Effects of GRP78 on Endoplasmic Reticulum Stress and Inflammatory Response in Macrophages of Large Yellow Croaker (Larimichthys crocea)

Endoplasmic reticulum (ER) homeostasis plays a vital role in cell physiological functions. Various factors can destroy the homeostasis of the ER and cause ER stress. Moreover, ER stress is often related to inflammation. Glucose-regulated protein 78 (GRP78) is an ER chaperone, which plays a vital role in maintaining cellular homeostasis. Nevertheless, the potential effects of GRP78 on ER stress and inflammation is still not fully elucidated in fish. In the present study, ER stress and inflammation was induced by tunicamycin (TM) or palmitic acid (PA) in the macrophages of large yellow croakers. GRP78 was treated with an agonist/inhibitor before or after the TM/PA treatment. The results showed that the TM/PA treatment could significantly induce ER stress and an inflammatory response in the macrophages of large yellow croakers whereas the incubation of the GRP78 agonist could reduce TM/PA-induced ER stress and an inflammatory response. Moreover, the incubation of the GRP78 inhibitor could further induce TM/PA-induced ER stress and an inflammatory response. These results provide an innovative idea to explain the relationship between GRP78 and TM/PA-induced ER stress or inflammation in large yellow croakers.


Introduction
The endoplasmic reticulum (ER) is essential for regulating calcium storage, protein folding, and the biosynthesis of lipids, steroids, and carbohydrates [1,2]. Cellular stress conditions such as exposure to free radicals, a glucose deficiency, and the accumulation of misfolded or unfolded proteins can cause ER stress and activate an unfolded protein response (UPR) [3][4][5][6]. One of the most essential protective mechanisms induced by the UPR is the upregulation of the expression of glucose-regulated protein 78 (GRP78).
GRP78 is also commonly referred to as BiP, the immunoglobulin heavy chain binding protein, which belongs to the heat shock protein 70 (HSP70) family. In non-stressed cells, GRP78 is bound to all three UPR transmembrane stress sensors, which are maintained in an inactive state, including double-stranded RNA-dependent protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein 1α (IRE1α). Upon ER stress, all three sensors become active after being released from GRP78. The UPR active form then downregulates the translation of protein and increases the correct folding [7].
All three UPR sensors have been found to regulate the inflammatory response [8][9][10][11]; for example, to activate TNFα and NF-κB. GRP78 also has been found to regulate inflammation. GRP78 is known as part of the resolution-associated molecular patterns (RAMPs) family that can be anti-inflammatory and promote the resolution of inflammation [12][13][14].

TM Treatment Induces ER Stress and an Inflammatory Response in the Macrophages of Large Yellow Croakers
The results indicated that TM incubation for different time points significantly upregulated the mRNA expression of grp78, xbp1, xbp1s, chop, atf6, and atf4 compared with the control group (p < 0.05) ( Figure 1A). Additionally, the protein levels of XBP1s and GRP78 as well as the phosphorylation levels of PERK and eif2α were significantly higher than those in the control group ( Figure 1B) whereas the protein level of CHOP was not remarkably different. We then analyzed the effect of ER stress on inflammation in the macrophages. TM incubation significantly increased the mRNA levels of proinflammatory genes, including il-8, il-6, il-1β, cox2, tnfα, and ifnγ (p < 0.05) ( Figure 1C). Moreover, TM incubation significantly upregulated the phosphorylation levels of JNK, ERK, and p38 ( Figure 1B). These results demonstrated that the TM treatment could induce ER stress and an inflammatory response in the macrophages of large yellow croakers.

BiP Inducer X (GRP78 Agonist) Treatment Induces ER Stress and an Inflammatory Response in Macrophages
The results showed that BiP inducer X (BiP) treatments at different concentrations significantly upregulated the mRNA expression of grp78 at 4 h and 8 h; however, at 12 h, only the 10 µM BiP incubation significantly upregulated the mRNA expression of grp78 (p < 0.05) (Figure 2A). Moreover, the BiP treatments for different concentrations at 4 h significantly upregulated the mRNA expression of xbp1, xbp1s, chop, atf6, and atf4 compared with the control group (p < 0.05) ( Figure 2B). Additionally, the 10 µM BiP incubation for 4 h and 8 h significantly upregulated the protein levels of XBP1s and GRP78, and at 4 h significantly upregulated the phosphorylation levels of eif2α (p < 0.05) ( Figure 2D). Different concentrations of the BiP treatment for 4 h significantly increased the mRNA levels of proinflammatory genes, including il-6, il-1β, cox2, tnfα, and ifnγ (p < 0.05) ( Figure 2C). Furthermore, BiP incubation significantly upregulated the phosphorylation levels of JNK, ERK, and p38 (p < 0.05) ( Figure 2D). These results indicated that the BiP treatment could induce ER stress and an inflammatory response in the macrophages of large yellow croakers.

BiP Inducer X (GRP78 Agonist) Treatment Induces ER Stress and an Inflammatory Response in Macrophages
The results showed that BiP inducer X (BiP) treatments at different concentrations significantly upregulated the mRNA expression of grp78 at 4 h and 8 h; however, at 12 h, Values are presented as mean ± SEM (n = 3). * Significant difference (p < 0.05); ** highly significant difference (p < 0.01).

HM03 (GRP78 Inhibitor) Treatment Induces ER Stress and an Inflammatory Response in Macrophages
The results indicated that HM03 incubations for different concentrations significantly decreased the mRNA level of grp78 at 3 h and 5 h (p < 0.05) ( Figure 3A). HM03 incubation significantly upregulated the mRNA expression of xbp1, xbp1s, chop, atf6, and atf4 compared with the control group (p < 0.05) ( Figure 3A). Additionally, the 10 µM HM03 incubation for 5 h significantly decreased the protein level of GRP78 (p < 0.05) ( Figure 3C). Moreover, HM03 incubation significantly decreased the protein level of XBP1s and upregulated the phosphorylation level of eif2α at 3 h (p < 0.05) ( Figure 3C). The 10 µM HM03 incubation significantly increased the mRNA levels of proinflammatory genes, including il-1β, il-6, il-8, cox2, tnfα, and ifnγ (p < 0.05) ( Figure 3B). Furthermore, HM03 incubation significantly upregulated the phosphorylation levels of JNK, ERK, and p38 (p < 0.05) ( Figure 3C). These results demonstrated that the HM03 treatment could induce ER stress and an inflammatory response in the macrophages of large yellow croakers.
only the 10 μM BiP incubation significantly upregulated the mRNA expression of (p < 0.05) (Figure 2A). Moreover, the BiP treatments for different concentrations significantly upregulated the mRNA expression of xbp1, xbp1s, chop, atf6, and atf4 pared with the control group (p < 0.05) ( Figure 2B). Additionally, the 10 μM BiP incub for 4 h and 8 h significantly upregulated the protein levels of XBP1s and GRP78, an h significantly upregulated the phosphorylation levels of eif2α (p < 0.05) ( Figure 2D ferent concentrations of the BiP treatment for 4 h significantly increased the mRNA of proinflammatory genes, including il-6, il-1β, cox2, tnfα, and ifnγ (p < 0.05) (Figur Furthermore, BiP incubation significantly upregulated the phosphorylation levels o ERK, and p38 (p < 0.05) ( Figure 2D). These results indicated that the BiP treatment induce ER stress and an inflammatory response in the macrophages of large yellow c ers.    Values are presented as mean ± SEM (n = 3). * Significant difference (p < 0.05); ** highly significant difference (p < 0.01).

Effect of GRP78 on TM-Induced ER Stress and Inflammation in Macrophages
To confirm the effect of GRP78 on TM-induced ER stress and inflammation, 10 µM BiP and HM03 were incubated for 4 h before or after 1 µM TM incubation for 16 h. The results showed that the BiP treatment before TM incubation significantly upregulated the mRNA expression of grp78, xbp1, xbp1s, chop, atf6, and atf4 compared with the TM treatment group (p < 0.05) ( Figure 4A). Moreover, the proinflammatory genes, including il-6, il-1β, cox2, and ifnγ, were significantly increased (p < 0.05) ( Figure 4B). BiP incubation significantly upregulated the phosphorylation level of ERK (p < 0.05) ( Figure 4C). The BiP treatment after TM incubation significantly downregulated the mRNA expression of grp78, xbp1s, atf4, and atf6 compared with the TM treatment group (p < 0.05) ( Figure 5A). Additionally, the proinflammatory genes, including il-8 and il-1β, were significantly downregulated (p < 0.05) ( Figure 5B). Furthermore, the phosphorylation levels of JNK and ERK were significantly decreased (p < 0.05) ( Figure 5C). The HM03 treatment before TM incubation significantly upregulated the mRNA expression of grp78, xbp1, chop, atf6, and atf4 compared with the TM treatment group (p < 0.05) ( Figure 6A). Moreover, the proinflammatory genes, including il-6, il-1β, cox2, tnfα, and ifnγ, were significantly increased (p < 0.05) ( Figure 6B). Additionally, the protein level of XBP1s and the phosphorylation levels of JNK, ERK, and eif2α were significantly upregulated (p < 0.05) ( Figure 6C). The HM03 treatment after TM incubation significantly upregulated the mRNA expression of grp78, xbp1, xbp1s, chop, and atf4 compared with the TM treatment group (p < 0.05) ( Figure 7A). Moreover, the proinflammatory genes, including il-1β, cox2, ifnγ, and tnfα, were significantly upregulated (p < 0.05) ( Figure 7B). Furthermore, the phosphorylation level of JNK was significantly increased (p < 0.05) ( Figure 7C). These results demonstrated that the BiP treatment after TM incubation could reduce the TM-induced ER stress and inflammatory response whereas the HM03 treatment could further induce the TM-induced ER stress and inflammatory response in the macrophages of large yellow croakers.

PA Treatment Induces ER Stress and an Inflammatory Response in Macrophages
The results indicated that PA incubation for different time points significantly upregulated the mRNA expression of grp78, xbp1, xbp1s, chop, atf6, and atf4 compared with the control group (p < 0.05) ( Figure 8A). Additionally, the protein levels of XBP1s and GRP78 were significantly upregulated compared with those in the control group ( Figure 8B). Moreover, PA incubation significantly increased the mRNA levels of proinflammatory genes, including il-1β, il-6, il-8, cox2, tnfα, and ifnγ (p < 0.05) ( Figure 8C). Furthermore, PA incubation significantly upregulated the phosphorylation levels of JNK and ERK ( Figure 8B). These results indicated that the PA treatment could induce ER stress and an inflammatory response in the macrophages of large yellow croakers.

Effect of GRP78 on PA-Induced ER Stress and Inflammation in Macrophages
To confirm the effect of GRP78 on PA-induced ER stress and inflammation, 10 µM BiP and HM03 were incubated for 4 h before or after 500 µM PA incubation for 6 h. The results showed that the BiP treatment before PA incubation significantly downregulated the mRNA expression of grp78, xbp1, xbp1s, chop, atf6, and atf4 compared with the PA treatment group (p < 0.05) ( Figure 9A). Moreover, the proinflammatory genes, including tnfα and ifnγ, were significantly decreased (p < 0.05) ( Figure 9B). The BiP treatment after PA incubation significantly downregulated the mRNA expression of xbp1, xbp1s, chop, atf6, and atf4 compared with the PA treatment group (p < 0.05) ( Figure 10A). Furthermore, the proinflammatory genes, including il-8, cox2, and tnfα, were significantly downregulated (p < 0.05) ( Figure 10B). The HM03 treatment before PA incubation significantly upregulated the mRNA expression of grp78, xbp1, chop, and atf4 compared with the PA treatment group (p < 0.05) ( Figure 11A). Moreover, the proinflammatory genes, including il-8, il-6, il-1β, cox2, tnfα, and ifnγ, were significantly upregulated (p < 0.05) ( Figure 11B). The HM03 treatment after PA incubation significantly upregulated the mRNA expression of grp78, xbp1, chop, and atf4 compared with the PA treatment group (p < 0.05) ( Figure 12A). Additionally, the proinflammatory genes, including il-8, il-6, il-1β, cox2, tnfα, and ifnγ, were significantly upregulated (p < 0.05) ( Figure 12B). These results demonstrated that the BiP treatment could reduce the PA-induced ER stress and inflammatory response whereas the HM03 treatment could further induce the PA-induced ER stress and inflammatory response in the macrophages of large yellow croakers.     Moreover, PA incubation significantly increased the mRNA levels of proinflammato genes, including il-1β, il-6, il-8, cox2, tnfα, and ifnγ (p < 0.05) ( Figure 8C). Furthermore, P incubation significantly upregulated the phosphorylation levels of JNK and ERK (Figu 8B). These results indicated that the PA treatment could induce ER stress and an infla matory response in the macrophages of large yellow croakers.  Additionally, the proinflammatory genes, including il-8, il-6, il-1β, cox2, tnfα, and ifnγ, were significantly upregulated (p < 0.05) ( Figure 12B). These results demonstrated that the BiP treatment could reduce the PA-induced ER stress and inflammatory response whereas the HM03 treatment could further induce the PA-induced ER stress and inflammatory response in the macrophages of large yellow croakers.

Discussion
The endoplasmic reticulum (ER) is the central organelle of the secretory pathways, which is the major place for synthesizing and exporting both lipids and proteins [32]. However, misfolded/unfolded proteins accumulate in the ER lumen, causing ER stress [33], which is a defense system for dealing with that situation. Previous studies have shown that ER stress is usually related to the inflammatory response [34,35]. Tunicamycin (TM) is a mixture of homologous nucleoside antibiotics, which can cause unfolded proteins to accumulate in the ER and induce ER stress. In the present study, TM incubation could significantly induce the expression of ER stress-related genes and proteins, which was consistent with previous studies [17][18][19][20]. Meanwhile, similar to previous studies [19,[21][22][23], the TM treatment induced the expression of proinflammatory genes.
In this study, the data indicated that palmitic acid (PA) incubation increased ER stress, which was consistent with previous studies in mammals [36][37][38]. PA induces ER stress by increasing the content of saturated ER membrane phospholipids [39], perturbing ER calcium homeostasis [40] and modulating protein folding [41]. Furthermore, the PA treatment induced an inflammatory response, which was consistent with previous studies [24,[27][28][29][30]. A study reported that dietary palm oil that contained a high level of PA could increase the inflammatory response by activating the TLR-NF-κB pathway [27]. Zhang [29] demonstrated that the IRE1α-XBP1s signaling pathway participated in PA-induced Values are presented as mean ± SEM (n = 3). * Significant difference (p < 0.05); ** highly significant difference (p < 0.01).

Discussion
The endoplasmic reticulum (ER) is the central organelle of the secretory pathways, which is the major place for synthesizing and exporting both lipids and proteins [32]. However, misfolded/unfolded proteins accumulate in the ER lumen, causing ER stress [33], which is a defense system for dealing with that situation. Previous studies have shown that ER stress is usually related to the inflammatory response [34,35]. Tunicamycin (TM) is a mixture of homologous nucleoside antibiotics, which can cause unfolded proteins to accumulate in the ER and induce ER stress. In the present study, TM incubation could significantly induce the expression of ER stress-related genes and proteins, which was consistent with previous studies [17][18][19][20]. Meanwhile, similar to previous studies [19,[21][22][23], the TM treatment induced the expression of proinflammatory genes.
In this study, the data indicated that palmitic acid (PA) incubation increased ER stress, which was consistent with previous studies in mammals [36][37][38]. PA induces ER stress by increasing the content of saturated ER membrane phospholipids [39], perturbing ER calcium homeostasis [40] and modulating protein folding [41]. Furthermore, the PA treatment induced an inflammatory response, which was consistent with previous studies [24,[27][28][29][30]. A study reported that dietary palm oil that contained a high level of PA could increase the inflammatory response by activating the TLR-NF-κB pathway [27]. Zhang [29] demonstrated that the IRE1α-XBP1s signaling pathway participated in PA-induced inflammation.
In order to figure out the relationship between glucose-regulated protein 78 (GRP78) and TM/PA-induced ER stress or inflammation, we used a GRP78 agonist (BiP inducer X) or inhibitor (HM03). In the present study, BiP inducer X (BiP) significantly upregulated the expression of GRP78 mRNA and the protein level. Similar to a previous study that observed that the rise of GRP78 mRNA by BiP was transient, the treatment for 4 h reached a peak, but the levels of GRP78 protein continued to upregulate up to 12 h [42]. Meanwhile, the BiP treatment increased the expression of ER stress-related genes and proteins, which was different from a previous study [42]. To be more specific, in that previous study, BiP preferentially increased GRP78 and slightly induced CHOP, calreticulin, and GRP94 mediated by the ATF6 pathway; however, BiP did not affect XBP1 or eif2α [42]. HM03 significantly decreased the GRP78 mRNA and protein levels; furthermore, the HM03 treatment increased the expression of ER stress-related genes.
In the present study, the BiP treatment after TM/PA incubation significantly decreased ER stress-related genes such as xbp1s, atf6, and atf4 compared with the TM/PA treatment group. Consistent with previous studies that BiP is an effective ER stress inhibitor [43], BiP is known to significantly downregulate ER stress through an induced GRP78 mRNA level in animal models and in various cell lines [42][43][44][45]. Previous studies have indicated that the overexpression of GRP78 can reduce ER stress and increase thermogenesis [46]; moreover, the genetic overexpression of GRP78 was competent to reduce ER stress and to revert the metabolic and obese phenotype [47]. The results of the BiP treatment before PA incubation was consistent with the post-treatment of BiP. However, the BiP treatment before TM incubation significantly upregulated the mRNA expression of xbp1, xbp1s, chop, atf6, and atf4 compared with the TM treatment group. These results may be because the BiP treatment could significantly increase ER stress-related genes, then TM incubation after the BiP treatment further induced the gene expression. Different from that under TMinduced ER stress, the overexpression of GRP78 through the BiP treatment could alleviate ER stress. These results also suggested that ER stress and inflammation induced by TM and PA were different. The HM03 treatment before or after TM/PA incubation significantly induced the expression of ER stress-related genes. Previous studies have shown that GRP78 siRNA increased the expression of UPR-induced genes such as chop [48]. An electronmicroscopic analysis of the structure of intracellular organelles showed that the ER was greatly disorganized and expanded in the cells where GRP78 was downregulated [49]; in the cells where the GRP78 expression was downregulated by siRNA, the UPR pathways were activated.
Furthermore, the results from the present study indicated that the inflammatory response under different treatments was consistent with the ER stress response. GRP78 has been indicated to be anti-inflammatory and promote the resolution of inflammation [12][13][14]. A previous study revealed that GRP78 mediated the endocytosis of TLR4 by targeting CD14, which is conducive to the regression of an inflammatory response [13]. Moreover, an adoptive GRP78-DC transfer was essential to resolve the inflammatory response in NOD mice [14]. The results suggested that the inflammatory response may be regulated by GRP78 on the one hand and be regulated by ER stress on the other hand.
In conclusion, we found that GRP78 could regulate TM/PA-induced ER stress or inflammation. To be more specific, the overexpression of GRP78 could reduce the TM/PAinduced ER stress and inflammatory response. These results provide a novel idea to explain the relationship between GRP78 and TM/PA-induced ER stress or inflammation in large yellow croakers.

Cell Culture and Treatment
The macrophage line of large yellow croakers was obtained from our colleagues [50]. The macrophages were seeded into 6-well plates at a density of 2.0 × 10 6 cells/well and were maintained in a DMEM/F12 medium (BI, Kibbutz Beit-Haemek, Israel) containing 15% FBS (BI, Kibbutz Beit-Haemek, Israel) and antibiotics (Solarbio, Beijing, China) in a 5% CO 2 atmosphere at 28 • C. To figure out the effects of TM on ER stress and the inflammatory response in the macrophages, the cells were incubated with 1 µM TM for different time points. The control group was treated with DMSO of the same concentration (Solarbio, Beijing, China). The cells were incubated with PA to confirm the effects of PA on ER stress and the inflammatory response in the macrophages. PA (Sigma, St. Louis, MO, USA) was supplemented to the cells with the forms of BSA/fatty acid complexes at 500 µM for different time points. The solvent group was set as the control group. To investigate the role of GRP78 in TM/PA-induced ER stress and the inflammatory response, a GRP78 agonist (BiP inducer X; MCE, Jersey City, NJ, USA) and a GRP78 inhibitor (HM03; MCE, Jersey City, NJ, USA) were treated with a concentration of 10 µM for 4 h before or after the TM/PA treatment. After incubation, the cells were lysed in the wells and harvested for the further analysis.

RNA Extraction and RT-qPCR
The harvested macrophage cells were added to a Trizol reagent (Takara, Dalian, China). The total RNA was then extracted according to the manufacturer's instructions. The quality of RNA was detected by electrophoresis using 1.2% denatured agarose gel. The RNA was reversed to cDNA by a PrimeScript RT reagent kit (Takara, Dalian, China) following the manufacturer's protocol. The mRNA expression levels were quantified in a quantitative thermal cycler (Bio-rad, Hercules, CA, USA) with an SYBR Green real-time PCR kit (Takara, Dalian, China). The normalization of the gene expression in different tissues of large yellow croakers was detected by rpl17, gapdh, and β-actin to test their suitability. Among all treatments, no significant differences in β-actin expression were detected, suggesting that β-actin could be used as a reference gene. Further, the mRNA expression of each gene was normalized to that of β-actin using the 2 −∆∆ct method [51].

Western Blot
Western blotting was carried out following the methods in previous studies [30,52,53].

Statistical Analysis
All data were presented as means ± SEM. All data were subjected to independent t-tests with the help of SPSS 19.0. A p < 0.05 was applied as a significant difference and a p < 0.01 as a highly significant difference.