Features of amphibian embryos that have served so well to elucidate the genetics of vertebrate development also enable detailed analysis of the physics that shape morphogenesis and regulate development. Biophysical tools are revealing how genes control mechanical properties of the embryo. The same tools that describe and control mechanical properties are being turned to reveal how dynamic mechanical information and feedback regulate biological programs of development. In this review we outline efforts to explore the various roles of mechanical cues in guiding cilia biology, axonal pathfinding, goblet cell regeneration, epithelial-to-mesenchymal transitions in neural crest, and mesenchymal-to-epithelial transitions in heart progenitors. These case studies reveal the power of Xenopus experimental embryology to expose pathways integrating mechanical cues with programs of development, organogenesis, and regeneration.

Mechanical forces between cells and their microenvironment critically regulate the asymmetric morphogenesis and physiological functions in vascular systems. Here, we investigated the asymmetric cell alignment and cellular forces simultaneously in micropatterned endothelial cell ring-shaped sheets and studied how the traction and intercellular forces are involved in the asymmetric vascular morphogenesis. Tuning the traction and intercellular forces using different topographic geometries of symmetric and asymmetric ring-shaped patterns regulated the vascular asymmetric morphogenesis in vitro. Moreover, pharmacologically suppressing the cell traction force and intercellular force disturbed the force-dependent asymmetric cell alignment. We further studied this phenomenon by modeling the vascular sheets with a mechanical force-propelled active particle model and confirmed that mechanical forces synergistically drive the asymmetric endothelial cell alignments in different tissue geometries. Further study using mouse diabetic aortic endothelial cells indicated that diseased endothelial cells exhibited abnormal cell alignments, traction, and intercellular forces, indicating the importance of mechanical forces in physiological vascular morphogenesis and functions. Overall, we have established a controllable micromechanical platform to study the force-dependent vascular asymmetric morphogenesis and thus provide a direct link between single-cell mechanical processes and collective behaviors in a multicellular environment.

Our lab has previously shown that β‐catenin regulates liver regeneration in a partial hepatectomy (Phx) model, under the control of Wnt and the canonical signaling pathway. This is true at early timepoints up to 40–72 hours. Using cell‐specific Wntless (Wls) knockout mouse models, we have previously determined epithelial cells are not the source of these Wnts. However, macrophage‐specific Wls knockouts (MP‐KO) have a regeneration deficit at 40 hours that is not seen earlier, suggesting they are a contributing source of Wnts in a temporal manner. We sought to further elucidate the source, identity, and initiation of Wnts using a Tamoxifen‐inducible endothelial cell‐specific Wls knockout model (EC‐KO). At basline, EC‐KO showed decrease in pericentral b‐catenin target genes such as GS, Cyp2e1, and notably Cyclin D1, suggesting a contribution to zonation and potentially regeneration. Since b‐catenin activation occurs early during regeneration, we also wanted to investigate the most proximal event that could be stimulating Wnt expression and secretion from relevant cells after hepatectomy. We hypothesized that shear stress immediately after Phx may initiate Wnt expression and secretion. Using an in vitro system, a preliminary analysis demonstrates that shear stress can initiate Wnt expression in EC, but not in MP or hepatocytes. In vivo, MP and EC demonstrate high levels of Wnt2 and 9b at baseline, which significantly increases after Phx. Taken together, we predict shear stress initiates secretion of Wnt2 and Wnt9b from EC at early timepoints, and MP secrete Wnt2 and Wnt9b at later timepoints, thus temporally regulating b‐catenin and contributing to liver regeneration.Support or Funding Information5T32HL094295‐05

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate‐tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up‐regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low‐density lipoprotein‐related protein 5/6, transgenic mice expressing stable β‐catenin, and in vitro studies, we show a role of Wnt signaling through β‐catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a β‐catenin‐independent manner. Conclusion: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both β‐catenin‐dependent and ‐independent manners. (Hepatology 2016;64:1652‐1666)

Our lab has previously shown that β‐catenin regulates liver regeneration in a partial hepatectomy (Phx) model, under the control of Wnt and the canonical signaling pathway. This is true at early timepoints up to 40–72 hours. Using cell‐specific Wntless (Wls) knockout mouse models, which lack Wls and prevent Wnt secretion, we have previously determined epithelial cells are not the source of these Wnts. However, macrophage‐specific Wls knockouts (MP‐KO) have a regeneration deficit at 40 hours that is not seen earlier, suggesting they are a contributing source of Wnts in a temporal manner. Considering removing Wls from all endothelial cells was embryonic lethal, we sought to elucidate contributions of Wnts using hepatic endothelial cell‐specific Wls knockouts, EC‐KO1 affecting venous endothelial cells, and EC‐KO2 affecting sinusoidal endothelial cells. Initially, EC‐KO1 showed minimal deficits in β‐catenin target genes and zonation markers GS, Cyp2e1, and Cyp1a2 by protein level. EC‐KO1 show reduction in Cyclin D1 24 hours after PHx which rebounded by 48 hours. Intriguingly, EC‐KO2 demonstrate a marked reduction in zonation markers at baseline, which has not yet been reported using protein levels. After PHx, EC‐KO2 display less proliferation than littermate controls, overall emphasizing the role of endothelial cell‐specific Wnts in liver regeneration. In vivo , MP and EC demonstrate high levels of Wnt2 and 9b at baseline, which significantly increase after Phx. There is an increase in portal pressure and shear stress after Phx, so we tested whether shear stress was sufficient to increase Wnt mRNA in vitro , and saw an increase in mRNA in three separate endothelial cell types including primary liver endothelial cells. Taken together, we hypothesize Wnt2 and Wnt9b secretion is initiated early after Phx in part via shear stress, to temporally regulate β‐catenin's contribution to liver regeneration.

Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

From the Departments of Pathology* and Developmental Biology,y University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; the Department of Pharmacology,z University of Virginia, Charlottesville, Virginia; the Department of Molecular Pharmacology and Toxicology,x School of Pharmacy, Keck School of Medicine, University of Southern California, Los Angeles, California; the Division of Cell Regulation Systems,{ Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; and the Department of Health Services and Health Administration,k University of Southern Indiana, Evansville, Indiana

β‐catenin plays a critical role in triggering liver regeneration (LR) after partial hepatectemy (PH). β‐catenin can be activated by numerous pathways, the primary one being Wnt signaling. Defect of β‐catenin leads to delay of LR in hepatocyte specific β‐catenin knockout mice (KO1). Ablation of Wnt‐dependent β‐catenin signaling in hepatocyte specific LRP5/6 double knockouts (KO2) also results in suboptimal LR after PH, which phenocopies KO1. Therefore, Wnt signaling is the major signaling pathway that activates β‐catenin during LR. To further elucidate the source of Wnts in the liver, we studied Wnts secretion from different hepatic cell population after PH. Wingless (Wls) is a critical regulator of Wnt secretion. We generated hepatocyte and macrophage specific Wls knockout mice (KO3&KO4, respectively), which lack the ability of hepatocytes and macrophages to secrete Wnts, respectively. KO3 had normal initiation of LR, while depletion of Wls from macrophages leads to suboptimal LR and impaired β‐catenin activation. Therefore, macrophages but not hepatocytes are responsible for Wnt secretion and β‐catenin activation during LR after PH.

Diethoxycarbonyl dihydrocollidine (DDC) diet induces significant bile duct and hepatocyte injury mimicking human cholestatic liver disease. Following DDC injury, repair is often observed via ductular proliferation of expansion of biliary markers in the hepatocytes. However, the role of Wnt secretion by hepatocytes during DDC injury and repair remains elusive. Alb‐Cre+/‐;Wlsflox/flox mice, which lack the ability to secrete Wnt from hepatocytes, were exposed to DDC diet, and pathological characteristics were compared between wild type (WT) and hepatocyte‐specific Wls knock out (KO) mice. Expression of Wnt7A, Wnt7B and Wnt10B were induced by DDC treatment in WT mice. WT had more abundant A6 positive ductular cells indicating higher ductular proliferation as compared to KO. WT showed more CD45+ cells and collagen deposits than KO. KO showed higher serum total bilirubin level, and poor survival, resulting in worse prognosis than WT. Wnt secreted by hepatocytes might be crucial factor to induce ductular proliferation. This response is important repair mechanism after DDC injury.

Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate Wnt/β-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect Wnt signaling by disrupting β-catenin-CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced β-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 μmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited Wnt target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased β-catenin-CREB binding protein interactions without affecting total β-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the β-catenin/Tcf reporter led to a notable decrease in β-catenin activity. The PMED effect on β-catenin signaling lasted from 12 to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of Wnt signaling that may have implications for HCC treatment.

We sought to identify a secreted biomarker for β-catenin activation commonly seen in hepatocellular carcinoma (HCC). By examination of our previously published genearray of hepatocyte-specific β-catenin knockout (KO) livers, we identified secreted factors whose expression may be β-catenin-dependent. We verified expression and secretion of the leading factor in HCC cells transfected with mutated (Hep3BS33Y)-β-catenin. Serum levels of biomarker were next investigated in a mouse model of HCC with β-catenin gene (Ctnnb1) mutations and eventually in HCC patients. Leukocyte cell-derived chemotaxin-2 (LECT2) expression was decreased in KO livers. Hep3BS33Y expressed and secreted more LECT2 in media as compared to Hep3BWT. Mice developing HCC with Ctnnb1 mutations showed significantly higher serum LECT2 levels. However patients with CTNNB1 mutations showed LECT2 levels of 54.28 ± 22.32 ng/mL (Mean ± SD; n = 8) that were insignificantly different from patients with non-neoplastic chronic liver disease (32.8 ± 21.1 ng/mL; n = 15) or healthy volunteers (33.2 ± 7.2 ng/mL; n = 11). Intriguingly, patients without β-catenin mutations showed significantly higher serum LECT2 levels (54.26 ± 22.25 ng/mL; n = 46). While β-catenin activation was evident in a subset of non-mutant β-catenin HCC group with high LECT2 expression, serum LECT2 was unequivocally similar between β-catenin-active and -normal group. Further analysis showed that LECT2 levels greater than 50 ng/ml diagnosed HCC in patients irrespective of β-catenin mutations with specificity of 96.1% and positive predictive value of 97.0%. Thus, LECT2 is regulated by β-catenin in HCC in both mice and men, but serum LECT2 reflects β-catenin activity only in mice. Serum LECT2 could be a potential biomarker of HCC in patients.

Liver-specific beta-catenin knockout (beta-catenin-LKO) mice have revealed an essential role of beta-catenin in metabolic zonation where it regulates pericentral gene expression and in initiating liver regeneration (LR) after partial hepatectomy (PH), by regulating expression of Cyclin-D1. However, what regulates beta-catenin activity in these events remains an enigma. Here we investigate to what extent beta-catenin activation is Wnt-signalingdependent and the potential cell source of Wnts. We studied liver-specific Lrp5/6 KO (Lrp-LKO) mice where Wnt-signaling was abolished in hepatocytes while the beta-catenin gene remained intact. Intriguingly, like beta-catenin-LKO mice, Lrp-LKO exhibited a defect in metabolic zonation observed as a lack of glutamine synthetase (GS), Cyp1a2, and Cyp2e1. Lrp-LKO also displayed a significant delay in initiation of LR due to the absence of beta-catenin-TCF4 association and lack of Cyclin-D1. To address the source of Wnt proteins in liver, we investigated conditional Wntless (Wls) KO mice, which lacked the ability to secrete Wnts from either liver epithelial cells (Wls-LKO), or macrophages including Kupffer cells (Wls-MKO), or endothelial cells (Wls-EKO). While Wls-EKO was embryonic lethal precluding further analysis in adult hepatic homeostasis and growth, Wls-LKO and Wls-MKO were viable but did not show any defect in hepatic zonation. Wls-LKO showed normal initiation of LR; however, Wls-MKO showed a significant but temporal deficit in LR that was associated with decreased beta-catenin-TCF4 association and diminished Cyclin-D1 expression. Conclusion: Wnt-signaling is the major upstream effector of beta-catenin activity in pericentral hepatocytes and during LR. Hepatocytes, cholangiocytes, or macrophages are not the source of Wnts in regulating hepatic zonation. However, Kupffer cells are a major contributing source of Wnt secretion necessary for beta-catenin activation during LR.

Hepatocellular cancer (HCC) is the third cause of death by cancer worldwide. In the current study we target β- catenin, an oncogene mutated and constitutively active in 20-30% of HCCs, via a novel, cell permeable gamma guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide designed against either the transcription or the translation start site of the human β-catenin gene. Using TOPflash, a luciferase reporter assay, we show that γGPNA targeting the transcription start site showed more robust activity against β-catenin activity in liver tumor cells that harbor β-catenin gene mutations (HepG2 & Snu-449). We identified concomitant suppression of β-catenin expression and of various Wnt targets including glutamine synthetase (GS) and cyclin-D1. Concurrently, γGPNA treatment reduced proliferation, survival and viability of HCC cells. Intriguingly, an angiogenesis quantitative Real-Time-PCR array identified decreased expression of several pro-angiogenic secreted factors such as EphrinA1, FGF-2, and VEGF-A upon β-catenin inhibition in liver tumor cells. Conversely, transfection of stabilized-β-catenin mutants enhanced the expression of angiogenic factors like VEGF-A. Conditioned media from HepG2 cells treated with β-catenin but not the mismatch γGPNA significantly diminished spheroid and tubule formation by SK-Hep1 cells, an HCC-associated endothelial cell line. Thus, we report a novel class of cell permeable and efficacious γGPNAs that effectively targets β-catenin, a known oncogene in the liver. Our study also identifies a novel role of β-catenin in liver tumor angiogenesis through paracrine mechanisms in addition to its roles in proliferation, survival, metabolism and cancer stem cell biology, thus further strengthening its effectiveness as a therapeutic target in HCC.

β-Catenin signaling is implicated in hepatocellular carcinoma (HCC), although its role in inflammation, fibrosis, and proliferation is unclear. Commercially available HCC tissue microarray (TMA) of 89 cases was assessed for β-catenin, one of its transcriptional targets glutamine synthetase (GS), proliferation (PCNA), inflammation (CD45), and fibrosis (Sirius Red). HCC cells transfected with wild-type (WT) or mutant-β-catenin were evaluated for β-catenin-T cell factor transactivation by TOPFlash reporter activity and expression of certain targets. Hepatocyte-specific-serine-45-mutated β-catenin transgenic mice (TG) and controls (Con) were used to study thioacetamide (TAA)-induced hepatic fibrosis and tumorigenesis. Sustained β-catenin activation was only observed in mutant-, not WT-β-catenin transfected HCC cells. Aberrant intratumoral β-catenin stabilization was evident in 33% cases with 9% showing predominant nuclear with some cytoplasmic (N/C) localization and 24% displaying predominant cytoplasmic with occasional nuclear (C/N) localization. N/C β-catenin was associated with reduced fibrosis (p=0.017) and tumor-wide GS staining (p<0.001) while C/N correlated with increased intratumoral inflammation (p=0.064) and proliferation (p=0.029). A small subset of HCC patients (15.5%) lacked β-catenin staining and exhibited low inflammation and fibrosis (p<0.05). TG and Con mice exposed to TAA showed comparable development of fibrosis and progression to cirrhosis and HCC. Taken together the data suggests a complex relationship of β-catenin, inflammation, fibrosis and HCC. GS staining is highly sensitive in identifying HCC with nuclear β-catenin, which may in turn represent β-catenin mutations, and does so with high negative predictive value. Also, β-catenin mutations and cirrhosis do not appear to cooperate in HCC pathogenesis in mice and men.

β‐catenin plays a critical role in liver homeostasis and during liver regeneration (LR). Numerous pathways can activate β‐catenin, the primary one being Wnt signaling. In this study we investigated the source of Wnts and to what extent β‐catenin signaling in liver is Wnt‐dependent. LRP5/6 are the co‐receptors for Wnts. We generated hepatocyte specific LRP5/6 double knockout mice (KO1) where Wnt‐signaling was abolished while Wnt‐independent signaling can still activate β‐catenin, and compared KO1 with hepatocyte specific β‐catenin knockout mice (KO2). We found that KO1 phenocopied KO2 in defective hepatic zonation and delay in LR. β‐catenin activation was impaired after partial hepatectomy (PH) in KO1 due to the lack of Wnt‐signaling. Given the importance of Wnt‐signaling in LR, we further investigated the source of Wnts. Evenness interrupted (Evi) regulates Wnt secretion. We generated hepatocyte specific Evi knockout mice (KO3), which lack the ability of hepatocytes to secrete Wnts. KO3 had normal hepatic zonation and initiation of LR; however, KO3 had sustained proliferation after PH at 72 & 96h. In WT, we found an induction of Wnt5a along with an increase in ROR2, an inhibitor of β‐catenin signaling, which is absent in KO3. Our study suggests that Wnt‐signaling is the major determinant controlling β‐ catenin activation in liver. Further, hepatocytes are not the source of canonical Wnts that activate β‐catenin, but rather produce noncanonical Wnt5a that terminates β‐catenin signaling during late LR.

Constitutive androstane receptor (CAR) is a nuclear orphan receptor. The translocation of CAR from cytoplasm to nucleus triggers the expression of its downstream targets, cytochrome P450 (CYP) enzymes, which play a critical role in the metabolism of drugs and xenobiotics. A potent synthetic inducer, 1,4‐bis‐ [2‐(3,5, ‐dichloropyridyloxy)] benzene (TCPOBOP), induces the activity of CAR. Once stimulated, CAR increases the expression of CYP genes. Activation of CAR also induces non‐compensatory liver growth, and it has been well established that wnt/β‐catenin signaling pathway is a key determinant in liver growth and regeneration, however, the interplay between β‐catenin and CAR signaling is not yet clear. In the current study, we investigated the interaction between β‐catenin and CAR. CAR reporter assay showed that knocking down β‐catenin by siRNA increased CAR transcriptional activity, and this induction effect was more potent than treatment with TCPOBOP. Further, we found that in liver tissue of liver specific β‐catenin knockout mice, CYP2b10, a downstream target of CAR, was up‐regulated in both mRNA and protein level. We conclude that CAR is a regulator of β‐catenin activity, while knocking down β‐catenin serves as a negative feedback to CAR, which further induces the expression of its downstream target, CYP2b10. Funded by 1R01DK62277 and 1R01CA124414 to SPM