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. 2017 Apr 18;8(16):27105-27119.
doi: 10.18632/onco_target.15637.

Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors

Jiaming Fan  1   2 , Qiang Wei  2   3 , Junyi Liao  2   3 , Yulong Zou  1   2 , Dongzhe Song  2   4 , Dongmei Xiong  1 , Chao Ma  2   5 , Xue Hu  2   3 , Xiangyang Qu  2   3 , Liqun Chen  2   3 , Li Li  2   6 , Yichun Yu  2   7 , Xinyi Yu  2   3 , Zhicai Zhang  2   8 , Chen Zhao  2   3 , Zongyue Zeng  2   3 , Ruyi Zhang  2   3 , Shujuan Yan  2   3 , Tingting Wu  2   5 , Xingye Wu  2   3 , Yi Shu  2   3 , Jiayan Lei  2   3 , Yasha Li  2   3 , Wenwen Zhang  2   9 , Rex C Haydon  2 , Hue H Luu  2 , Ailong Huang  1 , Tong-Chuan He  2 , Hua Tang  1
Affiliations

Noncanonical Wnt signaling plays an important role in modulating canonical Wnt-regulated stemness, proliferation and terminal differentiation of hepatic progenitors

Jiaming Fan et al. Onco_target. .

Abstract

The liver provides vital metabolic, exocrine and endocrine functions in the body as such pathological conditions of the liver lead to high morbidity and mortality. The liver is highly regenerative and contains facultative stem cells that become activated during injury to replicate to fully recover mass and function. Canonical Wnt/β-catenin signaling plays an important role in regulating the proliferation and differentiation of liver progenitor cells during liver regeneration. However, possible roles of noncanonical Wnts in liver development and regeneration remain undefined. We previously established a reversibly-immortalized hepatic progenitor cell line (iHPx), which retains hepatic differentiation potential. Here, we analyze the expression pattern of the essential components of both canonical and noncanonical Wnt signaling pathways at different postnatal stages of mouse liver tissues and iHPx cells. We find that noncanonical Wnt4, Wnt5a, Wnt9b, Wnt10a and Wnt10b, are highly expressed concordantly with the high levels of canonical Wnts in late stages of liver tissues. Wnt5a, Wnt9b, Wnt10a and Wnt10b are able to antagonize Wnt3a-induced β-catenin/TCF activity, reduce the stemness of iHPx cells, and promote hepatic differentiation of liver progenitors. Stem cell implantation assay demonstrates that Wnt5a, Wnt9b, Wnt10a and Wnt10b can inhibit cell proliferation and promote hepatic differentiation of the iHPx progenitor cells. Our results strongly suggest that noncanonical Wnts may play an important role in fine-tuning Wnt/β-catenin functions during liver development and liver regeneration. Thus, understanding regulatory mechanisms governing proliferation and differentiation of liver progenitor cells may hold great promise to facilitate liver regeneration and/or progenitor cell-based therapies for liver diseases.

Keywords: Wnt/β-catenin; liver cancer; liver regeneration; liver stem cells; noncanonical Wnt signaling.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression patterns of the essential components of Wnt signaling pathway during mouse liver development
Total RNA was isolated from mouse liver tissues at 0 (0D), 14 (14D), 28 (28D) and 180 (180D) days after birth. TqPCR analysis was carried out to detect the expression of 19 Wnts (A), Wnt co-receptor LRP5 and LRP6 (B), Wnt receptor Fzds (C), and Wnt ligand/receptor-associated factors (D) All samples were normalized with Gapdh levels. Each assay condition was done in triplicate.
Figure 2
Figure 2. Expression patterns of the negative regulators of Wnt signaling pathway during mouse liver development
Total RNA was isolated from mouse liver tissues as described in Figure 1. TqPCR analysis was performed to detect the expression of Fzd antagonists Sfrps (A), Wnt co-receptor antagonists Dkks (B), and several well-characterized negative regulators of Wnt signaling (C) All samples were normalized with Gapdh. Each assay condition was done in triplicate.
Figure 3
Figure 3. Expression patterns of the essential Wnt signaling components and regulators in the iHPx cells with or without the removal of the immortalizing gene SV40 T Antigen
The reversibly immortalized iHPx cells were infected with Ad-GFP (iHPx GFP) or Ad-Cre (iHPx Cre) for 48h. Total RNA was isolated from the infected cells and subjected to reverse transcription. TqPCR analysis was carried out to detect the expression of the Wnt ligands (A), co-receptors Lrp5 and 6 (B), Wnt receptor Fzds (C), Fzd antagonists Sfrps (D), LRP co-receptor antagonists Dkks (E), and other Wnt signaling regulators (F) All samples were normalized with Gapdh. Each assay condition was done in triplicate. “**” p < 0.001, “*” p < 0.05, Ad-GFP group vs. Ad-Cre group.
Figure 4
Figure 4. Non-canonical Wnts effectively inhibit the signaling activities of canonical Wnts in iHPx cells
A & B. Subconfluent iHPx cells were infected with Ad-TOP-Luc for 16h, replated into 24-well cell culture plates, and infected with canonical Ad-Wnts or Ad-GFP (A), or co-infected with Ad-Wnt3a and non-canonical Wnts or Ad-GFP (B). At 36h and 60h after infection, cells were lysed and collected for luciferase activity assay using the Firefly Luciferase Assay Kit (Mirus Bio LLC, Madison, WI, USA). C. Subconfluent iHPx cells were infected with Ad-Wnt5a, Wnt9b, Wnt10a, Wnt10b, or Ad-GFP, respectively. At 36h (a) or 60h (b) after infection, total RNA was isolated and subjected to TqPCR analysis of the expression of canonical Wnt-regulated genes, Axin2, c-Myc, and cyclin D1. All samples were normalized with Gapdh. Each assay condition was done in triplicate. Relative expression was calculated by dividing the relative expression values (i.e., gene/Gapdh) in Wnt-treated group with that from the GFP-treated group. “**” p < 0.001, “*” p < 0.05, Ad-GFP group vs. Ad-Wnt group.
Figure 5
Figure 5. The effect of non-canonical Wnts on the expression of liver stem cell markers and hepatic differentiation-associated genes in the iHPx cells
Subconfluent iHPx cells were infected with Ad-Wnt5a, Wnt9b, Wnt10a, Wnt10b, or Wnt-GFP for 36h (a) and 60h (b), respectively. Total RNA was isolated and subjected to TqPCR analysis of the expression of the liver stemness-related markers (A), hepatic regulators and associated genes (B), and mature hepatocyte markers (C) All samples were normalized with Gapdh. Each assay condition was done in triplicate. Relative expression was calculated by dividing the relative expression values (i.e., gene/Gapdh) in non-canonical Wnt-treated group with that from the GFP-treated group. “**” p < 0.001, “*” p < 0.05, Ad-GFP group vs. Ad-Wnt group.
Figure 6
Figure 6. The effect of Cre-mediated removal of SV40 T antigen on the basal expression of liver stemness markers and hepatic differentiation-associated genes in the iHPx cells
Subconfluent iHPx cells were infected with Ad-Cre or Ad-GFP for 48h. Total RNA was isolated and subjected to TqPCR analysis of the expression of the liver stemness-related markers (A), hepatic regulators and associated genes (B), and mature hepatocyte markers (C) All samples were normalized with Gapdh. Each assay condition was done in triplicate. Relative expression was calculated by dividing the relative expression values (i.e., gene/Gapdh) in non-canonical Wnt-treated group with that from the GFP-treated group. “**” p < 0.001, “*” p < 0.05, Ad-GFP group vs. Ad-Cre group.
Figure 7
Figure 7. Non-canonical Wnts promote ICG uptake and glycogen synthesis/storage in iHPx cells
A. NoncanonicalWnt-induced Indocyanine Green (ICG) uptake. Subconfluent iHPx cells were co-infected with Ad-GFP (a) or Ad-Cre (b) and the indicated Ad-Wnts for 10 days. Cells were washed with PBS and incubated with ICG (1mg/mL in complete DMEM) at 37°C for 1h, followed by PBS washes. ICG uptake was documented under a microscope. Each assay conditions were done in triplicate. Representative images are shown. Representative positive stains are indicated by arrows. B. NoncanonicalWnt-induced PAS staining for glycogen storage. Subconfluent iHPx cells were co-infected with Ad-GFP (a) or Ad-Cre (b) and the indicated Ad-Wnts for 10 days. Cells were fixed with 4% paraformaldehyde and stained with 0.5% periodic acid solution and the Schiff's reagent. Each assay condition was done in triplicate. Representative results are shown. Representative positive stains are indicated by arrows.
Figure 8
Figure 8. Nonecanonical Wnts induce cells differentiation, inhibit cell proliferation and downregulate β-catenin expression of the iHPx cells in the in vivo stem cell implantation assay
Subconfluent iHPx cells were infected with Ad-Wnt3a, Ad-Wnt5a, Ad-Wnt9b, Ad-Wnt10a, Ad-Wnt10b, or Ad-GFP for 36h. The cells were collected and injected into athymic nude mice subcutaneously for 10 days. Masses at the injection sites were retrieved, fixed, paraffin-embedded, and subjected to H & E staining (A). Sections were further subjected to immunohistochemical staining using either PCNA (B) or β-catenin (C) antibody (both from Santa Cruz Biotechnology). Control IgG and minus primary antibodies were used as negative controls (not shown). Representative images are shown.
Figure 9
Figure 9. Nonecanonical Wnts modulates canonical Wnt-regulated cell proliferation and differentiation of liver progenitor cells
Canonical Wnts promote the proliferation, stemness and expansion of hepatic progenitors, which is tightly controlled by negative regulators, including noncanonical Wnts and naturally occurring antagonists (e.g., sFRPs) and inhibitors (e.g., DKKs and WIFs). Hepatic differentiation may be further regulated by other signaling pathways, such as BMPs, FGFs and retinoic acid receptors.
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