Yes-Associated Protein Is Crucial for Constitutive Androstane Receptor-Driven Hepatocyte Proliferation But Not for Induction of Drug Metabolism Genes in Mice.

BACKGROUND AND AIMS: Constitutive androstane receptor (CAR) agonists, such as 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes without any associated liver injury. Yes-associated protein (Yap) is a key transcription regulator that tightly controls organ size, including that of liver. Our and other previous studies suggested increased nuclear localization and activation of Yap after TCPOBOP treatment in mice and the potential role of Yap in CAR-driven proliferative response. Here, we investigated a direct role of Yap in CAR-driven hepatomegaly and hepatocyte proliferation using hepatocyte-specific Yap-knockout (KO) mice. APPROACH AND RESULTS: Adeno-associated virus 8-thyroxine binding globulin promoter-Cre recombinase vector was injected to Yap-floxed mice for achieving hepatocyte-specific Yap deletion followed by TCPOBOP treatment. Yap deletion did not decrease protein expression of CAR or CAR-driven induction of drug metabolism genes (including cytochrome P450 [Cyp] 2b10, Cyp2c55, and UDP-glucuronosyltransferase 1a1 [Ugt1a1]). However, Yap deletion substantially reduced TCPOBOP-induced hepatocyte proliferation. TCPOBOP-driven cell cycle activation was disrupted in Yap-KO mice because of delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma protein. Furthermore, the induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2, and B1), and important mitotic regulators (such as Aurora B kinase and polo-like kinase 1) was remarkably reduced in Yap-KO mice. Microarray analysis revealed that 26% of TCPOBOP-responsive genes that were mainly related to proliferation, but not to drug metabolism, were altered by Yap deletion. Yap regulated these proliferation genes through alerting expression of Myc and forkhead box protein M1, two critical transcriptional regulators of CAR-mediated hepatocyte proliferation. CONCLUSIONS: Our study revealed an important role of Yap signaling in CAR-driven hepatocyte proliferation; however, CAR-driven induction of drug metabolism genes was independent of Yap.

Cellular Location of HNF4α is Linked With Terminal Liver Failure in Humans.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post-translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure.

Correction: Synthesis of IL-6 by Hepatocytes Is a Normal Response to Common Hepatic Stimuli.

[This corrects the article DOI: 10.1371/journal.pone.0096053.].

Pharmacologic Inhibition of Epidermal Growth Factor Receptor Suppresses Nonalcoholic Fatty Liver Disease in a Murine Fast-Food Diet Model.

Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoyl-coenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD.

TCPOBOP-Induced Hepatomegaly and Hepatocyte Proliferation are Attenuated by Combined Disruption of MET and EGFR Signaling.

TCPOBOP (1,4-Bis [2-(3,5-Dichloropyridyloxy)] benzene) is a constitutive androstane receptor (CAR) agonist that induces robust hepatocyte proliferation and hepatomegaly without any liver injury or tissue loss. TCPOBOP-induced direct hyperplasia has been considered to be CAR-dependent with no evidence of involvement of cytokines or growth factor signaling. Receptor tyrosine kinases (RTKs), MET and epidermal growth factor receptor (EGFR), are known to play a critical role in liver regeneration after partial hepatectomy, but their role in TCPOBOP-induced direct hyperplasia, not yet explored, is investigated in the current study. Disruption of the RTK-mediated signaling was achieved using MET knockout (KO) mice along with Canertinib treatment for EGFR inhibition. Combined elimination of MET and EGFR signaling [MET KO + EGFR inhibitor (EGFRi)], but not individual disruption, dramatically reduced TCPOBOP-induced hepatomegaly and hepatocyte proliferation. TCPOBOP-driven CAR activation was not altered in [MET KO + EGFRi] mice, as measured by nuclear CAR translocation and analysis of typical CAR target genes. However, TCPOBOP-induced cell cycle activation was impaired in [MET KO + EGFRi] mice due to defective induction of cyclins, which regulate cell cycle initiation and progression. TCPOBOP-driven induction of FOXM1, a key transcriptional regulator of cell cycle progression during TCPOBOP-mediated hepatocyte proliferation, was greatly attenuated in [MET KO + EGFRi] mice. Interestingly, TCPOBOP treatment caused transient decline in hepatocyte nuclear factor 4 alpha expression concomitant to proliferative response; this was not seen in [MET KO + EGFRi] mice. Transcriptomic profiling revealed the vast majority (~40%) of TCPOBOP-dependent genes primarily related to proliferative response, but not to drug metabolism, were differentially expressed in [MET KO + EGFRi] mice. Conclusion: Taken together, combined disruption of EGFR and MET signaling lead to dramatic impairment of TCPOBOP-induced proliferative response without altering CAR activation.

Combined Systemic Disruption of MET and Epidermal Growth Factor Receptor Signaling Causes Liver Failure in Normal Mice.

MET and epidermal growth factor receptor (EGFR) tyrosine kinases are crucial for liver regeneration and normal hepatocyte function. Recently, we demonstrated that in mice, combined inhibition of these two signaling pathways abolished liver regeneration after hepatectomy, with subsequent hepatic failure and death at 15 to 18 days after resection. Morbidity was associated with distinct and specific alterations in important downstream signaling pathways that led to decreased hepatocyte volume, reduced proliferation, and shutdown of many essential hepatocyte functions, such as fatty acid synthesis, urea cycle, and mitochondrial functions. Herein, we explore the role of MET and EGFR signaling in resting mouse livers that are not subjected to hepatectomy. Mice with combined disruption of MET and EGFR signaling were noticeably sick by 10 days and died at 12 to 14 days. Mice with combined disruption of MET and EGFR signaling mice showed decreased liver/body weight ratios, increased apoptosis in nonparenchymal cells, impaired liver metabolic functions, and activation of distinct downstream signaling pathways related to inflammation, cell death, and survival. The present study demonstrates that, in addition to controlling the regenerative response, MET and EGFR synergistically control baseline liver homeostasis in normal mice in such a way that their combined disruption leads to liver failure and death.

Oct4 Is Crucial for Transdifferentiation of Hepatocytes to Biliary Epithelial Cells in an In Vitro Organoid Culture Model.

Hepatocyte to biliary transdifferentiation has been documented in various models of bile duct injury. In this process, mature hepatocytes transform into mature biliary epithelial cells by acquiring biliary phenotypic markers. Several signaling pathways including PI3 kinase, Notch, Hes1, Sox9, and Hippo are shown to be involved in the process. However, whether Oct4 is involved in hepatocyte to biliary transdifferentiation is unknown. We investigated the role of Oct4 in hepatocyte to biliary transdifferentiation utilizing an in vitro organoid culture system as a model of transdifferentiation. Oct4 was inhibited using adenovirus containing Oct4 shRNA. Hepatocyte-specific HNF-4α and biliary-specific HNF-1ß and CK19 expression were assessed to gauge the extent of transdifferentiation. Oct4 was induced during hepatocyte to biliary transdifferentiation. Oct4 inhibition significantly downregulated the appearance of biliary cells from hepatocytes. This was accompanied by a significant downregulation of signaling pathways including Notch, Sox9, and Hippo. Our findings suggest that Oct4 is crucial for hepatocyte to biliary transdifferentiation and maturation and that it acts upstream of Notch, Sox9, and Hippo signaling in this model. This finding identifies new signaling through Oct4 in plasticity between hepatocytes and biliary epithelial cells, which can be potentially utilized to identify new strategies in chronic biliary diseases.

Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation.

Receptor tyrosine kinases MET and epidermal growth factor receptor (EGFR) are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was, however, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers. CONCLUSION: MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals is not tolerated; the results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation. (Hepatology 2016;64:1711-1724).

Synthesis and Structure-Activity Relationship Analysis of 5-HT7 Receptor Antagonists: Piperazin-1-yl Substituted Unfused Heterobiaryls.

A series of piperazin-1-yl substituted unfused heterobiaryls was synthesized as ligands of the 5-HT7 receptors. The goal of this project was to elucidate the structural features that affect the 5-HT7 binding affinity of this class of compounds represented by the model ligand 4-(3-furyl)-2-(4-methylpiperazin-1-yl)pyrimidine (2). The SAR studies included systematical structural changes of the pyrimidine core moiety in 2 to quinazoline, pyridine and benzene, changes of the 3-furyl group to other heteroaryl substituents, the presence of various analogs of the 4-methylpiperazin-1-yl group, as well as additional substitutions at positions 5 and 6 of the pyrimidine. Substitution of position 6 of the pyrimidine in the model ligand with an alkyl group results in a substantial increase of the binding affinity (note a change in position numbers due to the nomenclature rules). It was also demonstrated that 4-(3-furyl) moiety is crucial for the 5-HT7 binding affinity of the substituted pyrimidines, although, the pyrimidine core can be replaced with a pyridine ring without a dramatic loss of the binding affinity. The selected ethylpyrimidine (12) and butylpyrimidine (13) analogs of high 5-HT7 binding affinity showed antagonistic properties in cAMP functional test and varied selectivity profile-compound 12 can be regarded as a dual 5-HT7/5-HT2AR ligand, and 13 as a multi-receptor (5-HT7, 5-HT2A, 5-HT6 and D2) agent.

Leukocyte-specific protein 1: a novel regulator of hepatocellular proliferation and migration deleted in human hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is the most commonly diagnosed form of liver cancer with high morbidity and mortality. Copy number variation (CNV) analysis of human HCC revealed that leukocyte-specific protein 1 (LSP1) had the highest number of cases with CNV. LSP1, a F-actin-binding protein, is expressed in hematopoietic cells and interacts with kinase suppressor of Ras (KSR), a scaffold for the extracellular signal-related kinase/mitogen-activated protein kinase pathway. Expression of LSP1 in liver, and its role in normal hepatocellular function and carcinogenesis, remains unknown. Therefore, LSP1 messenger RNA and protein levels were analyzed in normal hepatocytes in culture, rat liver following partial hepatectomy (PHx), and hepatoma cell lines. In culture and after PHx, LSP1 increased after the termination of hepatocyte proliferation. To investigate LSP1 function in HCC, short hairpin RNA was utilized to stably knock down LSP1 expression in the JM1 rat hepatoma cell line. Loss of LSP1 in JM1 cells resulted in dramatic up-regulation of cyclin D1 and phosphorylated ERK2, increased cell proliferation, and migration. Coimmunoprecipitation and immunofluorescence analysis displayed an interaction and colocalization between LSP1, KSR, and F-actin in JM1 cells and liver during regeneration. Conversely, expression of LSP1 in the JM2 rat hepatoma cell line led to decreased proliferation. Enhanced expression of LSP1 in mouse hepatocytes during liver regeneration after injection of an LSP1 expression plasmid also led to decreased hepatocyte proliferation. CONCLUSION: LSP1 is expressed in normal hepatocytes and liver after PHx after termination of proliferation. In rat hepatoma cell lines and mouse liver in vivo, LSP1 functions as a negative regulator of proliferation and migration. Given the high frequency of LSP1 CNV in human HCC, LSP1 may be a novel target for diagnosis and treatment of HCC.

Synthesis of IL-6 by hepatocytes is a normal response to common hepatic stimuli.

Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response.

Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo.

UNLABELLED: Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor RE-1 silencing transcription factor (REST) maintains self-renewal and pluripotency in mouse embryonic stem cells by maintaining the expression of Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is up-regulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in down-regulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also up-regulated after 70% partial hepatectomy in vivo. CONCLUSION: These findings show that genes inducing the iPS phenotype, even though expressed at lower levels than embryonic stem cells, nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role in such processes during liver regeneration.

Hepatocyte proliferation and hepatomegaly induced by phenobarbital and 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene is suppressed in hepatocyte-targeted glypican 3 transgenic mice.

UNLABELLED: Glypican 3 (GPC3) is a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. Loss-of-function mutations of GPC3 cause Simpson-Golabi-Behmel syndrome characterized by overgrowth of multiple organs, including liver. Our previous study showed that in GPC3 transgenic (TG) mice, hepatocyte-targeted overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration after partial hepatectomy and alters gene expression profiles and potential cell cycle-related proteins. This study investigates the role of GPC3 in hepatocyte proliferation and hepatomegaly induced by the xenobiotic mitogens phenobarbital (PB) and TCPOBOP (1, 4-bis [2-(3, 5-dichloropyridyloxy)] benzene). Wildtype (WT) and GPC3 TG mice were given 0.1% PB in drinking water for 10 days or a single dose of TCPOBOP (3 mg/kg) by oral gavage. At day 5 the WT mice showed a 2.2- and 3.0-fold increase in liver weight, whereas the GPC3 TG mice showed a 1.3- and 1.6-fold increase in liver weight after PB and TCPOBOP administration, respectively. There was a significant suppression of proliferative response in the GPC3 TG mice, as assessed by percent of Ki67-positive hepatocyte nuclei. Moreover, gene array analysis showed a panel of changes in the gene expression profile of TG mice, both before and after administration of the xenobiotic mitogens. Expression of cell cycle-related genes in the TG mice was also decreased compared to the WT mice. CONCLUSION: Our results indicate that in GPC3 TG mice, hepatocyte-targeted overexpression of GPC3 plays an important role for regulation of liver size and termination of hepatocyte proliferation induced by the xenobiotic mitogens PB and TCPOBOP, comparable to the effects seen in the GPC3 TG mice during liver regeneration after partial hepatectomy.

Suppression of liver regeneration and hepatocyte proliferation in hepatocyte-targeted glypican 3 transgenic mice.

UNLABELLED: Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol-anchored, cell-surface heparan sulfate proteoglycans. GPC3 is overexpressed in hepatocellular carcinoma. Loss-of-function mutations of GPC3 result in Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by overgrowth of multiple organs, including the liver. Our previous study showed that GPC3 plays a negative regulatory role in hepatocyte proliferation, and this effect may involve CD81, a cell membrane tetraspanin. To further investigate GPC3 in vivo, we engineered transgenic (TG) mice overexpressing GPC3 in the liver under the control of the albumin promoter. GPC3 TG mice with hepatocyte-targeted, overexpressed GPC3 developed normally in comparison with their nontransgenic littermates but had a suppressed rate of hepatocyte proliferation and liver regeneration after partial hepatectomy. Moreover, gene array analysis revealed a series of changes in the gene expression profiles in TG mice (both in normal mice and during liver regeneration). In unoperated GPC3 TG mice, there was overexpression of runt related transcription factor 3 (7.6-fold), CCAAT/enhancer binding protein alpha (2.5-fold), GABA A receptor (2.9-fold), and wingless-related MMTV integration site 7B (2.8-fold). There was down-regulation of insulin-like growth factor binding protein 1 (8.4-fold), Rab2 (5.6-fold), beta-catenin (1.7-fold), transforming growth factor beta type I (3.1-fold), nodal (1.8-fold), and yes-associated protein (1.4-fold). Changes after hepatectomy included decreased expression in several cell cycle-related genes. CONCLUSION: Our results indicate that in GPC3 TG mice, hepatocyte overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration and alters gene expression profiles, and potential cell cycle-related proteins and multiple other pathways are involved and affected.

RNA interference against hepatic epidermal growth factor receptor has suppressive effects on liver regeneration in rats.

Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP beta. The decrease in the ratio of C/EBP alpha to C/EBP beta known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines.

Investigation of the role of glypican 3 in liver regeneration and hepatocyte proliferation.

Glypicans are heparan sulfate proteoglycans that are bound to the cell surface by glycosylphosphatidylinositol. While six members of the glypican family are known in mammals, our study focused on glypican 3 (GPC3). Loss-of-function mutations of GPC3 result in the Simpson-Golabi-Behmel syndrome, an X-linked disorder characterized by pre- and postnatal liver and other organ overgrowth. GPC3 is overexpressed in human hepatocellular carcinoma; however, its role in normal liver regeneration and hepatocyte proliferation is unknown. Here we investigated the role of GPC3 in hepatocyte proliferation. GPC3 mRNA and protein levels begin to increase 2 days after hepatectomy with peak expression levels by day 5. In hepatocyte cultures, GPC3 reaches a plateau when hepatocyte proliferation decreases. In vitro studies using Morpholino oligonucleotides showed that blocking GPC3 expression promoted hepatocyte growth. Yeast two-hybrid assays revealed that GPC3 interacts with CD81, a member of the tetraspanin family that is reported to be involved in hepatitis C virus infection and cell proliferation. We found that CD81 levels also increased 2 days after partial hepatectomy and toward the end of regeneration. Immunofluorescence showed that CD81 and GPC3 colocalize by 2 and 6 days after hepatectomy. Co-immunoprecipitation validated the interaction of GPC3 and CD81. Our results indicate that GPC3 may be a negative regulator of liver regeneration and hepatocyte proliferation, and that this regulation may involve CD81.

Cell cycle effects resulting from inhibition of hepatocyte growth factor and its receptor c-Met in regenerating rat livers by RNA interference.

UNLABELLED: Hepatocyte growth factor (HGF) and its receptor c-Met are involved in liver regeneration. The role of HGF and c-Met in liver regeneration in rat following two-thirds partial hepatectomy (PHx) was investigated using RNA interference to silence HGF and c-Met in separate experiments. A mixture of 2 c-Met-specific short hairpin RNA (ShRNA) sequences, ShM1 and ShM2, and 3 HGF-specific ShRNA, ShH1, ShH3, and ShH4, were complexed with linear polyethylenimine. Rats were injected with the ShRNA/PEI complex 24 hours before and at the time of PHx. A mismatch and a scrambled ShRNA served as negative controls. ShRNA treatment resulted in suppression of c-Met and HGF mRNA and protein compared with that in controls. The regenerative response was assessed by PCNA, mitotic index, and BrdU labeling. Treatment with the ShHGF mixture resulted in moderate suppression of hepatocyte proliferation. Immunohistochemical analysis revealed severe suppression of incorporation of BrdU and complete absence of mitosis in rats treated with ShMet 24 hours after PHx compared with that in controls. Gene array analyses indicated abnormal expression patterns in many cell-cycle- and apoptosis-related genes. The active form of caspase 3 was seen to increase in ShMet-treated rats. The TUNEL assay indicated a slight increase in apoptosis in ShMet-treated rats compared with that in controls. CONCLUSION: The data indicated that in vivo silencing of c-Met and HGF mRNA by RNA interference in normal rats results in suppression of mRNA and protein, which had a measurable effect on proliferation kinetics associated with liver regeneration.

High throughput screening of methylation status of genes in prostate cancer using an oligonucleotide methylation array.

Recent work using high-throughput microarray technology has discovered altered expression of a large number of genes in prostate cancer. Many of these alterations may be the consequence of changes in methylation status in the CpG islands of promoter or exon 1 regions of these genes. In order to determine the methylation status of a large number of genes and ESTs we combined the principle of match/mismatch hybridization with the technique of whole genome labeling to develop a highly specific oligonucleotide-based methylation microarray. Using this array, we analyzed the methylation status of 105 genes and ESTs in three prostate cancer cell lines. Between 32 and 47% of these genes and ESTs were methylated in these cell lines. By correlating the methylation status of this array with the results of Affymetrix expression arrays of three prostate cancer cell lines, we determined that methylation of genes played a significant role (37%) in down-regulating the expression of certain genes in prostate cancer. We also tested this array on a number of primary prostate tissue samples. Our results indicated that a subset of genes in this microarray (25/105) were methylated in all prostate cancer samples but not in normal prostate, suggesting the potential significance of alterations in the methylation status of certain genes in the development of prostate cancer.

Persistent HIV type 1 infection in semen and blood compartments in patients after long-term potent antiretroviral therapy.

HIV-1 RNA levels in semen and blood compartments decrease below detection limits during highly active antiretroviral therapy. Despite these therapeutic effects, it is clear that persistent, latent HIV-1 reservoirs are capable of rebounding in the absence of drug treatment or by evolution of escape mutants remain. The current study was designed to examine the presence of latent virus in semen and blood compartments and its evolution following potent combination therapy with indinavir (protease inhibitor) and efavirenz [nonnucleoside reverse transcriptase (RT) inhibitor]. Using an ultrasensitive in situ hybridization assay HIV-1 mRNA was detected in cultured seminal and blood mononuclear cells in all patients up to 1789 days posttherapy. Higher levels of HIV-1 mRNA were consistently detected in seminal mononuclear cells as compared to peripheral blood mononuclear cells (PBMC) in all time points analyzed posttherapy. Analysis of viral RNA from cultured PBMC before and after therapy displayed no evidence of therapy-induced drug resistance in the viral polymerase gene in the majority of patients. However, distinct envelope populations were detected in these viral RNA populations following therapy, indicating possible selection of quasispecies. The observed ongoing replication and evolution in the PBMC viral envelope sequences likely occurred in the seminal compartment HIV populations, given that the seminal cells showed the ability to express HIV-1 mRNA following cultivation. This together with our previous studies (Gupta P, et al.: J Infect Dis 2000;182:79-87) suggest that the genital and blood compartments likely serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy.

Subcompartmentalization of HIV-1 quasispecies between seminal cells and seminal plasma indicates their origin in distinct genital tissues.

The mononuclear cells and plasma components of semen from HIV-infected subjects have been shown to contain HIV-1. However, there is very little information as to whether distinct HIV-1 population are present in these two seminal compartments or as to their tissue of origin. Phylogenetic analysis of nucleotide sequences of the C2-V5 region of the HIV-1 gp120 from HIV-1 RNA isolated from seminal cells and seminal plasma of five subjects indicates that the HIV-1 population derived from seminal plasma was distinct from that present in seminal cells. Such subcompartmentalization of HIV-1 between seminal cells and seminal plasma was detected as early as 3 months after seroconversion and persisted up to 38 months following seroconversion. Furthermore, comparison of HIV-1 sequences between testis and prostate tissues showed distinct HIV-1 populations in these tissue compartments. In situ real-time (Taqman) PCR analysis of prostate and testis tissues indicated that T lymphocytes were the predominant cells infected with HIV-1 in both of these tissues. Since seminal plasma is derived from prostate and most of the seminal cells originate from the rete testis and epididymis, these results are consistent with the idea that HIV-1 in seminal plasma is derived from the prostate, while HIV-1-infected cells in semen originate mostly from the rete testis and epididymis. These findings provide for the first time evidence of subcompartmentalization of HIV-1 in male genital organs and suggest that intervention strategies such as vasectomy may not prevent sexual transmission.