Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution

  1. Jennifer L Mamrosh
  2. Jae Man Lee
  3. Martin Wagner
  4. Peter J Stambrook
  5. Richard J Whitby
  6. Richard N Sifers
  7. San-Pin Wu
  8. Ming-Jer Tsai
  9. Francesco J DeMayo
  10. David D Moore  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. University of Cincinnati, United States
  3. University of Southampton, United Kingdom
8 figures, 2 tables and 1 additional file

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. …

https://doi.org/10.7554/eLife.01694.003
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 …

https://doi.org/10.7554/eLife.01694.004
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 …

https://doi.org/10.7554/eLife.01694.005
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 …

https://doi.org/10.7554/eLife.01694.006
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 …

https://doi.org/10.7554/eLife.01694.007
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

https://doi.org/10.7554/eLife.01694.009
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 …

https://doi.org/10.7554/eLife.01694.010
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 …

https://doi.org/10.7554/eLife.01694.012
Mechanism of LRH-1’s requirement in ER stress resolution.
https://doi.org/10.7554/eLife.01694.013

Tables

Table 1

Genes differentially induced by TM between control and Lrh-1-null mice and their regulation by ATF2 and LRH-1

https://doi.org/10.7554/eLife.01694.008
GeneKnown target of
ATF2LRH-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
  1. 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.

  2. *

    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

https://doi.org/10.7554/eLife.01694.011
Transcription factor name:Known phosphorylation by PLK3:Overlap of target genes with our gene set (Table 1):Dataset used for analysis:
ATF2T71 (Wang et al., 2011b)3.83E-08ATF2 binding in G12878 cells (ENCODE EH002306)
Lrh-1none1.40E-04Lrh-1 binding in mouse liver (Chong et al., 2012)
p53S20 (Xie et al., 2001)1.37E-04p53 binding in U2OS cells treated with Nutlin-3 (Menendez et al., 2013), which results in S20 phosphorylation (Valentine et al., 2011)
c-JunS63 and S73 (Wang et al., 2011a)2.95E-02c-Jun binding in CH12 cells (ENCODE EM001943), in which S63 may be constitutively phosphorylated like other B-lymphoma lines (Gururajan et al., 2005)
NRF2NoneNSNRF2 binding in lymphoid cell lines treated with sulforaphane (Chorley et al., 2012)
  1. 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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