The effect of hepatic progenitor cells on experimental hepatocellular carcinoma in the regenerating liver.

OBJECTIVE: Liver regeneration following hepatectomy can stimulate the growth of hepatocellular carcinoma (HCC), and major hepatectomy can be associated with activation of hepatic progenitor cells (HPCs). The aim of this study was to evaluate how HPCs influence the malignant potential of tumor cells in vitro and HCC tumor growth after surgery in a rodent model. MATERIAL AND METHODS: Hepatoma cells (JM1) were cultured with conditioned medium (CM) from syngeneic HPCs (WB-F344). Growth rate, resistance to Adriamycin, and expression patterns for invasiveness and stemness were compared with naïve JM1. Microscopic HCC tumors from naïve JM1 or JM1 cultured with CM were inoculated in Fischer 344 rats undergoing 70% hepatectomy with or without simultaneous infusion of WB-F344. Tumor growth and invasiveness-related factors were compared. Buffalo rats were induced with Morris hepatoma cells. Liver tissue from both in vivo models was examined with regard to activation of cells with progenitor-like phenotype. RESULTS: Co-culture with CM resulted in an increased resistance to Adriamycin and enhanced expressions of α-FP, MMP9, ABCG2, CD133, and SOX2, as well as the activation of ERK, AKT, WNT, and TGF-ß1 pathways. Tumor size and metastases were significantly higher in groups with co-cultured cells or HPCs infusion. After 70% hepatectomy and tumor implantation, cells positive for α-FP, CK19, and CD133 were found, thus suggesting a progenitor-like phenotype in the setting of epithelial-mesenchymal transition. CONCLUSION: HPCs have a marked effect on HCC cells in vitro and appear to stimulate the growth and malignant potential of experimental HCC tumors.

Deletion of the γ-aminobutyric acid transporter 2 (GAT2 and SLC6A13) gene in mice leads to changes in liver and brain taurine contents.

The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [(3)H]taurine. Furthermore, most of the uptake of [(3)H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

FAK regulates Cdk2 in EGF-stimulated primary cultures of hepatocytes.

In this study, we report a novel role of FAK as a regulator of Cdk2 in anchorage-dependent primary cultured hepatocytes. In response to EGF, we found that S-phase entry was reduced upon FAK inhibition. This correlated with decreased protein expression and nuclear accumulation of the G1/S-phase regulator Cdk2. Further, nuclear accumulation of the Cdk2 partner cyclinE, was reduced, but not its protein level. Also, protein levels of Cdk2 were inversely linked with increased expression of the Cdk2 inhibitor p27, known to be degraded in a Cdk2-dependent manner. Also, cyclinD1 was regulated by FAK, but to a lesser extent than Cdk2. To assess the mechanism in which FAK mediates Cdk2-regulation, FAK mutants were used: FAKY397F, mutated at its integrin-regulated site, and two others mutated at docking sites for Grb2-ERK-activation (FAKY925F) and for p130Cas-Rac1-activation (FAKY861F). All three sites were central for EGF-induced ERK-activity and Cdk2 expression. In addition, FAK was important for HGF-mediated proliferation, suggesting a general mechanism for anchorage-dependent growth. Moreover, growth factor-induced cell spreading, but not survival, required FAK. Hence, integrins and growth factors cooperate in anchorage-dependent signaling events leading to proliferation and motility. In conclusion, our data suggest that FAK acts as a central coordinator of integrin and growth factor-mediated S-phase entry by its ability to regulate Cdk2.

EGF activates autocrine TGFα to induce prolonged egf receptor signaling and hepatocyte proliferation.

BACKGROUND/AIMS: EGF receptor is a main participant in the regulation of liver regeneration. In primary hepatocyte cultures, EGF or TGFα binding to EGF receptor activates Erk1/2 and PI3K pathways, induces cyclin D1 and thus initiates DNA synthesis. We have explored mechanisms by which prolonged EGF receptor activation induces hepatocyte proliferation. METHODS: EGF receptor activation, as well as Erk1/2 and PI3K signaling were explored in EGF-stimulated primary hepatocyte cultures by Western blotting and immunocytochemistry. TGFα release to the medium was quantified by ELISA. Effects of a neutralizing antibody to TGFα on EGF receptor signaling and proliferation were explored. RESULTS: Inhibitors of PI3K or Erk1/2 inhibited cyclin D1 expression and G1 progression when added 12 hours after EGF stimulation, whereas depletion of EGF from the medium at this time point did not. ELISA demonstrated that EGF induced TGFα release to the medium. Cyclin D1 induction and cellular proliferation were efficiently inhibited when a neutralizing antibody to TGFα was added to the medium. This also occurred when the antibody was added 12 hours after EGF stimulation. CONCLUSION: Sustained EGF receptor activity and signaling through both Erk1/2 and PI3K pathways were necessary for proliferation. This was achieved by EGF activation of autocrine TGFα.

RAF-targeted therapy for hepatocellular carcinoma in the regenerating liver.

BACKGROUND: Post-operative liver regeneration may contribute to tumor recurrence. There is a theoretical need for an adjuvant therapy that can suppress tumor growth without adversely affecting post-operative liver regeneration. OBJECTIVE: To evaluate the effect of RAF inhibitor Sorafenib on cell viability and proliferation of hepatoma cells and hepatocytes in vitro and in an in vivo rat model. METHODS: Cell viability, DNA synthesis, and RAF/MAPK kinase activity in the primary hepatocyte and hepatoma cell lines were investigated after Sorafenib exposure. Sequence analysis of the B-RAF gene in hepatic cells was determined. Tumor markers were compared within the rats after 70% hepatectomy with or without daily oral gavages of Sorafenib. Liver regeneration was assessed by liver function tests and proliferation markers. RESULTS: Primary hepatocytes showed higher cell viability, proliferation rate, and stronger RAF/MAPK kinase activity compared with hepatoma cell lines. The in vivo tumor volumes, size, and metastases were significantly decreased (P < 0.05) whereas no significant change in liver regeneration related to Sorafenib exposure was found (P > 0.05). B-RAF V600E mutation was not detected neither in the hepatic cells nor untransformed hepatocytes. CONCLUSIONS: The RAF targeted inhibitor can reduce tumor growth without retarding liver regeneration in this experiment.

EGF-induced ERK-activation downstream of FAK requires rac1-NADPH oxidase.

Reactive oxygen species (ROS) function as signaling molecules mainly by reversible oxidation of redox-sensitive target proteins. ROS can be produced in response to integrin ligation and growth factor stimulation through Rac1 and its effector protein NADPH oxidase. One of the central roles of Rac1-NADPH oxidase is actin cytoskeletal rearrangement, which is essential for cell spreading and migration. Another important regulator of cell spread is focal adhesion kinase (FAK), a coordinator of integrin and growth factor signaling. Here, we propose a novel role for NADPH oxidase as a modulator of the FAK autophosphorylation site. We found that Rac1-NADPH oxidase enhanced the phosphorylation of FAK at Y397. This site regulates FAK's ability to act as a scaffold for EGF-mediated signaling, including activation of ERK. Accordingly, we found that EGF-induced activation of FAK at Y925, the following activation of ERK, and phosphorylation of FAK at the ERK-regulated S910-site depended upon NADPH oxidase. Furthermore, the inhibition of NADPH oxidase caused excessive focal adhesions, which is in accordance with ERK and FAK being modulators of focal adhesion dissociation. Our data suggest that Rac1 through NADPH oxidase is part of the signaling pathway constituted by FAK, Rac1, and ERK that regulates focal adhesion disassembly during cell spreading.

Growth of hepatocellular carcinoma in the regenerating liver.

Liver resection and liver transplantation are the treatment modalities with the greatest potential for curing hepatocellular carcinoma (HCC). Tumor recurrence after resection for HCC is, however, a major problem, and an increased rate of recurrence after living donor transplantation versus cadaveric whole liver transplantation has been suggested. Factors involved in liver regeneration may stimulate the growth of occult tumors. The aim of this project was to test the hypothesis that a microscopic HCC tumor in the setting of partial hepatectomy would show enhanced growth and signs of increased invasiveness corresponding to the size of the liver resection. Hepatectomy was performed to various degrees in groups of Buffalo rats with the concomitant implantation of a fixed number of hepatoma cells in the remnant liver; a control group underwent only resection. After 21 days, the sizes and numbers of the tumors and the expression of alpha-fetoprotein (AFP), cyclin D1, calpain small subunit 1 (CAPNS1), CD34 (a microvessel density marker), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) were evaluated and compared between the groups. The tumor volume and number increased significantly with the size of the partial hepatectomy (P < 0.05). The largest resections were also associated with increased hepatoma cell infiltration in the lungs and significant up-regulation of cyclin D1, AFP, CAPNS1, CD34, VEGF, and VEGFR2. The results suggest that liver regeneration after partial hepatectomy facilitates the growth and malignant transformation of microscopic HCC, and this could be significant for liver resection and partial liver transplantation strategies for HCC.

Identification and characterization of two putative nuclear localization signals (NLS) in the DNA-binding protein NUCKS.

Immunofluorescence analyses show that the vertebrate specific and DNA-binding protein NUCKS is distributed throughout the cytoplasm in mitotic cells and targeted to the reforming nuclei in late telophase of the cell cycle. Computer analysis of the primary structure of NUCKS revealed the presence of two regions of highly charged, basic residues, which were identified as potential nuclear localization signals (NLSs). One of these signals (NLS1) is highly conserved between the species investigated, and fits to the description of being a classical bipartite NLS. The other amino acid motif (NLS2) is less conserved and does not constitute a classical bipartite NLS consensus sequence. We have shown that each of the two putative NLSs is capable of translocating green fluorescent protein (GFP) into the nucleus. The highly conserved NLS1 is monopartite, resembling the signals of c-Myc and RanBP3. Surprisingly, a natural occurring splice variant of NUCKS lacking 40 amino acids including NLS1, is not capable of translocating a corresponding NUCKS-GFP fusion protein into the nucleus, indicating that NLS1 is the main nuclear localization signal in NUCKS. This is also confirmed by site-directed mutagenesis of the full-length protein. By GFP-immunoprecipitation and GST-pull down experiments, we show that NUCKS binds to importin alpha3 and importin alpha5 in vitro, suggesting that the nuclear targeting of NUCKS follows a receptor-mediated and energy-dependent import mechanism.

Distinct functions of H-Ras and K-Ras in proliferation and survival of primary hepatocytes due to selective activation of ERK and PI3K.

Ras proteins mediate signals both via extracellular signal-regulated kinase 1 and 2 (ERK), and phosphoinositide 3-kinase (PI3K). These signals are key events in cell protection and compensatory cell growth after exposure to cell damaging and pro-apoptotic stimuli, thus maintaining homeostasis. By transfection techniques, we found that both H-Ras and K-Ras were expressed and appeared functionally active in primary hepatocytes. We compared the ability of H-Ras and K-Ras homologues to preferentially activate one of the two pathways, thereby differentially controlling cell survival and growth. We found that ectopic expression of dominant negative (DN) H-RasN17, but not DN K-RasN17, efficiently inhibited both phosphorylation and translocation of ERK to the nuclear compartment, which are prerequisites for cell cycle progression. Furthermore, ectopic expression of constitutive active (CA) H-RasV12, but not CA K-RasV12, potentiated EGF-induced proliferation. We also found that expression of CA mutants of either H-Ras or K-Ras protected hepatocytes from transforming growth factor-beta1 (TGF-beta1)-induced apoptosis. However, H-Ras-induced survival was mediated by ERK/RSK as well as by PI3K, whereas K-Ras-induced survival was mediated by PI3K only. In conclusion, H-Ras and K-Ras had differential functions in proliferation and survival of primary hepatocytes. H-Ras was the major mediator of ERK-induced proliferation and survival, whereas H-Ras and K-Ras both mediated PI3K-induced survival.

Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit.

BACKGROUND: Epidermal Growth Factor Receptor (EGFR) is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA) induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. RESULTS: We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Calpha and Cbeta in two different models. The first model used targeted disruption of either Calpha or Cbeta in mice whereas the second model used Calpha and Cbeta RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. CONCLUSION: Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

Nondysplastic Ulcerative Colitis Has High Levels of the Homologous Recombination Repair Protein NUCKS1 and Low Levels of the DNA Damage Marker Gamma-H2AX.

Background: The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. Methods: NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Results: Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Conclusions: Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.

Evidence of selective activation of aryl hydrocarbon receptor nongenomic calcium signaling by pyrene.

In its classical genomic mode of action, the aryl hydrocarbon receptor (AhR) acts as a ligand activated transcription factor regulating expression of target genes such as CYP1A1 and CYP1B1. Some ligands may also trigger more rapid nongenomic responses through AhR, including calcium signaling (Ca2+). In the present study we observed that pyrene induced a relatively rapid increase in intracellular Ca2+-concentrations ([Ca2+]i) in human microvascular endothelial cells (HMEC-1) and human embryonic kidney cells (HEK293) that was attenuated by AhR-inhibitor treatment and/or transient AhR knockdown by RNAi. In silico molecular docking based on homology models, suggested that pyrene is not able to bind to the human AhR in the agonist conformation. Instead, pyrene docked in the antagonist conformation of the AhR PAS-B binding pocket, although the interaction differed from antagonists such as GNF-351 and CH223191. Accordingly, pyrene did not induce CYP1A1 or CYP1B1, but suppressed CYP1-expression by benzo[a]pyrene (B[a]P) in HMEC-1 cells, confirming that pyrene act as an antagonist of AhR-induced gene expression. Use of pharmacological inhibitors and Ca2+-free medium indicated that the pyrene-induced AhR nongenomic [Ca2+]i increase was initiated by Ca2+-release from intracellular stores followed by a later phase of extracellular Ca2+-influx, consistent with store operated calcium entry (SOCE). These effects was accompanied by an AhR-dependent reduction in ordered membrane lipid domains, as determined by di-4-ANEPPDHQ staining. Addition of cholesterol inhibited both the pyrene-induced [Ca2+]i-increase and alterations in membrane lipid order. In conclusion, we propose that pyrene binds to AhR, act as an antagonist of the canonical genomic AhR/Arnt/CYP1-pathway, reduces ordered membrane lipid domains, and activates AhR nongenomic Ca2+-signaling from intracellular stores.

Structure function analysis of SH2D2A isoforms expressed in T cells reveals a crucial role for the proline rich region encoded by SH2D2A exon 7.

BACKGROUND: The activation induced T cell specific adapter protein (TSAd), encoded by SH2D2A, interacts with and modulates Lck activity. Several transcript variants of TSAd mRNA exist, but their biological significance remains unknown. Here we examined expression of SH2D2A transcripts in activated CD4+ T cells and used the SH2D2A variants as tools to identify functionally important regions of TSAd. RESULTS: TSAd was found to interact with Lck in human CD4+ T cells ex vivo. Three interaction modes of TSAd with Lck were identified. TSAd aa239-256 conferred binding to the Lck-SH3 domain, whereas one or more of the four tyrosines within aa239-334 encoded by SH2D2A exon 7 was found to confer interaction with the Lck-SH2-domain. Finally the TSAd-SH2 domain was found to interact with Lck. The SH2D2A exon 7 encoding TSAd aa 239-334 was found to harbour information essential not only for TSAd interaction with Lck, but also for TSAd modulation of Lck activity and translocation of TSAd to the nucleus. All five SH2D2A transcripts were found to be expressed in CD3 stimulated CD4+ T cells. CONCLUSION: These data show that TSAd and Lck may interact through several different domains and that Lck TSAd interaction occurs in CD4+ T cells ex vivo. Alternative splicing of exon 7 encoding aa239-334 results in loss of the majority of protein interaction motives of TSAd and yields truncated TSAd molecules with altered ability to modulate Lck activity. Whether TSAd is regulated through differential alternative splicing of the SH2D2A transcript remains to be determined.

Serine mutations that abrogate ligand-induced ubiquitination and internalization of the EGF receptor do not affect c-Cbl association with the receptor.

In the present study, we examined EGF-induced internalization, degradation and trafficking of the epidermal growth factor receptor (EGFR) mutated at serines 1046, 1047, 1057 and 1142 located in its cytoplasmic carboxy-terminal region. We found the serine-mutated EGFR to be inhibited in EGF-induced internalization and degradation in NIH3T3 cells. We therefore tested the hypothesis that these mutations affect ligand-induced c-Cbl association with the receptor, leading to inhibited receptor ubiquitination. EGF was unable to induce ubiquitination of the serine-mutated EGFR, yet EGF-induced phosphorylation of the c-Cbl-binding site at tyrosine 1045, and c-Cbl-EGFR association, was unaffected. To compare the relevance of these serine residues with tyrosine 1045 in their regulation of c-Cbl binding and receptor ubiquitination, we analysed an EGFR mutated at tyrosine 1045 (Y1045F). EGF-induced c-Cbl-EGFR binding was partially inhibited, and receptor ubiquitination was abrogated in cells expressing Y1045F-EGFR. In contrast, ligand-induced internalization and degradation of the Y1045F mutant was similar to that of wild-type EGFR. Together, our data indicate that the serine residues and tyrosine 1045 are essential for EGF-induced receptor ubiquitination, but only the serine residues are critical for EGFR internalization and degradation.

Identification of 14-3-3zeta as an EGF receptor interacting protein.

The 14-3-3 proteins are known to interact with a number of proteins involved in the regulation of cell signaling. Here, we describe an association of 14-3-3zeta with the epidermal growth factor receptor (EGFR) that is rapidly induced by EGF. The 1028-EGFR truncated mutant which lacks the cytoplasmic tail from amino acids 1029-1186 identified the binding site for 14-3-3 to be between amino acid 1028 and the receptor carboxyl terminus. Mutational deletion of serine residues 1046, 1047, 1057 and 1142 did not inhibit EGF-induced 14-3-3 association with the receptor. Immunofluorescence microscopy indicated an EGF-induced co-localization of EGFR and HA-14-3-3zeta along the plasma membrane. Our finding adds to the growing complexity of EGF receptor signaling and indicates a role for 14-3-3 proteins in EGF receptor signaling or regulation.

Fluorescent histochemical techniques for analysis of intracellular signaling.

Intracellular signaling relies on the orchestrated cooperation of signaling proteins and modules, their intracellular localization, and membrane trafficking. Recently, a repertoire of fluorescence-based techniques, which significantly increases our potential for detailed studies of the involved mechanisms, has been introduced. Microscopic techniques with increased resolution have been combined with improved techniques for detection of signaling proteins. Transfections of fluorescently tagged proteins have allowed in vivo microscopy of their trafficking and interactions with other proteins and intracellular structures. We present an overview of general signaling principles and a description of techniques based on fluorescent microscopy suited for studies of signaling mechanisms.

UV-radiation-induced internalization of the epidermal growth factor receptor requires distinct serine and tyrosine residues in the cytoplasmic carboxy-terminal domain.

The mechanism of UV-radiation-induced EGF receptor (EGFR) internalization remains to be established. In the present study, we found UV-radiation-mediated internalization of the EGFR to be dependent on the cytoplasmic carboxy-terminal region. UV radiation was unable to induce internalization of EGFR carboxy-terminal truncation mutants where all or four of the five major autophosphorylation sites were missing (963- and 1028-EGFR, respectively). Mutational removal of serine residues 1046, 1047, 1057 and 1142 within the carboxy-terminal receptor region was also sufficient to abolish UV-radiation-induced internalization of the EGFR. Furthermore, the UV-radiation-induced internalization was abrogated for an EGFR mutated in tyrosine 1045 (Y1045F), the major c-Cbl binding site. However, UV radiation did not induce phosphorylation at tyrosine 1045, in contrast to the prominent phosphorylation induced by EGF. Our results suggest a mechanism for UV-radiation-induced internalization of EGFR involving a conformational change that is dependent on structural elements formed by specific serine and tyrosine residues in the carboxy-terminal domain.