Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Notch signaling inactivation by small molecule γ-secretase inhibitors restores the multiciliated cell population in the airway epithelium.

Multiciliated cell loss is a hallmark of airway epithelial remodeling in chronic inflammatory airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. It disrupts mucociliary clearance, which fuels disease progression. Effective clearance requires an optimal proportion of multiciliated and secretory cells. This is controlled by Notch signaling such that between two adjacent cells the one that activates Notch becomes a secretory cell and the one that avoids Notch activation becomes a multiciliated cell. Consequently, blocking Notch by a small molecule inhibitor of the γ-secretase enzyme that cleaves the Notch receptor for signal activation directs differentiation toward the multiciliated lineage. Thus, γ-secretase inhibitor (GSI) treatment may alleviate multiciliated cell loss in lung disease. Here, we demonstrate the therapeutic restoration of multiciliated cells by the GSI LY450139 (semagacestat). LY450139 increased multiciliated cell numbers in a dose-dependent manner in healthy primary human nasal epithelial cells (HNECs) during differentiation and in mature cultures, but not when applied during early epithelialization of progenitors. LY450139 did not impact stem cell proliferation. Basal and apical administration were equally effective. In healthy adult mice, LY450139 increased multiciliated cell numbers without detectible toxicity. LY450139 also increased multiciliated cells and decreased excess mucus secretory cells in CF HNECs and IL-13 remodeled healthy HNECs. LY450139 normalized multiciliated cell numbers in CF HNECs without interfering with the activity of CFTR modulator compounds. In summary, we demonstrate that GSI administration is a promising therapeutic to restore multiciliated cells and potentially improve epithelial function in a wide range of chronic lung diseases.NEW & NOTEWORTHY Our findings show that low-dose, short-term topical or systemic γ-secretase inhibitor treatment may lead to restoration of multiciliated cells without toxicity and potentially improve epithelial function in a wide range of chronic lung diseases.

Shc Is Implicated in Calreticulin-Mediated Sterile Inflammation in Alcoholic Hepatitis.

BACKGROUND & AIMS: Src homology and collagen (Shc) proteins are major adapters to extracellular signals, however, the regulatory role of Shc isoforms in sterile inflammatory responses in alcoholic hepatitis (AH) has not been fully investigated. We hypothesized that in an isoform-specific manner Shc modulates pre-apoptotic signals, calreticulin (CRT) membrane exposure, and recruitment of inflammatory cells. METHODS: Liver biopsy samples from patients with AH vs healthy subjects were studied for Shc expression using DNA microarray data and immunohistochemistry. Shc knockdown (hypomorph) and age-matched wild-type mice were pair-fed according to the chronic-plus-binge alcohol diet. To analyze hepatocyte-specific effects, adeno-associated virus 8-thyroxine binding globulin-Cre (hepatocyte-specific Shc knockout)-mediated deletion was performed in flox/flox Shc mice. Lipid peroxidation, proinflammatory signals, redox radicals, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratio, as well as cleaved caspase 8, B-cell-receptor-associated protein 31 (BAP31), Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist killer (Bak), were assessed in vivo. CRT translocation was studied in ethanol-exposed p46ShcẟSH2-transfected hepatocytes by membrane biotinylation in conjunction with phosphorylated-eukaryotic initiation factor 2 alpha, BAP31, caspase 8, and Bax/Bak. The effects of idebenone, a novel Shc inhibitor, was studied in alcohol/pair-fed mice. RESULTS: Shc was significantly induced in patients with AH (P < .01). Alanine aminotransferase, reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide ratios, production of redox radicals, and lipid peroxidation improved (P < .05), and interleukin 1ß, monocyte chemoattractant protein 1, and C-X-C chemokine ligand 10 were reduced in Shc knockdown and hepatocyte-specific Shc knockout mice. In vivo, Shc-dependent induction, and, in hepatocytes, a p46Shc-dependent increase in pre-apoptotic proteins Bax/Bak, caspase 8, BAP31 cleavage, and membrane translocation of CRT/endoplasmic reticulum-resident protein 57 were seen. Idebenone protected against alcohol-mediated liver injury. CONCLUSIONS: Alcohol induces p46Shc-dependent activation of pre-apoptotic pathways and translocation of CRT to the membrane, where it acts as a damage-associated molecular pattern, instigating immunogenicity. Shc inhibition could be a novel treatment strategy in AH.

Distinct overlapping functions for Prickle1 and Prickle2 in the polarization of the airway epithelium.

Planar cell polarity (PCP) signaling polarizes cells within the plane of an epithelium. In the airways, planar cell polarity signaling orients the directional beating of motile cilia required for effective mucociliary clearance. The planar cell polarity signaling mechanism is best understood from work in Drosophila, where it has been shown to both coordinate the axis of polarity between cells and to direct the morphological manifestations of polarization within cells. The 'core' planar cell polarity signaling mechanism comprises two protein complexes that segregate to opposite sides of each cell and interact with the opposite complex in neighboring cells. Proper subcellular localization of core planar cell polarity proteins correlates with, and is almost certainly responsible for, their ability to direct polarization. This mechanism is highly conserved from Drosophila to vertebrates, though for most of the core genes, mammals have multiple paralogs whereas Drosophila has only one. In the mouse airway epithelium, the core protein Prickle2 segregates asymmetrically, as is characteristic for core proteins, but is only present in multiciliated cells and is absent from other cell types. Furthermore, Prickle2 mutant mice show only modest ciliary polarity defects. These observations suggest that other Prickle paralogs might contribute to polarization. Here, we show that Prickle1 segregates asymmetrically in multiciliated and nonciliated airway epithelial cell types, that compared to Prickle2, Prickle1 has different spatial and temporal expression dynamics and a stronger ciliary polarity phenotype, and that Prickle1 and Prickle2 mutants genetically interact. We propose distinct and partially overlapping functions for the Prickle paralogs in polarization of the airway epithelium.

A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production.

Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

Non-alcoholic Fatty Liver Disease and Liver Fibrosis during Aging.

Non-alcoholic fatty liver disease (NAFLD) and its progressive form non-alcoholic steatohepatitis (NASH) have emerged as the leading causes of chronic liver disease-related mortality. The prevalence of NAFLD/NASH is expected to increase given the epidemics of obesity and type 2 diabetes mellitus. Older patients are disproportionally affected by NASH and related complications such as progressive fibrosis, cirrhosis and hepatocellular carcinoma; however, they are often ineligible for liver transplantation due to their frailty and comorbidities, and effective medical treatments are still lacking. In this review we focused on pathways that are key to the aging process in the liver and perpetuate NAFLD/NASH, leading to fibrosis. In addition, we highlighted recent findings and cross-talks of normal and/or senescent liver cells, dysregulated nutrient sensing, proteostasis and mitochondrial dysfunction in the framework of changing metabolic milieu. Better understanding these pathways during preclinical and clinical studies will be essential to design novel and specific therapeutic strategies to treat NASH in the elderly.

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called "chicken wire," which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson's trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis. The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

A human multi-lineage hepatic organoid model for liver fibrosis.

To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFß pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

Odf2 haploinsufficiency causes a new type of decapitated and decaudated spermatozoa, Odf2-DDS, in mice.

Outer dense fibre 2 (Odf2 or ODF2) is a cytoskeletal protein required for flagella (tail)-beating and stability to transport sperm cells from testes to the eggs. There are infertile males, including human patients, who have a high percentage of decapitated and decaudated spermatozoa (DDS), whose semen contains abnormal spermatozoa with tailless heads and headless tails due to head-neck separation. DDS is untreatable in reproductive medicine. We report for the first time a new type of Odf2-DDS in heterozygous mutant Odf2+/- mice. Odf2+/- males were infertile due to haploinsufficiency caused by heterozygous deletion of the Odf2 gene, encoding the Odf2 proteins. Odf2 haploinsufficiency induced sperm neck-midpiece separation, a new type of head-tail separation, leading to the generation of headneck sperm cells or headnecks composed of heads with necks and neckless tails composed of only the main parts of tails. The headnecks were immotile but alive and capable of producing offspring by intracytoplasmic headneck sperm injection (ICSI). The neckless tails were motile and could induce capacitation but had no significant forward motility. Further studies are necessary to show that ICSI in humans, using headneck sperm cells, is viable and could be an alternative for infertile patients suffering from Odf2-DDS.

Claudin-3 regulates bile canalicular paracellular barrier and cholesterol gallstone core formation in mice.

BACKGROUND & AIMS: Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. METHODS: We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. RESULTS: In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. CONCLUSION: We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. LAY SUMMARY: Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Disruption of Core Planar Cell Polarity Signaling Regulates Renal Tubule Morphogenesis but Is Not Cystogenic.

Oriented cell division (OCD) and convergent extension (CE) shape developing renal tubules, and their disruption has been associated with polycystic kidney disease (PKD) genes, the majority of which encode proteins that localize to primary cilia. Core planar cell polarity (PCP) signaling controls OCD and CE in other contexts, leading to the hypothesis that disruption of PCP signaling interferes with CE and/or OCD to produce PKD. Nonetheless, the contribution of PCP to tubulogenesis and cystogenesis is uncertain, and two major questions remain unanswered. Specifically, the inference that mutation of PKD genes interferes with PCP signaling is untested, and the importance of PCP signaling for cystogenic PKD phenotypes has not been examined. We show that, during proliferative stages, PCP signaling polarizes renal tubules to control OCD. However, we find that, contrary to the prevailing model, PKD mutations do not disrupt PCP signaling but instead act independently and in parallel with PCP signaling to affect OCD. Indeed, PCP signaling that is normally downregulated once development is completed is retained in cystic adult kidneys. Disrupting PCP signaling results in inaccurate control of tubule diameter, a tightly regulated parameter with important physiological ramifications. However, we show that disruption of PCP signaling is not cystogenic. Our results suggest that regulating tubule diameter is a key function of PCP signaling but that loss of this control does not induce cysts.

Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains.

Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188-806, whereas basal foot formation required aa 1-59 and 188-806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

Involvement of IQGAP3, a regulator of Ras/ERK-related cascade, in hepatocyte proliferation in mouse liver regeneration and development.

The spatio-temporal regulation of hepatocyte proliferation is a critical issue in liver regeneration. Here, in normal and regenerating liver as well as in developing liver, we examined its expression/localization of IQGAP3, which was most recently reported as a Ras/Rac/Cdc42-binding proliferation factor associated with cell-cell contacts in epithelial-type cells. In parallel, the expression/localization of Rac/Cdc42-binding IQGAP1/2 was examined. IQGAP3 showed a specific expression in proliferating hepatocytes positive for the proliferating marker Ki-67, the levels of expressions of mRNAs and proteins were significantly increased in hepatocytes in liver regeneration and development. In immunofluorescence, IQGAP3 was highly enriched at cell-cell contacts of hepatocytes. IQGAP1 and IQGAP2 were exclusively expressed in Kupffer and sinusoidal endothelial cells, respectively, in normal, regenerating, and developing liver. The expression of IQGAP1, but not of IQGAP2, was increased in CCl4-induced (but not in partial hepatectomy-induced) liver regeneration. Exclusive expression/localization of IQGAP3 to hepatocytes in the liver likely reflects the specific involvement of the IQGAP3/Ras/ERK signaling cascade in hepatocyte proliferation in addition to the previously identified signaling pathways, possibly by integrating cell-cell contact-related proliferating signaling events. On the other hand, the Rac/Cdc42-binding properties of IQGAP1/2/3 may be related to the distinct modes of remodeling due to the different strategies which induced proliferation of liver cells; partial hepatectomy, CCl4 injury, or embryonic development. Thus, the functional orchestration of Ras and the Ras homologous (Rho) family proteins Rac/Cdc42 likely plays a critical role in liver regeneration and development.