Downregulation of pathways implicated in liver inflammation and tumorigenesis of glycogen storage disease type Ia mice receiving gene therapy

Abstract

Glycogen storage disease type Ia (GSD-Ia) is characterized by impaired glucose homeostasis and long-term risks of hepatocellular adenoma (HCA) and carcinoma (HCC). We have shown that the non-tumor-bearing (NT), recombinant adeno-associated virus (rAAV) vector-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic glucose-6-phosphatase-α activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction (CR). We now show that in AAV-NT mice, the signaling pathways of the CR mediators, AMP-activated protein kinase (AMPK) and sirtuin-1 are activated. AMPK/sirtuin-1 inhibit the activity of STAT3 (signal transducer and activator of transcription 3) and NFκB (nuclear factor κB), the pro-inflammatory and cancer-promoting transcription factors. Sirtuin-1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFκB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFκB targets and β-catenin targets, all of which were consistent with the promotion of tumorigenesis. AAV-NT mice also expressed increased levels of E-cadherin and fibroblast growth factor 21 (FGF21), targets of sirtuin-1, and β-klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a sirtuin-1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/sirtuin-1 and FGF21/β-klotho signaling pathways combined with down-regulation of STAT3/NFκB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice.

Figure 1

 Analysis of hepatic AMPK, SIRT1 and STAT3 signaling in 66–88 week-old wild-type and rAAV-treated G6pc-/- mice. For quantitative RT-PCR, the data were analyzed for wild-type (+/+, n = 13) and AAV-NT (n = 22) mice. (A) Western-blot analysis of AMPK, p-AMPK-T172, SIRT1 and β-actin with quantification of protein levels by densitometry in wild-type (n = 8), AAV/3–63% (n = 4) and AAV/0.9–2.4%-NT (n = 4) mice. (B) Hepatic NAD⁺ levels in wild-type (n = 13), AAV/3–63% (N = 8), and AAV/0.9–2.4%-NT (n = 8) mice. (C) Blood IL-6 levels in wild-type (n = 13) and AAV-NT (n = 16) mice. (D) Quantification of Stat3 mRNA by real-time RT-PCR; Western blot analysis of p-STAT3-Y705, STAT3 and β-actin with quantification of protein levels by densitometry from 4 pairs of wild-type/AAV-NT mice. The AAV-NT analyzed included AAV/3–63% (n = 2) and AAV/0.9–2.4%-NT (n = 2) mice. (E) Immunofluorescence analysis of hepatic STAT3 nuclear localization and quantification of nuclear STAT3-translocated cells. Representative plates shown are analyzed for wild-type mice (n = 6) and AAV-NT (n = 5) mice. Scale bar: 25 μm. Arrows denote STAT3-positive nuclei. Data represent the mean ± SEM; **P < 0.005.

Figure 2

 Analysis of hepatic NFκB signaling and inflammatory markers in 66–88 week-old wild-type and AAV-NT mice. For quantitative RT-PCR, the data were analyzed for wild-type (+/+, n = 13) and AAV-NT (n = 22) mice. For densitometry analysis, the data were analyzed from 4 pairs of wild-type/AAV-NT mice. The AAV-NT analyzed included AAV/3–63% (n = 2) and AAV/0.9–2.4%-NT (n = 2) mice. For blood MCP-1 and CRP levels, the data were analyzed for wild-type (+/+, n = 12) and AAV-NT (n = 22) mice. (A) Quantification Nfκb-p65 mRNA by real-time RT-PCR; Western blot analysis of NFκB-p65, Ac-NFκB-p65-K310 and β-actin with quantification of protein levels by densitometry. (B) Quantification of mRNA for STAT3/NFκB targets by real-time RT-PCR; Western-blot analysis of NOS2, survivin and β-actin with quantification of protein levels by densitometry. (C) Blood MCP-1 levels. (D) Immunofluorescence analysis of hepatic cells stained positive for F4/80 that detects macrophages and quantification of F4/80-positive cells. Representative plates shown are analyzed for wild-type (n = 6) and AAV-NT (n = 5) mice. Scale bar: 25 μm. (E) Quantification of Crp and Saa2 mRNA by real-time RT-PCR and blood CRP levels. Data represent the mean ± SEM; *P < 0.05; **P < 0.005.

Figure 3

Analysis of hepatic E-cadherin, mesenchymal markers, FGF21, β-klotho and β-catenin targets in 66–88 week-old wild-type and AAV-NT mice. For quantitative RT-PCR, the data were analyzed for wild-type (+/+, n = 13) and AAV-NT (n = 22) mice. For densitometry analysis, the data were analyzed from 4 pairs of wild-type/AAV-NT mice. The AAV-NT analyzed included AAV/3-63% (n = 2) and AAV/0.9–2.4%-NT (n = 2) mice. (A) Quantification of E-cadherin, N-cadherin, vimentin, Slug and Snail mRNA by real-time RT-PCR. (B) Western blot analysis of E-cadherin, N-cadherin, vimentin, Slug and β-actin with quantification of protein levels by densitometry. (C) Quantification β-Klotho, Fgf21 and Cyp7a1 mRNA by real-time RT-PCR; Western blot analysis of FGF21, β-klotho, CYP7A1 and β-actin; and quantification of protein levels by densitometry. (D) Quantification of β-Catenin, Glul, Lgr5, Cyclin D1 and Lef1 mRNA by real-time RT-PCR; Western-blot analysis of β-catenin, GLUL and β-actin; and quantification protein levels by densitometry. Data represent the mean ± SEM; *P < 0.05; **P < 0.005.

Figure 4

 Effects of EX-527 on AMPK/STAT3/NFκB signaling in wild-type and AAV-NT mice. Wild-type and AAV-NT mice at age 64–74 weeks were treated daily for four weeks with EX-527 and their phenotype analyzed at age 68–78 weeks. For quantitative RT-PCR, the data were analyzed for wild-type (+/+, n = 8), wild-type-EX-527 (+/+/EX-527, n = 8), AAV-NT (n = 8) and AAV-NT-EX-527 (n = 8) mice. For densitometry analysis, the data were analyzed from 4 pairs of wild-type/AAV-NT and wild-type-EX-527/AAV-NT-EX-527 mice. (A) Body weight, body fat and fasting blood glucose levels of mice before and after EX-527 treatment. (B) Western blot analysis of p-AMPK-T172, AMPK and β-actin with quantification of protein levels by densitometry. (C) Western-blot analysis of p-STAT3-Y705, STAT3, NFκB-p65, Ac-NFκB-p65-K310 and β-actin and quantification protein levels by densitometry. Data represent the mean ± SEM; *P < 0.05; **P < 0.005.

Figure 5

 Effects of EX-527 on hepatic STAT3/NFκB targets in wild-type and AAV-NT mice. Wild-type and AAV-NT mice at age 64–74 weeks were treated with EX-527 for 4 weeks, and their phenotype analyzed at age 68–78 weeks. For densitometry analysis, the data were analyzed from 4 pairs of wild-type/AAV-NT and wild-type-EX-527/AAV-NT-EX-527 mice. (A) Western blot analysis of NOS2, survivin, and β-actin with quantification of protein levels by densitometry. (B) Immunofluorescence analysis of hepatic cells stained positive for F4/80 that detects macrophages and quantification of F4/80-positive cells. Representative plates shown are analyzed for 4 pairs of wild-type/AAV-NT and wild-type-EX-527/AAV-NT-EX-527 mice. Scale bar: 25 μm. Data represent the mean ± SEM; *P < 0.05; **P < 0.005.

Figure 6

 Effects of EX-527 on hepatic E-cadherin, mesenchymal markers, FGF21 and β-klotho in wild-type and AAV-NT mice. Wild-type and AAV-NT mice at age 64–74 weeks were treated with EX-527 for 4 weeks, and their phenotype analyzed at age 68–78 weeks. For densitometry analysis, the data were analyzed from 4 pairs of wild-type/AAV-NT and wild-type-EX-527/AAV-NT-EX-527 mice. (A) Western blot analysis of E-cadherin, N-cadherin, vimentin, slug and β-actin with quantification of protein levels by densitometry. (B) Western-blot analysis of FGF21, β-klotho, β-catenin, GLUL and β-actin and quantification protein levels by densitometry. Data represent the mean ± SEM; *P < 0.05; **P < 0.005.

Figure 7

 Hepatic signaling pathways underlying the absence of HCA/HCC in G6pc-/- mice receiving gene therapy and living under calorie restriction. Several signaling pathways implicated in inflammation and tumorigenesis are downregulated in the liver of AAV-NT mice, contributing to the absence of hepatic tumors. The increases in total AMPK and p-AMPK-T172 activate AMPK that suppresses phosphorylation and activation of STAT3 signaling. The increase in hepatic NAD⁺ levels activates SIRT1 that suppresses NFκB signaling via deacetylation of the p65 subunit of NFκB. The down-regulation of hepatic STAT3/NFκB signaling leads to reduced expression of inflammatory and tumorigenic STAT3/NFκB targets. SIRT1 activation also negatively regulates tumor metastasis by increasing the expression of E-cadherin and reducing the expression of mesenchymal markers and the EMT transcription factors. The increase in E-cadherin also inhibits β-catenin signaling that plays a key role in the pathogenesis of HCC. CR also activates FGF21/β-klotho signaling that decreases the expression of β-catenin targets and prevents hepatocarcinogenesis.