New Era of Immune-Based Therapy in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (CC) accounts for approximately 20% of all biliary tract cancer (BTC) cases and 10-15% of all primary liver cancer cases. Many patients are diagnosed with unresectable BTC, and, even among patients with resectable BTC, the 5-year survival rate is approximately 20%. The BTC incidence rate is high in Southeast and East Asia and has increased worldwide in recent years. Since 2010, cytotoxic chemotherapy, particularly combination gemcitabine + cisplatin (ABC-02 trial), has been the first-line therapy for patients with BTC. In 2022, a multicenter, double-blind, randomized phase 3 trial (TOPAZ-1 trial) examined the addition of programmed death-ligand 1 immunotherapy (durvalumab) to combination gemcitabine + cisplatin for BTC treatment, resulting in significantly improved survival without notable additional toxicity. As a result of this trial, this three-drug combination has become the new standard first-line therapy, leading to notable advances in BTC management for the first time since 2010. The molecular profiling of BTC has continued to drive the development of new targeted therapies for use when first-line therapies fail. Typically, second-line therapy decisions are based on identified genomic alterations in tumor tissue. Mutations in fibroblast growth factor receptor 1/2/3, isocitrate dehydrogenase 1/2, and neurotrophic tyrosine receptor kinase A/B/C are relatively frequent in intrahepatic CC, and precision medicines are available that can target associated pathways. In this review, we suggest strategies for systemic pharmacotherapy with a focus on intrahepatic CC, in addition to presenting the results and safety outcomes of clinical trials evaluating immune checkpoint inhibitor therapies in BTC.

Hypo-osmolarity induces apoptosis resistance via TRPV2-mediated AKT-Bcl-2 pathway.

In cirrhosis, several molecular alterations such as resistance to apoptosis could accelerate carcinogenesis. Recently, mechanotransduction has been attracting attention as one of the causes of these disturbances. In patients with cirrhosis, the serum sodium levels progressively decrease in the later stage of cirrhosis, and hyponatremia leads to serum hypo-osmolality. Since serum sodium levels in patients with cirrhosis with liver cancer are inversely related to cancer's number, size, stage, and cumulative survival, we hypothesized that hypo-osmolality-induced mechanotransduction under cirrhotic conditions might contribute to oncogenesis and/or progression of hepatocellular carcinoma (HCC). In this study, we adjusted osmosis of culture medium by changing the sodium chloride concentration and investigated the influence of hypotonic conditions on the apoptosis resistance of an HCC cell line, HepG2, using a serum-deprivation-induced apoptosis model. By culturing the cells in a serum-free medium, the levels of an antiapoptotic protein Bcl-2 were downregulated. In contrast, the hypotonic conditions caused apoptosis resistance by upregulation of Bcl-2. Next, we examined which pathway was involved in the apoptosis resistance. Hypotonic conditions enhanced AKT signaling, and constitutive activation of AKT in HepG2 cells led to upregulation of Bcl-2. Moreover, we revealed that the enhancement of AKT signaling was caused by intracellular calcium influx via a mechanosensor, TRPV2. Our findings suggested that hyponatremia-induced serum hypotonic in patients with cirrhosis promoted the progression of hepatocellular carcinoma.NEW & NOTEWORTHY Our study first revealed that hypo-osmolarity-induced mechanotransduction enhanced calcium-mediated AKT signaling via TRPV2 activation, resulting in contributing to apoptosis resistance. The finding indicates a possible view that liver cirrhosis-induced hyponatremia promotes hepatocellular carcinogenesis.

Nitric Oxide Derived from Cytoglobin-Deficient Hepatic Stellate Cells Causes Suppression of Cytochrome c Oxidase Activity in Hepatocytes.

Aims: Cell-cell interactions between hepatocytes (Hep) and other liver cells are key to maintaining liver homeostasis. Cytoglobin (CYGB), expressed exclusively by hepatic stellate cells (HSC), is essential in mitigating mitochondrial oxidative stress. CYGB absence causes Hep dysfunction and evokes hepatocarcinogenesis through an elusive mechanism. CYGB deficiency is speculated to hinder nitric oxide dioxygenase (NOD) activity, resulting in the elevated formation and release of nitric oxide (NO). Hence, we hypothesized that NO accumulation induced by the loss of NOD activity in CYGB-deficient HSC could adversely affect mitochondrial function in Hep, leading to disease progression. Results: NO, a membrane-permeable gas metabolite overproduced by CYGB-deficient HSC, diffuses into the neighboring Hep to reversibly inhibit cytochrome c oxidase (CcO), resulting in the suppression of respiratory function in an electron transport chain (ETC). The binding of NO to CcO is proved using purified CcO fractions from Cygb knockout (Cygb-/-) mouse liver mitochondria. Its inhibitory action toward CcO-specific activity is fully reversed by the external administration of oxyhemoglobin chasing away the bound NO. Thus, these findings indicate that the attenuation of respiratory function in ETC causes liver damage through the formation of excessive reactive oxygen species. Treating Cygb-/- mice with an NO synthase inhibitor successfully relieved NO-induced inhibition of CcO activity in vivo. Innovation and Conclusion: Our findings provide a biochemical link between CYGB-absence in HSC and neighboring Hep dysfunction; mechanistically the absence of CYGB in HSC causes mitochondrial dysfunction of Hep via the inhibition of CcO activity by HSC-derived NO. Antioxid. Redox Signal. 38, 463-479.

Cancer cells produce liver metastasis via gap formation in sinusoidal endothelial cells through proinflammatory paracrine mechanisms.

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.

Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21.

Cholangiocarcinoma (CC) has a poor prognosis and different driver genes depending on the site of onset. Intrahepatic CC is the second-most common liver cancer after hepatocellular carcinoma, and novel therapeutic targets are urgently needed. The present study was conducted to identify novel therapeutic targets by exploring differentially regulated genes in human CC. MicroRNA (miRNA) and mRNA microarrays were performed using tissue and serum samples obtained from 24 surgically resected hepatobiliary tumor cases, including 10 CC cases. We conducted principal component analysis to identify differentially expressed miRNA, leading to the identification of miRNA-3648 as a differentially expressed miRNA. We used an in silico screening approach to identify its target mRNA, the tumor suppressor Sloan Kettering Institute (SKI). SKI protein expression was decreased in human CC cells overexpressing miRNA-3648, endogenous SKI protein expression was decreased in human CC tumor tissues, and endogenous SKI mRNA expression was suppressed in human CC cells characterized by rapid growth. SKI-overexpressing OZ cells (human intrahepatic CC cells) showed upregulation of cyclin-dependent kinase inhibitor p21 mRNA and protein expression and suppressed cell proliferation. Nuclear expression of CDT1 (chromatin licensing and DNA replication factor 1), which is required for the G1/S transition, was suppressed in SKI-overexpressing OZ cells. SKI knockdown resulted in the opposite effects. Transgenic p21-luciferase was activated in SKI-overexpressing OZ cells. These data indicate SKI involvement in p21 transcription and that SKI-p21 signaling causes cell cycle arrest in G1, suppressing intrahepatic CC cell growth. Therefore, SKI may be a potential therapeutic target for intrahepatic CC.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

Glass-patternable notch-shaped microwave architecture for on-chip spin detection in biological samples.

We report a notch-shaped coplanar microwave waveguide antenna on a glass plate designed for on-chip detection of optically detected magnetic resonance (ODMR) of fluorescent nanodiamonds (NDs). A lithographically patterned thin wire at the center of the notch area in the coplanar waveguide realizes a millimeter-scale ODMR detection area (1.5 × 2.0 mm2) and gigahertz-broadband characteristics with low reflection (∼8%). The ODMR signal intensity in the detection area is quantitatively predictable by numerical simulation. Using this chip device, we demonstrate a uniform ODMR signal intensity over the detection area for cells, tissue, and worms. The present demonstration of a chip-based microwave architecture will enable scalable chip integration of ODMR-based quantum sensing technology into various bioassay platforms.

Wide-field fluorescent nanodiamond spin measurements toward real-time large-area intracellular thermometry.

Measuring optically detected magnetic resonance (ODMR) of diamond nitrogen vacancy centers significantly depends on the photon detectors used. We study camera-based wide-field ODMR measurements to examine the performance in thermometry by comparing the results to those of the confocal-based ODMR detection. We show that the temperature sensitivity of the camera-based measurements can be as high as that of the confocal detection and that possible artifacts of the ODMR shift are produced owing to the complexity of the camera-based measurements. Although measurements from wide-field ODMR of nanodiamonds in living cells can provide temperature precisions consistent with those of confocal detection, the technique requires the integration of rapid ODMR measurement protocols for better precisions. Our results can aid the development of camera-based real-time large-area spin-based thermometry of living cells.

Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Stress can attenuate hepatic lipid accumulation via elevation of hepatic ß-muricholic acid levels in mice with nonalcoholic steatohepatitis.

Stress can affect our body and is known to lead to some diseases. However, the influence on the development of nonalcohol fatty liver disease (NAFLD) remains unknown. This study demonstrated that chronic restraint stress attenuated hepatic lipid accumulation via elevation of hepatic ß-muricholic acid (ßMCA) levels in the development of nonalcoholic steatohepatitis (NASH) in mice. Serum cortisol and corticosterone levels, i.e., human and rodent stress markers, were correlated with serum bile acid levels in patients with NAFLD and methionine- and choline-deficient (MCD) diet-induced mice, respectively, suggesting that stress is related to bile acid (BA) homeostasis in NASH. In the mouse model, hepatic ßMCA and cholic acid (CA) levels were increased after the stress challenge. Considering that a short stress enhanced hepatic CYP7A1 protein levels in normal mice and corticosterone increased CYP7A1 protein levels in primary mouse hepatocytes, the enhanced Cyp7a1 expression was postulated to be involved in the chronic stress-increased hepatic ßMCA level. Interestingly, chronic stress decreased hepatic lipid levels in MCD-induced NASH mice. Furthermore, ßMCA suppressed lipid accumulation in mouse primary hepatocytes exposed to palmitic acid/oleic acid, but CA did not. In addition, Cyp7a1 expression seemed to be related to lipid accumulation in hepatocytes. In conclusion, chronic stress can change hepatic lipid accumulation in NASH mice, disrupting BA homeostasis via induction of hepatic Cyp7a1 expression. This study discovered a new ßMCA action in the liver, indicating the possibility that ßMCA is available for NAFLD therapy.

Anti-fibrotic treatments for chronic liver diseases: The present and the future.

Liver fibrosis reflects tissue scarring in the liver due to the accumulation of excessive extracellular matrix in response to chronically persistent liver injury. Hepatocyte cell death can trigger capillarization of liver sinusoidal endothelial cells, stimulation of immune cells including macrophages and Kupffer cells, and activation of hepatic stellate cells (HSCs), resulting in progression of liver fibrosis. Liver cirrhosis is the terminal state of liver fibrosis and is associated with severe complications, such as liver failure, portal hypertension, and liver cancer. Nevertheless, effective therapy for cirrhosis has not yet been established, and liver transplantation is the only radical treatment for severe cases. Studies investigating HSC activation and regulation of collagen production in the liver have made breakthroughs in recent decades that have advanced the knowledge regarding liver fibrosis pathophysiology. In this review, we summarize molecular mechanisms of liver fibrosis and discuss the development of novel anti-fibrotic therapies.

Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin-Containing Adherens Junctions with Hepatocytes.

Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride-induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin-containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-ß, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes.

TGF-ß1-driven reduction of cytoglobin leads to oxidative DNA damage in stellate cells during non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species. The molecular role of CYGB in human hepatic stellate cell (HSC) activation and human liver disease remains uncharacterised. The aim of this study was to reveal the mechanism by which the TGF-ß1/SMAD2 pathway regulates the human CYGB promoter and the pathophysiological function of CYGB in human non-alcoholic steatohepatitis (NASH). METHODS: Immunohistochemical staining was performed using human NASH biopsy specimens. Molecular and biochemical analyses were performed by western blotting, quantitative PCR, and luciferase and immunoprecipitation assays. Hydroxyl radicals (•OH) and oxidative DNA damage were measured using an •OH-detectable probe and 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA. RESULTS: In culture, TGF-ß1-pretreated human HSCs exhibited lower CYGB levels - together with increased NADPH oxidase 4 (NOX4) expression - and were primed for H2O2-triggered •OH production and 8-OHdG generation; overexpression of human CYGB in human HSCs reversed these effects. Electron spin resonance demonstrated the direct •OH scavenging activity of recombinant human CYGB. Mechanistically, pSMAD2 reduced CYGB transcription by recruiting the M1 repressor isoform of SP3 to the human CYGB promoter at nucleotide positions +2-+13 from the transcription start site. The same repression did not occur on the mouse Cygb promoter. TGF-ß1/SMAD3 mediated αSMA and collagen expression. Consistent with observations in cultured human HSCs, CYGB expression was negligible, but 8-OHdG was abundant, in activated αSMA+pSMAD2+- and αSMA+NOX4+-positive hepatic stellate cells from patients with NASH and advanced fibrosis. CONCLUSIONS: Downregulation of CYGB by the TGF-ß1/pSMAD2/SP3-M1 pathway brings about •OH-dependent oxidative DNA damage in activated hepatic stellate cells from patients with NASH. LAY SUMMARY: Cytoglobin (CYGB) is a respiratory protein that acts as a scavenger of reactive oxygen species and protects cells from oxidative DNA damage. Herein, we show that the cytokine TGF-ß1 downregulates human CYGB expression. This leads to oxidative DNA damage in activated hepatic stellate cells. Our findings provide new insights into the relationship between CYGB expression and the pathophysiology of fibrosis in patients with non-alcoholic steatohepatitis.

Adrenoceptor-related decrease in serum triglycerides is independent of PPARα activation.

Adrenoceptor (AR)-linked pathways belong to the major components of the stress response system and are associated with the pathophysiology of diseases within the spectrum of metabolic syndrome. In this study, the role of adrenoceptor stimulation in serum triglyceride (TG) regulation in mice was investigated. For this purpose, α1 -ARs were activated with phenylephrine (PH) and ß1/2 -ARs with isoprenaline (ISOP). Both AR agonists markedly reduced serum TG levels independently of PPARα activation. These drugs also significantly activated the hormone-sensitive lipase in the white adipose tissue indicating increased mobilization of TGs in this tissue. In addition, PH and ISOP up-regulated Lpl, Nr4A, Dgat1, Mttp, Aadac and Cd36 genes, critical in TG regulation, whereas the observed decrease in serum TG levels was independent of the hepatic very low-density lipoprotein (VLDL)-TG secretion. Interestingly, PH and ISOP also inactivated the hepatic insulin/PI3k/AKT/FoxO1 signaling pathway, holding a critical role in the regulation of genes involved in TG synthesis. Taken together, the findings of the present study indicate that stimulation of α1 - and ß1/2 -ARs markedly reduced serum TG steady-state levels as a result of alterations in TG synthesis, uptake, transport, hydrolysis, metabolism and clearance, an effect induced by PPARα independent mechanisms.

Adrenoceptor-stimulated inflammatory response in stress-induced serum amyloid A synthesis.

RATIONALE: Stressful life events are suggested to contribute to the development of various pathologies, such as cardiovascular disorders, whose etiopathogenesis is highly associated with elevated levels of serum amyloid A (SAA) proteins. SAA synthesis in the liver is regulated by a complex network of cytokines acting independently or in concert with various hormones/stimulants including the stress-activated sympathetic nervous system. OBJECTIVE: This study aims to investigate the underlying mechanisms that regulate the stress-induced hepatic synthesis of SAA, with particular focus on adrenoceptors (AR), major components of the sympathoadrenal response to stress. METHODS AND RESULTS: We demonstrated that repeated stress elevates IL-1ß, IL-6, and TNFα serum levels in mice, accompanied by increased synthesis and secretion of hepatic SAA1/2 and SAA3, an effect that was blocked by AR antagonists. Moreover, stimulation of α1- and ß1/2-ARs mimics the stress effect on SAA1/2 regulation, whereas α2-AR stimulation exhibits a relatively weak impact on SAA. In support of the essential cytokine contribution in the AR-agonist induced SAA production is the fact that the anti-inflammatory drug, sodium salicylate, prevented the AR-stimulated hepatic SAA1/2 synthesis by reducing IL-1ß levels, whereas IL-1ß inhibition with Anakinra mimics this sodium salicylate preventive effect, thus indicating a crucial role for IL-1ß. Interestingly, the AR-driven SAA3 synthesis was elevated by sodium salicylate in a TNFα-dependent way, supporting diverse and complex regulatory roles of cytokines in SAA production. In contrast to α1/α2-AR, the ß1/2-AR-mediated SAA1/2 and SAA3 upregulation cannot be reversed by fenofibrate, a hypolipidemic drug with anti-inflammatory properties. CONCLUSION: Taken together, these findings strongly support a critical role of the AR-stimulated inflammatory response in the hepatic SAA production under stressful conditions, highlighting distinct AR type-specific mechanisms that regulate the hepatic synthesis of SAA1/2 and SAA3.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated ß-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling.

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN-targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5'-TGA(C/G)TCA), located -218 to -222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct-ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.

Lack of epithelial PPARγ causes cystic adenomatoid malformations in mouse fetal lung.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (PpargΔLuEpC) were developed using Cre-Lox system. PpargΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of PpargΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of PpargΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the PpargΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system.

St. John's Wort Attenuates Colorectal Carcinogenesis in Mice through Suppression of Inflammatory Signaling.

Despite widespread use as well as epidemiologic indications, there have been no investigations into the effect of St. John's wort (SJW) extract on colorectal carcinogenesis in vivo. This study reports a systematic evaluation of the impact of dietary supplementation of SJW extract on azoxymethane-induced colorectal carcinogenesis in mice. Mice were fed with either AIN-93G (control) diet or SJW extract-supplemented diet (SJW diet) prior to azoxymethane treatment. SJW diet was found to significantly improve the overall survival of azoxymethane-treated mice. Pretreatment with the SJW diet significantly reduced body weight loss as well as decrease of serum albumin and cholesterol levels associated with azoxymethane-induced colorectal tumorigenesis. SJW diet-fed mice showed a significant decrease in tumor multiplicity along with a decrease in incidence of large tumors and a trend toward decreased total tumor volume in a dose-dependent manner. A short-term study, which examined the effect of SJW prior to rectal bleeding, also showed decrease in colorectal polyps in SJW diet-fed mice. Nuclear factor kappa B (NF-κB) and extracellular signal-regulated kinase (ERK1/2) pathways were attenuated by SJW administration. SJW extract resulted in early and continuous attenuation of these pathways in the colon epithelium of SJW diet-fed mice under both short-term and long-term treatment regimens. In conclusion, this study demonstrated the chemopreventive potential of SJW extract against colorectal cancer through attenuation of proinflammatory processes.

Fat-Specific Protein 27/CIDEC Promotes Development of Alcoholic Steatohepatitis in Mice and Humans.

BACKGROUND & AIMS: Alcoholic steatohepatitis (ASH) is the progressive form of alcoholic liver disease and may lead to cirrhosis and hepatocellular carcinoma. We studied mouse models and human tissues to identify molecules associated with ASH progression and focused on the mouse fat-specific protein 27 (FSP-27)/human cell death-inducing DFF45-like effector C (CIDEC) protein, which is expressed in white adipose tissues and promotes formation of fat droplets. METHODS: C57BL/6N mice or mice with hepatocyte-specific disruption of Fsp27 (Fsp27(Hep-/-) mice) were fed the Lieber-Decarli ethanol liquid diet (5% ethanol) for 10 days to 12 weeks, followed by 1 or multiple binges of ethanol (5 or 6 g/kg) during the chronic feeding. Some mice were given an inhibitor (GW9662) of peroxisome proliferator-activated receptor γ (PPARG). Adenoviral vectors were used to express transgenes or small hairpin (sh) RNAs in cultured hepatocytes and in mice. Liver tissue samples were collected from ethanol-fed mice or from 31 patients with alcoholic hepatitis (AH) with biopsy-proved ASH and analyzed histologically and immunohistochemically and by transcriptome, immunoblotting, and real-time PCR analyses. RESULTS: Chronic-plus-binge ethanol feeding of mice, which mimics the drinking pattern of patients with AH, produced severe ASH and mild fibrosis. Microarray analyses revealed similar alterations in expression of many hepatic genes in ethanol-fed mice and humans with ASH, including up-regulation of mouse Fsp27 (also called Cidec) and human CIDEC. Fsp27(Hep-/-) mice and mice given injections of adenovirus-Fsp27shRNA had markedly reduced ASH following chronic-plus-binge ethanol feeding. Inhibition of PPARG and cyclic AMP-responsive element binding protein H (CREBH) prevented the increases in Fsp27α and FSP27ß mRNAs, respectively, and reduced liver injury in this chronic-plus-binge ethanol feeding model. Overexpression of FSP27 and ethanol exposure had synergistic effects in inducing production of mitochondrial reactive oxygen species and damage to hepatocytes in mice. Hepatic CIDEC mRNA expression was increased in patients with AH and correlated with the degree of hepatic steatosis and disease severity including mortality. CONCLUSIONS: In mice, chronic-plus-binge ethanol feeding induces ASH that mimics some histological and molecular features observed in patients with AH. Hepatic expression of FSP27/CIDEC is highly up-regulated in mice following chronic-plus-binge ethanol feeding and in patients with AH; this up-regulation contributes to alcohol-induced liver damage.