Dual modulation of human hepatic zonation via canonical and non-canonical Wnt pathways

Abstract

The hepatic lobule is divided into three zones along the portal-central vein axis. Hepatocytes within each zone exhibit a distinctive gene expression profile that coordinates their metabolic compartmentalization. The zone-dependent heterogeneity of hepatocytes has been hypothesized to result from the differential degree of exposure to oxygen, nutrition and gut-derived toxins. In addition, the gradient of Wnt signaling that increases towards the central vein seen in rodent models is believed to play a critical role in shaping zonation. Furthermore, hepatic zonation is coupled to the site of the homeostatic renewal of hepatocytes. Despite its critical role, the regulatory mechanisms that determine the distinctive features of zonation and its relevance to humans are not well understood. The present study first conducted a comprehensive zone-dependent transcriptome analysis of normal human liver using laser capture microdissection. Upstream pathway analysis revealed the signatures of host responses to gut-derived toxins in the periportal zone, while both the canonical Wnt pathway and the xenobiotic response pathway govern the perivenular zone. Furthermore, we found that the hypoxic environment of the perivenular zone promotes Wnt11 expression in hepatocytes, which then regulates unique gene expression via activation of the non-canonical Wnt pathway. In summary, our study reports the comprehensive zonation-dependent transcriptome of the normal human liver. Our analysis revealed that the LPS response pathway shapes the characteristics of periportal hepatocytes. By contrast, the perivenular zone is regulated by a combination of three distinct pathways: the xenobiotic response pathway, canonical Wnt signaling, and hypoxia-induced noncanonical Wnt signaling.

Figure 1

Laser capture microdissection (LCM) approach for the collection of zonation-based normal human liver tissue. (a) FFPE liver tissue was analyzed with HE staining. Representative histology of a normal human hepatic lobule of the enrolled subject is shown (left), with a solid line that connects PV and CV for the demonstration of zones 1, 2, and 3. The portal triad is within the dotted line and contains PV, HA, BD (right, top). CV is located in the center of the hepatic lobule and can be distinguished from PV by the lack of adjacent HA and BD (left, bottom). (b) Representative image of LCM of the fresh frozen normal human liver tissue stained with Hematoxylin. The selected area of liver tissue, zones-1, 2 and 3 (top), were subjected to LCM-based tissue collection (bottom). (c) Total RNA extracted from each zone via LCM was quality-controlled with a bioanalyzer. The displayed results are from one representation of one subjected enrolled in this study. (d) The quality and quantity control of the cDNA library synthesized with a PCR approach using polyA tailed RNA extracted from each zone is shown. The displayed result is from one representation of zone-1 cDNA library obtained from one subject enrolled in this study. *: indicates signal from internal control. PV: portal vein, HA: hepatic artery, BD: bile duct, CV: central vein.

Figure 2

Zone-dependent distinctive gene expression profile of normal human liver tissue. (a) Volcano plot illustrates the analysis of variance (ANOVA)analysis result, which revealed 139 transcripts (shown as red dots) that are differentially expressed between zones 1 and 3. Each dot represents a single transcript. Discrimination (false discovery rate (FDR)) and the expression change in fold index are indicated on the y axis and the x axis, respectively. (b) Hierarchical clustering of the 139 genes that are differentially expressed between zone-1 and zone-3 identified by ANOVA with cutoff values of at least a 2.5-fold change, FDR<0.01.

Figure 3

The contribution of active β-catenin to regulation zonation of the human liver. (a) Ingenuity pathways analysis (IPA) of genes that are differentially expressed in zone-1 and zone-3. The genes shown in red indicate upregulation in zone-3, while genes in green indicate upregulation in zone-1. Genes in white circles are the predicted upstream regulators. (b) IHC analysis of FFPE liver tissue obtained from the enrolled subjects for the expression of pan-β-Catenin (upper panel) or the active form (non-phosphorylation at Serine 45) of β catenin (lower panel). The scale bar for lower magnification (× 10, left) and higher magnification (× 40, middle and right) span 200 and 50 μm, respectively. PV, portal vein, CV, central vein.

Figure 4

Upregulation of Wnt11 in the perivenular zone. The relative abundance of the indicated Wnts was assessed, with the mean of the biological triplicates in individual zones identified via RNA sequencing. The expression of each Wnt in zone-1 was used to normalize. *P<0.01.

Figure 5

Wnt11 regulation of zone-3 signature genes through a non-canonical pathway. (a) RNA extracted from mouse primary hepatocytes treated with phosphate-buffered saline (PBS), Wnt11 (50 ng ml⁻¹) or GSK3β inhibitor (5 μM) was subjected to quantitative RT-PCR array of zone-3 genes. The heat map represents the relative abundance of the indicated genes. The scale bar indicates the expression change in fold index. (b) Huh7 cells were cotransfected with TOPFLASH, a firefly luciferase reporter regulated by β-Catenin, and a renilla luciferase vector. Sixteen hours after transfection, cells were treated with the indicated ligands for 20 h followed by a dual luciferase assay. The relative intensity of canonical Wnt signaling activity is shown as TOPFLASH relative luciferase unit (RLU). *P<0.01. (c, d) Huh7 cells were treated with the indicated Wnts (50 ng ml⁻¹) for 24 h followed by immunoblot analysis of the indicated protein.

Figure 6

Hypoxia augments the expression of Wnt11 and zone-3 signature genes. (a) RNA extracted from mouse primary hepatocytes treated with PBS, GSK3β inhibitor (5 μM), TCDD (10 nM), or CoCl₂ (100 μM) were subjected to quantitative RT-PCR for the assessment of Wnt11 expression. (b) RNA extracted from mouse primary hepatocytes treated with PBS, TCDD (10 nM) or Wnt11 (50 ng ml⁻¹) was subjected to quantitative reverse transcription (RT-PCR) array of zone-3 genes. The heat map represents the relative abundance of the indicated genes. The scale bar indicates the expression change in fold index. (c) RNA extracted from hepatocyte-specific Vhl deficient liver tissue and its control mouse was used for the RT-qPCR analysis of the indicated genes. *P<0.01. (d) Proposed model of the multifaceted regulatory mechanism of hepatic zonation. This model is described in the text in more detail.