Functional analysis of the Vsx2 super-enhancer uncovers distinct cis-regulatory circuits controlling Vsx2 expression during retinogenesis.

Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation, but the molecular mechanisms underlying its developmental roles are unclear. Here, we have profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal genetic programs associated with VSX2 during development. VSX2 binds and transactivates its enhancer in association with the transcription factor PAX6. Mice harboring deletions in the Vsx2 regulatory landscape exhibit specific abnormalities in retinal proliferation and in bipolar cell differentiation. In one of those deletions, a complete loss of bipolar cells is associated with a bias towards photoreceptor production. VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in the adult mouse and human retina, including a conserved region nearby Prdm1, a factor implicated in the specification of rod photoreceptors and suppression of bipolar cell fate. VSX2 interacts with the transcription factor OTX2 and can act to suppress OTX2-dependent enhancer transactivation of the Prdm1 enhancer. Taken together, our analyses indicate that Vsx2 expression can be temporally and spatially uncoupled at the enhancer level, and they illuminate important mechanistic insights into how VSX2 is engaged with gene regulatory networks that are essential for retinal proliferation and cell fate acquisition.

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.

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.

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.

Inhibition of Heat Shock Factor 1 Signaling Decreases Hepatoblastoma Growth via Induction of Apoptosis.

Although rare compared with adult liver cancers, hepatoblastoma (HB) is the most common pediatric liver malignancy, and its incidence is increasing. Currently, the treatment includes surgical resection with or without chemotherapy, and in severe cases, liver transplantation in children. The effort to develop more targeted, HB-specific therapies has been stymied by the lack of fundamental knowledge about HB biology. Heat shock factor 1 (HSF1), a transcription factor, is a canonical inducer of heat shock proteins, which act as chaperone proteins to prevent or undo protein misfolding. Recent work has shown a role for HSF1 in cancer beyond the canonical heat shock response. The current study found increased HSF1 signaling in HB versus normal liver. It showed that less differentiated, more embryonic tumors had higher levels of HSF1 than more differentiated, more fetal-appearing tumors. Most strikingly, HSF1 expression levels correlated with mortality. This study used a mouse model of HB to test the effect of inhibiting HSF1 early in tumor development on cancer growth. HSF1 inhibition resulted in fewer and smaller tumors, suggesting HSF1 is needed for aggressive tumor growth. Moreover, HSF1 inhibition also increased apoptosis in tumor foci. These data suggest that HSF1 may be a viable pharmacologic target for HB treatment.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver.

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/ß-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.

Fusion Gene Detection in Prostate Cancer Samples Enhances the Prediction of Prostate Cancer Clinical Outcomes from Radical Prostatectomy through Machine Learning in a Multi-Institutional Analysis.

Prostate cancer remains one of the most fatal malignancies in men in the United States. Predicting the course of prostate cancer is challenging given that only a fraction of prostate cancer patients experience cancer recurrence after radical prostatectomy or radiation therapy. This study examined the expressions of 14 fusion genes in 607 prostate cancer samples from the University of Pittsburgh, Stanford University, and the University of Wisconsin-Madison. The profiling of 14 fusion genes was integrated with Gleason score of the primary prostate cancer and serum prostate-specific antigen level to develop machine-learning models to predict the recurrence of prostate cancer after radical prostatectomy. Machine-learning algorithms were developed by analysis of the data from the University of Pittsburgh cohort as a training set using the leave-one-out cross-validation method. These algorithms were then applied to the data set from the combined Stanford/Wisconsin cohort (testing set). The results showed that the addition of fusion gene profiling consistently improved the prediction accuracy rate of prostate cancer recurrence by Gleason score, serum prostate-specific antigen level, or a combination of both. These improvements occurred in both the training and testing cohorts and were corroborated by multiple models.

In vitro study to identify ligand-independent function of estrogen receptor-α in suppressing DNA damage-induced chondrocyte senescence.

In osteoarthritis (OA), chondrocytes undergo many pathological alternations that are linked with cellular senescence. However, the exact pathways that lead to the generation of a senescence-like phenotype in OA chondrocytes are not clear. Previously, we found that loss of estrogen receptor-α (ERα) was associated with an increased senescence level in human chondrocytes. Since DNA damage is a common cause of cellular senescence, we aimed to study the relationship among ERα levels, DNA damage, and senescence in chondrocytes. We first examined the levels of ERα, representative markers of DNA damage and senescence in normal and OA cartilage harvested from male and female human donors, as well as from male mice. The influence of DNA damage on ERα levels was studied by treating human chondrocytes with doxorubicin (DOX), which is an often-used DNA-damaging agent. Next, we tested the potential of overexpressing ERα in reducing DNA damage and senescence levels. Lastly, we explored the interaction between ERα and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Results indicated that the OA chondrocytes contained DNA damage and displayed senescence features, which were accompanied by significantly reduced ERα levels. Overexpression of ERα reduced the levels of DNA damage and senescence in DOX-treated normal chondrocytes and OA chondrocytes. Moreover, DOX-induced the activation of NF-κB pathway, which was partially reversed by overexpressing ERα. Taken together, our results demonstrated the critical role of ERα in maintaining the health of chondrocytes by inhibiting DNA damage and senescence. This study also suggests that maintaining the ERα level may represent a new avenue to prevent and treat OA.

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.

TMEM16A partners with mTOR to influence pathways of cell survival, proliferation, and migration in cholangiocarcinoma.

Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl- channel activity. The Cl- channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA.NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.

NOTCH-YAP1/TEAD-DNMT1 Axis Drives Hepatocyte Reprogramming Into Intrahepatic Cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is a devastating liver cancer with extremely high intra- and inter-tumoral molecular heterogeneity, partly due to its diverse cellular origins. We investigated clinical relevance and the molecular mechanisms underlying hepatocyte (HC)-driven ICC development. METHODS: Expression of ICC driver genes in human diseased livers at risk for ICC development were examined. The sleeping beauty and hydrodynamic tail vein injection based Akt-NICD/YAP1 ICC model was used to investigate pathogenetic roles of SRY-box transcription factor 9 (SOX9) and yes-associated protein 1 (YAP1) in HC-driven ICC. We identified DNA methyltransferase 1 (DNMT1) as a YAP1 target, which was validated by loss- and gain-of-function studies, and its mechanism addressed by chromatin immunoprecipitation sequencing. RESULTS: Co-expression of AKT and Notch intracellular domain (NICD)/YAP1 in HC yielded ICC that represents 13% to 29% of clinical ICC. NICD independently regulates SOX9 and YAP1 and deletion of either, significantly delays ICC development. Yap1 or TEAD inhibition, but not Sox9 deletion, impairs HC-to-biliary epithelial cell (BEC) reprogramming. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directs HC-to-BEC/ICC fate switch through the repression of HC-specific genes regulated by master regulators for HC differentiation, including hepatocyte nuclear factor 4 alpha, hepatocyte nuclear factor 1 alpha, and CCAAT/enhancer-binding protein alpha/beta. DNMT1 loss prevented NOTCH/YAP1-dependent HC-driven cholangiocarcinogenesis, and DNMT1 re-expression restored ICC development following TEAD repression. Co-expression of DNMT1 with AKT was sufficient to induce tumor development including ICC. DNMT1 was detected in a subset of HCs and dysplastic BECs in cholestatic human livers prone to ICC development. CONCLUSION: We identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-to-BEC/ICC conversion, which may be relevant in cholestasis-to-ICC pathogenesis in the clinic.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

BACKGROUND: In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. METHODS: We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. RESULTS: TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. CONCLUSIONS: Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Western diet unmasks transient low-level vinyl chloride-induced tumorigenesis; potential role of the (epi-)transcriptome.

BACKGROUND & AIMS: Vinyl chloride (VC) monomer is a volatile organic compound commonly used in industry. At high exposure levels, VC causes liver cancer and toxicant-associated steatohepatitis. However, lower exposure levels (i.e., sub-regulatory exposure limits) that do not directly damage the liver, enhance injury caused by Western diet (WD). It is still unknown if the long-term impact of transient low-concentration VC enhances the risk of liver cancer development. This is especially a concern given that fatty liver disease is in and of itself a risk factor for the development of liver cancer. METHODS: C57Bl/6 J mice were fed WD or control diet (CD) for 1 year. During the first 12 weeks of feeding only, mice were also exposed to VC via inhalation at sub-regulatory limit concentrations (<1 ppm) or air for 6 h/day, 5 days/week. RESULTS: Feeding WD for 1 year caused significant hepatic injury, which was exacerbated by VC. Additionally, VC increased the number of tumors which ranged from moderately to poorly differentiated hepatocellular carcinoma (HCC). Transcriptomic analysis demonstrated VC-induced changes in metabolic but also ribosomal processes. Epitranscriptomic analysis showed a VC-induced shift of the modification pattern that has been associated with metabolic disease, mitochondrial dysfunction, and cancer. CONCLUSIONS: These data indicate that VC sensitizes the liver to other stressors (e.g., WD), resulting in enhanced tumorigenesis. These data raise concerns about potential interactions between VC exposure and WD. It also emphasizes that current safety restrictions may be insufficient to account for other factors that can influence hepatotoxicity.

12-h clock regulation of genetic information flow by XBP1s.

Our group recently characterized a cell-autonomous mammalian 12-h clock independent from the circadian clock, but its function and mechanism of regulation remain poorly understood. Here, we show that in mouse liver, transcriptional regulation significantly contributes to the establishment of 12-h rhythms of mRNA expression in a manner dependent on Spliced Form of X-box Binding Protein 1 (XBP1s). Mechanistically, the motif stringency of XBP1s promoter binding sites dictates XBP1s's ability to drive 12-h rhythms of nascent mRNA transcription at dawn and dusk, which are enriched for basal transcription regulation, mRNA processing and export, ribosome biogenesis, translation initiation, and protein processing/sorting in the Endoplasmic Reticulum (ER)-Golgi in a temporal order consistent with the progressive molecular processing sequence described by the central dogma information flow (CEDIF). We further identified GA-binding proteins (GABPs) as putative novel transcriptional regulators driving 12-h rhythms of gene expression with more diverse phases. These 12-h rhythms of gene expression are cell autonomous and evolutionarily conserved in marine animals possessing a circatidal clock. Our results demonstrate an evolutionarily conserved, intricate network of transcriptional control of the mammalian 12-h clock that mediates diverse biological pathways. We speculate that the 12-h clock is coopted to accommodate elevated gene expression and processing in mammals at the two rush hours, with the particular genes processed at each rush hour regulated by the circadian and/or tissue-specific pathways.

Dual ß-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-ß and Hepatocyte Nuclear Factor 4α Signaling.

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of ß-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of ß-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-ß signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of ß-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by ß-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

Nuclear factor erythroid 2-related factor 2 and ß-Catenin Coactivation in Hepatocellular Cancer: Biological and Therapeutic Implications.

BACKGROUND AND AIMS: HCC remains a major unmet clinical need. Although activating catenin beta-1 (CTNNB1) mutations are observed in prominent subsets of HCC cases, these by themselves are insufficient for hepatocarcinogenesis. Coexpression of mutant CTNNB1 with clinically relevant co-occurrence has yielded HCCs. Here, we identify cooperation between ß-catenin and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling in HCC. APPROACH AND RESULTS: Public HCC data sets were assessed for concomitant presence of CTNNB1 mutations and either mutations in nuclear factor erythroid-2-related factor-2 (NFE2L2) or Kelch like-ECH-associated protein 1 (KEAP1), or Nrf2 activation by gene signature. HCC development in mice and similarity to human HCC subsets was assessed following coexpression of T41A-CTNNB1 with either wild-type (WT)-, G31A-, or T80K-NFE2L2. Based on mammalian target of rapamycin complex 1 activation in CTNNB1-mutated HCCs, response of preclinical HCC to mammalian target of rapamycin (mTOR) inhibitor was investigated. Overall, 9% of HCC cases showed concomitant CTNNB1 mutations and Nrf2 activation, subsets of which were attributable to mutations in NFE2L2/KEAP1. Coexpression of mutated CTNNB1 with mutant NFE2L2, but not WT-NFE2L2, led to HCC development and mortality by 12-14 weeks. These HCCs were positive for ß-catenin targets, like glutamine synthetase and cyclin-D1, and Nrf2 targets, like NAD(P)H quinone dehydrogenase 1 and peroxiredoxin 1. RNA-sequencing and pathway analysis showed high concordance of preclinical HCC to human HCC subset showing activation of unique (iron homeostasis and glioblastoma multiforme signaling) and expected (glutamine metabolism) pathways. NFE2L2-CTNNB1 HCC mice were treated with mTOR inhibitor everolimus (5-mg/kg diet ad libitum), which led to >50% decrease in tumor burden. CONCLUSIONS: Coactivation of ß-catenin and Nrf2 is evident in 9% of all human HCCs. Coexpression of mutant NFE2L2 and mutant CTNNB1 led to clinically relevant HCC development in mice, which responded to mTOR inhibitors. Thus, this model has both biological and therapeutic implications.

The Thyromimetic Sobetirome (GC-1) Alters Bile Acid Metabolism in a Mouse Model of Hepatic Cholestasis.

Chronic cholestasis results from bile secretory defects or impaired bile flow with few effective medical therapies available. Thyroid hormone triiodothyronine and synthetic thyroid hormone receptor agonists, such as sobetirome (GC-1), are known to impact lipid and bile acid (BA) metabolism and induce hepatocyte proliferation downstream of Wnt/ß-catenin signaling after surgical resection; however, these drugs have yet to be studied as potential therapeutics for cholestatic liver disease. Herein, GC-1 was administered to ATP binding cassette subfamily B member 4 (Abcb4-/-; Mdr2-/-) knockout (KO) mice, a sclerosing cholangitis model. KO mice fed GC-1 diet for 2 and 4 weeks had decreased serum alkaline phosphatase but increased serum transaminases compared with KO alone. KO mice on GC-1 also had higher levels of total liver BA due to alterations in expression of BA detoxification, transport, and synthesis genes, with the net result being retention of BA in the hepatocytes. Interestingly, GC-1 does not induce hepatocyte proliferation or Wnt/ß-catenin signaling in KO mice, likely a result of decreased thyroid hormone receptor ß expression without Mdr2. Therefore, although GC-1 treatment induces a mild protection against biliary injury in the early stages of treatment, it comes at the expense of hepatocyte injury and is suboptimal because of lower expression of thyroid hormone receptor ß. Thus, thyromimetics may have limited therapeutic benefits in treating cholestatic liver disease.

Impaired Bile Secretion Promotes Hepatobiliary Injury in Sickle Cell Disease.

BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Inhibition of Estrogen Sulfotransferase (SULT1E1/EST) Ameliorates Ischemic Acute Kidney Injury in Mice.

BACKGROUND: Studies have suggested that estrogens may protect mice from AKI. Estrogen sulfotransferase (SULT1E1, or EST) plays an important role in estrogen homeostasis by sulfonating and deactivating estrogens, but studies on the role of SULT1E1 in AKI are lacking. METHODS: We used the renal ischemia-reperfusion model to investigate the role of SULT1E1 in AKI. We subjected wild-type mice, Sult1e1 knockout mice, and Sult1e1 knockout mice with liver-specific reconstitution of SULT1E1 expression to bilateral renal ischemia-reperfusion or sham surgery, either in the absence or presence of gonadectomy. We assessed relevant biochemical, histologic, and gene expression markers of kidney injury. We also used wild-type mice treated with the SULT1E1 inhibitor triclosan to determine the effect of pharmacologic inhibition of SULT1E1 on AKI. RESULTS: AKI induced the expression of Sult1e1 in a tissue-specific and sex-specific manner. It induced expression of Sult1e1 in the liver in both male and female mice, but Sult1e1 induction in the kidney occurred only in male mice. Genetic knockout or pharmacologic inhibition of Sult1e1 protected mice of both sexes from AKI, independent of the presence of sex hormones. Instead, a gene profiling analysis indicated that the renoprotective effect was associated with increased vitamin D receptor signaling. Liver-specific transgenic reconstitution of SULT1E1 in Sult1e1 knockout mice abolished the protection in male mice but not in female mice, indicating that Sult1e1's effect on AKI was also tissue-specific and sex-specific. CONCLUSIONS: SULT1E1 appears to have a novel function in the pathogenesis of AKI. Our findings suggest that inhibitors of SULT1E1 might have therapeutic utility in the clinical management of AKI.

Blocking integrin α4ß7-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis.

BACKGROUND & AIMS: The heterodimeric integrin receptor α4ß7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4ß7-mediated recruitment of CD4 T cells to the intestine and liver in NASH. METHODS: Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r-/- mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. RESULTS: WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4ß7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4ß7+ CD4 T cells was associated with markedly higher expression of the α4ß7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4ß7 blockade in WD-fed knockout mice significantly decreased α4ß7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and ß7 integrins. CONCLUSIONS: These findings establish α4ß7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. LAY SUMMARY: Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4ß7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4ß7 antibody in the treatment of human NASH.