The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size

Abstract

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/β-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/β-catenin signalling gradient, but the instructive mechanism controlling its spatiotemporal regulation is not known. We have now identified the RSPO-LGR4/5-ZNRF3/RNF43 module as a master regulator of Wnt/β-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/β-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/β-catenin signalling gradient in a reversible and LGR4/5-dependent manner. Recombinant RSPO1 protein increased liver size and improved liver regeneration, whereas LGR4/5 LOF caused the opposite effects, resulting in hypoplastic livers. Furthermore, we show that LGR4(+) hepatocytes throughout the lobule contribute to liver homeostasis without zonal dominance. Taken together, our results indicate that the RSPO-LGR4/5-ZNRF3/RNF43 module controls metabolic liver zonation and is a hepatic growth/size rheostat during development, homeostasis and regeneration.

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“…Particularly, this pathway is important in liver homeostasis: from embryonic liver to its functional maturation, which reinforces its role in hepatoblastoma tumorigenesis (18)(19)(20). In the adult liver, canonical Wnt signaling is only activated in the centrizonal region of the hepatic lobule as proven by positive nuclear and cytoplasmic β-catenin in this area (20,21). It is known to regulate genes involved in ammonia and xenobiotic metabolism (22).…”

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“…Conversely, in skin and kidney where Lgr5 null embryos show a subtle phenotype as compared to the dominant cognate Lgr4 receptor, double knockout Lgr4/Lgr5 embryos exhibit a worsened phenotype, suggesting that these two receptors both contribute to the stem cell pools in these tissues [28,29]. Similarly, in adult intestine and liver, conditional ablation of Lgr5 has no overt phenotype but conditional ablation of both Lgr4 and Lgr5 receptors further aggravates the phenotype induced by Lgr4 deficiency [7,35]. In sum, in the absence of additional data on the spectrum of the regulatory cascades they control, the question of functional redundancy of Lgr4 and Lgr5 in vivo is still open.…”

“…In digits, Lgr5 expression is detected in the dermis [52]. Of relevance, Lgr5 is detected in cells also expressing the paralogue receptors Lgr4 and/or Lgr6, thus rendering the analysis of individual receptor function quite complex [7,28,30,35,53].…”

“…Moreover, though premature differentiation has been reported in Lgr4-deficient embryos [17,21,30], Lgr4 deficiency has been mainly described to be associated with impaired or retarded cell differentiation [23,24,28,29,[31][32][33]. Such phenotypes have been demonstrated to be associated with decreased Wnt signaling in intestinal, liver and dental epithelia as well as in peritubular myoid cells in testis [7,23,28,31,34,35]. Accordingly, experiments using GSK3β inhibitors (LiCl, CHIR99201) or in an Apc min/+ background efficiently restored Wnt activity in the intestine of Lgr4-knockout homozygous mice ex vivo and in vivo [7,28,34].…”

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“…119 This E3 ubiquitin ligase serves as an important component of the Rspo-LGR4/5-ZnRF3/RNF43 module that acts as a regulator of the Wnt/b-cateninmediated metabolic liver zonation and controls hepatic growth and size during development, homeostasis, and regeneration. 120 Human ZnRF3 (UniProt ID: Q9ULT6) is a singlepass transmembrane protein containing N-terminal signal peptide (residues 1-55), extracellular domain (residues 56-219), transmembrane helix (residues 220-240), and a cytoplasmic domain (residues 241-936), where the zinc finger domain (RING-type, residues 293-334) is embedded. This protein exists in 2 isoforms, the full-length canonical form (936 residues) and alternatively spliced isoform #2 that differs from the canonical form by missing first 100 residues.…”