Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

Sulfur dioxide may predominate in the adverse effects of ambient air pollutants on semen quality among the general population in Hefei, China.

Previous studies have reported potential adverse effects of exposure to ambient air pollutants on semen quality in infertile men, but studies on the general population have been limited and inconsistent, and the pollutants that play a major role remain unclear. This study aimed to explore the potential association between exposure to six air pollutants (PM2.5, PM10, NO2, SO2, O3 and CO) during different sperm development periods and semen quality among the general population, and to explore the interaction between different air pollutant exposures. We included 1515 semen samples collected from the Human Sperm Bank. We improved individuals' exposure level estimation by combining inverse distance weighting (IDW) interpolation with satellite remote sensing data. Multivariate linear regression models, restricted cubic spline functions and double-pollutant models were used to assess the relationship between exposure to six air pollutants and sperm volume, concentration, total sperm number and sperm motility. A negative association was found between SO2 exposure and progressive motility and total motility during 0-90 lag days and 70-90 lag days, and SO2 exposure during 10-14 lag days adversely affected sperm concentration and total sperm number. Sensitive analyses for qualified sperm donors and the double-pollutant models obtained similar results. Additionally, there were nonlinear relationships between exposure to PM, NO2, O3, CO and a few semen parameters, with NO2 and O3 exposure above the threshold showing negative correlations with total motility and progressive motility, respectively. Our study suggested that SO2 may play a dominant role in the adverse effects of ambient air pollutants on semen quality in the general population by decreasing sperm motility, sperm concentration and total sperm number. Also, even SO2 exposure lower than the recommended standards of the World Health Organization (WHO) could still cause male reproductive toxicity, which deserves attention.

Overexpression of long non-coding RNA ACTA2-AS1 inhibits the viability, proliferation, migration and invasion of colorectal cancer cells.

The role of long non-coding RNA ACTA2 antisense RNA 1 (LncRNA ACTA2-AS1) in colorectal cancer (CRC) was awaited to be elucidated. Clinical specimen and data on ACTA2-AS1 expression in colon adenocarcinoma (COAD) were collected, followed by in situ hybridization. Transfected CRC cell viability, proliferation, migration, and invasion were determined with Cell Counting Kit-8, colony formation, Scratch, and Transwell assays, respectively. Relative expressions of ACTA2-AS1, PCNA, Bcl-2, MMP-2 and MMP-9 were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ACTA2-AS1 expression was downregulated in CRC. Overexpressed ACTA2-AS1 repressed the cell viability, proliferation, migration and invasion, increased cleaved caspase-3 level yet decreased PCNA, Bcl-2, MMP-2 and MMP-9 levels. ACTA2-AS1 silencing, however, did oppositely. Collectively, ACTA2-AS1 inhibits the viability, proliferation, migration and invasion of CRC cells to effectively suppress the progression of CRC.

Low expression of moonlight gene ALAD is correlated with poor prognosis in hepatocellular carcinoma.

BACKGROUND: Moonlighting genes may involve in the progression of hepatocellular carcinoma (HCC), and the establishment of a prognostic signature based on moonlighting genes may help predict the prognosis of HCC patients. METHODS: This study aimed to construct a prognostic signature based on moonlighting genes in HCC and determine whether there is a correlation with tumor microenvironment or immune responses. Then we used HCC cell lines and an HCC cDNA microarray to illuminate the role of moonlighting gene in prognosis of HCC. RESULTS: We constructed an original prognostic signature based on eight moonlighting genes (ABCB1, S100A9, NCL, PRDX6, ALAD, YBX1, POU2F1, RPL5) with strong prognosis prediction capability. The prognostic signature may demonstrate the immune status of patients with HCC, because high-risk subgroups had significantly higher scores for regulatory T cells, dendritic cells, T follicular helper cells, macrophages, and major histocompatibility complex-I, and different expression levels of immune checkpoint molecules. Importantly, patients in the high-risk subgroup exhibited higher tumor immune dysfunction and exclusion scores, suggesting that they might be less sensitive to immunotherapy. The roles of ABCB1, S100A9, NCL, PRDX6, YBX1, and POU2F1 in HCC have been reported. However, there have been no reports on the association between ALAD and HCC. Then we used bioinformatics to confirm that ALAD expression was lower in HCC and low expression of ALAD was an indicator of poor prognosis. Moreover, we found that ALAD expression was lower in HCC cells than that in normal human hepatocytes or tumor-adjacent tissues, it was negatively correlated with the pathological grade, and low expression of ALAD was related to poor prognosis in patients with HCC. CONCLUSION: We have successfully established a novel prognostic signature based on moonlighting genes, with a strong predictive capability for prognosis, immune status, and possible response to immunotherapy. Additionally, we have identified ALAD as a prognostic biomarker for HCC.

Homozygous mutation in SLO3 leads to severe asthenoteratozoospermia due to acrosome hypoplasia and mitochondrial sheath malformations.

BACKGROUND: Potassium channels are important for the structure and function of the spermatozoa. As a potassium transporter, the mSlo3 is essential for male fertility as Slo3 knockout male mice were infertile with the series of functional defects in sperm cells. However, no pathogenic variant has been detected in human SLO3 to date. Here we reported a human case with homozygous SLO3 mutation. The function of SLO3 in human sperm and the corresponding assisted reproductive strategy are also investigated. METHODS: We performed whole-exome sequencing analysis from a large cohort of 105 patients with asthenoteratozoospermia. The effects of the variant were investigated by quantitative RT-PCR, western blotting, and immunofluorescence assays using the patient spermatozoa. Sperm morphological and ultrastructural studies were conducted using haematoxylin and eosin staining, scanning and transmission electron microscopy. RESULTS: We identified a homozygous missense variant (c.1237A > T: p.Ile413Phe) in the sperm-specific SLO3 in one Chinese patient with male infertility. This SLO3 variant was rare in human control populations and predicted to be deleterious by multiple bioinformatic tools. Sperm from the individual harbouring the homozygous SLO3 variant exhibited severe morphological abnormalities, such as acrosome hypoplasia, disruption of the mitochondrial sheath, coiled tails, and motility defects. The levels of SLO3 mRNA and protein in spermatozoa from the affected individual were reduced. Furthermore, the acrosome reaction, mitochondrial membrane potential, and membrane potential during capacitation were also afflicted. The levels of acrosome marker glycoproteins and PLCζ1 as well as the mitochondrial sheath protein HSP60 and SLO3 auxiliary subunit LRRC52, were significantly reduced in the spermatozoa from the affected individual. The affected man was sterile due to acrosome and mitochondrial dysfunction; however, intra-cytoplasmic sperm injection successfully rescued this infertile condition. CONCLUSIONS: SLO3 deficiency seriously impact acrosome formation, mitochondrial sheath assembly, and the function of K+ channels. Our findings provided clinical implications for the genetic and reproductive counselling of affected families.

Severe interstitial pneumonia caused by cetuximab: a case report and review of the literature.

Cetuximab is an IgG1 chimeric mAb against epidermal growth factor receptor, which can be used for chemotherapy failure or tolerance in patients with epidermal growth factor receptor expressed RAS wild-type metastatic colorectal cancer. We report on a patient who developed rapid-onset interstitial pneumonia while being treated with cetuximab plus XELOX (oxaliplatin, capecitabine) for metastatic colorectal cancer. A 75-year-old man patient was administered cetuximab plus XELOX regularly. After his cetuximab schedule was adjusted from 1 to 2 weeks, he rapidly developed interstitial pneumonia which led to acute respiratory distress syndrome. Our literature review indicated that, for patients with risk factors, a 2-week regimen of cetuximab might lead to interstitial pneumonia. Clinicians should closely monitor patients for adverse drug reactions to improve drug safety.

Curcumin suppresses LGR5(+) colorectal cancer stem cells by inducing autophagy and via repressing TFAP2A-mediated ECM pathway.

Colorectal cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation. And LGR5 is a stem cell marker gene of colorectal cancer. Curcumin can suppress oncogenicity of many cancer cells, yet the effect and mechanism of curcumin in LGR5(+) colorectal cancer stem cells (CSCs) have not been studied. In this study, we studied the effect of curcumin on LGR5(+) colorectal CSCs using the experiments of tumorsphere formation, cell viability and cell apoptosis. Then autophagy analysis, RNA-Seq, and real-time PCR were used to identify the mechanism responsible for the inhibition of LGR5(+) colorectal CSCs. Our results showed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner, and also promoted apoptosis of LGR5(+) colorectal CSCs. Next, we found curcumin induced autophagy of LGR5(+) colorectal CSCs. When LGR5(+) colorectal CSCs were co-treated with curcumin and the autophagy inhibitor (hydroxychloroquine), curcumin-induced cell proliferation inhibition decreased. In addition, we also found that curcumin inhibited the extracellular matrix (ECM)-receptor interaction pathway via the downregulation of the following genes: GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC. Further, these genes were transcriptionally regulated by TFAP2A, and the high expression of TFAP2A was associated with poor prognosis in colorectal cancer. In conclusion, curcumin suppressed LGR5(+) colorectal CSCs, potentially by inducing autophagy and repressing the oncogenic TFAP2A-mediated ECM pathway.

Identification of differentially methylated genes as diagnostic and prognostic biomarkers of breast cancer.

BACKGROUND: Aberrant DNA methylation is significantly associated with breast cancer. METHODS: In this study, we aimed to determine novel methylation biomarkers using a bioinformatics analysis approach that could have clinical value for breast cancer diagnosis and prognosis. Firstly, differentially methylated DNA patterns were detected in breast cancer samples by comparing publicly available datasets (GSE72245 and GSE88883). Methylation levels in 7 selected methylation biomarkers were also estimated using the online tool UALCAN. Next, we evaluated the diagnostic value of these selected biomarkers in two independent cohorts, as well as in two mixed cohorts, through ROC curve analysis. Finally, prognostic value of the selected methylation biomarkers was evaluated breast cancer by the Kaplan-Meier plot analysis. RESULTS: In this study, a total of 23 significant differentially methylated sites, corresponding to 9 different genes, were identified in breast cancer datasets. Among the 9 identified genes, ADCY4, CPXM1, DNM3, GNG4, MAST1, mir129-2, PRDM14, and ZNF177 were hypermethylated. Importantly, individual value of each selected methylation gene was greater than 0.9, whereas predictive value for all genes combined was 0.9998. We also found the AUC for the combined signature of 7 genes (ADCY4, CPXM1, DNM3, GNG4, MAST1, PRDM14, ZNF177) was 0.9998 [95% CI 0.9994-1], and the AUC for the combined signature of 3 genes (MAST1, PRDM14, and ZNF177) was 0.9991 [95% CI 0.9976-1]. Results from additional validation analyses showed that MAST1, PRDM14, and ZNF177 had high sensitivity, specificity, and accuracy for breast cancer diagnosis. Lastly, patient survival analysis revealed that high expression of ADCY4, CPXM1, DNM3, PRDM14, PRKCB, and ZNF177 were significantly associated with better overall survival. CONCLUSIONS: Methylation pattern of MAST1, PRDM14, and ZNF177 may represent new diagnostic biomarkers for breast cancer, while methylation of ADCY4, CPXM1, DNM3, PRDM14, PRKCB, and ZNF177 may hold prognostic potential for breast cancer.

Video Education Reduces Pain and Anxiety Levels in Cancer Patients Who First Use Fentanyl Transdermal Patch: A Randomized Controlled Trial.

OBJECTIVE: We sought to evaluate the efficacy of using a quick response (QR) code within video education to guide proper use of fentanyl transdermal patches and control pain, depression, and anxiety levels in cancer patients. METHODS: Patients using a fentanyl transdermal patch for the first time were enrolled in the study and then given an information leaflet as well as an informed consent form. We asked them to complete the first questionnaire (Q1) prior to first use of the fentanyl transdermal patch, and then used a random number table to randomize those who completed it into two groups. Participants in group A received a QR code (to make it easier for them to obtain additional video information) and a traditional information leaflet, whereas those in group B (control group) only received a traditional information leaflet. Thereafter, we requested all participants to complete standard questionnaires, which comprised a Numeric Rating Scale (NRS), a Spielberger State-Trait Anxiety Inventory (STAI), as well as a Hospital Anxiety and Depression Scale (HADS). The resulting continuous (with a normal distribution) and categorical data were analyzed using Student's t- and chi-square tests, respectively. We also recorded parameters such as NRS, STAI, and HADS, as well as the frequency of rescue medication in both groups. RESULTS: A total of 154 cancer patients who first used a fentanyl transdermal patch were recruited during the study period, from April to May 2020. Among these, 138 completed follow-up, with 70 and 68 in group A and B, respectively. Participants in both groups had similar baseline and clinical characteristics, whereas significant differences were observed between the groups with regard to the other parameters. Specifically, participants in group A recorded a lower STAI state (38.2 vs 38.9, P=0.027) and HADS (3.9 vs 4.2, P=0.001) anxiety scores, as well as NRS (2.1 vs 2.4, P=0.025) and frequency of rescue medication (0.4 vs 1.4, P<0.001) than those in group B, following 14 days of using a fentanyl transdermal patch. CONCLUSION: Our results indicated that incorporating a QR code within additional video education leads to proper use of a fentanyl transdermal patch and relieves pain and anxiety levels in patients with cancer. Based on this, we recommend a new style of education during care of cancer patients who first use a fentanyl transdermal patch.

Bi-allelic Loss-of-function Variants in CFAP58 Cause Flagellar Axoneme and Mitochondrial Sheath Defects and Asthenoteratozoospermia in Humans and Mice.

Multiple morphological abnormalities of the sperm flagella (MMAF) is a severe form of asthenoteratozoospermia. Although recent studies have revealed several MMAF-associated genes and demonstrated MMAF to be a genetically heterogeneous disease, at least one-third of the cases are still not well understood for their etiology. Here, we identified bi-allelic loss-of-function variants in CFAP58 by using whole-exome sequencing in five (5.6%) unrelated individuals from a cohort of 90 MMAF-affected Chinese men. Each of the men harboring bi-allelic CFAP58 variants presented typical MMAF phenotypes. Transmission electron microscopy demonstrated striking flagellar defects with axonemal and mitochondrial sheath malformations. CFAP58 is predominantly expressed in the testis and encodes a cilia- and flagella-associated protein. Immunofluorescence assays showed that CFAP58 localized at the entire flagella of control sperm and predominantly concentrated in the mid-piece. Immunoblotting and immunofluorescence assays showed that the abundances of axoneme ultrastructure markers SPAG6 and SPEF2 and a mitochondrial sheath protein, HSP60, were significantly reduced in the spermatozoa from men harboring bi-allelic CFAP58 variants. We generated Cfap58-knockout mice via CRISPR/Cas9 technology. The male mice were infertile and presented with severe flagellar defects, consistent with the sperm phenotypes in MMAF-affected men. Overall, our findings in humans and mice strongly suggest that CFAP58 plays a vital role in sperm flagellogenesis and demonstrate that bi-allelic loss-of-function variants in CFAP58 can cause axoneme and peri-axoneme malformations leading to male infertility. This study provides crucial insights for understanding and counseling of MMAF-associated asthenoteratozoospermia.

Effects of a patient-derived de novo coding alteration of CACNA1I in mice connect a schizophrenia risk gene with sleep spindle deficits.

CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.

UTS2B Defines a Novel Enteroendocrine Cell Population and Regulates GLP-1 Secretion Through SSTR5 in Male Mice.

The gut-pancreas axis plays a key role in the regulation of glucose homeostasis and may be therapeutically exploited to treat not only type 2 diabetes but also hypoglycemia and hyperinsulinemia. We identify a novel enteroendocrine cell type expressing the peptide hormone urotensin 2B (UTS2B). UTS2B inhibits glucagon-like peptide-1 (GLP-1) secretion in mouse intestinal crypts and organoids, not by signaling through its cognate receptor UTS2R but through the activation of the somatostatin receptor (SSTR) 5. Circulating UTS2B concentrations in mice are physiologically regulated during starvation, further linking this peptide hormone to metabolism. Furthermore, administration of UTS2B to starved mice demonstrates that it is capable of regulating blood glucose and plasma concentrations of GLP-1 and insulin in vivo. Altogether, our results identify a novel cellular source of UTS2B in the gut, which acts in a paracrine manner to regulate GLP-1 secretion through SSTR5. These findings uncover a fine-tuning mechanism mediated by a ligand-receptor pair in the regulation of gut hormone secretion, which can potentially be exploited to correct metabolic unbalance caused by overactivation of the gut-pancreas axis.

Effects of nursing intervention on lung infection prevention in patients with tracheotomy.

BACKGROUND: This study will evaluate the effects of nursing intervention (NIV) on lung infection prevention (LIP) in patients with tracheotomy. METHODS: The electronic databases of MEDLINE, Cochrane Library, EMBASE, Web of Science, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure will be retrieved from inception to the June 1, 2019 for randomized controlled trials investigating the effects of NIV on LIP in patients with tracheotomy without any language limitations. In addition, we will also search grey literature to avoid missing any potential studies. Two independent authors will perform study selection, data extraction, and risk of bias evaluation. RESULTS: This study will investigate the effects of NIV on LIP in patients with tracheotomy. The primary outcome is incidence of lung infection. The secondary outcomes include pulmonary function, quality of life, and complications post-surgery. CONCLUSION: The results of this study will summarize recent evidence for the effects of NIV on LIP in patients with tracheotomy.No ethic approval is needed in this study, because it will not need any individual data. The results of this study will be published at a peer-reviewed journal.

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced ß-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate ß-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146.

Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of ß-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of ß-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

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

LGR4/5 receptors and their cognate RSPO ligands potentiate Wnt/ß-catenin signalling and promote proliferation and tissue homeostasis in epithelial stem cell compartments. In the liver, metabolic zonation requires a Wnt/ß-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/ß-catenin-mediated metabolic liver zonation. Liver-specific LGR4/5 loss of function (LOF) or RSPO blockade disrupted hepatic Wnt/ß-catenin signalling and zonation. Conversely, pathway activation in ZNRF3/RNF43 LOF mice or with recombinant RSPO1 protein expanded the hepatic Wnt/ß-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.