Elucidation of a universal size-control mechanism in Drosophila and mammals

Abstract

Coordination of cell proliferation and cell death is essential to attain proper organ size during development and for maintaining tissue homeostasis throughout postnatal life. In Drosophila, these two processes are orchestrated by the Hippo kinase cascade, a growth-suppressive pathway that ultimately antagonizes the transcriptional coactivator Yorkie (Yki). Here we demonstrate that a single phosphorylation site in Yki mediates the growth-suppressive output of the Hippo pathway. Hippo-mediated phosphorylation inactivates Yki by excluding it from the nucleus, whereas loss of Hippo signaling leads to nuclear accumulation and therefore increased Yki activity. We further delineate a mammalian Hippo signaling pathway that culminates in the phosphorylation of YAP, the mammalian homolog of Yki. Using a conditional YAP transgenic mouse model, we demonstrate that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results uncover a universal size-control mechanism in metazoan.

Figure 1. Hippo Signaling Phosphorylates Yki S168 and Promotes Its Cytoplasmic Localization

(A) S2 cells expressing HA-Yki (left) or HA-Yki plus Hpo, Sav, and Wts plasmids (right) were stained with α-HA antibody. Nuclear HA-Yki signal is detected in 100% of cells expressing HA-Yki alone, but in only 9% of cells coexpressing Hpo-Sav-Wts. (B) Cells were transfected as in (A) and analyzed by subcellular fractionation. Note the decrease of nuclear Yki under active Hippo signaling (compare lanes 2 and 4). dSREBP (nuclear) and tubulin (cytosolic) were used as quality control for fractionation. C, cytoplasmic; N, nuclear. (C) Cells were transfected as in (A), and α-HA immunoprecipitates were probed with α-14-3-3 (top gel). Cell lysates were also probed with the indicated antibodies (middle and bottom gels). Yki, but not Yki^S168A, immunoprecipitated 14-3-3 under active Hippo signaling. (D) Cells were transfected as in (A), and α-HA immunoprecipitates were probed with α-P-Yki(S168) and α-HA antibodies (top two gels). Cell lysates were also probed with the indicated antibodies (bottom three gels). Yki, but not Yki^S168A, showed S168 phosphorylation, which was further increased under active Hippo signaling. Also note that Yki, but not Yki^S168A, showed mobility shift under active Hippo signaling (compare lanes 2 and 4; indicated by white and black dots, respectively). (E) Wts phosphorylates Yki at S168 in vitro. V5-tagged Wts (or kinase-dead Wts^KD) was expressed alone or together with Hpo-Sav in S2 cells, immunoprecipitated, and incubated with GST-Yki (or GST-Yki^S168A), and the reaction products were probed with α-P-Yki(S168). The input kinase and substrate are also shown (bottom two gels). Note that our GST-Yki preparation contains two Yki-related bands (Huang et al., 2005). Strong S168 phosphorylation was detected when Wts (lane 3), but not Wts^KD (lane 4), was coexpressed with Hpo-Sav. No S168 phosphorylation was detected using GST-Yki^S168A as a substrate (lane 5). (F) Subcellular fractionation of Yki^S168A mutant. The relative proportion of cytoplasmic and nuclear YkiS^168A was not changed under active Hippo signaling. (G) S2 cells expressing HA-Yki or HA-Yki^S168A were treated with or without insulin. α-HA immunoprecipitates were probed with the indicated antibodies (top two gels). Cell lysates were also probed with the P-S6K or P-Akt antibodies (bottom two gels). Arrowheads mark the phospho-Akt signals. Insulin stimulates the phosphorylation of Akt and S6K, but not that of Yki (compare lanes 1 and 2). (H) S2 cells expressing HA-Yki or HA-Yki^S168A plus a constitutively active Akt mutant (myr-Akt) were analyzed as in (G). Similar P-Yki(S168) levels were seen in the presence or absence of myr-Akt (compare lanes 1 and 2).

Figure 2. S168 Phosphorylation Mediates the Growth-Suppressive Output of Hippo Signaling In Vivo

(A and B) A flp-out strategy to assay the activity of Yki (A) and Yki^S168A (B) in vivo. (C) Adult flies carrying Tub > y⁺ > yki (left) or Tub > y⁺ > yki^S168A (right) and an eye-specific FLP recombinase. The flies were photographed together to show their relative size. Note the enlarged and folded eyes (arrow) and excess head cuticle (arrowhead) in flies expressing Tub > yki^S168A (right). (D and E) Eye discs carrying Tub > y⁺ > yki (D) or Tub > y⁺ > yki^S168A (E) and an eye-specific FLP recombinase. The images were taken under the same magnification. (F and G) Adult flies carrying Tub > y⁺ > yki (F) or Tub > y⁺ > yki^S168A (G) and a heat-shock-inducible FLP source, and heat shocked at first instar larval stage. The wings had been removed for photographic purpose. Note the presence of thoracic overgrowth (arrowheads) in (G) only. (H and H′) Eye disc containing Tub > y⁺ > yki flpout clones, stained for Yki (red) and Diap1 (green). The circled area in H indicates a flpout clone, which expressed a higher level of Yki. Diap1 levels were not affected in the clone (arrow in H′). (I and I′) similar to H and H′ except that Tub > y⁺ > yki^S168A clones (outlined by circles in [I]) were analyzed. Note the increased levels of Diap1 in the clones (arrows in [I′]).

Figure 3. Inactivation of Hippo Signaling Leads to Nuclear Accumulation of Yki In Vivo

In all panels, three images are shown, one of GFP (panels A, B, C, and D) (green), one of Yki (panels A′, B′, C′, and D′) (red), and one of superimposed GFP and Yki (panels A″, B″, C″, and D″). The GFP marker used here is concentrated in the nucleus. (A–A″) Third instar wing disc containing yki^B5 clones and stained with α-Yki. −/− clones were marked by the absence of GFP (white arrow) while the +/+ twin spots were marked by 2×GFP signal (darker green, yellow arrow). Note the absence of Yki staining in the −/− clones and increased Yki staining in the twin spots, as well as the relative absence of Yki signal in the GFP-marked cell nuclei. (B–B″) Third instar eye disc. Note the relative absence of Yki signal in the GFP-marked cell nucleus. Arrowhead marks the MF. (C–C″) Third instar eye discs containing wts^X1 clones. Note the relative absence of nuclear Yki in wild-type cells. In contrast, Yki is present throughout the cytoplasm and the nucleus in wts^X1 clones (arrow). (D–D″) Third instar wing discs containing hpo^42–47 clones. Note the relative absence of nuclear Yki in wild-type cells. In contrast, Yki is present throughout the cytoplasm and the nucleus in hpo^42–47 clones (arrows).

Figure 4. Delineation of a Mammalian Hippo Signaling Pathway Leading to YAP S127 Phosphorylation

(A) HEK293 cells were transfected with epitope-tagged constructs as indicated. Note the increased YAP(S127) phosphorylation and mobility retardation resulting from Lats1/2 (lane 2), Mst1/2 (lane 4), or both (lane 6), but not the respective kinase-dead forms or their combinations (lanes 3, 5, and 7). The slower-migrating species of YAP was indicated by a small dot to the right of the protein band. (B) HEK293 cells expressing the indicated plasmids were analyzed. The slower-migrating species of YAP was indicated by a small dot to the right of the protein band. (C) ACHN cells expressing the indicated plasmids were analyzed. Note the failure of Mst1/2 (lane 3), but not Lats1/2 (lane 5), to induce YAP(S127) phosphorylation, and the rescue of this defect by hWW45 (lane 4). Also note that hWW45 alone (lane 2) can induce YAP(S127) phosphorylation. (D) Various cell lines were probed with α-P-YAP(S127) and α-YAP, and analyzed by RT-PCR for the expression of indicated genes. Note the absence of hWW45 expression and the much diminished levels of YAP(S127) phosphorylation in ACHN cells (lane 4). (E) HPNE cells stably expressing YAP (middle), but not those expressing an empty vector (left), grew in soft agar to form colonies. HPNE cells stably expressing YAP^S127A grew to significantly larger colonies (right). Similar results were obtained with multiple independently established cell lines. (F) Adult heads from flies expressing YAP (left) or YAP^S127A (right) in the eye. The genotypes are: GMRGal4/UASYAP (left) and GMRGal4/UASYAP^S127A (right). The heads were photographed together to show their relative size.

Figure 5. Reversible Control of Mammalian Organ Size by Conditional Activation/Inactivation of YAP

(A) Schematic of the ApoE/rtTA-YAP mice. (B) RT-PCR analysis of hYAP transgene expression in control and transgenic livers in the absence or presence of Dox for 1 week. Two mice were used for each genotype/condition. (C and D) Livers from control (left) and ApoE/rtTA-YAP (right) mice kept on Dox for 1 (C) or 4 (D) weeks, starting at 3 weeks of age. (E) The temporal course of YAP-induced hepatomegaly. YAP was induced as in (C) and (D), and the liver and body weight was measured at the indicated time. Values represent mean ± SD (n = 4; ***p < 0.001, t test). (F and G) Hematoxylin and eosin (H&E) staining of control (F) and transgenic (G) livers after 4 weeks of Dox exposure. Both images were taken under the same magnification. Note the higher cell density in the transgenic liver. (H and I) Reversal of liver overgrowth by Dox withdrawal. Three-week-old ApoE/rtTA-YAP mice were first kept on Dox for 2 (H) or 8 (I) weeks, followed by withdrawal of Dox from the drinking water. Values represent mean ± SD (n = 4).

Figure 6. Coordinate Regulation of Cell Proliferation and Apoptosis by the Mammalian Hippo Pathway

(A) Quantitative real-time RT-PCR analysis of selected genes from control (white columns) and ApoE/rtTA-YAP (black columns) livers after 2 weeks of Dox exposure. Gene expression was measured in triplicate and expressed as mean ± SEM. The subtle changes of Cyclin E1 and Cyclin E2 are insignificant (p = 0.1, t test). (B and C) YAP overexpression drives cell proliferation. Control (B) and ApoE/rtTA-YAP (C) livers after 1 week of Dox exposure were analyzed for BrdU incorporation (red), counter-stained with DAPI (blue). (D–H) YAP overexpression confers potent resistance to apoptosis. Control and ApoE/rtTA-YAP littermates were kept on Dox for 1 week, injected with Jo-2, and analyzed 3 hr postinjection by H&E staining (D and E), TUNEL (F and G), and western blotting for cell death markers (H). Note the widespread hemorrhage (asterisk) and apoptotic nuclei (arrowhead) in the control (D), but not the transgenic livers (E). Also note the extensive TUNEL staining in the control (F), but not the transgenic livers (G). In (H), cleavage of caspase-3 (Casp3) and PARP was detected in the control, but not the transgenic, livers (arrowhead marks the cleaved PARP product). Three animals were analyzed for each genotype. (I) RNAi knockdown of BIRC5/survivin reduces the transforming activity of YAP. Left: western blots showing increased BIRC5/survivin expression in YAP-HPNE cells compared to mock transfected cells, and the knockdown of BIRC5/survivin expression in YAP-HPNE cells stably expressing BIRC5/survivin shRNA. Right: soft agar assay showing anchorage-independent growth of YAP-HPNE cells, a feature that is not observed in the mock-transfected HPNE cells. Note that YAP-HPNE cells stably expressing BIRC5/survivin shRNA formed reduced numbers of colonies.

Figure 7. Dysregulation of the Mammalian Hippo Pathway Leads to Tumorigenesis In Vivo

(A) Liver from an ApoE/rtTA-YAP mouse raised on Dox for 8 weeks, starting at 3 weeks after birth. Note the presence of discrete nodules scattered throughout the liver (arrowheads). (B) Liver from an ApoE/rtTA-YAP mouse raised on Dox for 3 months, starting at birth. Note the widespread development of HCC throughout the liver. (C–E) Histolopathologic examination of murine liver nodules reveals characteristics of hepatocellular carcinoma. Mice were fed Dox-water as in (A). (C) shows cellular pleiomorphism of YAP-induced HCC, with a large cell (arrow) surrounded by smaller cells. (D) shows loss of cytoplasmic staining (arrows), or the so called “clear cell change.” (E) shows expanded hepatic plates. A reticulin stain highlights the edges of the hepatic plates (indicated by parallel lines), which are wider in HCC than the typical 1 to 2 cells in a nonneoplastic liver. (F) Survival curves of control (Non-Tg) and ApoE/rtTA-YAP (Tg) littermates raised on Dox, starting at 3 or 8 weeks of age as indicated. (G) Conserved Hippo kinase cascade in Drosophila and mammals. The corresponding proteins in Drosophila and mammals are indicated by matching colors and shapes. The critical phosphorylation site on Yki and YAP is also indicated. “X” denotes unknown DNA-binding protein(s) that partner with Yki or YAP to regulate target gene transcription.