Impaired Hippo signaling promotes Rho1-JNK-dependent growth

Abstract

The Hippo and c-Jun N-terminal kinase (JNK) pathway both regulate growth and contribute to tumorigenesis when dysregulated. Whereas the Hippo pathway acts via the transcription coactivator Yki/YAP to regulate target gene expression, JNK signaling, triggered by various modulators including Rho GTPases, activates the transcription factors Jun and Fos. Here, we show that impaired Hippo signaling induces JNK activation through Rho1. Blocking Rho1-JNK signaling suppresses Yki-induced overgrowth in the wing disk, whereas ectopic Rho1 expression promotes tissue growth when apoptosis is prohibited. Furthermore, Yki directly regulates Rho1 transcription via the transcription factor Sd. Thus, our results have identified a novel molecular link between the Hippo and JNK pathways and implicated the essential role of the JNK pathway in Hippo signaling-related tumorigenesis.

Keywords: Drosophila; Hippo; JNK; Rho1; growth.

Fig. 1.

Yki activates JNK signaling in Drosophila. Third instar wing discs are shown. Ectopic Yki activates JNK target puc expression. puc-LacZ expression is shown in sd > GFP control (A), sd>Hep^WT (B), and sd > Yki (C) wing discs. Compared with control clones (D and H), impaired Hippo signaling activates MMP1 expression and JNK phosphorylation in wts mutant clones (E and I), or clones expressing Yki (F and J) or Yki^S168A (G and K). Clones were labeled by the presence (D, F, G, H, J, and K) or absence (E and I) of GFP expression. See also Figs. S1 and S2.

Fig. 2.

JNK is required for Yki-induced overgrowth and Wg expression. (A–J) Yki induces JNK-dependent overgrowth in wing discs. Third instar wing discs of ptc > GFP (A), ptc> Bsk^DN (B), ptc > Yki^S168A + LacZ (C), or ptc > Yki^S168A + Bsk^DN (D) were stained for PH3 and DNA (DAPI). (E) Average PH3⁺ cells within ptc expression domain in A–D are shown. **P < 0.01, *P < 0.05 (mean + SD, n ≥ 5). (F–I) Third instar wing discs bearing flip-out clones expressing GFP without (F) or with Bsk^DN (G), Yki^S168A + LacZ (H), or Yki^S168A + Bsk^DN (I). (J) Statistical analyses of clone sizes in F–I. ***P < 0.001 (mean ± SD, n = 15). (K–N) Blocking JNK activity suppressed Yki-induced expansion of the intervein region between L3 and L4 in adult wings. Light micrographs of adult wings from ptc > GFP (K), ptc> Bsk^DN (L), ptc > Yki^S168A, tub-Gal80^ts + LacZ (M), or ptc > Yki^S168A, tub-Gal80^ts + Bsk^DN (N) flies. (O) Average ratio of a/b in K–N. a, distance between L3 and L4; b, length of posterior cross-vein between L4 and L5. Positions of a and b are shown in K. ***P < 0.001 (mean + SD, n = 10). (P–S) JNK is required for Yki-induced ectopic Wg expression. Third instar wing discs of ptc > GFP (P), ptc > Yki^S168A + LacZ (Q), ptc > Yki^S168A + Bsk^DN (R), or ptc> Bsk^DN (S) were stained with DNA (DAPI) and Wg. See also Figs. S3–S5.

Fig. 3.

Rho1 is required for Yki-induced overgrowth and JNK activation. (A–E) Third instar wing discs of control (A) or expressing Yki^S168A + LacZ (B), Yki^S168A + hep RNAi (C), Yki^S168A + dTAK1 RNAi (D), and Yki^S168A + Rho1 RNAi (E) are shown. (F–K) Third instar wing discs stained with X-Gal (F and G), MMP1 (H and J), or Wg (I and K). Yki-induced expansion of GFP⁺ region (H and I), JNK target gene expression (F and H), and Wg expression (I) were all suppressed by knocking down Rho1 (G, J, and K). (L and M) Third instar wing disk stained with cleaved caspase 3. Strong apoptosis was induced by ptc > Rho1 (M), but not ptc > Yki^S168A (L). (N–P) Blocking Rho1-triggered apoptosis by P35-stimulated Wg production (O) and cell proliferation (P). See also Figs. S6 and S7.

Fig. 4.

Yki induces Sd-dependent JNK activation. Knocking down sd suppressed Yki-induced JNK activation. Third instar wing discs were stained for MMP1. Yki^S168A-induced MMP1 up-regulation (A) is suppressed by expression of sd RNAi (B), but not that of mad RNAi (C and D). Genotype: ptc > Yki^S168A + LacZ (A), ptc > Yki^S168A + sd RNAi (B), ptc > Yki^S168A + mad RNAi (C), and ptc> mad RNAi (D). See also Figs. S8 and S9.

Fig. 5.

Yki–Sd directly induces Rho1 transcription. (A–F) Knocking down sd suppresses Yki-induced Rho1 transcription. In situ hybridization to Rho1 mRNA of third instar wing discs (A–C) or salivary gland (D–F) expressing GFP (A and D), Yki^S168A + LacZ (B and E), Yki^S168A + sd RNAi (C and F) driven by ptc–Gal4. (G and H) Ectopic Yki up-regulates Rho1 mRNA (G) and protein (H) levels, as shown by quantitative PCR and Western blot, respectively. (n = 200, mean + SD, ***P < 0.001) (I) Scheme of the Rho1 locus showing the first intron. The Sd binding motif (CATTCCA) is indicated. E1, E2, and E3 were used to drive luciferase expression; A and B represent the control and target region of ChIP assays, respectively. (J) Luciferase assay in Drosophila S2 cells. (n = 3, mean + SD, **P < 0.01) (K–N) The Sd binding motif is required for Yki-induced target gene expression in vivo. Compared with the controls (K and M), ectopic expression of Yki^S168A along the A-P compartment boundary results in dramatic up-regulation of E2–LacZ (L), but not E3–LacZ (N). (O and P) Drosophila S2 cells transfected with Sd and Yki–HA were used for quantification of ChIP-PCRs. (n = 3, mean + SD).