a crucial regulator of ER stress-induced UPR signaling.
mRNA expression levels of ERDJ4 and HERP (c) or CHOP (d) were analyzed using qRT-PCR. The mRNA levels are shown as fold changes as compared to control vector cells. (e) Apoptosis assay of overexpressed Prx1 in HEK293 cells incubated with or without 300 nM Tg for 24 h using the annexin V/PI staining assay. Quantification of annexin V/PI positive cells was expressed as fold change compared to control vector cells. The cleaved PARP-1 protein levels were analyzed using WB. The band intensity of the cleaved PARP-1 level was normalized to β-actin using ImageJ software. Data are indicated as the mean ± SD of three independent experiments (* p < 0.05).
To examine the involvement of Prx1 in Tg-induced apoptosis, we measured the number of apoptotic cells using flow cytometry. When HEK293 cells were treated with Tg, the proportion of apoptotic cells increased by 2.6-fold. After 24 h of Tg treatment, the proportion of apoptotic cells was 23% lower in Prx1-overexpressed cells than that in control cells (2.02 ± 0.08 vs. 2.62 ± 0.12, p < 0.05) ( Figure 2E). Similarly, Prx1 overexpression inhibited Tg-induced PARP-1 cleavage ( Figure 2E). These results indicated that Prx1 overexpression inhibits UPR activation, CHOP expression, and apoptosis.
To confirm whether Prx1 mediated regulation of Tg-induced apoptosis via UPR signaling, we induced Prx1 knockdown using siRNA and analyzed apoptosis and mRNA levels of ERDJ4 and HERP. Prx1 knockdown increased Tg-induced upregulation of ERDJ4 and HERP mRNA levels ( Figure 3A). In addition, CHOP mRNA expression levels were upregulated by Prx1 silencing after Tg treatment ( Figure 3B). The annexin V/PI staining assay demonstrated that Tg-mediated apoptotic cell induction was significantly higher in Prx1-depleted cells ( Figure 3C). Furthermore, we showed that depletion of Prx1 induced the cleavage of PARP-1 protein compared to Tg-treated control cells ( Figure 3D). Consequently, Prx1 is a crucial regulator of ER stress-induced UPR signaling.

Prx1 Overexpression Inhibits ER Stress-Induced ROS Production
We investigated the effect of Tg on the expression levels and localization of Prx1 in HEK293 cells to determine if the changes in Prx1 expression and localization were associated with the ER stress-induced UPR signaling. After the cells were treated with Tg for 0, 6, 12, or 24 h, the Prx1 mRNA and protein levels were measured using qPCR and Western blotting, respectively. As shown in Figure 4a, the expression levels of Prx1 mRNA and protein did not alter in response to Tg treatment. To study the Prx1 localization, HeLa cells were exposed to Tg for 12 h and assessed via immunofluorescence. Prx1 was mainly distributed in the cytoplasm of the control cells; however, it was increased in the nucleus of Tg-treated cells (Figure 4b). Thus, the Tg-induced effect of Prx1 may be involved in the control of ER stress-induced UPR signaling. distributed in the cytoplasm of the control cells; however, it was increased in the nucleus of Tg-treated cells ( Figure 4B). Thus, the Tg-induced effect of Prx1 may be involved in the control of ER stress-induced UPR signaling.
To investigate the effect of Prx1 on ER stress-induced ROS accumulation, we examined Tg-induced ROS production in Prx1 overexpression cells using DCF staining. After the overexpression of Prx1 in HeLa cells, Tg was treated for 12 h. In the absence of Tg treatment, overexpression of Prx1 reduced intracellular ROS levels. In comparison to control cells, DCF fluorescence was 3.77-fold higher in Tg-treated cells (377 ± 22 vs. 100 ± 5, p < 0.01). In Prx1-overexpressed cells, DCF fluorescence in Tg-treated cells was 2.25-fold higher than that in untreated cells (159 ± 12 vs. 71 ± 5, p < 0.01). Moreover, ER stress-induced ROS production was 40% lower in Prx1-overexpressed cells than in control cells ( Figure 4C). These results indicate that Prx1 is involved in the suppression of ROS production during ER stress. The ratio (%) of the average intensities of the Prx1 signals in the nucleus and cytoplasm was determined using ImageJ software. (c) DCF-DA staining of HeLa cells with control vector or Myc-Prx1 exposed to 300 nM Tg for 12 h. The DCF fluorescence intensity was assessed using ImageJ software. Scale bar, 20 μm. Data are expressed as the mean ± SD of three independent experiments (* p < 0.05 and ** p < 0.01). Myc-Prx1 exposed to 300 nM Tg for 12 h. The DCF fluorescence intensity was assessed using ImageJ software. Scale bar, 20 µm. Data are expressed as the mean ± SD of three independent experiments (* p < 0.05 and ** p < 0.01).
To investigate the effect of Prx1 on ER stress-induced ROS accumulation, we examined Tg-induced ROS production in Prx1 overexpression cells using DCF staining. After the overexpression of Prx1 in HeLa cells, Tg was treated for 12 h. In the absence of Tg treatment, overexpression of Prx1 reduced intracellular ROS levels. In comparison to control cells, DCF fluorescence was 3.77-fold higher in Tg-treated cells (377 ± 22 vs. 100 ± 5, p < 0.01). In Prx1-overexpressed cells, DCF fluorescence in Tg-treated cells was 2.25-fold higher than that in untreated cells (159 ± 12 vs. 71 ± 5, p < 0.01). Moreover, ER stress-induced ROS production was 40% lower in Prx1-overexpressed cells than in control cells (Figure 4c). These results indicate that Prx1 is involved in the suppression of ROS production during ER stress.

ER Stress-Induced ROS Production Is Associated with Apoptosis but Does Not Affect UPR Signaling
Prx1 not only inhibited ER stress-induced ROS production but also XBP1 and CHOP pathways, as well as apoptosis (Figures 2 and 3). Therefore, to determine whether the inhibition of ROS production by Prx1 affects UPR signaling and apoptosis during ER stress, we examined the effect of the ROS scavenger NAC on HEK293 cells. First, we analyzed the DCF fluorescence intensity following NAC pretreatment to determine intracellular ROS production by ER stress. The cells treated with Tg for 12 h increased intracellular ROS levels by 3.4-fold, whereas NAC-treated cells inhibited ROS production (Figure 5a). Second, we examined whether the NAC-mediated reduction in ROS could restore XBP-1 and CHOP expression. However, NAC was unable to inhibit the Tg-induced nuclear translocation of sXBP1 (Figure 5b). NAC treatment also failed to suppress the Tg-induced expression of its target genes (ERDJ4 and HERP) and CHOP (Figure 5c,d). These results indicated that ROS is neither involved in ER stress-induced UPR activation nor CHOP expression.
Prx1 not only inhibited ER stress-induced ROS production but also XBP1 and CHOP pathways, as well as apoptosis (Figures 2 and 3). Therefore, to determine whether the inhibition of ROS production by Prx1 affects UPR signaling and apoptosis during ER stress, we examined the effect of the ROS scavenger NAC on HEK293 cells. First, we analyzed the DCF fluorescence intensity following NAC pretreatment to determine intracellular ROS production by ER stress. The cells treated with Tg for 12 h increased intracellular ROS levels by 3.4-fold, whereas NAC-treated cells inhibited ROS production ( Figure  5A). Second, we examined whether the NAC-mediated reduction in ROS could restore XBP-1 and CHOP expression. However, NAC was unable to inhibit the Tg-induced nuclear translocation of sXBP1 ( Figure 5B). NAC treatment also failed to suppress the Tginduced expression of its target genes (ERDJ4 and HERP) and CHOP ( Figure 5C,D). These results indicated that ROS is neither involved in ER stress-induced UPR activation nor CHOP expression. We further demonstrated apoptosis in HEK293 cells by pretreating them with NAC before Tg treatment to investigate whether ROS production is associated with ER stressinduced apoptosis. NAC pretreatment efficiently reduced the proportion of apoptotic cells compared to Tg-treated cells ( Figure 5E). In addition, Western blot analysis showed a significant decrease in cleaved PARP-1 after NAC pretreatment. These findings showed We further demonstrated apoptosis in HEK293 cells by pretreating them with NAC before Tg treatment to investigate whether ROS production is associated with ER stressinduced apoptosis. NAC pretreatment efficiently reduced the proportion of apoptotic cells compared to Tg-treated cells (Figure 5e). In addition, Western blot analysis showed a significant decrease in cleaved PARP-1 after NAC pretreatment. These findings showed that ER stress induces apoptosis through ROS production, which can be prevented by the ROS scavenger NAC.
To determine whether Prx1 is ROS-independently involved in regulating UPR activation, we investigated the levels of ERDJ4, HERP, and CHOP with or without NAC treatment under ER stress. NAC treatment had no effect on UPR activation and CHOP expression inhibited by Prx1 ( Figure 6). Therefore, these results indicated that Prx1 regulates ROS-independent UPR activation during ER stress.
To determine whether Prx1 is ROS-independently involved in regulating UPR activation, we investigated the levels of ERDJ4, HERP, and CHOP with or without NAC treatment under ER stress. NAC treatment had no effect on UPR activation and CHOP expression inhibited by Prx1 ( Figure 6). Therefore, these results indicated that Prx1 regulates ROS-independent UPR activation during ER stress.

Discussion
This study investigated the role of Prx1 and ROS in response to ER stress. We demonstrated that ER stress induced Prx1 to inhibit the activation of UPR signaling, CHOP expression, and apoptosis. Prx1 may control ER stress-induced UPR and CHOP activation by mechanisms other than the regulation of ROS levels.
Moreover, we discovered that apoptosis enhanced ER stress-induced ROS production; however, this did not contribute to the activation of UPR signaling. During protein folding in the lumen of the ER, ROS production is increased by protein disulfide isomerase (PDI), ER membrane-associated protein, and endoplasmic reticulum oxidoreductin-1 (ERO1) [21]. In some cases, ROS generation triggers UPR activation [25]. According to our findings, Prx1 overexpression suppresses ROS generation and UPR activation. Although apoptosis was reduced by ER stress-induced ROS, the ROS scavenger NAC did not affect the activation of the UPR and CHOP pathways. These results suggest that the peroxidase activity of Prx1 is not involved in the signal activation of UPR and CHOP and directly inhibits apoptosis by reducing ER stress-induced ROS. These findings demonstrate that the regulation of UPR activation by Prx1 is a novel mechanism that is not mediated by ROS. This possibility is supported by the variation in the cellular location of Prx1 through immunofluorescence but not its Tg-treated mRNA and protein expression levels.
Furthermore, Prx1 is entirely found in the cytosol under normal conditions [2]. When exposed to Tg, Prx1 translocates into the nucleus. This finding supports the hypothesis that Prx1 regulates UPR signaling via other mechanisms. During ER stress, cytosolic Prx1 suppresses the nuclear translocation of XBP via unknown mechanisms, such as physical interaction, and can inhibit UPR signaling. In addition, nuclear-translocated Prx1 can inhibit UPR signaling through modulation of ER stress-related factors, such as CHOP.
Previous studies have revealed that the translocation of XBP-1 was interrupted by CDK5 and PI3K [26,27]. According to our data, Prx1 does not affect the splicing of XBP1 but inhibits migration to the nucleus in the presence of Tg. This result suggested that nuclear Prx1 may directly or indirectly affect XBP1 regulation in response to Tg-induced ER stress.

Discussion
This study investigated the role of Prx1 and ROS in response to ER stress. We demonstrated that ER stress induced Prx1 to inhibit the activation of UPR signaling, CHOP expression, and apoptosis. Prx1 may control ER stress-induced UPR and CHOP activation by mechanisms other than the regulation of ROS levels.
Moreover, we discovered that apoptosis enhanced ER stress-induced ROS production; however, this did not contribute to the activation of UPR signaling. During protein folding in the lumen of the ER, ROS production is increased by protein disulfide isomerase (PDI), ER membrane-associated protein, and endoplasmic reticulum oxidoreductin-1 (ERO1) [21]. In some cases, ROS generation triggers UPR activation [25]. According to our findings, Prx1 overexpression suppresses ROS generation and UPR activation. Although apoptosis was reduced by ER stress-induced ROS, the ROS scavenger NAC did not affect the activation of the UPR and CHOP pathways. These results suggest that the peroxidase activity of Prx1 is not involved in the signal activation of UPR and CHOP and directly inhibits apoptosis by reducing ER stress-induced ROS. These findings demonstrate that the regulation of UPR activation by Prx1 is a novel mechanism that is not mediated by ROS. This possibility is supported by the variation in the cellular location of Prx1 through immunofluorescence but not its Tg-treated mRNA and protein expression levels.
Furthermore, Prx1 is entirely found in the cytosol under normal conditions [2]. When exposed to Tg, Prx1 translocates into the nucleus. This finding supports the hypothesis that Prx1 regulates UPR signaling via other mechanisms. During ER stress, cytosolic Prx1 suppresses the nuclear translocation of XBP via unknown mechanisms, such as physical interaction, and can inhibit UPR signaling. In addition, nuclear-translocated Prx1 can inhibit UPR signaling through modulation of ER stress-related factors, such as CHOP.
Previous studies have revealed that the translocation of XBP-1 was interrupted by CDK5 and PI3K [26,27]. According to our data, Prx1 does not affect the splicing of XBP1 but inhibits migration to the nucleus in the presence of Tg. This result suggested that nuclear Prx1 may directly or indirectly affect XBP1 regulation in response to Tg-induced ER stress.
A previous study showed that when oxidative protein folding occurs in ER lumen, Prx4 eliminated the ER oxidase, ERO1-induced hydrogen peroxide, and protects cells against ER-stress-induced cell death [28,29]. Based on our data, we demonstrated that Prx1, unlike Prx4, inhibited ER stress-associated activation of UPR signaling but also ER stress-induced apoptosis.

Conclusions
Our findings emphasize that Prx1 responds to ER stress via mechanisms other than the elimination of ER stress-induced ROS. Finally, this study revealed that Prx1 plays a novel role as a therapeutic target for ER stress and is the main regulator of UPR signaling and apoptosis. Further studies should be conducted to investigate the effect of Prx1 on the interaction and expression of UPR signaling and the dependence of Prx1 on subcellular location through various experimental approaches.