Loss of ß-catenin reveals a role for glutathione in regulating oxidative stress during cholestatic liver disease.

BACKGROUND: Cholestasis is an intractable liver disorder that results from impaired bile flow. We have previously shown that the Wnt/ß-catenin signaling pathway regulates the progression of cholestatic liver disease through multiple mechanisms, including bile acid metabolism and hepatocyte proliferation. To further explore the impact of these functions during intrahepatic cholestasis, we exposed mice to a xenobiotic that causes selective biliary injury. METHODS: α-naphthylisothiocyanate (ANIT) was administered to liver-specific knockout (KO) of ß-catenin and wild-type mice in the diet. Mice were killed at 6 or 14 days to assess the severity of cholestatic liver disease, measure the expression of target genes, and perform biochemical analyses. RESULTS: We found that the presence of ß-catenin was protective against ANIT, as KO mice had a significantly lower survival rate than wild-type mice. Although serum markers of liver damage and total bile acid levels were similar between KO and wild-type mice, the KO had minor histological abnormalities, such as sinusoidal dilatation, concentric fibrosis around ducts, and decreased inflammation. Notably, both total glutathione levels and expression of glutathione-S-transferases, which catalyze the conjugation of ANIT to glutathione, were significantly decreased in KO after ANIT. Nuclear factor erythroid-derived 2-like 2, a master regulator of the antioxidant response, was activated in KO after ANIT as well as in a subset of patients with primary sclerosing cholangitis lacking activated ß-catenin. Despite the activation of nuclear factor erythroid-derived 2-like 2, KO livers had increased lipid peroxidation and cell death, which likely contributed to mortality. CONCLUSIONS: Loss of ß-catenin leads to increased cellular injury and cell death during cholestasis through failure to neutralize oxidative stress, which may contribute to the pathology of this disease.

A cis-regulatory module underlies retinal ganglion cell genesis and axonogenesis.

Atoh7 is transiently expressed in retinal progenitor cells (RPCs) and is required for retinal ganglion cell (RGC) differentiation. In humans, a deletion in a distal non-coding regulatory region upstream of ATOH7 is associated with optic nerve atrophy and blindness. Here, we functionally interrogate the significance of the Atoh7 regulatory landscape to retinogenesis in mice. Deletion of the Atoh7 enhancer structure leads to RGC deficiency, optic nerve hypoplasia, and retinal blood vascular abnormalities, phenocopying inactivation of Atoh7. Further, loss of the Atoh7 remote enhancer impacts ipsilaterally projecting RGCs and disrupts proper axonal projections to the visual thalamus. Deletion of the Atoh7 remote enhancer is also associated with the dysregulation of axonogenesis genes, including the derepression of the axon repulsive cue Robo3. Our data provide insights into how Atoh7 enhancer elements function to promote RGC development and optic nerve formation and highlight a key role of Atoh7 in the transcriptional control of axon guidance molecules.

Therapeutic potential of SOX9 dysruption in Combined Hepatocellular Carcinoma-Cholangiocarcinoma.

Combined hepatocellular carcinoma-cholangiocarcinoma (cHCC-CCA) represents a challenging subtype of primary liver cancer with limited treatment options and a poor prognosis. Recently, we and others have highlighted the context-dependent roles of the biliary-specific transcription factor SOX9 in the pathogenesis of liver cancers using various Cre applications in Sox9 (flox/flox) strains, to achieve elimination for exon 2 and 3 of the Sox9 gene locus as a preventive manner. Here, we reveal the contrasting responses of developmental Sox9 elimination using Alb-Cre;Sox9 (flox/flox) ( Sox9 LKO) versus CRISPR/Cas9 -based tumor specific acute Sox9 CKO in SB-HDTVI-based Akt-YAP1 and Akt-NRAS cHCC-CCA formation. Sox9 LKO specifically abrogates the Akt-YAP1 CCA region while robustly stimulating the proliferation of remaining poorly differentiated HCC pertaining liver progenitor cell characteristics, whereas Sox9 CKO potently prevents Akt-YAP1 and Akt-NRAS cHCC-CCA development irrespective of fate of tumor cells compared to respective controls. Additionally, we find that Akt-NRAS , but not Akt-YAP1 , tumor formation is partially dependent on the Sox9-Dnmt1 cascade. Pathologically, SOX9 is indispensable for Akt-YAP1 -mediated HC-to-BEC/CCA reprogramming but required for the maintenance of CCA nodules. Lastly, therapeutic elimination of Sox9 using the OPN-CreERT2 strain combined with an inducible CRISPR/Cas9 -based Sox9 iKO significantly reduces Akt-YAP1 cHCC-CCA tumor burden, similar to Sox9 CKO. Thus, we contrast the outcomes of acute Sox9 deletion with developmental Sox9 knockout models, emphasizing the importance of considering adaptation mechanisms in therapeutic strategies. This necessitates the careful consideration of genetic liver cancer studies using developmental Cre and somatic mutant lines, particularly for genes involved in hepatic commitment during development. Our findings suggest that SOX9 elimination may hold promise as a therapeutic approach for cHCC-CCA and underscore the need for further investigation to translate these preclinical insights into clinical applications.

IFDlong: an isoform and fusion detector for accurate annotation and quantification of long-read RNA-seq data.

Advancements in long-read transcriptome sequencing (long-RNA-seq) technology have revolutionized the study of isoform diversity. These full-length transcripts enhance the detection of various transcriptome structural variations, including novel isoforms, alternative splicing events, and fusion transcripts. By shifting the open reading frame or altering gene expressions, studies have proved that these transcript alterations can serve as crucial biomarkers for disease diagnosis and therapeutic targets. In this project, we proposed IFDlong, a bioinformatics and biostatistics tool to detect isoform and fusion transcripts using bulk or single-cell long-RNA-seq data. Specifically, the software performed gene and isoform annotation for each long-read, defined novel isoforms, quantified isoform expression by a novel expectation-maximization algorithm, and profiled the fusion transcripts. For evaluation, IFDlong pipeline achieved overall the best performance when compared with several existing tools in large-scale simulation studies. In both isoform and fusion transcript quantification, IFDlong is able to reach more than 0.8 Spearman's correlation with the truth, and more than 0.9 cosine similarity when distinguishing multiple alternative splicing events. In novel isoform simulation, IFDlong can successfully balance the sensitivity (higher than 90%) and specificity (higher than 90%). Furthermore, IFDlong has proved its accuracy and robustness in diverse in-house and public datasets on healthy tissues, cell lines and multiple types of diseases. Besides bulk long-RNA-seq, IFDlong pipeline has proved its compatibility to single-cell long-RNA-seq data. This new software may hold promise for significant impact on long-read transcriptome analysis. The IFDlong software is available at https://github.com/wenjiaking/IFDlong.

Selective Targeting of α4ß7/MAdCAM-1 Axis Suppresses Fibrosis Progression by Reducing Proinflammatory T Cell Recruitment to the Liver.

Integrin α4ß7+ T cells perpetuate tissue injury in chronic inflammatory diseases, yet their role in hepatic fibrosis progression remains poorly understood. Here, we report increased accumulation of α4ß7+ T cells in the liver of people with cirrhosis relative to disease controls. Similarly, hepatic fibrosis in the established mouse model of CCl4-induced liver fibrosis was associated with enrichment of intrahepatic α4ß7+ CD4 and CD8 T cells. Monoclonal antibody (mAb)-mediated blockade of α4ß7 or its ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 attenuated hepatic inflammation and prevented fibrosis progression in CCl4-treated mice. Improvement in liver fibrosis was associated with a significant decrease in the infiltration of α4ß7+ CD4 and CD8 T cells, suggesting that α4ß7/MAdCAM-1 axis regulates both CD4 and CD8 T cell recruitment to the fibrotic liver, and α4ß7+ T cells promote hepatic fibrosis progression. Analysis of hepatic α4ß7+ and α4ß7- CD4 T cells revealed that α4ß7+ CD4 T cells were enriched for markers of activation and proliferation, demonstrating an effector phenotype. The findings suggest that α4ß7+ T cells play a critical role in promoting hepatic fibrosis progression, and mAb-mediated blockade of α4ß7 or MAdCAM-1 represents a promising therapeutic strategy for slowing hepatic fibrosis progression in chronic liver diseases.

Utility Analyses of AVITI Sequencing Chemistry.

Background: DNA sequencing is a critical tool in modern biology. Over the last two decades, it has been revolutionized by the advent of massively parallel sequencing, leading to significant advances in the genome and transcriptome sequencing of various organisms. Nevertheless, challenges with accuracy, lack of competitive options and prohibitive costs associated with high throughput parallel short-read sequencing persist. Results: Here, we conduct a comparative analysis using matched DNA and RNA short-reads assays between Element Biosciences' AVITI and Illumina's NextSeq 550 chemistries. Similar comparisons were evaluated for synthetic long-read sequencing for RNA and targeted single-cell transcripts between the AVITI and Illumina's NovaSeq 6000. For both DNA and RNA short-read applications, the study found that the AVITI produced significantly higher per sequence quality scores. For PCR-free DNA libraries, we observed an average 89.7% lower experimentally determined error rate when using the AVITI chemistry, compared to the NextSeq 550. For short-read RNA quantification, AVITI platform had an average of 32.5% lower error rate than that for NextSeq 550. With regards to synthetic long-read mRNA and targeted synthetic long read single cell mRNA sequencing, both platforms' respective chemistries performed comparably in quantification of genes and isoforms. The AVITI displayed a marginally lower error rate for long reads, with fewer chemistry-specific errors and a higher mutation detection rate. Conclusion: These results point to the potential of the AVITI platform as a competitive candidate in high-throughput short read sequencing analyses when juxtaposed with the Illumina NextSeq 550.

Proteome characterization of liver-kidney comorbidity after microbial sepsis.

Sepsis is a life-threatening condition that occurs when the body responds to an infection but subsequently triggers widespread inflammation and impaired blood flow. These pathologic responses can rapidly cause multiple organ dysfunction or failure either one by one or simultaneously. The fundamental common mechanisms involved in sepsis-induced multiple organ dysfunction remain unclear. Here, employing quantitative global and phosphoproteomics, we examine the liver's temporal proteome and phosphoproteome changes after moderate sepsis induced by cecum ligation and puncture. In total, 4593 global proteins and 1186 phosphoproteins according to 3275 phosphosites were identified. To characterize the liver-kidney comorbidity after sepsis, we developed a mathematical model and performed cross-analyses of liver and kidney proteome data obtained from the same set of mice. Beyond immune response, we showed the commonly disturbed pathways and key regulators of the liver-kidney comorbidity are linked to energy metabolism and consumption. Our data provide open resources to understand the communication between the liver and kidney as they work to fight infection and maintain homeostasis.

Nuclear speckle rejuvenation alleviates proteinopathies at the expense of YAP1.

Current treatments targeting individual protein quality control have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common upstream druggable target capable of global proteostasis modulation. Building on our prior research establishing nuclear speckles as pivotal organelles responsible for global proteostasis transcriptional control, we aim to alleviate proteinopathies through nuclear speckle rejuvenation. We identified pyrvinium pamoate as a small-molecule nuclear speckle rejuvenator that enhances protein quality control while suppressing YAP1 signaling via decreasing the surface tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, pyrvinium pamoate reduced tauopathy and alleviated retina degeneration by promoting autophagy and ubiquitin-proteasome system. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity were also observed in human tauopathies. Our study uncovers novel therapeutic targets for tackling protein misfolding disorders within an expanded proteostasis framework encompassing nuclear speckles and YAP1.

Chloride/proton antiporters ClC3 and ClC5 support bone formation in mice.

Acid transport is required for bone synthesis by osteoblasts. The osteoblast basolateral surface extrudes acid by Na+/H+ exchange, but apical proton uptake is undefined. We found high expression of the Cl-/H+ exchanger ClC3 at the bone apical surface. In mammals ClC3 functions in intracellular vesicular chloride transport, but when we found Cl- dependency of H+ transport in osteoblast membranes, we queried whether ClC3 Cl-/H+ exchange functions in bone formation. We used ClC3 knockout animals, and closely-related ClC5 knockout animals: In vitro studies suggested that both ClC3 and ClC5 might support bone formation. Genotypes were confirmed by total exon sequences. Expression of ClC3, and to a lesser extent of ClC5, at osteoblast apical membranes was demonstrated by fluorescent antibody labeling and electron microscopy with nanometer gold labeling. Animals with ClC3 or ClC5 knockouts were viable. In ClC3 or ClC5 knockouts, bone formation decreased ~40 % by calcein and xylenol orange labeling in vivo. In very sensitive micro-computed tomography, ClC5 knockout reduced bone relative to wild type, consistent with effects of ClC3 knockout, but varied with specific histological parameters. Regrettably, ClC5-ClC3 double knockouts are not viable, suggesting that ClC3 or ClC5 activity are essential to life. We conclude that ClC3 has a direct role in bone formation with overlapping but probably slightly smaller effects of ClC5. The mechanism in mineral formation might include ClC H+ uptake, in contrast to ClC3 and ClC5 function in cell vesicles or other organs.

Serum Fusion Transcripts to Assess the Risk of Hepatocellular Carcinoma and the Impact of Cancer Treatment through Machine Learning.

Hepatocellular carcinoma (HCC) is one of the most fatal malignancies. Early diagnosis of HCC is crucial in reducing the risk for mortality. This study analyzed a panel of nine fusion transcripts in serum samples from 61 patients with HCC and 75 patients with non-HCC conditions, using TaqMan real-time quantitative RT-PCR. Seven of the nine fusions frequently detected in patients with HCC included: MAN2A1-FER (100%), SLC45A2-AMACR (62.3%), ZMPSTE24-ZMYM4 (62.3%), PTEN-NOLC1 (57.4%), CCNH-C5orf30 (55.7%), STAMBPL1-FAS (26.2%), and PCMTD1-SNTG1 (16.4%). Machine-learning models were constructed based on serum fusion-gene levels to predict HCC in the training cohort, using the leave-one-out cross-validation approach. One machine-learning model, called the four fusion genes logistic regression model (MAN2A1-FER≤40, CCNH-C5orf30≤38, SLC45A2-AMACR≤41, and PTEN-NOLC1≤40), produced accuracies of 91.5% and 83.3% in the training and testing cohorts, respectively. When serum α-fetal protein level was incorporated into the machine-learning model, a two fusion gene (MAN2A1-FER≤40, CCNH-C5orf30≤38) + α-fetal protein logistic regression model was found to generate an accuracy of 94.8% in the training cohort. The same model resulted in 95% accuracy in both the testing and combined cohorts. Cancer treatment was associated with reduced levels of most of the serum fusion transcripts. Serum fusion-gene machine-learning models may serve as important tools in screening for HCC and in monitoring the impact of HCC treatment.

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

Restoring glucose balance: Conditional HMGB1 knockdown mitigates hyperglycemia in a Streptozotocin induced mouse model.

Diabetes mellitus (DM) poses a significant global health burden, with hyperglycemia being a primary contributor to complications and high morbidity associated with this disorder. Existing glucose management strategies have shown suboptimal effectiveness, necessitating alternative approaches. In this study, we explored the role of high mobility group box 1 (HMGB1) in hyperglycemia, a protein implicated in initiating inflammation and strongly correlated with DM onset and progression. We hypothesized that HMGB1 knockdown will mitigate hyperglycemia severity and enhance glucose tolerance. To test this hypothesis, we utilized a novel inducible HMGB1 knockout (iHMGB1 KO) mouse model exhibiting systemic HMGB1 knockdown. Hyperglycemic phenotype was induced using low dose streptozotocin (STZ) injections, followed by longitudinal glucose measurements and oral glucose tolerance tests to evaluate the effect of HMGB1 knockdown on glucose metabolism. Our findings showed a substantial reduction in glucose levels and enhanced glucose tolerance in HMGB1 knockdown mice. Additionally, we performed RNA sequencing analyses, which identified potential alternations in genes and molecular pathways within the liver and skeletal muscle tissue that may account for the in vivo phenotypic changes observed in hyperglycemic mice following HMGB1 knockdown. In conclusion, our present study delivers the first direct evidence of a causal relationship between systemic HMGB1 knockdown and hyperglycemia in vivo, an association that had remained unexamined prior to this research. This discovery positions HMGB1 knockdown as a potentially efficacious therapeutic target for addressing hyperglycemia and, by extension, the DM epidemic. Furthermore, we have revealed potential underlying mechanisms, establishing the essential groundwork for subsequent in-depth mechanistic investigations focused on further elucidating and harnessing the promising therapeutic potential of HMGB1 in DM management.

Long-read single-cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells.

The protein diversity of mammalian cells is determined by arrays of isoforms from genes. Genetic mutation is essential in species evolution and cancer development. Accurate long-read transcriptome sequencing at single-cell level is required to decipher the spectrum of protein expressions in mammalian organisms. In this report, we developed a synthetic long-read single-cell sequencing technology based on LOOPSeq technique. We applied this technology to analyze 447 transcriptomes of hepatocellular carcinoma (HCC) and benign liver from an individual. Through Uniform Manifold Approximation and Projection analysis, we identified a panel of mutation mRNA isoforms highly specific to HCC cells. The evolution pathways that led to the hyper-mutation clusters in single human leukocyte antigen molecules were identified. Novel fusion transcripts were detected. The combination of gene expressions, fusion gene transcripts, and mutation gene expressions significantly improved the classification of liver cancer cells versus benign hepatocytes. In conclusion, LOOPSeq single-cell technology may hold promise to provide a new level of precision analysis on the mammalian transcriptome.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Defining spatiotemporal gene modules in liver regeneration using Analytical Dynamic Visual Spatial Omics Representation (ADViSOR).

BACKGROUND: The liver is the only organ with the ability to regenerate following surgical or toxicant insults, and partial hepatectomy serves as an experimental model of liver regeneration (LR). Dynamic changes in gene expression occur from the periportal to pericentral regions of the liver following partial hepatectomy; thus, spatial transcriptomics, combined with a novel computational pipeline (ADViSOR [Analytic Dynamic Visual Spatial Omics Representation]), was employed to gain insights into the spatiotemporal molecular underpinnings of LR. METHODS: ADViSOR, comprising Time-Interval Principal Component Analysis and sliding dynamic hypergraphs, was applied to spatial transcriptomics data on 100 genes assayed serially through LR, including key components of the Wnt/ß-catenin pathway at critical timepoints after partial hepatectomy. RESULTS: This computational pipeline identified key functional modules demonstrating cell signaling and cell-cell interactions, inferring shared regulatory mechanisms. Specifically, ADViSOR analysis suggested that macrophage-mediated inflammation is a critical component of early LR and confirmed prior studies showing that Ccnd1, a hepatocyte proliferative gene, is regulated by the Wnt/ß-catenin pathway. These findings were subsequently validated through protein localization, which provided further confirmation and novel insights into the spatiotemporal changes in the Wnt/ß-catenin pathway during LR. CONCLUSIONS: Thus, ADViSOR may yield novel insights in other complex, spatiotemporal contexts.

Hepatocyte CYR61 polarizes profibrotic macrophages to orchestrate NASH fibrosis.

Obesity is increasing worldwide and leads to a multitude of metabolic diseases, including cardiovascular disease, type 2 diabetes, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis (NASH). Cysteine-rich angiogenic inducer 61 (CYR61) is associated with the progression of NASH, but it has been described to have anti- and proinflammatory properties. We sought to examine the role of liver CYR61 in NASH progression. CYR61 liver-specific knockout mice on a NASH diet showed improved glucose tolerance, decreased liver inflammation, and reduced fibrosis. CYR61 polarized infiltrating monocytes promoting a proinflammatory/profibrotic phenotype through an IRAK4/SYK/NF-κB signaling cascade. In vitro, CYR61 activated a profibrotic program, including PDGFa/PDGFb expression in macrophages, in an IRAK4/SYK/NF-κB-dependent manner. Furthermore, targeted-antibody blockade reduced CYR61-driven signaling in macrophages in vitro and in vivo, reducing fibrotic development. This study demonstrates that CYR61 is a key driver of liver inflammation and fibrosis in NASH.

Calponin 2 regulates ketogenesis to mitigate acute kidney injury.

Calponin 2 (CNN2) is a prominent actin stabilizer. It regulates fatty acid oxidation (FAO) by interacting with estrogen receptor 2 (ESR2) to determine kidney fibrosis. However, whether CNN2 is actively involved in acute kidney injury (AKI) remains unclear. Here, we report that CNN2 was induced in human and animal kidneys after AKI. Knockdown of CNN2 preserved kidney function, mitigated tubular cell death and inflammation, and promoted cell proliferation. Distinct from kidney fibrosis, proteomics showed that the key elements in the FAO pathway had few changes during AKI, but we identified that 3-hydroxymethylglutaryl-CoA synthase 2 (Hmgcs2), a rate-limiting enzyme of endogenous ketogenesis that promotes cell self-renewal, was markedly increased in CNN2-knockdown kidneys. The production of ketone body ß-hydroxybutyrate and ATP was increased in CNN2-knockdown mice. Mechanistically, CNN2 interacted with ESR2 to negatively regulate the activities of mitochondrial sirtuin 5. Activated sirtuin 5 subsequently desuccinylated Hmgcs2 to produce energy for mitigating AKI. Understanding CNN2-mediated discrete fine-tuning of protein posttranslational modification is critical to optimize organ performance after AKI.

Inhibiting Wnt Signaling Reduces Cholestatic Injury by Disrupting the Inflammatory Axis.

BACKGROUND & AIMS: ß-Catenin, the effector molecule of the Wnt signaling pathway, has been shown to play a crucial role in bile acid homeostasis through direct inhibition of farnesoid X receptor (FXR), which has pleiotropic effects on bile acid homeostasis. We hypothesize that simultaneous suppression of ß-catenin signaling and activation of FXR in a mouse model of cholestasis will reduce injury and biliary fibrosis through inhibition of bile acid synthesis. METHODS: To induce cholestasis, we performed bile duct ligation (BDL) on wild-type male mice. Eight hours after surgery, mice received FXR agonists obeticholic acid, tropifexor, or GW-4064 or Wnt inhibitor Wnt-C59. Severity of cholestatic liver disease and expression of target genes were evaluated after either 5 days or 12 days of treatment. RESULTS: We found that although the FXR agonists worsened BDL-induced injury and necrosis after 5 days, Wnt-C59 did not. After 12 days of BDL, Wnt-C59 treatment, but not GW-4064 treatment, reduced both the number of infarcts and the number of inflammatory cells in liver. RNA sequencing analysis of whole livers revealed a notable suppression of nuclear factor kappa B signaling when Wnt signaling is inhibited. We then analyzed transcriptomic data to identify a cholangiocyte-specific signature in our model and demonstrated that Wnt-C59-treated livers were enriched for genes expressed in quiescent cholangiocytes, whereas genes expressed in activated cholangiocytes were enriched in BDL alone. A similar decrease in biliary injury and inflammation occurred in Mdr2 KO mice treated with Wnt-C59. CONCLUSIONS: Inhibiting Wnt signaling suppresses cholangiocyte activation and disrupts the nuclear factor kappa B-dependent inflammatory axis, reducing cholestatic-induced injury.

Loss of TAZ after YAP deletion severely impairs foregut development and worsens cholestatic hepatocellular injury.

BACKGROUND: We previously showed that loss of yes-associated protein 1 (YAP) in early liver development (YAPKO) leads to an Alagille syndrome-like phenotype, with failure of intrahepatic bile duct development, severe cholestasis, and chronic hepatocyte adaptations to reduce liver injury. TAZ, a paralog of YAP, was significantly upregulated in YAPKO hepatocytes and interacted with TEA domain family member (TEAD) transcription factors, suggesting possible compensatory activity. METHODS: We deleted both Yap1 and Wwtr1 (which encodes TAZ) during early liver development using the Foxa3 promoter to drive Cre expression, similar to YAPKO mice, resulting in YAP/TAZ double knockout (DKO) and YAPKO with TAZ heterozygosity (YAPKO TAZHET). We evaluated these mice using immunohistochemistry, serum biochemistry, bile acid profiling, and RNA sequencing. RESULTS: DKO mice were embryonic lethal, but their livers were similar to YAPKO, suggesting an extrahepatic cause of death. Male YAPKO TAZHET mice were also embryonic lethal, with insufficient samples to determine the cause. However, YAPKO TAZHET females survived and were phenotypically similar to YAPKO mice, with increased bile acid hydrophilicity and similar global gene expression adaptations but worsened the hepatocellular injury. TAZ heterozygosity in YAPKO impacted the expression of canonical YAP targets Ctgf and Cyr61, and we found changes in pathways regulating cell division and inflammatory signaling correlating with an increase in hepatocyte cell death, cell cycling, and macrophage recruitment. CONCLUSIONS: YAP loss (with or without TAZ loss) aborts biliary development. YAP and TAZ play a codependent critical role in foregut endoderm development outside the liver, but they are not essential for hepatocyte development. TAZ heterozygosity in YAPKO livers increased cell cycling and inflammatory signaling in the setting of chronic injury, highlighting genes that are especially sensitive to TAZ regulation.

Age-related decline in bone mineral transport and bone matrix proteins in osteoblasts from stromal stem cells.

We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFß pathway, and reduced ß-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.NEW & NOTEWORTHY This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.