HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation

The liver has the capacity to restore damaged or lost liver cell mass by cell proliferation (Bucher, 1963). The process of liver regeneration has been studied extensively in a rat model using 70% partial hepatectomy (PH), first described by Higgins and Anderson (Higgins and Anderson, 1931). After PH the remaining differentiated liver cells re-enter the cell cycle through transition from the quiescent G₀ phase to the G₁ phase (priming phase) followed by DNA synthesis and G₂-M phases. The initial rounds of cell division are synchronous and the first DNA replication occurs within 24 or 38 hours after surgery in rat and mouse, respectively (Fausto, 2000). After one or two replication rounds the hepatocytes return to the quiescent state. The regeneration process is completed within 10 to 12 days (Bucher, 1963). Hormones, growth factors, cytokines and their coupled signal transduction pathways have been implicated in governing hepatocyte proliferation, but the precise orchestration of these factors is still poorly understood (Cressman et al., 1996; Yamada et al., 1997; Michalopoulos and DeFrances, 1997; Fausto et al., 1995; Fausto, 2001). The increase of cellular cAMP, observed in the first hours after PH, is followed by a modification of protein kinase A expression (Ekanger et al., 1989; Diehl and Rai, 1996; Servillo et al., 2001; Servillo et al., 2002). A direct role for cAMP-responsive transcription in proliferation after PH has been established (Della Fazia et al., 1997). It has been shown that inducible cAMP early repressor (ICER) expression is strongly induced upon PH (Servillo et al., 1997) and that cAMP responsive element modulator (CREM) plays a critical role in the proliferative process through analysis of liver regeneration in CREM knockout mice (Servillo et al., 1998). Lack of CREM results in reduced proliferation and deregulation of the residual hepatocyte cell cycle. Studies on CREM knockout mice revealed delayed expression of various cyclin genes (Servillo et al., 1998).

Hepatocytes are resting cells in G₀ phase that after PH go through G₁ phase and progress to the cell cycle. The process that allows hepatocytes to pass from G₀ to G₁ phase has been described as priming. Growth factors such as hepatocyte growth factor (HGF), transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) act primarily on hepatocytes in liver regeneration (Fausto, 2000). Many researchers have shown that liver regeneration is a multistep process and that priming is necessary for hepatocyte proliferation. HGF, TGF-α and EGF alter gene expression in residual hepatocytes and in the subsequent proliferative steps. Following priming, two major events occur in residual hepatocytes after PH, activation of several genes in response to proliferation and restoration of the quiescent state of hepatocytes by activated genes.

To describe the mechanisms involved in the proliferative response, we focussed on the molecular changes occurring during liver regeneration following PH through characterization of novel genes activated in residual hepatocytes. A cDNA library was constructed with mRNAs derived from residual hepatocytes at different times following PH. We performed a rat regenerating liver cDNA library screening with cDNA-subtracted probes derived from rat regenerating liver cDNAs (2-18 hours after PH) and rat normal liver mRNAs. Screening allowed us to isolate up to 40 genes. All isolated genes were upregulated in the liver after PH and in hepatoma cells. One of these novel genes expressed in liver regeneration has been characterized previously (Della Fazia et al., 2002). This gene Lal-1 is involved during liver regeneration and in the proliferative process. At present our attention has been drawn to another of these genes that we named hepatocyte odd protein shuttling (Hops).

In this paper, we demonstrate that HOPS is a novel shuttling protein, which via CRM-1 (Fornerod et al., 1997), actively directs proliferation of cells controlling protein synthesis. Evidence is provided that cAMP governs HOPS export in hepatocytes of normal and regenerating liver. Following PH, HOPS is rapidly exported from the nucleus and is overexpressed during liver regeneration. It has been established that HOPS binds to elongation factor EF-1A and interferes with protein synthesis. Overexpression of HOPS in H-35 hepatoma cells and 3T3-NIH cells strongly reduces cell proliferation.

H-35 rat hepatoma and COS-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS; GIBCO Invitrogen) (Taub et al., 1987). NIH-3T3 cells were cultured in DMEM supplemented with BCS (GIBCO Invitrogen). The Phoenix cells were cultured in DMEM supplemented with FBS (GIBCO Invitrogen).

Experiments were performed on 3-month-old male animals: Sprague-Dawley rats and SVJ-129 mice. The animals were purchased from Harlan-Nossan and received human care according to NIH guidelines. Animals were maintained in a 12 hours:12 hours light:dark cycle with food and water ad libitum. Liver resection was performed between 8 a.m. and 12 a.m. removing about 70% of the liver mass (Higgins and Anderson, 1931). As control, sham operation by transverse abdominal incision followed by digital manipulation of the liver was performed. Rats were sacrificed at 2, 5, 8, 12, 18, 24, 48 and 72 hours after PH. Mice were sacrificed at 15, 30 minutes, 1, 2, 5, 8, 12, 18, 36, 38, 48, 60 and 72 hours after PH. Four animals per experimental group were used for each time point and livers were pooled prior to analysis.

Total RNA extraction was performed and analyzed by northern blot analysis as described previously (Sambrook et al., 1989; Della Fazia et al., 1992).

H-35 cell cycle was synchronized at the G₀/G₁ boundary in serum-free medium and in G₁/S phases by the double thymidine method. Briefly, H-35 were cultured in the absence of serum, arrested for 72 hours and released in fresh culture medium in the presence of serum. The time point at which H-35 received serum after starvation was considered time 0. The double thymidine method to arrest the cells at the G₁/S boundary was performed as described previously (Crosio et al., 2002). The time point, corresponding to the G₁/S transition, was considered time 0. The percentage of H-35 cells, labeled with propidium iodide, was determined at different phases of the cell cycle by flow cytometry (FACS analysis; Becton Dickinson FACStar Plus flow cytometer).

H-35 stable cell clones overexpressing HOPS were generated by transfection with pcDNA3 vector containing full-length Hops and by selection in culture medium with geneticin (0.4 mg/ml). H-35 stable cell clones generated by transfection with empty pcDNA3 vector were used as control. Thymidine incorporation in H-35 cells and H-35 stable cell clones was performed as described previously (Della Fazia et al., 2001).

pBabe-puro, MuLV-based retroviral vector, was used to transduce the Hops gene (Morgenstern et al., 1990). Wild-type Hops cDNA was inserted into pBabe-puro vector to produce pBabe-Hops.

Phoenix cells were plated at 1.5 ×10⁶ per plate 2 days before transfection. For each transfection, 2 μg of pBabe-puro or pBabe-Hops plasmid were used. Viral supernatants were concentrated and collected 48 hours after transfection according to Nolan laboratory protocol (www.stanford.edu/group/nolan/tutorials). Culture supernatants containing retroviral vectors were then added to NIH-3T3 with polybrene (8 μg/ml) (Sigma). Cells were cultured for 24 hours and selected in the same medium containing puromycin (2 μg/ml) for 4 days. Resistant cells (1 ×10⁴) were plated and counted every 2 days for 10 days after selection.

Hops gene isolation from a regenerating liver library was performed using a subtracted probes procedure. A number of positive clones were isolated and tested by northern blot analysis of the time course of RNA extracted at different times following PH. Selected cDNAs overexpressed during liver regeneration were isolated and sequenced. Hops DNA sequence and putative protein prediction procedures have been previously described (Della Fazia et al., 2002).

Polyclonal anti-HOPS was generated in our laboratory by immunizing rabbits with KHL-coupled peptide (H T T E S T D P L P Q S S G T T T P A Q P S E) corresponding to N-terminal sequence (aa 32-54) of the mouse HOPS protein. The serum of two rabbits was collected and immunopurified on a column Sulfo-Link coupling gel (Pierce) where the specific peptide had been immobilized. To test the specificity of the antibody, a quenching test was performed with different amounts of specific peptide in western blot and in immunohistochemistry analyses (Della Fazia et al., 2002).

Protein extracts were resolved by standard SDS-polyacrylamide gel electrophoresis from total liver and H-35 hepatoma rat cells. Liver and cells were minced immediately in RIPA buffer. Each sample (50 μg) was separated by gel electrophoresis and blotted onto a nitrocellulose membrane (Schleicher and Schuell). The blots were incubated with rabbit anti-HOPS polyclonal antibody and with rabbit anti-CREB polyclonal antibody (Cell Signaling Technology) the signals were detected using an ECL kit (Amersham Pharmacia Biotech).

The livers were embedded into OCT compound for cryosectioning. Sections (7 μm) were cut and mounted on slides. For histological analysis, sections were stained with Hematoxylin-Eosin. Slides were blocked with 3% BSA and then incubated with specific primary polyclonal anti-HOPS antibody. After three washes in PBS, slides were incubated with anti-rabbit Cy-3-conjugated secondary antibody in 3% BSA. DAPI was added at the final concentration of 10 ng/ml. Images were captured with a Zeiss Axioplan fluorescence microscope controlled by a Spot-2 cooled camera (Diagnostic Instruments) with a 40 × objective lens.

H-35 hepatoma cells were pretreated for 30 minutes with 10 ng/ml of cycloheximide (CHX; Sigma). The cells were then treated for an additional 6 hours with 20 ng/ml LMB. After CHX treatment PBS was added to the control cell population. At the end of treatment the cells were fixed in 4% paraformaldehyde and hybridized with specific anti-HOPS antibody overnight. The cells were examined using a fluorescence microscope with a standard filter for red fluorescence. Six random fields of cells stained for HOPS in the presence or absence of LMB were counted. The image was captured with a Zeiss Axioplan fluorescence microscope controlled by Spot-2 cooled camera (Diagnostic Instruments). Images were saved as TIFF-files.

Hops full-length cDNA was cloned into yeast expression vector pGBKT7. The pGBKT7-Hops plasmid was transformed into yeast strain AH109. Two-hybrid screening was carried out according to the manufacturer's protocol (Clontech) using a VP-16 DNA activation domain fusion library in an E9.5-12.5 mouse embryo cDNA library in the vector pASV3 (Le Douarin et al., 1995). The transformants were plated onto appropriate selective medium supplemented with 25 mM 3-amino-triazole. β-gal assays were performed on isolated clones and carried out in Y190 yeast strain. The results reported are in Miller units and are the means of triplicate measurements performed using three distinct transformations. The plasmids extracted by lysing cells with acid-washed beads were electroporated in E. coli bacterial strain HB101 and then plated onto M9 (–Leu) plates (Vojtek et al., 1993). The isolated clones were sequenced using the Sanger method.

H-35 cells were harvested, washed and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 2% Triton X-100) supplemented with protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride (PMSF). Immunoprecipitation was carried out as described (Bardoni et al., 1999) using the anti-eEF-1A (Upstate Biotech). The proteins bound to the beads were separated by electrophoresis on 10-12% SDS-PAGE and visualized by immunoblot using the anti-HOPS antibody. At the same time, the cell lysate was co-immunoprecipitated using anti-HOPS and detected with anti-eEF-1A.

The HOPS gene was cloned in pET-14Tb expression vector (Novagen). The resulting gene was expressed in BL21 (DE3) cells (Novagen) and after induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 hours at 25°C the recombinant protein was purified under denaturing conditions using His-Bind affinity chromatography. Expression and purification were carried out according to the manufacturer's protocol (Novagen).

An aliquot (1 μg) of luciferase cDNA (Promega) was added to 100 μl of a rabbit reticulocyte lysate in vitro translation reaction (Promega) in the presence of HOPS or GST purified recombinant proteins at different concentrations: 60, 120, 240, 360 and 420 nM. The reaction mixture was incubated at 30°C for 2 hours. Newly synthesized ³⁵S-labeled luciferase protein was analyzed by 10% SDS-PAGE and results were quantified using densitometry analysis with the Scion Image 4.0 program. Western blot analysis was performed using anti-eEF-1A as control.

We have cloned and characterized a novel gene named Hops. A cDNA library was constructed with mRNA isolated from residual hepatocytes at different times after the G₀/G₁ to S phase transition following PH. Screening of the rat regenerating liver cDNA library was performed using a subtracted probe derived from mRNA of normal liver and cDNA of regenerating liver that enabled us to isolate up to 40 novel genes. All isolated genes were tested by northern blot analysis to verify expression at different times following PH. Our interest was directed to one of the isolated genes overexpressed during liver regeneration, Hops. The HOPS nucleotide sequence and the deduced amino acid sequence were determined. Northern blot analysis showed that Hops mRNA was expressed in rat regenerating liver and the transcript length was estimated. Hops mRNA expression, almost undetectable in normal liver, presented a specific band of 1.5 kb corresponding in size to the Hops transcript. Liver mRNAs were evaluated at different times during liver regeneration and in sham-operated rats. Furthermore, induction of gene expression was detected in the first hours of liver regeneration after PH (Fig. 1A). No change in Hops gene expression from sham-operated animal mRNA was detected (data not shown). HOPS protein is composed of an ubiquitin-like domain (102-175 aa), a proline rich region (176-183 aa) and three leucine rich α-helixes: 13-32, 190-212, 197-209 (Fig. 1B,C). The rat, mouse and human HOPS genes were cloned and the nucleotide sequences have been submitted to the NCBI database under accession numbers AY603378, AY603379 and AY603380, respectively. The rat and mouse HOPS amino acid sequences showed 96% identity and 97% similarity. Human HOPS showed 90% identity with respect to mouse and rat (data not shown). The amino acid identity in the C-terminal of HOPS in the three species was about 97% (data not shown). Rat Hops spans over 1381 bp with a +1 ATG sequence at 132 bp showing an ORF of 735 bp (Fig. 1C).

To investigate HOPS expression and validate the results obtained in differential screening, we analyzed HOPS expression by western blot and immunolocalization analyses in mouse normal liver and regenerating liver. The specific anti-HOPS antibody was raised in rabbit against a peptide localized in the N terminus of HOPS. Transfection of CMV-Hops in COS-1 cells detected two specific bands of different molecular masses: 27 kDa corresponding to full-length Hops and an additional band of 24 kDa (see Fig. S1A in supplementary material). Western blot analysis performed on mouse normal liver showed an appreciable single band of 27 kDa corresponding to the translation of full-length Hops cDNA. During mouse liver regeneration HOPS expression gradually increased with a peak at 48 hours after PH in concurrence with the first mitotic wave of residual hepatocytes. In the following hours protein expression progressively decreased. The additional band of different molecular mass, 24 kDa, was detected 8 hours after PH (Fig. 1D). This additional band was associated with post-translation modifications that affected HOPS at the C-terminal of the protein. A fusion protein, HOPS-GFP, expressed in cells, with GFP placed at the C terminus of HOPS, showed release of GFP from HOPS. Western blot analysis with anti-GFP revealed a specific cleavage in the C terminus of HOPS (Fig. S1 in supplementary material).

Following western blot analysis where overexpression of HOPS was detected after PH, we tested HOPS immunolocalization in histological specimens of normal and regenerating mouse liver. In normal hepatocytes, HOPS was localized mainly in the nucleus with a small amount detectable in the cytoplasm (Fig. 2). At 30 minutes after PH in the residual hepatocytes, HOPS migrated to the cytoplasm. This phenomenon was observed until 8 hours after PH. At 12 hours the shuttling protein returned progressively to the nucleus and at 48 hours, in concurrence with the first mitotic wave of the residual hepatocytes, HOPS was strongly expressed in the nucleus. At 72 hours after PH, in synchronization with a second mild wave of proliferation, a slow migration pattern of HOPS was detected in cytoplasm (Fig. 2).

Following the results obtained in vivo in liver regeneration, the role of HOPS in H-35 rat hepatoma cells was examined. HOPS western blot analysis in H-35 proliferating cells showed two distinct bands, at 27 and 24 kDa (Fig. 3A). To investigate HOPS expression at different stages of the cell cycle, H-35 cells were synchronized using the double thymidine arrest technique. As demonstrated by flow cytometry, highly synchronized cells were obtained. Thymidine inhibited DNA synthesis and arrested cells on the G₁/S border. At the end of the double thymidine arrest (time 0), 90-92% of hepatoma cells arrested in G₁/S phase (Fig. 3A).

Protein extracts from H-35 synchronized cells were collected at different times after release from the double thymidine block. HOPS was strongly expressed at time 0 in relation to the arrest of the cell cycle of hepatoma cells and down-regulated rapidly after release (Fig. 3A). Similar results were obtained by blocking the cell cycle of H-35 cells in G₀/G₁ by serum deprivation. During cell starvation the progressive increase of HOPS expression was analyzed. When the cell cycle was arrested, at the end of 72 hours of serum deprivation, HOPS was overexpressed in the starved cells. Addition of serum to the culture medium down-regulated HOPS expression in the cells (Fig. 3A).

In light of these results, we investigated a possible change of HOPS localization in relation to the cell cycle. The progressive starvation of H-35 cells, following serum deprivation, allowed us to study the redistribution of HOPS localization during cell cycle arrest. HOPS was diffused in the nucleus and cytoplasm of wild-type hepatoma cells. The protein progressively migrated to the nucleus 12 hours after serum deprivation. In the following hours, cells progressively stopped proliferating and HOPS accumulated in the nuclei. At 72 hours after serum deprivation, when almost 90% of the cells were in G₀/G₁ phase (Fig. 3B) and HOPS was overexpressed, the protein was localized mainly in the nucleus. Addition of serum to the culture medium caused a rapid increase in cell proliferation, as demonstrated by flow cytometry, and redistribution of HOPS in the cells (Fig. 3C).

Rapid HOPS export from the nucleus to the cytoplasm in proliferating hepatocytes raised the question of whether there are agents that could affect shuttling. During liver regeneration different factors act on residual hepatocytes to modify expression and induce proliferation. In particular, our attention focused on two factors that act rapidly on residual hepatocytes following PH, EGF and cAMP. The effects of EGF and cAMP on HOPS shuttling in the liver were analyzed. In EGF-treated mice a small amount of HOPS migrated into the cytoplasm of hepatocytes at 30 minutes after treatment and almost all protein returned to the nucleus after 60 minutes. At 90 minutes HOPS was detected again in the nucleus (data not shown). In cAMP-treated mice the shuttling protein was detected in the nucleus at 15 and 30 minutes after treatment and migrated in part into the cytoplasm. At 60 minutes HOPS was exported completely into the cytoplasm. At 90 and 120 minutes HOPS returned progressively to the nucleus, showing a distribution pattern similar to normal hepatocytes (Fig. 4).

Analysis of the HOPS amino acid sequence revealed the motif LACLLVLALA in the N-terminal region, a typical nuclear export signal (NES) region, present in proteins that are exported from the nucleus to the cytoplasm via CRM-1 (Fig. 1B and Fig. 5A) (Fornerod et al., 1997; Fukuda et al., 1997; Macara, 2001).

To assess the export of HOPS via CRM-1, we investigated HOPS localization in H-35 hepatoma cells following treatment with leptomycin B (LMB), a specific inhibitor of nuclear export mediated by leucine-rich NES (Wolff et al., 1997). Nuclear export inhibition revealed enrichment of HOPS in the nucleus compared with the control, demonstrating that HOPS export acts via CRM-1 (Fig. 5B,C).

To gain further insight into the role of HOPS and its shuttling function, screenings were performed using the two-hybrid system in yeast; HOPS was used as bait to identify proteins that specifically bind it. Positive yeast clones were isolated from a cDNA library of total E9.5-12.5 embryos in selected medium during the two-hybrid system screening and all were positive for β-galactosidase activity. In addition, there was no β-galactosidase activity in the yeast strain transformed with the empty vector (pASV3; Fig. 6A).

Our attention was drawn to one of the isolated clones that is present in more than 10% of all positive yeast clones characterized. The clone displays high β-galactosidase activity and its sequence corresponds to eukaryotic elongation factor-1A (eEF-1A) (Fig. 6B). Immunoprecipitation studies conducted in endogenous protein on H-35 hepatoma cell lines and in liver confirmed the native interaction of HOPS with eEF-1A. Protein extracts were immunoprecipitated using polyclonal anti-HOPS followed by immunoblotting with the monoclonal anti-eEF-1A antibody (Fig. 6C). Similarly, immunoprecipitated protein extracts were tested with monoclonal anti-eIF2A antibody as control (Fig. 6C). The results confirmed the in vivo interaction between HOPS and eEF-1A.

Following immunoprecipitation studies on binding specificity between HOPS and eEF-1A, the possibility of a significant role for this interaction was analyzed. Because eEF-1A is essential in protein synthesis in peptide chain elongation, in vitro protein synthesis levels were evaluated in the presence of different HOPS recombinant protein concentrations. In vitro transcription and translation experiments were performed using luciferase cDNA as the reporter gene. The amount of luciferase protein was evaluated in the presence and absence of HOPS recombinant protein. HOPS recombinant protein was added to the in vitro translation (Fig. 7A) at different concentrations (60, 120, 240, 360 and 420 nM). There was a slight increase in protein synthesis when 60 nM of recombinant HOPS was added while 120 nM had no effect. Surprisingly, recombinant HOPS at 240 nM reduced protein synthesis to almost 50% (Fig. 7A). No synthesis of luciferase was detected using 360 and 420 nM (Fig. 7A,B). Analogous experiments performed with the same concentration of GST, used as protein control, had no significant effects on the synthesis of luciferase (Fig. 7B). In reticulate lysates with different HOPS concentrations the expression of eEF-1A was tested by western blot analysis. No differences in eEF-1A expression were detected in all samples examined (Fig. 7A).

The increased level of HOPS expression detected in H-35 cells induced to arrest proliferation or in residual hepatocytes following PH suggest a possible involvement of HOPS in proliferation. To test this hypothesis, H-35 stable cells overexpressing HOPS were generated. Stable cell cultures were analyzed by cytofluorimetric analysis and [³H]thymidine incorporation was evaluated. The study was performed on four different stable clones. The results showed that HOPS overexpression blocked H-35 cell growth. Colony growth assay showed that [³H]thymidine incorporation was drastically reduced in H-35 stable clones overexpressing HOPS with respect to H-35 stable clones used as control. In H-35 stable cells overexpressing HOPS the percentage of [³H]thymidine incorporation showed a reduction of about of 65% (Fig. 7C) with respect to controls. In H-35 stable clones HOPS is localized in the nucleus and cytoplasm (Fig. S2 in supplementary material). These results implicate the involvement of HOPS during cell proliferation. To further verify this hypothesis, experiments were performed in NIH-3T3 proliferating cells and the HOPS anti-proliferative effect was quantified. NIH-3T3 cells were infected with pBabe-puro or pBabe-Hops. A growth cell selection by puromycin was carried out for 5 days in both types of infected cells. The results indicate that HOPS inhibits proliferation in NIH-3T3 cells infected with pBabe-Hops. After puromycin selection the number of cells at day 2 decreased in both types of infected cells. In the following days, a stronger proliferation of cells infected with pBabe-puro was observed than in cells infected with pBabe-Hops. The number of the cells switched from approximately 58 ×10⁴ with pBabe-puro to about 22 ×10⁴ with pBabe-Hops, showing a reduction of almost 65% (Fig. 7D).

In this paper, the role played during cell proliferation of a newly identified gene, Hops, isolated in regenerating liver, is described. Evidence is provided that HOPS is a nucleocytoplasmic shuttling protein that contributes to the control of cell proliferation by regulating protein synthesis. In liver regeneration, early after PH, many factors such as hormones and growth factors act on residual hepatocytes that rearrange gene expression and protein synthesis. Following PH, the residual hepatocytes begin to proliferate in synchrony. Recent studies speculate that a crucial decision regarding growth and proliferation arrest must be taken in the cell after cell division. In fact, studies indicate that external and internal factors act on the cell directing quiescence or proliferation (Malumbres and Barbacid, 2001).

In liver regeneration many factors act on residual hepatocytes to organize the reconstitution of the original liver mass. After PH, change occur in stable hepatocytes at many levels (protein synthesis, energy requirement, proliferation) arranging their cell program on the basis of actual needs. It has been demonstrated that the growth factors EGF, TGF-α and HGF work as priming factors on the residual hepatocytes in the first hours after PH, and increased levels in cAMP concentration have been detected in the first hours after PH. We observed in vivo that the increased level of cAMP allows HOPS to export in proliferating hepatocytes following PH or in normal hepatocytes after cAMP intraperitoneal injection in mice. Our results show that the rapid export of HOPS from the nucleus to cytoplasm is ascribed to high levels of cAMP in the cells. The return of HOPS to the hepatocyte nucleus, 90 minutes after cAMP injection compared with regenerating hepatocytes where HOPS returns 12 hours after PH, would suggest that HOPS stimulated by cAMP migrates into the cytoplasm, but proliferation of regenerating liver retains HOPS in the cytoplasm.

The compartmentalization of proteins and their regulation in export and import mechanisms from the nucleus to cytoplasm is an important system of control in cell functions. The presence of a NES domain in the HOPS sequence and specific protein accumulation in hepatoma cell nucleus after treatment with LMB indicates an involvement of CRM-1 in nuclear export of HOPS protein. Based on these data we speculate that CRM-1 is responsible for the nuclear export of HOPS and in turn regulates HOPS cytoplasmic functions.

The identification of eEF-1A as a molecular partner binding HOPS in liver and in hepatoma cells shows a specific functional interaction between the two proteins. eEF-1A plays a key role in protein synthesis and controls the first step of elongation of the growing peptide. In the cells, eEF-1A is located predominantly in the cytoplasm. Recent studies showed that eEF-1A is actively exported from the nucleus to keep the nuclear eEF-1A concentration down to 1/100 with respect to the cytoplasmic concentration, preventing eventual nuclear translation (Bohnsack et al., 2002; Calado et al., 2002). During cell proliferation, translation machinery of protein synthesis rapidly increases and protein synthesis factors, ribosomes and regulating factors guarantee a rapid processing of transcripts (Thomas, 2000; Ruggero and Pandolfi, 2003).

In the first hours following PH, in the period preceding cell mitosis, the residual hepatocytes are hypertrophic and protein synthesis is strongly activated. Our findings suggest a molecular mechanism in which, during residual hepatocyte proliferation after PH, HOPS shuttles from the nucleus to cytoplasm playing a pivotal role in the control of protein synthesis by eEF-1A activity regulation (Fig. 8). These assumptions are supported by results of in vitro translation assay in which the addition of recombinant HOPS regulates protein synthesis.

During liver regeneration HOPS rapidly migrates from the nucleus to the cytoplasm 12 hours after PH and the shuttling protein returns to the nucleus where it is overexpressed until 72 hours. In hepatoma cells, the cell cycle is arrested by starvation and HOPS is progressively overexpressed and accumulates in the nucleus. Upon analysis the two phenomena may appear incongruous. HOPS is overexpressed in proliferating cells, such as residual hepatocytes in liver regeneration, and in hepatoma cells induced to arrest proliferation. After PH the residual hepatocytes are programmed to perform one or two rounds of the cell cycle, unlike hepatoma cells that lack the capacity to control the proliferative process. It could be that proliferative signals act, on residual hepatocytes in the first hours following PH in vivo, and signals from the serum act on hepatoma cells allowing HOPS to shuttle from the nucleus to cytoplasm. The absence of these signals, in the late phases of liver regeneration or in serum-deprived cells, induces HOPS migration into the nucleus and its overexpression. These events precede the proliferative arrest in residual hepatocytes after a round of proliferation as well as in starved hepatoma cells. This hypothesis is supported by experiments performed in the H-35 hepatoma cells. H-35 stable clones overexpressing HOPS have a strong reduction in thymidine uptake and a delay in growth. Furthermore, our data confirm that HOPS overexpression in NIH-3T3 cells is able to strongly reduce cell proliferation. These findings suggest that cAMP and/or other proliferative signals act on HOPS to regulate shuttling and expression. We believe that HOPS is a protein that finely regulates cell proliferation at the level of protein synthesis. HOPS exerts its role by affecting eEF-1A in cytoplasm thus regulating cell proliferation.

Recently it has been suggested that a key role is played by translation factors and protein synthesis in the transformation and regulation of cell proliferation (Caraglia et al., 2000; Ruggero and Pandolfi, 2003). Alterations in eEF-1A expression correlate with cancer and potential metastatic activity of mammary adenocarcinoma (Edmonds et al., 1996). Furthermore, the oncogene PTI1 (prostate tumor inducing gene), a hybrid molecule containing a truncated form eEF-1A, appears to play an important role in prostate cancer (Gopalkrishnan et al., 1999).

The implication of translation factors in protein synthesis in cancer cells may facilitate identification of novel therapeutic agents that act on protein synthesis.

The authors thank Stefano Brancorsini, Emira Ayroldi, Eileen Mahoney Zannetti and all the members of the Servillo laboratory for help, fruitful discussion and for critical reading of the manuscript, and Silvano Pagnotta and Maria Luisa Alunni for their excellent technical assistance. This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Ministero dell'Università edella Ricerca Scientifica 2003-2004, Cofin prot. 2003063402_004, Italy.