LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling

Abstract

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

Keywords: YAP; Hippo signaling; LRP6; O-GlcNAcylation; Starvation.

Figure EV1. The amount of LRP6 was reduced in nutrient starvation

1. A

   HEK293T cells were seeded to low‐density or high‐density conditions. One day after the cells were lysed and the lysates were analyzed by immunoblotting.

2. B

   The loss of LRP6 was due to a lack of nutrients. HEK293T cells were seeded to low‐density or high‐density conditions. One day after, medium for the cells at high density was replaced with fresh medium for 2 h. Cells were then lysed, and the lysates were analyzed by immunoblotting.

3. C

   The loss of LRP6 level was not due to lower pH. HEK293 cells were treated in pH‐modified medium for 2 h. Medium was titrated with HCl, and pH was measured by pH meter (Mettler Toledo). Cells were lysed, and the lysates were analyzed by immunoblotting.

4. D

   The amount of LRP6 decreased as the glucose level was reduced. HEK293 cells were incubated for 4 h with concentrations of glucose and mannitol as indicated in the figure. Cells were lysed, and the lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β‐actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t‐test was used for statistical analysis.

5. E

   A competitive inhibitor of glucose processing reduced the amount of LRP6. HEK293 cells were incubated in 2‐DG (25 mM)‐contained medium for 4 h. Cells were lysed, and the lysates were analyzed by immunoblotting.

6. F

   Serum starvation reduced the amount of both LRP5 and LRP6. HEK293 cells were incubated in serum‐free medium for 4 h. Cell lysates were immunoblotted with antibodies as indicated in the figure.

7. G

   The mRNA level of LRP6 was not reduced under serum starvation. We used a quantitative real‐time PCR assay for measuring the expression of LRP6 and ANKRD1 mRNA in HEK293 cells cultured under serum‐starved conditions for 4 h. Quantification of LRP6 and ANKRD1 mRNA was normalized to the level of β‐actin. Error bars indicate standard deviation of technical triplicate measurements. **P < 0.01 and ***P < 0.001. Student's t‐test was used for statistical analysis.

8. H

   The loss of LRP6 during serum starvation was not mediated by the proteasomal degradation pathway. HEK293 cells were incubated in serum‐free medium for 4 h with DMSO or MG132 (25 μM). Cell lysates were analyzed by immunoblotting.

9. I, J

   The loss of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum‐free medium for 4 h with DMSO, pepstatin A (20 μg/ml)/E64d (10 nM) (I), or ammonium chloride (2 mM) (J). Cells were lysed, and the lysates were analyzed by immunoblotting.

10. K

    The loss of LRP6 by serum starvation was not mediated via autophagy. HEK293 cells were incubated in serum‐free medium for 4 h with DMSO or wortmannin (1 μM). Cells were lysed, and the lysates were analyzed by immunoblotting.

11. L

    The loss of LRP6 by serum starvation was not mediated via autophagy. HEK293 cells were transfected with GFP or ATG7 siRNA and were incubated in serum‐free medium for 4 h. Cells were lysed, and the lysates were analyzed by immunoblotting.

12. M, N

    The mRNA levels of clathrin and caveolin‐1 were properly reduced by the respective siRNAs. We used quantitative real‐time PCR assay for measuring expression of Clathrin and Caveolin‐1 mRNAs. Quantification of the mRNAs was normalized to the level of β‐actin. Error bars indicate standard deviation of technical triplicate measurements. ***P < 0.001. Student's t‐test was used for statistical analysis.

13. O

    Components of serum such as cytokines and hormones were dispensable for maintaining LRP6. HEK293 cells were incubated in serum‐free medium with dialyzed FBS for 4 h. Cells were lysed, and the lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β‐actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S, non‐significant and *P < 0.05. Student's t‐test was used for statistical analysis.

Figure 1. The level of LRP6 was reduced in nutrient starvation via endocytosis‐mediated lysosomal‐dependent degradation

1. A

   LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum‐free medium for the indicated hours. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

2. B

   Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum‐free medium for 3 h. Then, cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed, and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β‐actin and p‐YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non‐significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t‐test was used for statistical analysis.

3. C

   Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum‐free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.

4. D

   Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose‐free medium for 4 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

5. E

   The decrease in LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum‐free medium for 4 h with DMSO or bafilomycin A1 (100 nM). Cells were lysed, and the cell lysates were analyzed by immunoblotting.

6. F, G

   Loss of LRP6 by serum starvation was mediated in a clathrin or caveolin‐1-dependent manner. HEK293 cells were transfected with clathrin (F) or caveolin (G) siRNA and incubated in serum‐free medium for 4 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

7. H

   Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum‐free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β‐actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non‐significant and *P < 0.05. Student's t‐test was used for statistical analysis.

Figure 2. LRP6 regulated the phosphorylation, localization, and transcriptional activity of YAP

1. The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation. HEK293 cells were incubated in serum‐free medium for the indicated times. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

2. Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient‐rich condition. HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient‐rich conditions. The ratio of p‐YAP/total‐YAP of three independent immunoblot bands was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t‐test was used for statistical analysis.

3. Overexpression of LRP6 blocked the increase in YAP phosphorylation under serum starvation conditions. HEK293T cells were transfected with empty vector (−) or LRP6‐EGFP as indicated in the figure. One day after transfection, cells were incubated in serum‐free medium for 4 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β‐actin and p‐YAP/YAP of three independent immunoblots was quantified (middle and right panel, respectively). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05 and ***P < 0.001. Student's t‐test was used for statistical analysis.

4. Knockdown of LRP6 reduced YAP reporter activity. HEK293T cells were transfected with GFP siRNA as a control, LRP6 siRNA, pRL‐TK, and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.005. Student's t‐test was used for statistical analysis.

5. Knockdown of LRP6 reduced the expression of YAP target genes. Quantitative real‐time PCR assay measuring the expression of CTGF, ANKRD1, INHBA, and LRP6 mRNA in GFP and LRP6 siRNA‐transfected HEK293T cells was performed. Quantification of CTGF, ANKRD1, INHBA, and LRP6 mRNA was normalized against the level of β‐actin. Error bars indicate standard deviation of technical triplicate measurements. *P < 0.05, **P < 0.01 and ***P < 0.001. Student's t‐test was used for statistical analysis.

6. Knockdown of LRP6 reduced the interaction between YAP and TEAD. HEK293T cells were transfected with EGFP‐YAP, Myc‐TEAD4, and siRNA for LRP6. Cells were lysed, and the cell lysates were immunoprecipitated with anti‐Myc antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

7. Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions. HEK293T cells were transfected with an empty vector (−) or VSVG‐LRP6, and with pRL‐TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were incubated in serum‐free medium for 4 h and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.001. Student's t‐test was used for statistical analysis.

8. Knockdown of LRP6 induced cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP or LRP6, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear‐localized YAP per image (n = 8). Error bars indicate standard deviation. ***P < 0.001. Student's t‐test was used for statistical analysis.

9. Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions. HEK293A cells were transfected with LRP6‐EGFP and 1 day after transfected cells were incubated with or without serum‐containing medium for 4 h, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear‐localized YAP per image (n = 6). Error bars indicate standard deviation. *P < 0.05. Student's t‐test was used for statistical analysis.

Figure EV2. LRP6 regulated the phosphorylation, localization, and transcriptional activity of YAP

1. Conditional knockout of LRP6 in MEF resulted in down‐regulation of endogenous expression of YAP target genes. For conditional knockout of Lrp6, Lrp6‐floxed MEF cells were transduced with Cre‐expressing adenovirus for 48 h. GFP‐expressing adenovirus was used as a control. Quantitative real‐time PCR assay for measuring the levels of Ctgf, Cyr61, and Lrp6 mRNA in WT and Lrp6 KO MEF cells was performed. Error bars indicate standard deviation of four measurements (technical replicates). *P < 0.05 and ***P < 0.001. Student's t‐test was used for statistical analysis.

2. Overexpression of LRP6 rescued the reduced expression of YAP target gene under serum starvation. We used quantitative real‐time PCR assay for measuring expression of ANKRD1 and LRP6 mRNA in empty vector (−) or VSVG‐LRP6 transfected HEK293T cells. After transfection, serum was removed as indicated in the figure. Quantification of ANKRD1 and LRP6 mRNA was normalized to the level of β‐actin. Error bars indicate standard deviation of four measurements (technical replicates). *P < 0.05 and ***P < 0.001. Student's t‐test was used for statistical analysis.

3. Knockdown of either LRP5 or LRP6 reduced YAP reporter activity. HEK293T cells were transfected with siRNA for GFP, LRP5, and LRP6 and with pRL‐TK and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05, **P < 0.01 and ***P < 0.001. Student's t‐test was used for statistical analysis.

4. Knockdown of LRP6 enhanced the level of phosphorylated YAP. HEK293T cells were transfected with siRNA for GFP and LRP6. One day after transfection, cells were dissociated and seeded again at low‐density or high‐density condition. For the low‐density condition, cells were seeded on a 60‐mm dish to be 50% confluent; for high‐density condition, twice the number of cells were seeded on a 35‐mm dish. One day after seeding, cells were lysed and the lysates were analyzed by immunoblotting.

5. Overexpression of DVL1 enhanced Wnt‐reporter activity in LRP6 knockdown. HEK293T cells were transfected with siRNA for GFP and LRP6, empty vector (−), and Flag‐DVL1, and with pRL‐TK and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate the standard deviation of biological triplicate measurements. ***P < 0.001. Student's t‐test was used for statistical analysis.

6. Overexpression of DVL1 did not rescue the reduced YAP reporter activity caused by LRP6 knockdown. HEK293T cells were transfected with siRNAs for GFP and LRP6, empty vector (−), and Flag‐DVL1, and with pRL‐TK and pSuper‐TOP luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. N.S: non‐significant and ***P < 0.001. Student's t‐test was used for statistical analysis.

Figure 3. Regulation of YAP phosphorylation and activity by LRP6 requires Merlin and LATS1/2

1. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.

2. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (−) or VSVG‐LRP6. One day after transfection, cells were incubated in serum‐free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.

3. Merlin and LATS1/2 were necessary for the LRP6‐knockdown‐mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

4. Merlin and LATS1/2 were necessary for the LRP6‐knockdown‐mediated decrease in YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. After 1 day, these cells were transfected again with pRL‐TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01 and N.S: non‐significant. Student's t‐test was used for statistical analysis.

5. Merlin and LATS1/2 were necessary for the LRP6 knockdown‐mediated‐cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.

6. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 day were transfected again with HA‐Merlin and HA‐MerlinΔFERM. Cells were lysed, and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA‐Merlin, and the arrowhead indicates HA‐MerlinΔFERM.

7. MST1/2 were necessary for the LRP6‐knockdown‐mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MST1/2. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

8. MAP4K4/6/7 were necessary for the LRP6 knockdown‐mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

Figure EV3. Regulation of YAP phosphorylation and activity by LRP6 requires Merlin and LATS1/2

1. Overexpression of DVL1 did not inhibit LRP6‐knockdown‐mediated activation of LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6, empty vector (−), and Flag‐DVL1. Cells were lysed, and the lysates were analyzed by immunoblotting.

2. A quantification was performed by counting cells that have nuclear‐localized YAP per image (n = 8). Error bars indicate standard deviation. N.S: non‐significant and ***P < 0.001. Student's t‐test was used for statistical analysis.

3. The mRNA levels of MAP4K4/6/7 were properly reduced by siRNAs. We used quantitative real‐time PCR assay for measuring expression of MAP4K4, MAP4K6, and MAP4K7 mRNA in GFP and MAP4K4/6/7 siRNA‐transfected HEK293T cells. The quantification was normalized to the level of β‐actin. Error bars indicate standard deviation of technical triplicate measurements. *P < 0.05 and ***P < 0.005. Student's t‐test was used for statistical analysis.

4. Knockdown of LRP6 did not induce a mobility shift of MAP4K4. HEK293 cells were transfected with siRNA for GFP or LRP6. Cells were lysed and the lysates were analyzed by immunoblotting.

5. Serum starvation did not induce a mobility shift of MAP4K4. HEK293 cells were incubated in serum‐free medium for 4 h. Cells were lysed, and the lysates were analyzed by immunoblotting.

6. Knockdown of LRP6 did not induce phosphorylation of MST1. HEK293 cells were transfected with EGFP‐MST1 and siRNA for GFP or LRP6. Cells were lysed, and the lysates were analyzed by immunoblotting (left panel). The ratio of p‐MST1/MST1 of three independent immunoblots was quantified (biological replicates, right panel). N.S: non‐significant. Error bars indicate standard deviation of biological triplicate measurements. Student's t‐test was used for statistical analysis.

7. Serum starvation did not induce phosphorylation of MST1. HEK293 cells were transfected with EGFP‐MST1 and incubated in serum‐free medium for 4 h. Cells were lysed, and the lysates were analyzed by immunoblotting (left panel). The ratio of p‐MST1/MST1 of three independent immunoblots was quantified (biological replicates, right panel). N.S: non‐significant. Error bars indicate the standard deviation of biological triplicate measurements. Student's t‐test was used for statistical analysis.

Figure 4. Merlin changed binding partners from LRP6 to LATS under serum starvation

1. A

   The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag‐Merlin and LRP6‐EGFP. After transfection, cells were incubated in serum‐free medium for overnight with bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole‐cell lysates.

2. B, C

   The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum‐free medium for 4 h with bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti‐LRP6 (B) or anti‐Merlin (C) antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole‐cell lysates.

3. D

   The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc‐LATS1 and EGFP‐Merlin. After transfection, cells were incubated in serum‐free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

4. E

   Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc‐LATS1, EGFP‐Merlin, and LRP6 siRNA. Cells were lysed, and the cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

5. F

   Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti‐Merlin antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

6. G

   Schematic summary of Fig 4 experimental results.

Figure 5. O‐GlcNAcylation of LRP6 was regulated by nutrient status and modulation of global O‐GlcNAcylation affected the interaction between Merlin and LATS

1. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed, and the cell lysates were immunoprecipitated with anti‐LRP6 antibody and blotted with OGT. WCL, whole‐cell lysates.

2. LRP6 is O‐GlcNAcylated. LRP6‐EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI‐1 (50 μM) for 6 h with bafilomycin A1 (100 nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

3. LRP6 is O‐GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA beads were precipitated and analyzed by immunoblotting. WCL, whole‐cell lysates.

4. Blocking of O‐GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI‐1 (50 μM) and bafilomycin A1 (100 nM) for 6 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting.

5. The O‐GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6‐EGFP. Cells were incubated in serum‐free medium with bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates. The ratio of O‐GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t‐test was used for statistical analysis.

6. Increase in O‐GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6‐EGFP and Flag‐Merlin transfected HEK293T cells were incubated in serum‐free medium with bafilomycin A1 (20 nM) and Thiamet G (30 μM) overnight. Cells were lysed, and the cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

7. Enhancement of global O‐GlcNAcylation blocked the serum starvation‐mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc‐LATS1 and Flag‐Merlin. After transfection, cells were incubated in serum‐free medium with Thiamet G (30 μM) and bafilomycin A1 (20 nM) overnight. Cells were lysed, and the cell lysates were immunoprecipitated with anti‐Myc antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

Figure EV4. LRP6 is O‐GlcNAcylated, and the level of LRP6 O‐GlcNAcylation is reduced in nutrient starvation

1. LRP6 binds to OGT. HEK293T cells were transfected with LRP6‐EGFP, Flag‐OGT, and Flag‐Merlin. Cells were lysed, and the lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates. The interaction between LRP6‐EGFP and Flag‐Merlin was used as a positive control.

2. LRP6 is O‐GlcNAcylated. HEK293T cells were transfected with VSVG‐LRP6, Flag‐OGT, and Flag‐OGA. Cells were lysed, and the lysates were immunoprecipitated with anti‐VSVG antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

3. O‐GlcNAcylation of LRP6 was detected after the removal of N‐glycosylation. HEK293T cells were transfected with LRP6‐EGFP. Cells were lysed, and the lysates were incubated with PNGase F. The cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. Upper and lower arrowheads indicate glycosylated and un‐glycosylated LRP6, respectively.

4. Inhibition of global O‐GlcNAcylation leads to less LRP6. HEK293 cells were treated with OSMI‐1 (50 μM) for indicated times. Cells were lysed, and the lysates were analyzed by immunoblotting.

5. The decrease in LRP6 by OSMI‐1 treatment is mediated by the lysosomal degradation pathway. HEK293 cells were treated with OSMI‐1 (50 μM), bafilomycin A1 (100 nM), and MG132 (25 μM) for 6 h. Cells were lysed, and the lysates were analyzed by immunoblotting.

6. Membrane OGT was reduced during serum starvation. HEK293 cells were incubated in serum‐free medium for the indicated times. Cells were lysed, and membrane fractionation was performed. Proteins were analyzed by immunoblotting.

7. The interaction between overexpressed LRP6 and OGT was decreased under serum starvation conditions. HEK293T cells were transfected with LRP6‐EGFP and Flag‐OGT. Cells were incubated in serum‐free medium with bafilomycin A1 (20 nM) overnight. Cells were lysed, and the lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

8. The O‐GlcNAcylation of LRP6 was reduced under glucose starvation. HEK293T cells were transfected with LRP6‐EGFP. Cells were incubated in glucose‐free medium with bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

9. The phosphorylation of LRP6 at S1490 site was unchanged by serum starvation. HEK293T cells were transfected with LRP6‐EGFP. Cells were incubated in serum‐free medium with bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

10. The serine phosphorylation of LRP6 is reduced by serum starvation. HEK293T cells were transfected with LRP6‐EGFP. Cells were incubated in serum‐free medium with bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the lysates were immunoprecipitated with anti‐EGFP antibody and analyzed by immunoblotting. WCL, whole‐cell lysates.

Figure 6. Nutrient starvation reduced the level of LRP6 and its O‐GlcNAcylation in vivo

1. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h, and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t‐test was used for statistical analysis.

2. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h, and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t‐test was used for statistical analysis.

3. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved, and brain tissues were analyzed by immunoblotting.

4. LRP6 was O‐GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA beads were precipitated and analyzed by immunoblotting. WCL, whole‐cell lysates.

5. O‐GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA beads for 3 h. After incubation, sWGA beads were precipitated and analyzed by immunoblotting. WCL, whole‐cell lysates.

Figure EV5. Graphical summary of LRP6‐YAP signaling pathway

Under nutrient‐rich conditions, LRP6 is O‐GlcNAcylated and it maintains its interaction with Merlin. In this situation, YAP is activated and its target genes are expressed. In nutrient starvation, the O‐GlcNAcylation of LRP6 is lower and Merlin is detached from LRP6, followed by lysosomal degradation of LRP6. Merlin then binds to LATS1/2, allowing LATS1/2 to be activated by upstream kinases such as MST1/2 and MAP4K4/6/7. Activated LATS1/2 phosphorylates and inactivates YAP, thereby decreasing the expression of YAP target genes.