doi: 10.1038/ncomms4315.
A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response
Affiliations
- PMID: 24525530
- PMCID: PMC3962829
- DOI: 10.1038/ncomms4315
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A functional interaction between Hippo-YAP signalling and FoxO1 mediates the oxidative stress response
Nat Commun.
2014.
Abstract
The Hippo pathway is an evolutionarily conserved regulator of organ size and tumorigenesis that negatively regulates cell growth and survival. Here we report that Yes-associated protein (YAP), the terminal effector of the Hippo pathway, interacts with FoxO1 in the nucleus of cardiomyocytes, thereby promoting survival. YAP and FoxO1 form a functional complex on the promoters of the catalase and manganese superoxide dismutase (MnSOD) antioxidant genes and stimulate their transcription. Inactivation of YAP, induced by Hippo activation, suppresses FoxO1 activity and decreases antioxidant gene expression, suggesting that Hippo signalling modulates the FoxO1-mediated antioxidant response. In the setting of ischaemia/reperfusion (I/R) in the heart, activation of Hippo antagonizes YAP-FoxO1, leading to enhanced oxidative stress-induced cell death through downregulation of catalase and MnSOD. Conversely, restoration of YAP activity protects against I/R injury. These results suggest that YAP is a nuclear co-factor of FoxO1 and that the Hippo pathway negatively affects cardiomyocyte survival by inhibiting the function of YAP-FoxO1.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
a, The localization of FoxO1 and YAP was examined. Myocytes were stained with FoxO1 (red), YAP (green) and DAPI (blue). Scale bar, 10 μm. b, Cells were stained with Rabbit anti-FoxO1 antibody and/or Mouse anti-YAP antibody, and in vivo protein–protein interaction between FoxO1 and YAP (red dots) was detected with secondary proximity probes, anti-Rabbit PLUS and anti-Mouse MINUS, using the Duolink in situ PLA detection kit. Scale bar, 10 μm. c, Neonatal cardiomyocyte lysates were prepared and immunoprecipitated with FoxO1 antibody or control IgG. The interaction between FoxO1 and YAP was examined. Immunoblots of input lysate controls (5% of inputs) are also shown. d, Recombinant GST-YAP and myc-FoxO1 were used to examine the direct interaction between YAP and FoxO1. e, Cardiomyocytes were co-transfected with 1 μg FoxO1-luc vector and the indicated expression vectors. After 48 hours, the luciferase activity was measured, demonstrating that YAP enhanced FoxO1 transcriptional activity (*p<0.05 vs. empty vector, n=3). f, Overexpression of Lats2 inhibited, whereas knockdown of Lats2 enhanced, FoxO1 activity. Cardiomyocytes were transfected with 1 μg FoxO1-luc vector. After 6 hours, myocytes were transduced with the indicated virus and luciferase activity was measured (*p<0.05 vs. LacZ or sh-con, n=3). Data shown as mean±s.e.m.. P values were determined using unpaired Student’s t-test.
a, The catalase and MnSOD promoters contain two potential FoxO1 DNA-binding elements, referred to as DBE1 and DBE2. b, ChIP analysis of in vivo YAP and FoxO1 binding to the catalase and MnSOD promoters. Protein-bound chromatin was prepared from H9C2 myoblasts and immunoprecipitated with YAP or FoxO1 antibodies. For sequential ChIP, the protein-bound chromatin was first immunoprecipitated with the FoxO1 antibody. The second ChIP was then performed on the chromatin eluted from the first ChIP by immunoprecipitating it with the YAP antibody. Normal IgG was used as a negative control. The immunoprecipitated DNA was analyzed by PCR using primers flanking each of the DBEs. Results are representative of three individual experiments. c, The minimal catalase promoter containing FoxO1 DBE2 was cloned into a luciferase vector. Cardiomyocytes were co-transfected with the catalase-luc vector and the indicated expression vectors. After 6 hours, myocytes were transduced with the indicated virus and, after 72 hours, the luciferase activity was measured (*p<0.05 vs. empty vector, #p<0.05 vs. 20ng FoxO1 or YAP, Δp<0.05 vs. 50ng FoxO1 or YAP, n=3). Data shown as mean±s.e.m.. P values were determined using one-way ANOVA followed by a Newman-Keuls comparison test.
a, ChIP analysis of in vivo YAP binding to the catalase and MnSOD promoters. H9C2 myoblasts were transduced with the indicated adenovirus for 48 hours. Protein-bound chromatin was prepared and immunoprecipitated with IgG and YAP antibodies. The relative occupancy on the promoters was compared with the input signal (*p<0.05 vs. LacZ, n=3). b–c, Overexpression of Lats2 reduced, whereas knockdown of Lats2 enhanced, catalase and MnSOD protein and mRNA levels. Cardiomyocytes were transduced with the indicated adenovirus. b, The mRNA levels of catalase and MnSOD were examined by quantitative RT-PCR. The relative copy number was normalized with β-actin (*p<0.05 vs. LacZ or sh-con, #p<0.01 vs. LacZ, n=4). c, Lysates were used for immunoblot analysis of catalase, MnSOD and α-tubulin. Results are representative of three individual experiments. d, Cardiomyocytes were transfected with the catalase-luc vector. After 6 hours, myocytes were transduced with the indicated virus and, after 72 hours, luciferase activity was measured (*p<0.05 vs. sh-con/sh-Lats2(−), #p<0.05 vs. sh-con/sh-Lats2(+), n=3). e, Downregulation of Lats2 prevented H2O2-induced cell death. Cardiomyocytes transduced with the indicated adenovirus were treated with 100 μM H2O2, and TUNEL-positive cells were examined (*p<0.05 vs. sh-con/sh-Lats2(−), #p<0.05 vs. sh-con/sh-Lats2(+), n=4). Data shown as mean±s.e.m.. P values were determined using unpaired Student’s t-test or one-way ANOVA followed by a Newman-Keuls comparison test.
a, Representative immunoblots indicating that I/R activated Hippo signaling. Left, WT mice were subjected to ischemia for 45 minutes followed by 2 hours of reperfusion. Sham and ischemic area samples were used for immunoblot analysis for p-Lats2 (T1041 and S872), Lats2, p-Mst1 (Thr183), and Mst1. Right, Tg-DN-Lats2 and control WT mice were subjected to ischemia for 45 minutes followed by 2 hours of reperfusion. Sham and ischemic area samples were used for immunoblot analysis for p-YAP (S127), YAP, Lats2, and GAPDH. The enhancement in YAP phosphorylation during I/R was attenuated in Tg-DN-Lats2, confirming that Lats2 activation was suppressed. b, Tg-DN-Lats2 and control WT mice were subjected to ischemia for 45 minutes followed by 2 hours of reperfusion. The cytosolic and nuclear fractions of heart homogenates were prepared and subjected to immunoblot analysis of p-YAP (S127), YAP, Lats2, GAPDH, and Lamin A/C (SE, short exposure; LE, long exposure). c–d, Activation of Lats2 decreased antioxidant protein levels and enhanced oxidative stress during I/R. Tg-DN-Lats2 and control WT mice were subjected to ischemia for 45 minutes followed by 24 hours of reperfusion. c, Sham and ischemic area samples were used for immunoblot analysis for catalase, MnSOD, and α-tubulin. d, The antioxidant capacity of the myocardium was examined (*p<0.05 vs. WT/sham, #p<0.05 vs. WT/I/R, n=4). Data shown as mean±s.e.m.. P values were determined using one-way ANOVA followed by a Newman-Keuls comparison test.
a–b, Purified adenovirus (1 × 109 opu) was administered by direct injection to the LV free wall (two sites, 25 μl/site) of Tg-DN-Lats2 and WT mice. I/R surgery was performed 5 days after injection. Mice were subjected to ischemia for 45 minutes followed by 24 hours of reperfusion. a, Gross appearance of LV tissue sections after Alcian blue and triphenyltetrazolium chloride staining demonstrates that injection of ad-sh-FoxO1 abolished the protection observed in Tg-DN-Lats2 mice. Quantitative measurement of myocardial infarct area/area at risk (% infarct size/AAR) was performed (*p<0.05 vs. WT/sh-con, #p<0.05 vs. Tg-DN-Lats2/sh-FoxO1, n=8). Scale bar, 1 mm. b, LV myocardial sections were subjected to TUNEL staining. The number of TUNEL-positive myocytes was expressed as a percentage of total nuclei detected by DAPI staining (*p<0.05 vs. WT/sh-con, #p<0.05 vs. Tg-DN-Lats2/sh-FoxO1, n=5–8). c–e, Purified adenovirus (1 × 109 opu) was administered by direct injection to the LV free wall (two sites, 25 μl/site). I/R surgery was performed 3 days after injection. Mice were subjected to ischemia for 30 minutes followed by 24 hours of reperfusion. c, Sham and ischemic area samples were used for immunoblot analysis for catalase, MnSOD, and GAPDH. FLAG was also used to examine exogenous YAP gene expression. Statistical analyses of densitometric measurements of catalase and MnSOD are shown (*p<0.05 vs. LacZ/sham, #p<0.05 vs. LacZ/I/R, n=5). d, Gross appearance of LV tissue sections after Alcian blue and triphenyltetrazolium chloride staining demonstrates that injection of YAP or YAP S127A decreased the infarct area. Quantitative measurement of myocardial infarct area/area at risk (% infarct size/AAR) was performed (*p<0.05 vs. LacZ, n=8). Scale bar, 1 mm. e, LV myocardial sections were subjected to TUNEL staining. The number of TUNEL-positive myocytes was expressed as a percentage of total nuclei detected by DAPI staining (*p<0.05 vs. LacZ, n=5–6). Data shown as mean±s.e.m.. P values were determined using one-way ANOVA followed by a Newman-Keuls comparison test.
In cardiomyocytes, YAP and FoxO1 form a functional complex that mediates catalase and MnSOD expression. In response to I/R, activation of the Hippo signaling cascade induces YAP inactivation, which leads to antioxidant gene suppression, ROS accumulation, and cell death.
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