Lithium Suppresses Hedgehog Signaling via Promoting ITCH E3 Ligase Activity and Gli1-SUFU Interaction in PDA Cells

Abstract

Dysregulation of Hedgehog (Hh) signaling pathway is one of the hallmarks of pancreatic ductal adenocarcinoma (PDA). Lithium, a clinical mood stabilizer for the treatment of mental disorders, is known to suppress tumorigenic potential of PDA cells by targeting the Hh/Gli signaling pathway. In this study, we investigated the molecular mechanism of lithium induced down-regulation of Hh/Gli1. Our data show that lithium promotes the poly-ubiquitination and proteasome-mediated degradation of Gli1 through activating E3 ligase ITCH. Additionally, lithium enhances interaction between Gli1 and SUFU via suppressing GSK3β, which phosphorylates SUFU and destabilizes the SUFU-Gli1 inhibitory complex. Our studies illustrate a novel mechanism by which lithium suppresses Hh signaling via simultaneously promoting ITCH-dependent Gli1 ubiquitination/degradation and SUFU-mediated Gli1 inhibition.

Keywords: GSK3β; Gli1; ITCH; hedgehog signaling; lithium; pancreatic cancer; ubiquitination.

FIGURE 1

Lithium induces the up-regulation of ITCH level and the down-regulation of Gli1. (A) The relative mRNA level of ITCH in PANC-1 cells was up-regulated in a dose-dependent manner by lithium treatment for 24 h. (B) The relative mRNA level of ITCH in PANC-1 cells was up-regulated in a time-dependent manner when treated by 10 mM LiCl. (C) The Gli1 protein level was down-regulated accompanied by the up-regulation of ITCH in a dose-dependent manner when using lithium to treat PANC-1 cells for 24 h. Histogram represented summarized results from three independent experiments. (D) The Gli1 protein level was down-regulated accompanied by the up-regulation of ITCH in a time-dependent manner by the treatment of 10 mM lithium. Histogram represented summarized results from three independent experiments. ^∗∗P < 0.01 and ^∗∗∗P < 0.005.

FIGURE 2

ITCH down-regulates the Gli1 level in PANC-1 cells. (A) The relative ITCH mRNA level in PANC-1 cells transfected with pCDNA3.1 or pCDNA3.1-ITCH. (B) The protein level of Gli1 and ITCH in PANC-1 cells after over-expressed with pCDNA3.1-ITCH. ^∗∗∗P < 0.005.

FIGURE 3

ITCH promotes ubiquitination and degradation of Gli1. (A) In vitro ubiquitination of Gli1 protein in the absent or present of Li treatment. PANC-1 cells were transfected with expression vectors encoding HA-ubiquitin and Myc-Gli1. More Gli1-HA-ubiquitin conjugates were detected when PANC-1 cells was treated by Li. (B) HA-ubiquitin and myc-Gli1 were co-expressed in PANC-1 cells, and more exogenous Myc-Gli1-HA-ubiquitin conjugates were detected with lithium treatment.

FIGURE 4

The identification of ubiquitylation sites in the C-terminus of Gli1. (A) The protein level of ΔGli1-His (755–1106 AA) was down-regulated when treated PANC-1 cells by 10, 20, or 40 mM lithium for 24 h. Histogram represented summarized results from three independent experiments. (B) The protein level of ΔGli1-His (755–1106 AA) was down-regulated when using 10 mM lithium to treated PANC-1 cells for 12, 24, or 48 h. Histogram represented summarized results from three independent experiments. (C) Three Gli1 mutants showed different levels of polyubiquitination, and K929A mutant was proved to be the key ubiquitylation site of Gli1 protein. ^∗∗∗P < 0.005.

FIGURE 5

The identification of ubiquitylation sites in the N-terminus of Gli1. (A) The protein level of myc-ΔGli1 (1–300 AA) was not obviously changed when PANC-1 cells treated by 10, 20, or 40 mM lithium for 24 h. Histogram represented summarized results from three independent experiments. (B) The protein level of myc-ΔGli1 (1–300 AA) was not obviously changed when using 10 mM lithium treating PANC-1 cells for 12, 24, or 48 h. Histogram represented summarized results from three independent experiments.

FIGURE 6

Lithium increases GSK3β Ser9 phosphorylation. Immunoblotting of Ser9 phosphorylated GSK3β showed an increase after 10 mM lithium treatment compared with total GSK3β and Tyr216 phosphorylated GSK3β, which remained constant. Histogram represented summarized results of p-GSK3β-Ser9, p-GSK3β-Tyr216, and GSK3β from three independent experiments. ^∗∗∗P < 0.005.

FIGURE 7

Lithium enhances Gli1-SUFU complex formation. Immunoblotting images of co-immunoprecipitation experiments, showing increased binding of Gli1 and SUFU after 10 mM lithium or 10 μM CHIR99021 treatment for 24 h in the presence of 2 μM E1 inhibitor PYR-41. (A) Using anti-Gli1 antibody to immunoprecipitation Gli1/SUFU complex. (B) Using anti-SUFU antibody to immunoprecipitation Gli1/SUFU complex showed increased Gli1 level.

FIGURE 8

Lithium reduces Gli11 level in nucleus. (A) Immunofluorescence staining showing a decreased Gli1 level in nucleus of PANC-1 cells after 10 mM lithium or 10 μM CHIR99021 treatment for 24 h in the presence of 2 μM E1 inhibitor PYR-41. (B) Integrated fluorescence density of Gli1 in nucleus analyzed by Image J software. ^∗∗∗P < 0.005.