Inhibits Hepatocyte Proliferation Research Articles

Prenatal dexamethasone exposure caused fetal rats liver dysplasia by inhibiting autophagy-mediated cell proliferation

As a synthetic glucocorticoid, dexamethasone has been widely used in the clinical treatment of premature birth and related pregnant diseases, but its clinical use is still controversial due to developmental toxicity. This study aimed to confirm the proliferation inhibitory effect of pregnant dexamethasone exposure (PDE) on fetal liver development and elucidate its molecular mechanism. In vitro studies, we found that dexamethasone inhibited hepatocyte proliferation through autophagy activated by glucocorticoid receptor (GR)-forkhead protein O1 (FOXO1) pathway. Subsequently, in vivo, we confirmed in a PDE rat model that male fetal liver proliferation was inhibited, and the expression of the GR-FOXO1 pathway and autophagy were increased. Taken together, PDE induces autophagy by activating the GR-FOXO1 pathway, which leads to fetal liver proliferation inhibition and dysplasia in offspring rats. This study confirmed that dexamethasone activates cell autophagy in utero through the GR-FOXO1 pathway, thereby inhibiting hepatocyte proliferation and liver development, which provides theoretical basis for understanding the developmental toxicity of dexamethasone and guiding the rational clinical use.

Hepatic Hippo signaling inhibits development of hepatocellular carcinoma.

Primary liver cancer is one of the most common cancer worldwide. Hepatocellular carcinoma (HCC) in particular, is the second leading cause of cancer deaths in the world. The Hippo signaling pathway has emerged as a major oncosuppressive pathway that plays critical roles inhibiting hepatocyte proliferation, survival, and HCC formation. A key component of the Hippo pathway is the inhibition of yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) transcription factors by the Hippo kinase cascade. Aberrant activation of YAP or TAZ has been found in several human cancers including HCC. It is also well established that YAP/TAZ activation in hepatocytes causes HCC in mouse models, indicating that YAP/TAZ are potential therapeutic targets for human liver cancer. In this review, we summarize the recent findings regarding the multifarious roles of Hippo/YAP/TAZ in HCC development, and focus on their cell autonomous roles in controlling hepatocyte proliferation, differentiation, survival and metabolism as well as their non-cell autonomous in shaping the tumor microenvironment.

Brg1 regulates murine liver regeneration by targeting miR-187-5p dependent on Hippo signalling pathway.

Brg1 and Hippo signalling pathway are abnormally expressed in many malignant tumours, especially in Hepatocellular carcinoma, but their role in liver regeneration (LR) is unknown. In our research, we investigated the role of Brg1 and Hippo signalling pathway in hepatocyte proliferation and LR. Following 2/3 partial hepatectomy (PH) in liver‐specific Brg1 deleted mice (Brg1−/−) (KO) mice and sex‐matched wild‐type (WT), depletion of Brg1 in mouse embryos caused liver cell growth disorders and significantly decreased expression of miR‐187‐5p. We identified LATS1 as a target gene of miR‐187‐5p and the introduction of miR‐187‐5p decrease the expression of LATS1 and inactivated the Hippo signalling pathway, which facilitated the expression of cell cycle‐related proteins, and rescues the inhibitory effect of Brg1 in LR. Taken together, our findings suggested that deletion of Brg1 inhibits hepatocyte proliferation and LR by targeting miR‐187‐5p dependent on Hippo signalling pathway.

Abstract IA11: Hippo signaling Is Intrinsically regulated during cell cycle progression

Abstract The Hippo-YAP/TAZ signaling pathway plays a pivotal role in growth control during development and regeneration, and its dysregulation is widely implicated in various cancers. Hippo signaling has been recently identified as a major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival, and hepatocellular carcinoma (HCC) formation. We have found that Hippo/YAP signaling cell-autonomously inhibits HCC proliferation by interacting with Wnt and Notch signaling. We have also found that Hippo/YAP signaling cell-nonautonomously inhibits HCC by shaping its inflammatory microenvironment. To further understand the cellular and molecular mechanisms underlying Hippo signaling regulation, we have found that activities of core Hippo signaling components, LATS kinases and YAP/TAZ transcription factors, are regulated during cell cycle progression by core cell cycle machinery components. Furthermore, we show in Drosophila eye and wing development that cell cycle is required in vivo to regulate Hippo signaling with a conserved mechanism. Cell cycle control of Hippo signaling represents a previously unappreciated and evolutionarily conserved new layer of Hippo signaling regulation. Citation Format: Yingzi Yang, Wantae Kim, Xiaohui Wang. Hippo signaling Is Intrinsically regulated during cell cycle progression [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr IA11.

A negative reciprocal regulatory axis between cyclin D1 and HNF4α modulates cell cycle progression and metabolism in the liver

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.

Tmub1 Suppresses Hepatocellular Carcinoma by Promoting the Ubiquitination of ΔNp63 Isoforms

Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) inhibits hepatocyte proliferation during liver regeneration, but its role in hepatocellular carcinoma (HCC) has yet to be revealed. In this study, we show that the levels of Tmub1 were significantly lower in HCC tissues and cells than they were in adjacent tissues and normal hepatic cells, and the low levels of Tmub1 indicated a poor prognosis in HCC patients. Xenograft growth assay revealed that Tmub1 represses HCC growth in vivo. In addition, Tmub1 formed a protein complex with apoptosis-associated protein tumor protein 63 (p63), especially with the ΔN isoforms (ΔNp63α, β, and γ). Further loss- and gain-of-function analyses indicated that Tmub1 promotes apoptosis of Hep3B and MHCC-LM3 cells. Tmub1 decreased the protein expression of ΔNp63, and the pro-apoptotic effect of Tmub1 can be reversed by ΔNp63 isoforms (α, β, and γ). Additionally, we report that Tmub1 promotes the ubiquitination and degradation of ΔNp63 proteins. Finally, we confirmed in HCC tissues that Tmub1 is negatively correlated with ΔNp63 and positively correlated with the level of apoptosis. Taken together, Tmub1 suppresses HCC by enhancing the ubiquitination and degradation of ΔNp63 isoforms to induce HCC cell apoptosis. These findings provide a potential strategy for the management of HCC.

CAMKK2‐dependent AMPK activation is required to drive cell cycle progression and hepatocyte proliferation after partial hepatectomy in the rat

Liver regeneration is a unique repair response to tissue damage where normally quiescent liver cells reenter the cell cycle and undergo cell division, regaining the damaged or lost tissue, while maintaining the differentiated function of the tissue. In previous studies, we and others demonstrated that 70% partial hepatectomy (PHx) in the rat leads to an instantaneous (within 30 sec) release of up to 25% of ATP from the remnant liver. Released ATP served to activate purinergic receptors that enhance cellular stress responses and immediate‐early gene expression. ATP loss was mediated at least in part by a transient opening of connexin hemichannels and activation of stress signals and cell cycle progression was inhibited by hemichannel blocking peptides. The low ATP levels persist through the cell cycle progression and tissue ATP levels did not recover until 24–48 hrs post‐PHx. In the current study, we assessed the intracellular signaling events initiated by ATP loss. The decline in tissue ATP levels was accompanied by a persistent increase in ATP/AMP ratio and a transient activation of AMPK in the remnant liver, which peaked within 1 min after PHx, but then declined to basal levels within 15 min, despite the persistence of a low ATP/AMP. A second, extended phase of AMPK activation occurred, which peaked at 12–24 hrs after 70% PHx without being accompanied by major changes in tissue ATP/AMP ratios. This secondary AMPK activation in the regenerating rat liver accompanied cell cycle progression and DNA synthesis through G1‐S phase. After 30% PHx, a model that exhibits the hepatocyte priming events, but does not display the synchronized cell cycle progression and proliferation response, the initial ATP release and signaling events were comparable, but the secondary peak of AMPK activation is absent in the 30% PHx. Treatment with STO‐609, a selective inhibitor of CAMKK2, suppressed the second AMPK activation peak and inhibited hepatocyte proliferation after 70% PHx, suggesting the importance of calcium signaling in the regenerating liver. We conclude that an AMP‐independent CAMKK2‐mediated activation of AMPK is essential for effective cell cycle progression after PHx.Support or Funding InformationSupported by AA018873This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatocyte Nuclear Factor 4 Alpha Activation Is Essential for Termination of Liver Regeneration in Mice.

Hepatocyte nuclear factor 4 alpha (HNF4α) is critical for hepatic differentiation. Recent studies have highlighted its role in inhibition of hepatocyte proliferation and tumor suppression. However, the role of HNF4α in liver regeneration (LR) is not known. We hypothesized that hepatocytes modulate HNF4α activity when navigating between differentiated and proliferative states during LR. Western blotting analysis revealed a rapid decline in nuclear and cytoplasmic HNF4α protein levels, accompanied with decreased target gene expression, within 1 hour after two-thirds partial hepatectomy (post-PH) in C57BL/6J mice. HNF4α protein expression did not recover to pre-PH levels until day 3. Hepatocyte-specific deletion of HNF4α (HNF4α-KO [knockout]) in mice resulted in 100% mortality post-PH, despite increased proliferative marker expression throughout regeneration. Sustained loss of HNF4α target gene expression throughout regeneration indicated that HNF4α-KO mice were unable to compensate for loss of HNF4α transcriptional activity. Deletion of HNF4α resulted in sustained proliferation accompanied by c-Myc and cyclin D1 overexpression and a complete deficiency of hepatocyte function after PH. Interestingly, overexpression of degradation-resistant HNF4α in hepatocytes delayed, but did not prevent, initiation of regeneration after PH. Finally, adeno-associated virus serotype 8 (AAV8)-mediated reexpression of HNF4α in hepatocytes of HNF4α-KO mice post-PH restored HNF4α protein levels, induced target gene expression, and improved survival of HNF4α-KO mice post-PH. Conclusion: In conclusion, these data indicate that HNF4α reexpression following initial decrease is critical for hepatocytes to exit from cell cycle and resume function during the termination phase of LR. These results indicate the role of HNF4α in LR and have implications for therapy of liver failure.

Evidence for a Novel Regulatory Interaction Involving Cyclin D1, Lipid Droplets, Lipolysis, and Cell Cycle Progression in Hepatocytes.

During normal proliferation, hepatocytes accumulate triglycerides (TGs) in lipid droplets (LDs), but the underlying mechanisms and functional significance of this steatosis are unknown. In the current study, we examined the coordinated regulation of cell cycle progression and LD accumulation. As previously shown, hepatocytes develop increased LD content after mitogen stimulation. Cyclin D1, in addition to regulating proliferation, was both necessary and sufficient to promote LD accumulation in response to mitogens. Interestingly, cyclin D1 promotes LD accumulation by inhibiting the breakdown of TGs by lipolysis through a mechanism involving decreased lipophagy, the autophagic degradation of LDs. To examine whether inhibition of lipolysis is important for cell cycle progression, we overexpressed adipose TG lipase (ATGL), a key enzyme involved in TG breakdown. As expected, ATGL reduced LD content but also markedly inhibited hepatocyte proliferation, suggesting that lipolysis regulates a previously uncharacterized cell cycle checkpoint. Consistent with this, in mitogen‐stimulated cells with small interfering RNA‐mediated depletion of cyclin D1 (which inhibits proliferation and stimulates lipolysis), concurrent ATGL knockdown restored progression into S phase. Following partial hepatectomy, a model of robust hepatocyte proliferation in vivo, ATGL overexpression led to decreased LD content, cell cycle inhibition, and marked liver injury, further indicating that down‐regulation of lipolysis is important for normal hepatocyte proliferation. Conclusion: We suggest a new relationship between steatosis and proliferation in hepatocytes: cyclin D1 inhibits lipolysis, resulting in LD accumulation, and suppression of lipolysis is necessary for cell cycle progression.

MiR-429 regulates rat liver regeneration and hepatocyte proliferation by targeting JUN/MYC/BCL2/CCND1 signaling pathway

Increasing evidence indicates that miR-429 is involved in tumor suppression in various human cancers. However, its role in liver regeneration remains unexplored. Liver regeneration is a highly orchestrated process that can be regulated by microRNAs (miRNAs), although the mechanisms are largely unclear. In this study, we aimed to identify the role of miR-429 in hepatocyte proliferation during liver regeneration. First, we performed microarray analysis and qRT-PCR. Results indicated that miR-429 level in rat liver markedly decreased 30 h after partial hepatectomy, and miR-429 overexpression disrupted BRL-3A proliferation and the transition of G1 to S phase in rat hepatocyte and promoted hepatocyte apoptosis. By contrast, miR-429 down-regulation had inverse effects. MiR-429 negatively regulated JUN expression in vitro and in vivo. After using JUN siRNA, we found that JUN inhibition mediates the effect of miR-429 in hepatocyte proliferation and growth and miR-429 negatively regulates JUN/MYC/BCL2/CCND1 signaling pathways. Our results also indicated that miR-429 inhibits hepatocyte proliferation and liver regeneration by targeting JUN/MYC/BCL2/CCND1.

Notch signaling pathway regulates cell cycle in proliferating hepatocytes involved in liver regeneration

It has been well documented that Notch signaling is involved in liver regeneration. However, the exact molecular mechanism mediating this process is not fully elucidated. The current study aimed to investigate the role of Notch signaling regulating cell cycle in proliferating hepatocytes in liver regeneration after partial hepatectomy (PHx, 67% resection) and the related molecular mechanism. Partial hepatectomy was performed in Sprague Dawley rats, and remnant livers were harvested 0, 1, 3, 5, and 7days after operation, and primary hepatocytes were isolated to investigate the molecular mechanism. Notch signaling activation and hepatocyte proliferation were significantly increased after PHx, while treatment with FLI-06, the inhibitor of γ-secreting enzyme, blocked these trends. Besides, inhibition of Notch signaling led to dysregulation of cell cycle and cell-cycle components. Furthermore, Akti-1/2 (a selective Akt inhibitor) and PX-478 (a selective Hif-1α inhibitor) inhibited hepatocyte proliferation and liver regeneration after PHx, and the effect of downstream molecules activation by Jagged-1 (Notch-1 ligand) in hepatocytes was abolished by FLI-06, Akti-1/2, and PX-478. The current study demonstrated for the first time that Notch signaling regulated cell cycle in proliferating hepatocytes involved in liver regeneration through NICD/Akt Akt/Hif-1α pathway.

Effect of perfluorooctane sulphonate-induced Kupffer cell activation on hepatocyte proliferation through the NF-κB/TNF-α/IL-6-dependent pathway

Perfluorooctane sulfonate (PFOS), one member of polyfluoroalkyl chemicals (PFASs), persist in the environment and are found in relatively high concentrations in animal livers. PFOS has been shown to induce tumour of the liver in rats following chronic dietary administration. However, the molecular mechanisms involved in PFOS-induced hepatocellular hypertrophy are still not well characterized. In this study, male Sprague-Dawley rats were daily gavaged with PFOS (1 or 10 mg/kg body weight) for 28 days. Rat primary cultured Kupffer cells or hepatocytes were exposed to 100 μM PFOS for 0–48 h. Our results showed that PFOS exposure caused serious hepatocellular damage and obvious inflammatory cell infiltration and increased serum tumour necrosis factor-ɑ (TNF-α) and interleukin-6 (IL-6) levels. Particularly, PFOS exposure triggered Kupffer cell activation and significantly upregulated the expression of proliferating cell nuclear antigen (PCNA), c-Jun, c-MYC and Cyclin D1 (CyD1) in liver. In vitro, PFOS significantly induced production of TNF-α and IL-6 in Kupffer cells and increased PCNA, c-Jun, c-MYC and CyD1 expression in the primary hepatocytes co-cultured with Kupffer cells. However, Kupffer cell activation was mostly abolished by anti-TNF-α or anti-IL6 treatment. Furthermore, blockage of TNF-α and IL-6 significantly inhibited hepatocyte proliferation by gadolinium chloride (GdCl3) pre-treatment in PFOS-treated mice and primary cultured Kupffer cells. On the other hand, NF-κB inhibitor (PDTC) and c-Jun amino-terminal kinase (JNK) inhibitor (SP600125) significantly inhibited production of PFOS-induced TNF-α and IL-6. Taken together, these data suggest that PFOS induces Kupffer cell activation, leading to hepatocyte proliferation by through the NF-κB/TNF-ɑ/IL-6-dependent pathway.

The Effects of Betaine on the Nuclear Fractal Dimension, Chromatin Texture, and Proliferative Activity in Hepatocytes in Mouse Model of Nonalcoholic Fatty Liver Disease.

The effects of betaine on hepatocytes chromatin architecture changes were examined by using fractal and gray-level co-occurrence matrix (GLCM) analysis in methionine/choline-deficient (MCD) diet-induced, nonalcoholic fatty liver disease (NAFLD). Male C57BL/6 mice were divided into groups: (1) Control: standard diet; (2) BET: standard diet and betaine supplementation through drinking water (solution 1.5%); (3) MCD group: MCD diet for 6 weeks; (4) MCD+BET: fed with MCD diet + betaine for 6 weeks. Liver tissue was collected for histopathology, immunohistochemistry, and determination of fractal dimension and GLCM parameters. MCD diet induced diffuse micro- and macrovesicular steatosis accompanied with increased Ki67-positive hepatocyte nuclei. Steatosis and Ki67 immunopositivity were less prominent in the MCD+BET group compared with the MCD group. Angular second moment (ASM) and inverse difference moment (IDM) (textural homogeneity markers) were significantly increased in the MCD+BET group versus the MCD group (p<0.001), even though no difference between the MCD and the control group was evident. Heterogeneity parameters, contrast, and correlation were significantly increased in the MCD group versus the control (p<0.001). On the other hand, betaine treatment significantly reduced correlation, contrast, and entropy compared with the MCD group (p<0.001). Betaine attenuated MCD diet-induced NAFLD by reducing fat accumulation and inhibiting hepatocyte proliferation. Betaine supplementation increased nuclear homogeneity and chromatin complexity with reduction of entropy, contrast, and correlation.

Hepatic Hippo signaling inhibits protumoural microenvironment to suppress hepatocellular carcinoma

ObjectiveHippo signalling is a recently identified major oncosuppressive pathway that plays critical roles in inhibiting hepatocyte proliferation, survival and hepatocellular carcinoma (HCC) formation. Hippo kinase (Mst1 and Mst2) inhibits HCC...

The Inhibition of Indoleamine 2,3-Dioxygenase Accelerates Early Liver Regeneration in Mice After Partial Hepatectomy.

The inflammatory response accelerates early liver regeneration after liver injury and resection. Recent studies have demonstrated that indoleamine 2,3-dioxygenase-1 (IDO1) suppresses the activation of inflammatory cells and induces immune tolerance. In this study, we examined the role of IDO1 in liver regeneration after partial hepatectomy (PHx). WT or IDO1-knockout (IDO1-KO) mice received 70% PHx. The liver-body weight ratio after PHx was measured and hepatocyte growth was assessed by immunostaining. The expression of cell cycle genes and pro-inflammatory cytokines in the liver was analyzed by quantitative RT-PCR. In addition, 1-methyl-DL-tryptophan (1-MT), which is an IDO1 inhibitory agent, was given to WT mice and the liver-body weight ratio was measured after PHx. The liver-body weight ratio was significantly increased in IDO1-KO mice compared with that in WT mice after PHx. More Ki-67-positive cells were present in IDO1-KO mice than in WT mice after PHx. The expression of cell cycle genes (cyclin D1, cyclin E) and pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) was up-regulated in the remnant liver of IDO1-KO mice compared with WT mice. Moreover, treatment with 1-MT promoted liver regeneration. IDO1 deficiency promoted early liver regeneration after PHx, indicating that IDO1 suppresses the production of inflammatory cytokines and subsequently inhibits hepatocyte proliferation during liver regeneration.

Endothelial transcription factor KLF2 negatively regulates liver regeneration via induction of activin A

Endothelial cells (ECs) not only are important for oxygen delivery but also act as a paracrine source for signals that determine the balance between tissue regeneration and fibrosis. Here we show that genetic inactivation of flow-induced transcription factor Krüppel-like factor 2 (KLF2) in ECs results in reduced liver damage and augmentation of hepatocyte proliferation after chronic liver injury by treatment with carbon tetrachloride (CCl4). Serum levels of GLDH3 and ALT were significantly reduced in CCl4-treated EC-specific KLF2-deficient mice. In contrast, transgenic overexpression of KLF2 in liver sinusoidal ECs reduced hepatocyte proliferation. KLF2 induced activin A expression and secretion from endothelial cells in vitro and in vivo, which inhibited hepatocyte proliferation. However, loss or gain of KLF2 expression did not change capillary density and liver fibrosis, but significantly affected hepatocyte proliferation. Taken together, the data demonstrate that KLF2 induces an antiproliferative secretome, including activin A, which attenuates liver regeneration.

Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.

Peroxisome proliferator-activated receptor γ activation inhibits liver growth through miR-122-mediated downregulation of cMyc

Although NR1C3 agonists inhibit cell growth, the molecular mechanism of their action has not been thoroughly characterized to date. A recent study demonstrated that NR1C3 can regulate miR-122 by binding to its promoter. Given that miR-122 can indirectly regulate cMyc-mediated promitogenic signaling by targeting E2f1, we hypothesized that NR1C3 activation inhibits hepatocyte proliferation through miR-122-mediated cMyc downregulation. In the present study, we examined if liver hyperplasia induced by a strong chemical mitogen for the liver, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is an agonist of NR1I3, can be repressed by NR1C3 activation through miR-122 upregulation. Acute TCPOBOP treatment caused a significant increase in liver-to-body weight ratio. The liver mass increase was accompanied with miR-122 downregulation. ChIP assays demonstrated that TCPOBOP-activated NR1I3 accumulated on the DR1 site in the pri-miR-122 promoter; and the NR1I3 accumulation is accompanied by a decrease in miR-122 and an increase in E2f1 and its transcription target cMyc. Rosiglitazone (Ros) treatment, which is an agonist of NR1C3, caused an opposite effect on liver-to-body weight ratio. When Ros was given with TCPOBOP, it attenuated the inhibitory effect of TCPOBOP on miR-122. Moreover, Ros treatment inhibited the NR1I3 binding with the DR1 site in the pri-miR-122 promoter. Furthermore, the increase of miR-122 produced by Ros was correlated with the downregulation of its targets, E2f1 and cMyc. Thus, our finding demonstrated that the liver growth inhibitory effect of NR1C3 activation was at least partly related to the decrease of cMyc though the activation of miR-122 and the downregulation of E2f1.

Contribution of Mature Hepatocytes to Biliary Regeneration in Rats with Acute and Chronic Biliary Injury.

Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine (DAPM+D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM+D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation.

Partial Hepatectomy Induced Long Noncoding RNA Inhibits Hepatocyte Proliferation during Liver Regeneration.

Liver regeneration after partial hepatectomy (PHx) is a complex and well-orchestrated biological process in which synchronized cell proliferation is induced in response to the loss of liver mass. To define long noncoding RNAs (lncRNAs) that participate in the regulation of liver regeneration, we performed microarray analysis and identified more than 400 lncRNAs exhibiting significantly altered expression. Of these, one lncRNA, LncPHx2 (Long noncoding RNA induced by PHx 2), was highly upregulated during liver regeneration. Depletion of LncPHx2 during liver regeneration using antisense oligonucleotides led to a transient increase in hepatocyte proliferation and more rapid liver regeneration. Gene expression analysis showed that LncPHx2 depletion resulted in upregulation of mRNAs encoding proteins known to promote cell proliferation, including MCM components, DNA polymerases, histone proteins, and transcription factors. LncPHx2 interacts with the mRNAs of MCM components, making it a candidate to regulate the expression of MCMs and other genes post-transcriptionally. Collectively, our data demonstrate that LncPHx2 is a key lncRNA that participates in a negative feedback loop modulating hepatocyte proliferation through RNA-RNA interactions.