Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling

Abstract

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

Figure 1. Serum induces dephosphorylation of YAP and TAZ

(A, B) Serum induces YAP and TAZ dephosphorylation. HEK293A cells were starved in serum-free medium for 12 h and then stimulated with 10% FBS for the indicated times (A) or with different concentrations of FBS for 1 h (B). Cell lysates were subjected to immunoblotting with the indicated antibodies. Where indicated, gels containing phos-tag were employed for assessment of YAP phosphorylation status (A, the bottom panel). l.e. denotes long exposure. (C) Serum reversibly regulates YAP/TAZ phosphorylation. Serum-starved HEK293A cells were treated with 10% FBS for 1 or 2 h as indicated. In the last three lanes, after 1 h stimulation FBS was removed for ¼, ½ or 1 h as indicated by upwards arrows. (D) Serum induces YAP nuclear localization in HEK293A and MCF10A cells. YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP (green) along with DAPI for DNA (blue). Serum stimulation (10% FBS, 1 h) is indicated. The data presented in this figure and all the subsequent figures are representative of at least three independent experiments. See also Figure S1.

Figure 2. Characterization of serum factor(s) responsible for YAP/TAZ dephosphorylation

(A) Serum contains a YAP-activating activity. HEK293A cells were treated with 10% of different brands of serum: FBS (from Omega Scientific or Hyclone (HC)), fetal calf serum (FCS), horse serum (HS), or 10% mTesr1. Total cell lysates were subjected to immunoblotting. (B) The YAP-activating activity in serum is protease-resistant. FBS that were pre-treated with pronase E or heat-inactivated pronase E (HI). The effectiveness of pronase E was demonstrated by Coomassie Blue staining (left panel). Cells were stimulated with control or pronase E treated FBS. (C) YAP-activating activity in BSA. Different BSA preparations (from Sigma Aldrich) were used to treat HEK293A cells. A3294 was prepared by heat shock, A7073 Fraction V (FV) and A6003 (fatty acid (FA)-free) were prepared by ethanol precipitation, and A2058 was prepared by chromatography. Protein contents of different BSA preparations were similar as indicated by Coomassie Blue staining (not shown). Serum-starved HEK293A cells were treated with 1 or 10 mg/ml BSA for 1 h before harvest. (D) Charcoal treatment depletes the YAP-activating activity in serum. 10% or 1% of regular or charcoal-stripped (Ch) FBS was used to stimulate serum-starved HEK293A cells for 1 h. (E) The YAP-activating activity in FBS is sensitive to organic extraction under acidic conditions. FBS was extracted using chloroform, methanol, or different ratios of chloroform and methanol mixture (CM, in the presence of HCl or NaOH); organic solvent was evaporated and materials extracted were dissolved in 2 mg/ml fatty acid-free BSA (FAF) and used to treat cells. (F) LPA induces YAP dephosphorylation. HEK293A cells were treated with 100 μM of various lipids. Full names of lipids used are shown in Extended Experimental Procedures. Also see Figure S2.

Figure 3. LPA and S1P activate YAP/TAZ by dephosphorylation

HEK293A cells were treated with 1 μM LPA (A) or S1P (B) for the indicated times. Cell lysates were subjected to immunoblotting with the indicated antibodies. (C) Serum and LPA stimulate YAP interaction with TEAD1 but inhibit YAP interaction with 14-3-3. Cells were treated with LPA or serum as indicated. Cell lysates were subjected to immunoprecipitation (IP) with control IgG or YAP antibody. The co-immunoprecipitated TEAD1 and 14-3-3 were detected by immunoblotting. (D) LPA treatment (1 μM, for 1 h) induces YAP nuclear localization in HEK293A and MCF10A cells. Also see Figures S2 and S3.

Figure 4. YAP/TAZ are required for LPA functions and are regulated by LPA signaling

(A) YAP/TAZ are required for LPA to induce gene expression. mRNA levels of the indicated genes were measured by quantitative PCR. LPA (1 μM) treatment was for 1 h. HEK293A cells with stable knockdown of YAP/TAZ or control cells were used. (B) Knockdown of YAP/TAZ blocks LPA-induced cell migration. Migration of MCF10A cells transfected with control siRNA or YAP/TAZ siRNA was assessed by transwell cell migration assays. (C) YAP/TAZ is required for LPA to stimulate cell proliferation. Control and YAP/TAZ knockdown HEK293A cells were cultured in the absence of FBS and treated with or without 10 μM LPA for 0, 1, 2 or 3 day as indicated. LPA was replenished every day. Cell number was then counted. (D) Hyperplasia caused by transgenic LPA1 and LPA2 expression. H & E staining. (E) LPA receptor transgenic mouse tissues exhibit increased TAZ nuclear localization. Immunofluorescence staining for TAZ (red) and DNA (blue). (F) LPA receptor transgenic mouse tissues exhibit decreased YAP/TAZ phosphorylation. Sample in each lane was from an individual mouse. Mammary tissues were analyzed in (D-F). Also see Figure S4.

Figure 5. LPA and S1P repress Lats kinase activity

(A) MST1/2 are not required for LPA-induced YAP dephosphorylation and CTGF induction in MEF cells. WT or knockout MEF cells at similar density were untreated or treated with 1 μM LPA for 1 h. YAP phosphorylation was assessed by immunoblotting in the presence of phos-tag. (B) Lats kinase activity is inhibited by LPA. Endogenous Lats1 was immunoprecipitated from HEK293A cells that had been treated with LPA at various times and doses of LPA, and Lats1 kinase activity was determined using GTS-YAP as a substrate. (C) Lats phosphorylation is repressed by LPA. Cell lysates from control or LPA-treated (1 μM for 1 h) cells were divided into two parts, one for IgG IP and the other for Lats1 IP. Endogenous Lats1 was immunoprecipitated and probed with phospho-specific antibodies. (D) Lats overexpression suppresses the effect of LPA on YAP phosphorylation. HEK293A cells were co-transfected with Flag-YAP and HA-Lats2 or HA-Mob. One day after transfection, cells were serum-starved for 24 h, and then treated with 1 μM LPA for 1 h. Also see also Figure S5.

Figure 6. LPA and S1P modulate YAP/TAZ through their membrane receptors and Rho GTPases

(A) LPA1/3 antagonist Ki16425 completely blocks LPA and partially blocks serum effects on YAP/TAZ phosphorylation. HEK293A cells were treated with Ki16425 (10 μM) or DMSO control for 30 min as indicated, then cells were stimulated with S1P, LPA or FBS for 1 h. (B). LPA and S1P receptor overexpression promotes YAP nuclear localization. Cells were transfected with HA-tagged LPA1, LPA4, or S1P2 as indicated. The transfected receptors were detected by HA antibody (red) and endogenous YAP was detected by YAP antibody (green). Note that the receptor expressing red cells have higher nuclear YAP. (C) Knockdown of G12 and G13 blocks the effect of LPA on YAP phosphorylation. HEK293A cells were transfected with control siRNA, a pool of siRNAs for G12 and G13, or a pool of siRNAs for Gq and G11, serum was removed at 48 h. Following 16 h serum starvation, cells were treated with 1 μM LPA for 1 h. (D) Inactivation of Rho by C3 toxin prevents YAP/TAZ dephosphorylation by LPA, S1P, and serum. HEK293A cells were pretreated with 2 μg/ml C3 for 4 h, then stimulated with LPA, S1P, or FBS for 1 h. (E), Disruption of actin cytoskeleton prevents YAP/TAZ dephosphorylation by LPA or serum. HEK293A cells were pretreated with 1 μg/ml LatB for 30 min, then stimulated with LPA or serum for 1 h. Also see Figure S6.

Figure 7. Stimulation of Gs coupled GPCRs increases YAP phosphorylation

(A) Epinephrine stimulates YAP phosphorylation. MDA-MB-231 cells were treated with indicated concentrations of epinephrine for 1 h. Phosphorylation of CREB was determined by immunoblotting with phospho-CREB-specific antibody (pCREB). B) Phosphorylation of YAP from the heart of mice injected with epinephrine is increased. Samples from three representative pairs (from strong to weak induction of YAP phosphorylation) of mice were shown. Epinephrine is known to increase blood glucose levels, which are indicated underneath each sample. (C) Dopamine agonist dihydrexidine stimulates YAP phosphorylation. U2OS cells were treated with 10 μM dihydrexidine for 1 h. YAP phosphorylation status was assessed. (D) Glucagon stimulates YAP phosphorylation. Primary mouse hepatocytes were treated with 2 μM glucagon for 1 h, and YAP phosphorylation status was determined. (E) Forskolin induces YAP phosphorylation. MDA-MB-231 cells were treated with different concentrations of Forskolin for 1 h. (F) Forskolin induces Lats1 phosphorylation. Endogenous Lats1 was immunoprecipitated from control cells and Forskolin (Fsk)-treated (10 μM for 1 h) HEK293A cells, and protein lysates were divided into two parts, one for IgG IP and the other for Lats1 IP. Proteins immunoprecipitated were probed with phospho-specific antibodies against S909 and T1079 of Lats1. (G) A proposed model for GPCRs and G-proteins in the regulation of Lats and YAP/TAZ activities. See discussion for details. Also see Figure S7 and Tables S1 and S2.