Yulia A. Nevzorova

The anti-fibrotic effects of CCN1/CYR61 in primary portal myofibroblasts are mediated through induction of reactive oxygen species resulting in cellular senescence, apoptosis and attenuated TGF-β signaling.

UNLABELLED Cysteine-rich protein 61 (CCN1/CYR61) is a CCN (CYR61, CTGF (connective tissue growth factor), and NOV (Nephroblastoma overexpressed gene)) family matricellular protein comprising six secreted CCN proteins in mammals. CCN1/CYR61 expression is associated with inflammation and injury repair. Recent studies show that CCN1/CYR61 limits fibrosis in models of cutaneous wound healing by inducing cellular senescence in myofibroblasts of the granulation tissue which thereby transforms into an extracellular matrix-degrading phenotype. We here investigate CCN1/CYR61 expression in primary profibrogenic liver cells (i.e., hepatic stellate cells and periportal myofibroblasts) and found an increase of CCN1/CYR61 expression during early activation of hepatic stellate cells that declines in fully transdifferentiated myofibroblasts. By contrast, CCN1/CYR61 levels found in primary parenchymal liver cells (i.e., hepatocytes) were relatively low compared to the levels exhibited in hepatic stellate cells and portal myofibroblasts. In models of ongoing liver fibrogenesis, elevated levels of CCN1/CYR61 were particularly noticed during early periods of insult, while expression declined during prolonged phases of fibrogenesis. We generated an adenovirus type 5 encoding CCN1/CYR61 (i.e., Ad5-CMV-CCN1/CYR61) and overexpressed CCN1/CYR61 in primary portal myofibroblasts. Interestingly, overexpressed CCN1/CYR61 significantly inhibited production of collagen type I at both mRNA and protein levels as evidenced by quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. CCN1/CYR61 further induces production of reactive oxygen species (ROS) leading to dose-dependent cellular senescence and apoptosis. Additionally, we demonstrate that CCN1/CYR61 attenuates TGF-β signaling by scavenging TGF-β thereby mitigating in vivo liver fibrogenesis in a bile duct ligation model. CONCLUSION In line with dermal fibrosis and scar formation, CCN1/CYR61 is involved in liver injury repair and tissue remodeling. CCN1/CYR61 gene transfer into extracellular matrix-producing liver cells is therefore potentially beneficial in liver fibrotic therapy.

Aberrant Cell Cycle Progression and Endoreplication in Regenerating Livers of Mice That Lack a Single E-Type Cyclin

BACKGROUND & AIMS E-cyclins control the transition of quiescent cells into the cell cycle. Two E-cyclins, CcnE1 and CcnE2, have been described, but their specific contributions to cell cycle reentry in vivo are poorly understood. Liver regeneration following partial hepatectomy is an excellent in vivo model for the study of cell cycle reentry of quiescent cells. We investigated the relevance of E-cyclins in directing resting hepatocytes into the cell cycle after partial hepatectomy using CcnE1 and CcnE2 knockout mice. METHODS Partial hepatectomy (70%) was performed in CcnE1 (E1(-/-)) and CcnE2 (E2(-/-)) knockout and wild-type mice. Liver regeneration was monitored by cell cycle markers for G(1)/S phase, S phase, and M phase as well as by determining the liver/body weight ratio after partial hepatectomy. Ploidy of hepatocytes was determined by fluorescence-activated cell sorting and fluorescent in situ hybridization. RESULTS CcnE1 deletion resulted in normal liver regeneration with a slight delay of the G(1)/S-phase transition and a defect in endoreplication of otherwise polyploid hepatocytes. Surprisingly, E2(-/-) mice displayed accelerated and sustained DNA synthesis after partial hepatectomy, excessive endoreplication in hepatocytes, and a liver mass that was 45% greater than that of wild-type mice after termination of the regeneration process. CcnE2 depletion induced overexpression of CcnE1 and prolonged cdk2 kinase activity after partial hepatectomy. CONCLUSIONS CcnE2 has an unexpected role in repressing CcnE1; the phenotype of E2(-/-) mice appears to result from CcnE1 overexpression and cdk2 hyperactivation. CcnE1 and CcnE2 therefore have nonredundant functions for S-phase entry and endoreplication during liver regeneration.

Overexpression of c-myc in hepatocytes promotes activation of hepatic stellate cells and facilitates the onset of liver fibrosis.

BACKGROUND Liver fibrosis is a consequence of chronic liver injury and can further progress to hepatocellular carcinoma (HCC). Fibrogenesis involves activation of hepatic stellate cells (HSC) and proliferation of hepatocytes upon liver injury. HCC is frequently associated with overexpression of the proto-oncogene c-myc. However, the impact of c-myc for initiating pathological precursor stages such as liver fibrosis is poorly characterized. In the present study we thus investigated the impact of c-myc for liver fibrogenesis. METHODS Expression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by quantitative real-time PCR (qPCR). Primary HSC were isolated from mice with transgenic overexpression of c-myc in hepatocytes (alb-myc(tg)) and wildtype (WT) controls and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Liver fibrosis in WT and alb-myc(tg) mice was induced by repetitive CCl4 treatment. RESULTS We detected strong up-regulation of hepatic c-myc in patients with advanced liver fibrosis. In return, overexpression of c-myc in alb-myc(tg) mice resulted in increased liver collagen deposition and induction of α-smooth-muscle-actin indicating HSC activation. Primary HSC derived from alb-myc(tg) mice showed enhanced proliferation and accelerated transdifferentiation into myofibroblasts in vitro. Accordingly, fibrosis initiation in vivo after chronic CCl4 treatment was accelerated in alb-myc(tg) mice compared to controls. CONCLUSION Overexpression of c-myc is a novel marker of liver fibrosis in man and mice. We conclude that chronic induction of c-myc especially in hepatocytes has the potential to prime resident HSC for activation, proliferation and myofibroblast differentiation.

Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice.

Liver fibrogenesis is associated with the transition of quiescent hepatocytes and hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E‐type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E‐type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild‐type (WT) livers after periodical administration of the profibrotic toxin, CCl4. To further evaluate the potential function of E‐type cyclins for liver fibrogenesis, we repetitively treated constitutive CcnE1−/− and CcnE2−/− knock‐out mice with CCl4 to induce liver fibrosis. Interestingly, CcnE1−/− mice were protected against CCl4‐mediated liver fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2−/− mice showed accelerated fibrogenesis after CCl4 treatment. We isolated primary HSCs from WT, CcnE1−/−, and CcnE2−/− mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1−/− HSCs showed dramatically impaired survival, cell‐cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2‐deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell‐cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis. (HEPATOLOGY 2012;56:1140–1149)

Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis

Objective The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. Design Jnk1f/f wildtype (WT), Jnk1−/− and Jnk1Δhepa (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. Results Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1−/− compared with WT and Jnk1Δhepa mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and α-smooth muscle actin expression were downregulated in Jnk1−/− compared with WT and Jnk1Δhepa livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow–derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1−/− livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. Conclusions Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.

Concurrent deletion of cyclin E1 and cyclin‐dependent kinase 2 in hepatocytes inhibits DNA replication and liver regeneration in mice

The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1‐S cell‐cycle transition of primed hepatocytes is regulated by complexes of cyclin‐dependent kinase 2 (Cdk2) with E‐type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E‐cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double‐ and triple‐knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre‐replication complex (pre‐RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre‐RC formation and further led to dramatically impaired S‐phase progression by down‐regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S‐phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. Conclusion: In the absence of Cdk2, CcnE1 performs crucial kinase‐independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S‐phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S‐phase machinery in vivo. (Hepatology 2014;59:651–660)

Loss of caspase‐8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Expression of a cyclin E1 isoform in mice is correlated with the quiescent cell cycle status of hepatocytes in vivo

Cyclin E1 controls G1/S phase transition of the eukaryotic cell cycle. We report the impact of alternative spliced cyclin E1 isoforms on cell cycle regulation in hepatocytes. We show that expression of new cyclin E1 mRNA variants IN3, Δ4, and Δ5 is associated with retarded proliferation in murine hepatocellular carcinoma. Additionally, we demonstrate that a new cyclin E1 isoform Δ3/8 lacking the central part of wild‐type mRNA is expressed predominantly in nonproliferating murine hepatocytes. Following partial hepatectomy, Δ3/8 is downregulated when hepatocytes enter the cell cycle from quiescence. The Δ3/8 protein does not exhibit any cyclin box motif but binds cyclin‐dependent kinase 2 without stimulating kinase activity. We demonstrate that Δ3/8 lacks any nuclear localization signal and is exclusively located in the cytoplasm. Overexpression of Δ3/8 in cultured cells leads to a delayed G0‐G1 transition, indicating that this splice variant helps to maintain a quiescent state of hepatocytes. In conclusion, we identified an isoform of cyclin E1 involved in G0 maintenance and suggest an additional mechanism for cell cycle control. (HEPATOLOGY 2006;44:164–173.)

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (NemoΔhepa) with constitutive TNFR1−/− and TRAIL−/− mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.

Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model

Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130Δhepa) and control animals (gp130f/f) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0–144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130Δhepa animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130Δhepa mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130f/f and gp130Δhepa animals. However, gp130Δhepa livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-β (TGFβ), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.