Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response
Abstract
:1. Introduction
2. Results
2.1. Repeated CCl4 Intoxication Induced ER Stress and UPR
2.2. Chronic Repeated CCl4 Administration Activated c-Jun N-Terminal Kinase (JNK) and Intrinsic Hepatocyte Apoptosis
2.3. Lcn2 Null Mice Developed Slightly More Steatosis Than Wild Type Mice
2.4. Strongest ER Stress Responses Were Observed in Lcn2−/− Hepatocytes That Also Expressed High Levels of Cyclic AMP-Responsive Element-Binding Protein 3-Like 3 (CREB3L3)
2.5. Pharmacological ER Stress Inducer Tunicamycin Activates TRAF2, c-Jun N-Terminal Kinase, NF-κB, and Mitochondrial Apoptotic Proteins in Hepatocytes
2.6. Chop−/− Alternates ER Stress Responses, Protects Against Tunicamycin-Activated ER Stress, and Induces Hepatocyte Apoptosis Through Delayed JNK Activation
2.7. ER Stress and UPR in Early Stage Of Tunicamycin-Stimulated Primary Hepatocytes
3. Discussion
4. Materials and Methods
4.1. Animal Experiments and Specimen Collections
4.2. RNA Isolation, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
4.3. SDS-PAGE and Western Blot Analysis
4.4. Immunohistochemistry
4.5. Terminal Transferase dUTP Nick End-Labeling Assay (TUNEL)
4.6. Oil Red O Staining
4.7. Primary Liver Cell Isolation and Culturing
4.8. Tunicamycin and Thapsigargin Treatment
4.9. Statistics
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ALT | alanine transaminase |
ASH | alcoholic steatohepatitis |
AST | aspartate transaminase |
ATF6 | activating transcription factor 6 |
BIM | Bcl2-interacting protein/Bcl2-like protein 11 |
BiP/GRP78 | binding immunoglobulin protein/glucose-regulated protein 78 |
BSA | bovine serum albumin |
CHOP/GADD153 | C/EBP homologous protein, growth arrest- and DNA damage-inducible gene |
CREBH/CREB3L3 | cyclic AMP response element-binding protein H/cyclic AMP-responsive element-binding protein 3-like protein 3 |
eIF2α | eukaryotic initiation factor 2α |
ER | endoplasmic reticulum |
GADD34 | growth arrest and DNA damage-inducible protein 34 |
GRP94 | Glucose-regulated protein, 94kD |
HCC | hepatocellular carcinoma |
HDL | high-density lipoprotein |
IRE1 | inositol-requiring enzyme 1 |
LCN2 | Lipocalin 2 |
LDL | low-density lipoprotein |
PC | hepatocyte(s) |
PBS | phosphate-buffered saline |
PCR | polymerase chain reaction |
PERK | protein kinase R (PKR)-like endoplasmic reticulum kinase |
PUMA | p53-upregulated modulator of apoptosis |
RT-qPCR | real-time quantitative polymerase chain reaction |
TG | thapsigargin |
TM | tunicamycin |
TUNEL | terminal deoxynucleotidyl transferase dUTP nick end labeling |
UPR | unfolded protein response |
Xbp1s | spliced X-box-binding protein 1 |
Xbp1u | unspliced X-box-binding protein 1 |
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Murine Gene | Acc. No. | Primer |
---|---|---|
Xbp-1 | NM_001271730 | for: 5′-GAACCAGGAGTTAAGAACACG-3′ |
rev: 5′-AGGCAACAGTGTCAGAGTCC-3′ | ||
Xbp1u = 205 bp, Xbp1s = 179 bp | ||
Gapdh | NM_008084 | for: 5′-TCGTGGATCTGACGTGCCGCCTG-3′ |
rev: 5′-CACCACCCTGTTGCTGTAGCCGTAT-3′ | ||
semi-qPCR = 251 bp | ||
Bip | NM_001163434 | for: 5′-CTGAGGCGTATTTGGGAAAG-3′ |
rev: 5′-TCATGACATTCAGTCCAGCAA-3′ | ||
Grp94 | NM_011631 | for: 5′-AGGGTCCTGTGGGTGTTG-3′ |
rev: 5′-CATCATCAGCTCTGACGAACC-3′ | ||
Chop | NM_007837 | for: 5′-GCGACAGAGCCAGAATAACA-3′ |
rev: 5′-GATGCACTTCCTTCTGGAACA-3′ | ||
Creb3L3 | NM_145365 | for: 5′-CTCCCGCTTCAACCTCACT-3′ |
rev: 5′-GCCAAGGAATGCTGTTGC-3′ | ||
Atf4 | NM_001287180 | for: 5′-GGCTGGTCGTCAACCTATAAA-3′ |
rev: 5′-CAGGCACTGCTGCCTCTAAT-3′ |
Antibody | Cat. No. | Supplier | Dilution |
---|---|---|---|
ATF2 | 242 | Santa Cruz, Santa Cruz Biotech, CA, USA | 1:1000 |
p-ATF2 | 8398 | Santa Cruz | 1:1000 |
ATF4 | sc-390063 | Santa Cruz | 1:1000 |
ATF6 | sc-166659 | Santa Cruz | 1:1000 |
β-actin | A5441 | Sigma, Taufkirchen, Germany | 1:10,000 |
Bak | 12105 | Cell Signaling, Darmstadt, Germany | 1:1000 |
Bax | 14796 | Cell Signaling | 1:1000 |
Bcl-xL | 2764 | Cell Signaling | 1:1000 |
Bcl2 | 3498 | Cell Signaling | 1:1000 |
BIM | 374358 | Santa Cruz | 1:1000 |
BiP | 3177 | Cell Signaling | 1:1000 |
c-Jun | 9165 | Cell Signaling | 1:1000 |
CHOP/GADD153 | sc-7351 | Santa Cruz | 1:1000 |
cleaved Caspase-3 | 9664 | Cell Signaling | 1:1000 |
cleaved Caspase-9 | 9507 | Cell Signaling | 1:1000 |
CREB3L3/CREBH | sc-377331 | Santa Cruz | 1:1000 |
Cytochrome C | 11940 | Cell Signaling | 1:1000 |
eIF2α | 9722 | Cell Signaling | 1:1000 |
p-eIF2α | 3597 | Cell Signaling | 1:1000 |
ERK1/2 | 9102 | Cell Signaling | 1:1000 |
p-ERK1/2 | 9101 | Cell Signaling | 1:1000 |
GADD34 | 373815 | Santa Cruz | 1:1000 |
GAPDH | sc-32233 | Santa Cruz | 1:1000 |
GRP94 | sc-393402 | Santa Cruz | 1:1000 |
IRE1α | 3294 | Cell Signaling | 1:1000 |
LCN2 | AF1857 | R&D Systems, Wiesbaden, Germany | 1:1000 |
p21 | 556430 | BD Bioscience, Heidelberg, Germany | 1:1000 |
p38 | 612168 | BD Biosciences | 1:2500 |
p-p38 | 612288 | BD Biosciences | 1:2500 |
p65 | sc-8008 | Santa Cruz | 1:1000 |
p-p65 | 3033 | Cell Signaling | 1:1000 |
p-c-Jun | 3270 | Cell Signaling | 1:1000 |
PERK | 377400 | Santa Cruz | 1:1000 |
PUMA | 374223 | Santa Cruz | 1:1000 |
SAPK/JNK2 | 9258 | Cell Signaling | 1:1000 |
p-SAPK/JNK | 4668 | Cell Signaling | 1:1000 |
TRAF2 | 4712 | Cell Signaling | 1:1000 |
p-TRAF2 | 13908 | Cell Signaling | 1:1000 |
TRB3 | 390242 | Santa Cruz | 1:1000 |
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Borkham-Kamphorst, E.; Haas, U.; Van de Leur, E.; Trevanich, A.; Weiskirchen, R. Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response. Int. J. Mol. Sci. 2020, 21, 5230. https://doi.org/10.3390/ijms21155230
Borkham-Kamphorst E, Haas U, Van de Leur E, Trevanich A, Weiskirchen R. Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response. International Journal of Molecular Sciences. 2020; 21(15):5230. https://doi.org/10.3390/ijms21155230
Chicago/Turabian StyleBorkham-Kamphorst, Erawan, Ute Haas, Eddy Van de Leur, Anothai Trevanich, and Ralf Weiskirchen. 2020. "Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response" International Journal of Molecular Sciences 21, no. 15: 5230. https://doi.org/10.3390/ijms21155230