GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin
Endoplasmic reticulum stress (ERS) is closely related to the occurrence and progression of metabolic liver disease. The treatment targeting glucose-regulated protein 94 (GRP94) for liver disease has gotten much attention, but the specific effect of GRP94 on hepatocyte apoptosis is still unclear. So...
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MDPI AG
2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| author | Xiaohong Wang Hairui Xin Chuang Zhang Xianhong Gu Yue Hao |
| author_facet | Xiaohong Wang Hairui Xin Chuang Zhang Xianhong Gu Yue Hao |
| author_sort | Xiaohong Wang |
| collection | DOAJ |
| description | Endoplasmic reticulum stress (ERS) is closely related to the occurrence and progression of metabolic liver disease. The treatment targeting glucose-regulated protein 94 (GRP94) for liver disease has gotten much attention, but the specific effect of GRP94 on hepatocyte apoptosis is still unclear. So far, all the studies on GRP94 have been conducted in mice or rats, and little study has been reported on pigs, which share more similarities with humans. In this study, we used low-dose (LD) and high-dose (HD) tunicamycin (TM) to establish ERS models on piglet livers and immortalized porcine hepatic stellate cells (HSCs). On the piglet ERS model we found that ERS could significantly (<i>p</i> < 0.01) stimulate the secretion and synthesis of insulin-like growth factor (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein (IGFBP)-1 and IGFBP-3; however, with the increase in ERS degree, the effect of promoting secretion and synthesis significantly (<i>p</i> < 0.01) decreased. In addition, the ubiquitin protein and ubiquitination-related gene were significantly increased (<i>p</i> < 0.05) in the LD group compared with the vehicle group. The protein level of Active-caspase 3 was significantly increased (<i>p</i> < 0.01) in the HD group, however, the TUNEL staining showed there was no significant apoptosis in the piglet liver ERS model. To explore the biofunction of ER chaperone GRP94, we used shRNA to knock down the expression of GRP94 in porcine HSCs. Interestingly, on porcine HSCs, the knockdown of GRP94 significantly (<i>p</i> < 0.05) decreased the secretion of IGF-1, IGFBP-1 and IGFBP-3 under ERS, but had no significant effect on these under normal condition, and knockdown GRP94 had a significant (<i>p</i> < 0.01) effect on the UBE2E gene and ubiquitin protein from the analysis of two-way ANOVA. On porcine HSCs apoptosis, the knockdown of GRP94 increased the cell apoptosis in TUNEL staining, and the two-way ANOVA analysis shows that knockdown GRP94 had a significant (<i>p</i> < 0.01) effect on the protein levels of Bcl-2 and Caspase-3. For CCK-8 assay, ERS had a significant inhibitory(<i>p</i> < 0.05) effect on cell proliferation when treated with ERS for 24 h, and both knockdown GRP94 and ERS had a significant inhibitory(<i>p</i> < 0.05) effect on cell proliferation when treated with ERS for 36 h and 48 h. We concluded that GRP94 can protect the cell from ERS-induced apoptosis by promoting the IGF-1 system and ubiquitin. These results provide valuable information on the adaptive mechanisms of the liver under ERS, and could help identify vital functional genes to be applied as possible diagnostic biomarkers and treatments for diseases induced by ERS in the future. |
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| spelling | doaj.art-141b0f6470244096af9038171a0b78ba2023-11-24T08:37:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123221405910.3390/ijms232214059GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and UbiquitinXiaohong Wang0Hairui Xin1Chuang Zhang2Xianhong Gu3Yue Hao4State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaState Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaEndoplasmic reticulum stress (ERS) is closely related to the occurrence and progression of metabolic liver disease. The treatment targeting glucose-regulated protein 94 (GRP94) for liver disease has gotten much attention, but the specific effect of GRP94 on hepatocyte apoptosis is still unclear. So far, all the studies on GRP94 have been conducted in mice or rats, and little study has been reported on pigs, which share more similarities with humans. In this study, we used low-dose (LD) and high-dose (HD) tunicamycin (TM) to establish ERS models on piglet livers and immortalized porcine hepatic stellate cells (HSCs). On the piglet ERS model we found that ERS could significantly (<i>p</i> < 0.01) stimulate the secretion and synthesis of insulin-like growth factor (IGF-1), IGF-1 receptor (IGF-1R), and IGF-binding protein (IGFBP)-1 and IGFBP-3; however, with the increase in ERS degree, the effect of promoting secretion and synthesis significantly (<i>p</i> < 0.01) decreased. In addition, the ubiquitin protein and ubiquitination-related gene were significantly increased (<i>p</i> < 0.05) in the LD group compared with the vehicle group. The protein level of Active-caspase 3 was significantly increased (<i>p</i> < 0.01) in the HD group, however, the TUNEL staining showed there was no significant apoptosis in the piglet liver ERS model. To explore the biofunction of ER chaperone GRP94, we used shRNA to knock down the expression of GRP94 in porcine HSCs. Interestingly, on porcine HSCs, the knockdown of GRP94 significantly (<i>p</i> < 0.05) decreased the secretion of IGF-1, IGFBP-1 and IGFBP-3 under ERS, but had no significant effect on these under normal condition, and knockdown GRP94 had a significant (<i>p</i> < 0.01) effect on the UBE2E gene and ubiquitin protein from the analysis of two-way ANOVA. On porcine HSCs apoptosis, the knockdown of GRP94 increased the cell apoptosis in TUNEL staining, and the two-way ANOVA analysis shows that knockdown GRP94 had a significant (<i>p</i> < 0.01) effect on the protein levels of Bcl-2 and Caspase-3. For CCK-8 assay, ERS had a significant inhibitory(<i>p</i> < 0.05) effect on cell proliferation when treated with ERS for 24 h, and both knockdown GRP94 and ERS had a significant inhibitory(<i>p</i> < 0.05) effect on cell proliferation when treated with ERS for 36 h and 48 h. We concluded that GRP94 can protect the cell from ERS-induced apoptosis by promoting the IGF-1 system and ubiquitin. These results provide valuable information on the adaptive mechanisms of the liver under ERS, and could help identify vital functional genes to be applied as possible diagnostic biomarkers and treatments for diseases induced by ERS in the future.https://www.mdpi.com/1422-0067/23/22/14059endoplasmic reticulum stresspiglet liverhepatic stellate cellGRP94IGF-1ubiquitin |
| spellingShingle | Xiaohong Wang Hairui Xin Chuang Zhang Xianhong Gu Yue Hao GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin International Journal of Molecular Sciences endoplasmic reticulum stress piglet liver hepatic stellate cell GRP94 IGF-1 ubiquitin |
| title | GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin |
| title_full | GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin |
| title_fullStr | GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin |
| title_full_unstemmed | GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin |
| title_short | GRP94 Inhabits the Immortalized Porcine Hepatic Stellate Cells Apoptosis under Endoplasmic Reticulum Stress through Modulating the Expression of IGF-1 and Ubiquitin |
| title_sort | grp94 inhabits the immortalized porcine hepatic stellate cells apoptosis under endoplasmic reticulum stress through modulating the expression of igf 1 and ubiquitin |
| topic | endoplasmic reticulum stress piglet liver hepatic stellate cell GRP94 IGF-1 ubiquitin |
| url | https://www.mdpi.com/1422-0067/23/22/14059 |
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