Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution
- Baylor College of Medicine, United States
- University of Cincinnati, United States
- University of Southampton, United Kingdom
Figures
Figure 1 with 1 supplement
Lrh-1 is required for ER stress resolution and for protection against stress-induced lipid accumulation and cell death.
(A) Macroscopic visualization of steatosis following ER stress in Lrh-1 LKO mice. Mice were i.p. injected with 1 mg/kg tunicamycin (TM) or vehicle and livers photographed following sacrifice. …
Figure 1—figure supplement 1
Loss of Lrh-1 does not result in loss of UPR target genes in response to stress.
Relative expression by quantitative PCR for genes dependent on each of the three UPR pathways. RNA was collected at designated timepoints for control and Lrh-1LKO mice treated with vehicle or 1 …
Figure 2
Loss of Lrh-1 sensitizes mice to ER stress resulting from chemical and physiological inducers.
(A) Immunoblot of nuclear spliced XBP-1, cleaved ATF6, and ATF4 for primary hepatocytes from control and Lrh-1LKO mice treated with 2 mM DTT or 0.05 µg/ml Brefeldin A (BFA). TBP was used as a …
Figure 3
An increase in LRH-1 transcriptional activity and expression is observed following ER stress, with heightened expression dependent on UPR components.
(A) Relative expression by quantitative PCR for Lrh-1. RNA was collected at designated timepoints for control and Lrh-1LKO mice treated with vehicle or 1 mg/kg tunicamycin (TM) (n = 3–6). Data were …
Figure 4
Microarray analysis suggests loss of ATF2 transcriptional ability in Lrh-1 deficient mice.
(A) Illumina Mouse Ref-8 arrays were performed for control and Lrh-1LKO mice treated with vehicle or tunicamycin (TM) for 24 hr (n = 3). Data were normalized and top 100 genes differentially induced …
Figure 5
Plk3 is a direct LRH-1 target that is required for induction of ATF2 target genes and ER stress resolution.
(A) Quantitative PCR for Plk3. RNA was collected at designated timepoints for control and Lrh-1LKO mice treated with vehicle or 1 mg/kg tunicamycin (TM) (n = 3–6). Data were normalized to Tbp …
Figure 6
Restoration of Plk3 induction rescues ATF2 phosphorylation and ER stress resolution in Lrh-1LKO mice, and loss or gain of ATF2 transcriptional activity also alters ER stress resolution capacity.
(A) Primary hepatocytes were prepared from Lrh-1f/f and Lrh-1LKO mice and transduced with Ad-Plk3 or Ad-control at a MOI of 100. Cells were treated 36 hr later with vehicle or tunicamycin (TM) (0.01 …
Figure 7
LRH-1 agonism promotes Plk3 and ATF2 target gene expression and increases resistance to ER stress independent of the UPR.
(A) Quantitative PCR for Plk3. RNA was collected at designated timepoints for primary hepatocytes isolated from hLrh-1 TG; mLrh-1 LKO and mLrh-1LKO mice treated with 10 uM RJW100 and/or 0.01 ug/ml …
Figure 8
Mechanism of LRH-1’s requirement in ER stress resolution.
https://doi.org/10.7554/eLife.01694.013Tables
Table 1
Genes differentially induced by TM between control and Lrh-1-null mice and their regulation by ATF2 and LRH-1
| Gene | Known target of | |
|---|---|---|
| ATF2 | LRH-1 | |
| PLK3 | ✓ | ✓ |
| GDF15 | ||
| FGF21 | * | |
| PMM1 | ||
| CRELD2 | ✓ | |
| NFIL3 | ||
| IGSF11 | ||
| GSTM2 | ||
| GOT1 | ||
| DPP9 | ✓ | ✓ |
| ST5 | ||
| CCBL1 | ✓ | ✓ |
| TMEM66 | ✓ | |
| KRTCAP2 | ✓ | ✓ |
| LRRC59 | ✓ | ✓ |
| CDK2AP2 | ✓ | ✓ |
| ST3GAL1 | ||
| TES | ✓ | |
| CCDC134 | ✓ | ✓ |
| UGT1L | ||
| ACOT2 | ✓ | |
| PLIN5 | ✓ | |
| LITAF | ✓ | |
| RPS13 | ✓ | |
| LGALS3BP | ||
| CRYM | ||
| SUPT5H | ✓ | |
| B3GAT3 | ✓ | |
| GRN | ✓ | |
| ARRDC4 | ||
| SLC30A7 | ✓ | |
| CRELD1 | ✓ | |
| NT5M | ||
| ARSG | ||
| SEP15 | ||
| MKNK1 | ✓ | |
| DDX52 | ||
| EXOSC5 | ✓ | |
| RABAC1 | ✓ | |
| RPS15 | ✓ | ✓ |
| D830014E11RIK | ||
| AVPI1 | ✓ | |
| RPS9 | ✓ | |
| CRAT | ✓ | |
| BHMT2 | ✓ | |
| B230217C12RIK | ||
| EIF3G | ✓ | ✓ |
| SLC25A28 | ✓ | |
| HR | ||
| CCL9 | ||
| ANXA4 | ✓ | ✓ |
| SMCO4 | ||
| SMOX | ||
| ARL14EP | ✓ | |
| SLC39A7 | ✓ | |
| ICA1 | ||
| ENTPD5 | ✓ | |
| PIWIL2 | ||
| ANG | ✓ | |
| MCFD2 | ✓ | ✓ |
| SOAT2 | ||
| SLC41A3 | ✓ | ✓ |
| MFGE8 | ||
| CYP4A14 | ||
| D12ERTD647E | ||
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Microarray analysis was performed for control and Lrh-1LKO mice treated with vehicle or 1 mg/kg tunicamycin for 24 hr (n = 3). Genes were screened for those induced at least 1.5 fold by TM in control mice with significantly different induction in Lrh-1LKO mice by t-test (p<0.05). This list was filtered for those with differential expression between genotypes with TM treatment by t-test (p<0.05). Previously published genome-wide ATF2 and LRH-1 binding datasets were analyzed to identify genes with ATF2 or LRH-1 binding sites −500 to +275 bp from the TSS. Genes in our set were compared with these sets and genes that contain ATF2 or LRH-1 binding sites meeting the above criteria are marked.
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*
No binding site for ENCODE data set but a known ATF2 direct target (Hondares et al., 2011).
Table 2
Enrichment of known transcription factor binding sites in our gene set (Table 1) of differentially regulated genes by ER stress between control and Lrh-1LKO mice
| Transcription factor name: | Known phosphorylation by PLK3: | Overlap of target genes with our gene set (Table 1): | Dataset used for analysis: |
|---|---|---|---|
| ATF2 | T71 (Wang et al., 2011b) | 3.83E-08 | ATF2 binding in G12878 cells (ENCODE EH002306) |
| Lrh-1 | none | 1.40E-04 | Lrh-1 binding in mouse liver (Chong et al., 2012) |
| p53 | S20 (Xie et al., 2001) | 1.37E-04 | p53 binding in U2OS cells treated with Nutlin-3 (Menendez et al., 2013), which results in S20 phosphorylation (Valentine et al., 2011) |
| c-Jun | S63 and S73 (Wang et al., 2011a) | 2.95E-02 | c-Jun binding in CH12 cells (ENCODE EM001943), in which S63 may be constitutively phosphorylated like other B-lymphoma lines (Gururajan et al., 2005) |
| NRF2 | None | NS | NRF2 binding in lymphoid cell lines treated with sulforaphane (Chorley et al., 2012) |
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Transcription factors known to be phosphorylated by PLK3 (ATF2, p53, and c-Jun) are summarized here, along with NRF2 to represent the oxidative stress response and LRH-1. Overlap of known transcription factor binding sites −500 to +b250 bp of the TSS for genes in Table 1 was calculated, and significance was determined using hypergeometric distribution tests.
Additional files
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Supplementary file 1
Primer sequences.
- https://doi.org/10.7554/eLife.01694.014
