Position-specific Gene Expression in the Liver Lobule Is Directed by the Microenvironment and Not by the Previous Cell Differentiation State*

Sanjeev GuptaDagger §parallel , Pankaj RajvanshiDagger , Rana P. SokhiDagger , Shilpa VaidyaDagger , Adil N. IraniDagger , and Giridhar R. GorlaDagger **

From the Dagger  Marion Bessin Liver Research Center and § Cancer Research Center, Departments of  Medicine and ** Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York 10461

    ABSTRACT
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Abstract
Introduction
References

Mechanisms directing position-specific liver gene regulation are incompletely understood. To establish whether this aspect of hepatic gene expression is an inveterate phenomenon, we used transplanted hepatocytes as reporters in dipeptidyl peptidase IV-deficient F344 rats. After integration in liver parenchyma, the position of transplanted cells was shifted from periportal to perivenous areas by targeted hepatic ablations with carbon tetrachloride. In controls, transplanted cells showed greater glucose-6-phosphatase and lesser glycogen content in periportal areas. This pattern was reversed when transplanted cells shifted from periportal to perivenous areas. Transplanted hepatocytes in perivenous areas exhibited inducible cytochrome P450 activity, which was deficient in periportal hepatocytes. Moreover, cytochrome P450 activity was rapidly extinguished in activated hepatocytes when these cells were transplanted into the nonpermissive liver of suckling rat pups. In cells isolated from the normal F344 rat liver, cytochrome P450 inducibility was originally greater in perivenous hepatocytes; however, periportal cells rapidly acquired this facility in culture conditions. These findings indicate that the liver microenvironment exerts supremacy over prior differentiation state of cells in directing position-specific gene expression. Therefore, persistence of specialized hepatocellular function will require interactions with regulatory signals and substrate availability, which bears upon further analysis of liver gene regulation, including in progenitor and/or stem cells.

    INTRODUCTION
Top
Abstract
Introduction
References

Hepatocytes show unique differences in gene expression patterns in the liver lobule. Some genes are expressed throughout the liver lobule, whereas others exhibit gradients between portal and perivenous areas (1-9). Associations between transcriptional gene switching, such as albumin and alpha -fetoprotein genes, and selective gene expression during development, as seen for cytochrome P450 genes have been interpreted in the context of cell differentiation mechanisms (10-12). Demonstrations of specialized functions along with other parameters in hepatocyte subsets, including those involved in drug disposition in perivenous areas, led to speculations concerning the presence of less differentiated hepatic progenitor cells in periportal areas with less comprehensive gene expression repertoire, followed by the acquisition of specialized functions in more differentiated hepatocytes in perivenous areas (12). This feature of hepatic gene expression was augmented by interspersed instances of markedly restricted gene expression in the liver lobule. For example, glutamine synthetase and GLUT-1 are expressed in only a small rim of hepatocytes immediately adjacent to the terminal hepatic venule (7, 9). The involvement of transcriptional, as well as post-transcriptional, mechanisms in the regulation of specific genes, such as glutamine synthetase, phenobarbitone-inducible cytochrome P450IIB1,2 genes, and GLUT-1 in perivenous areas, has been proposed to reflect compartmentation of specialized function in specific hepatocyte subsets (2). The possibility of other mechanisms, including substrate availability and regulation by soluble circulatory signals led to complex studies of vascular mechanisms, such as alterations in portal blood flow in intact animals (13).

The recent ability to repopulate the liver with genetically marked hepatocytes offers unique opportunities for studying physiological processes in the liver (14). One transplantation system utilizes F344 rats deficient in dipeptidyl peptidase IV enzyme activity (DPPIV-) as recipients of DPPIV+ syngeneic hepatocytes (15). In this system, transplanted hepatocytes can be conveniently localized by histochemical visualization of DPPIV and dual-label studies can be conducted to address gene expression or cell proliferation mechanisms (16). Transplanted hepatocytes are originally located in periportal areas due to their arrival in the liver lobule along with portal blood and entrapment in proximal hepatic sinusoids due to the smaller size of the latter. An important element concerns integration of transplanted hepatocytes in the liver parenchyma, as shown by the presence of hybrid plasma membrane structures, such as gap junctions and bile canaliculi among conjoint transplanted and host hepatocytes (14). Transplanted hepatocytes can be induced to replicate, especially under conditions involving loss of host hepatocytes, such that toxins producing perivenous liver injury that would spare transplanted cells in periportal areas cause these to proliferate, along with uninjured host hepatocytes (17). Repeated self-limited liver injury to the host can induce multiple proliferation cycles in transplanted hepatocytes. During these cycles of liver injury, transplanted hepatocytes migrate from periportal toward perivenous areas, in a process resembling "wound repair."

We considered that examination of position-dependent gene regulation mechanisms in the repopulated liver would be instructive. The fate of transplanted cells induced to change their position in the liver lobule could be compared with control animals receiving cells from the same donor animal but without undergoing any change from their original periportal location. This would allow analysis of cell age-related versus position-specific mechanisms in gene regulation. Also, we reasoned that transplantation of fully differentiated mature hepatocytes into another microenvironment, such as the liver of recent-born pups, would allow further analysis of position-specific gene expression. Finally, we asked whether gene expression could be regulated in similar fashions in hepatocytes isolated from periportal and perivenous areas of the liver lobule, if their microenvironment were altered. The latter studies were facilitated by our recent characterization of hepatocytes fractionated from periportal or perivenous areas of the same animal followed by analysis in cell culture (18).

    EXPERIMENTAL PROCEDURES

Animals-- Eight- to 10-week old DPPIV- F344 rats weighing 160-180 g and 3-day-old DPPIV- pups weighing 7-9 g were provided by the Special Animal Core of the Marion Bessin Liver Research Center. Normal F344 rats were obtained from the National Institutes of Health (Bethesda, MD) and weighed 250-300 g when used. Animals were utilized in groups of at least three to four animals each under protocols approved by the institutional Animal Care and Use Committee, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 86-23, revised 1985).

To induce perivenous hepatic injury (19), 1.45 ml/kg carbon tetrachloride (CCl4) was administered intramuscularly after diluting 1:1 (v/v) in mineral oil (Sigma). To induce CYP450 activity in hepatocyte recipients, 0.5 g/liter phenobarbitone (Sigma) was administered in drinking water for 4 days to adult rats, which amounted to 10-12.5 mg of phenobarbitone/rat/day as 20-25 ml water was consumed per 24 h. Hepatocyte donors were treated with phenobarbitone as above for 7 days. Rat pups received 1 mg of phenobarbitone intramuscularly for 7 days with continued nursing by mothers. The animals were housed in the Institute for Animal Studies at Albert Einstein College of Medicine under 14 h light, 10 h dark cycles and received standard pelleted food and water ad libitum.

Hepatocyte Isolation and Cell Fractionation-- Cells were isolated from F344 rats with two-step perfusion using 0.025% (w/v) collagenase, as described previously (15). The cell viability was tested by trypan blue dye exclusion and attachment to tissue culture plastic in RPMI 1640 medium containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). To fractionate cells, Percoll (Amersham Pharmacia Biotech, Uppsala, Sweden) was diluted in phosphate-buffered saline, pH 7.4 (PBS)1 containing 5 mg/ml bovine serum albumin (Sigma), as described (18). Hepatocytes were suspended in 1 ml of RPMI 1640 medium, and 1-1.5 × 107 cells were placed at the top of Percoll gradients in 15-ml polystyrene tubes, followed by centrifugation under 1000 × g for 30 min at 4 °C. Cells localizing in discrete bands were designated H4 (at the bottom) and H2 (toward the top). These cells were obtained by pipetting and washed twice in RPMI 1640 medium. Characterization of fractionated hepatocytes included flow cytometric determination of cell ploidy, analysis of gamma -glutamyl transpeptidase, glucose-6-phosphatase (Glu-6-P), glycogen, and P450 activities by histochemical and immunocytochemical methods, as described previously (18).

Hepatocyte Transplantation-- Animals were anesthetized with inhaled ether (Fisher). Adult rats received 2 × 107 cells, and pups received 0.5 × 106 cells via spleen, as described previously (15). After closing the abdomen, animals were monitored closely until full recovery. Pups were returned to the mothers immediately after recovery from anesthesia.

Histological Analysis for Transplanted Cell Localization-- To localize F344 rat hepatocytes in DPPIV- animals, histochemical staining was performed on frozen tissue sections as reported previously (15). Integrations of transplanted cells in the liver parenchyma were demonstrated by colocalization of DPPIV activity and bile canalicular ATPase activity. Tissues were counterstained with hematoxylin.

To document the position of transplanted cells in tissues, serial wedge liver biopsies were obtained from individual CCl4-treated adult rats. The position of transplanted cells in the liver of pups was verified by sampling of animals. To determine the position of transplanted cells in liver lobules, morphometric analysis was performed with multiple microphotographs using a standard magnification (× 40). At a minimum, 50 liver lobules were analyzed per animal with determination of the number of transplanted cells in various zones of the liver lobule. In addition, the number of cells in each liver lobule was determined. The actual distance of transplanted cells from the center of the nearest portal vein was measured in microphotographs. To document whether equivalent areas of the liver were analyzed, we measured portal vein diameters and the distance between portal and central veins.

Gene Expression in the Intact Liver-- Colocalization studies were performed to determine gene expression in transplanted cells. Glu-6-P activity was determined in unfixed cryostat tissue, followed by acetone-chloroform fixation and DPPIV histochemistry for localizing transplanted cells, as described previously (15). Tissue glycogen was determined by first performing DPPIV histochemistry in cryostat sections fixed with ethyl alcohol-acetic acid followed by incubation in 1% aqueous periodic acid for 5 min and Schiff's reagent for another 5 min (18). After washing repeatedly in 0.5% sodium bisulfite, slides were immersed in running tap water for 10 min. To verify the specificity of glycogen staining, control tissues were preincubated with salivary amylase. The intensity of staining for Glu-6-P and glycogen was arbitrarily graded from 0 (negative) to 4+ (maximally positive). To demonstrate differences in gene expression, at least 50 cells per condition were graded in each animal.

A polyclonal goat antibody directed against rat CYPIIB1 isoform (Gentest Inc., Woburn, MA) was used for analyzing phenobarbitone-induced P450 expression (18). Cryostat tissue sections or cytospun hepatocytes were fixed in alcohol-acetone (99:1 v/v) and ethanol alone at --20 °C for 5 min each. Tissues were air-dried, rehydrated in PBS, blocked with nonimmune goat serum (diluted 1:20 in PBS) for 20 min, and incubated with the primary antibody (diluted 1:20 in PBS) for 1 h at room temperature. After washing with PBS three times, antibody binding was localized with peroxidase-conjugated goat IgG for 30 min at room temperature using the diaminobenzidine substrate (Sigma).

In Vitro Analysis of P450 Activity-- Chemicals and reagents were from Sigma. Freshly isolated hepatocytes were cultured in 24-well tissue culture plates coated with rat tail collagen (0.5-1 × 105 cells/cm2). Cells were allowed to attach for 1-2 h in RPMI 1640 culture medium containing 10% fetal bovine serum, penicillin, streptomycin, and 10-8 M dexamethasone. To induce P450 activity, either 2 mM phenobarbitone or 2 µM 3-methylcholanthrene were added to the culture medium immediately after cell attachment, with replacements of these additives on days 2 and 3 of culture. P450 activity was assayed 1, 2, 3, and 5 days after initiating hepatocyte cultures, according to an assay described previously with minor modifications (20). Cells were washed twice with cold PBS followed by the addition of 8 µM 7-ethoxyresorufin and 10 µM dicumarol with incubation for 1 h at 37 °C. From some wells, culture medium and cells were harvested separately, whereas only medium was harvested from other wells for sequential studies. To 0.3-ml aliquots of culture medium, 0.2 ml of ethanol was added, and samples were centrifuged for 10 min in a microcentrifuge to solubilize resorufin products, followed by fluorescence measurements at 530 nm (excitation) and 590 nm (emission). Purified resorufin was used to generate standard curves in linear range and data normalized by measuring protein in aliquots with the Bio-Rad assay.

Experimental Design-- Studies using adult rats addressed gene expression in unprimed transplanted hepatocytes. The strategy was to transplant hepatocytes isolated from a single donor into 18 DPPIV- F344 rats. The animals were divided into two groups of nine rats each. One group served as controls with cells in periportal areas, since intrasplenic injection is known to deposit hepatocytes into these areas (15). The other animal group was given three doses of CCl4 at 10-day intervals to cause perivenous injury and shift the position of transplanted hepatocytes from periportal to perivenous areas during the ensuing liver repair (Fig. 1A).


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Fig. 1.   Experimental strategies for in vivo experiments. A shows the scheme of cell transplantation experiments in adult rats. Transplanted cells were allowed to engraft and integrate in the liver parenchyma for 2 months after which three cycles of CCl4 were administered separated by 10 days each. Serial biopsies were obtained from several animals before and after CCl4. Some animals were given phenobarbitone in drinking water for 4 days prior to sacrifice at 24 months after cell transplantation. B shows the scheme of cell transplantation into 3-day-old suckling pups. Hepatocytes were from animals treated with phenobarbitone for 7 days, which induced P450 activity. Pups intended for sacrifice after 1 week were treated with intramuscular phenobarbitone starting on the day of cell transplantation until sacrifice. Pups intended for sacrifice at 4 weeks were treated with intramuscular phenobarbitone for the preceding 7 days.

A second experiment concerned transplantation of hepatocytes into suckling DPPIV- rat pups with P450 induction by treating the donor rat with phenobarbitone for 7 days. These hepatocytes were transplanted into nine 3-day-old DPPIV- pups (Fig. 1B). The pups were killed in groups of 2-3 animals each at 2 h, 7 days, and 4 weeks after cell transplantation. The pups received phenobarbitone daily for 7 days each prior to being killed at 7 days and 4 weeks after cell transplantation.

Statistics-- Data are expressed as mean ± S.D. SigmaStat 2.0 software was used for data analysis (Jandel Corp., San Rafael, CA). The significance of differences was analyzed as appropriate by Student's t test, Chi-square test, Mann-Whitney rank sum tests, or analysis of variance.

    RESULTS

The experiments were first conducted in adult rats. After transplantation via spleen, hepatocytes engrafted in the liver with integrations in the hepatic parenchyma, which was documented by colocalization of bile canalicular DPPIV and ATPase activities (not shown).

Changing the Position of Transplanted Hepatocytes in the Adult Rat Liver-- In normal control rats, transplanted hepatocytes were distributed mainly in periportal areas of the liver. This pattern was generally unchanged throughout the 2-year duration of the studies (Fig. 2). The findings were compatible with previous studies of liver repopulation in normal mice (21). However, at 24 months, transplanted hepatocytes showed somewhat larger cell clusters in F344 rats, which indicated the possibility of spontaneous cell proliferation in this setting, although transplanted cells still remained in periportal locations. In contrast, in response to CCl4-induced liver injury, the position of transplanted cells changed progressively. In this situation, transplanted cells were localized in periportal areas (zone 1), deeper inside (zone 2), as well as perivenous areas (zone 3) of the liver lobule.


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Fig. 2.   Switching the position of transplanted hepatocytes in the adult rat liver. Control rat liver from an individual animal at 2 months (A), 3 months (B), and 24 months (C) after hepatocyte transplantation. Transplanted hepatocytes are visualized by histochemical localization of DPPIV activity. Note that transplanted cells (arrows) appear in periportal areas at all these times in control animals. The remaining panels show transplanted cells in an animal subjected to CCl4 treatments. D shows a liver biopsy at 2 months after cell transplantation, before CCl4 was given. Transplanted cells are in periportal locations, similar to control animals. E shows the liver following two cycles of CCl4, when the position of transplanted cells began to shift toward perivenous areas. F shows a large cluster of transplanted hepatocytes after cells shifted to the perivenous area at 24 months. Also, there is spontaneous proliferation of biliary cells in F, which is a known feature of the aging F344 rat liver. p = portal area; c = perivenous area.

Detailed morphometric analysis of sequential biopsies in three animals showed that at the end of all three CCl4 treatments, significantly fewer transplanted hepatocytes were in zone 1 of the liver lobule, whereas more hepatocytes were in zone 3 (Table I). To assure that various tissue samples were comparable, we evaluated the dimensions of the liver lobules under study by using portal vein diameters and the distance between portal and central veins as reference points. These parameters were not different in the tissues analyzed. The mean distance of transplanted cells from the center of portal vein shifted significantly after CCl4. Also, the overall number of transplanted hepatocytes increased in the liver, consistent with cell proliferation during recovery from CCl4-induced liver injury. Analysis of the number of transplanted cells showed 6 ± 2 cells per liver lobule before CCl4 and 15 ± 3 cells per liver lobule after the completion of all three CCl4 treatments, p < 0.02 (t test). However, some cell clusters in CCl4 recipients were large and contained >100-200 cells each. The findings were compatible with the ability to shift the position of transplanted hepatocytes from periportal areas to additional locations without altering the overall organization of the liver lobule.

                              
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Table I
Relative position of transplanted hepatocytes in the liver lobule

Liver Gene Regulation in Transplanted Cells Engrafted in the Adult Liver-- Although Glu-6-P and glycogen are present in hepatocytes throughout the liver lobule, during previous studies to demonstrate gene expression in transplanted cells, we recognized gradients of expression in the F344 rat liver. We found that while Glu-6-P showed greater histochemical activity in periportal areas (3+ to 4+ grade), compared with perivenous locations (0 to 2+ grade), this situation was reversed in the case of glycogen (Fig. 3). These findings allowed us to determine whether gene expression was regulated in transplanted hepatocytes following shifts in their position. In normal control animals, as well as in animals before CCl4 treatments, periportally located transplanted hepatocytes showed greater Glu-6-P activity (3+ to 4+), similar to adjacent host hepatocytes, compared with host hepatocytes in perivenous areas (0 to 2+). In contrast, when the position of transplanted hepatocytes was shifted toward perivenous areas, Glu-6-P expression began to progressively decline, such that in hepatocytes immediately adjacent to the terminal hepatic venule, Glu-6-P expression was markedly attenuated (p < 0.01, t test). Interestingly, in the CCl4-treated liver where transplanted cells were simultaneously localized elsewhere, there was much greater Glu-6-P activity in periportal areas, indicating that the change in gene expression was not simply due to some other cellular alteration during CCl4-induced proliferation. Furthermore, changes in the glycogen content of transplanted hepatocytes mirrored those of Glu-6-P. In transplanted hepatocytes situated in periportal areas, as well as adjacent host hepatocytes, less glycogen was visualized (1+ to 2+) compared with hepatocytes in perivenous areas (3+ to 4+), p < 0.01, t test. This situation became reversed when transplanted hepatocytes shifted to perivenous areas.


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Fig. 3.   Gene regulation in the adult liver. A, glycogen staining showing a gradient in the liver lobule with greater glycogen content in perivenous hepatocytes. B, Glu-6-P staining showing a reversed gradient, with greater expression in periportal areas. C, transplanted hepatocytes (arrow) in a control animal showing less glycogen content, similar to adjacent host hepatocytes in periportal areas compared with host hepatocytes in perivenous areas. D, Glu-6-P staining in liver from the same control animal showing a reversed pattern, with greater Glu-6-P expression in transplanted periportal hepatocytes compared with perivenous hepatocytes. E, glycogen staining in the liver from an animal subjected to repeated CCl4 treatments showing extensive glycogen expression in perivenous areas. F, attenuation of Glu-6-P expression in transplanted hepatocytes localized in perivenous areas with some hepatocytes immediately adjacent to the terminal hepatic venule showing very little Glu-6-P activity (short arrows). In contrast, transplanted hepatocytes situated in adjacent periportal areas show much greater Glu-6-P activity (long arrows).

To demonstrate regulation of cytochrome P450 system, we analyzed changes in response to induction by phenobarbitone. Hepatocyte recipients were given phenobarbitone for 4 days immediately before sacrificing animals at 24 months. Immunolocalization of CYPIIB1 activity, along with DPPIV staining to localize transplanted cells, showed that whereas host and transplanted hepatocytes in perivenous areas were induced by phenobarbitone, neither transplanted nor host hepatocytes in periportal areas could express CYPIIB1 activity (not shown). The findings were again compatible with regulation of gene expression in transplanted hepatocytes by the ambient microenvironment of the liver lobule.

Gene Regulation in Transplanted Hepatocytes in the Developing Rat Liver-- These studies were performed in suckling F344 rat pups to elucidate regulation of gene expression in hepatocytes isolated from adult rats. The premise was that transplanted hepatocytes would engraft and function in the developing liver. Immediately after injection of cells into the spleen, transplanted hepatocytes appeared in portal areas and hepatic sinusoids (Fig. 4). Dual staining for bile canalicular DPPIV and ATPase activities showed that transplanted hepatocytes were integrated in the liver parenchyma at 7 days of transplantation. The suckling rat liver showed a gradient of Glu-6-P, which was similar to the adult rat liver, and supported abundant expression of Glu-6-P activity in transplanted hepatocytes (3+ to 4+). In contrast, hepatic glycogen stores were less pronounced (1+) in the suckling rat liver and showed no gradients in the liver lobule at 7 days of age. This was reflected in transplanted hepatocytes, many of which showed very little glycogen. However, glycogen staining became more pronounced in both transplanted and host hepatocytes at the age of 4 weeks (2+ to 3+), consistent with further maturation of the liver.


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Fig. 4.   Gene regulation in the suckling liver. A, hepatocytes integrated in the liver of rat pups as shown here at 7 days after cell transplantation. The liver was first stained for DPPIV activity (red color, short arrows) and then for ATPase activity (brown color, long arrows) to demonstrate networks of hybrid bile canaliculi in liver plates. B, the gradient of Glu-6-P activity in the suckling rat liver, which was similar to the adult rat liver. The arrow is pointing to transplanted hepatocytes at 7 days after injection into the pup. C, higher magnification view of the same area as in B showing Glu-6-P expression in transplanted hepatocytes. D, glycogen staining in the 7-day-old suckling rat liver showing relatively faint staining compared with the adult liver and an absence of a gradient in the liver lobule. Transplanted hepatocytes (arrows) show little glycogen activity. E, at 4 weeks after cell transplantation, when animals had not yet been weaned, more glycogen was apparent in the liver. However, at this stage, glycogen staining was more pronounced in periportal areas compared with perivenous areas, which was in contradistinction with the adult liver. Transplanted hepatocytes (arrow) began to demonstrate greater glycogen content as well. F shows magnified view of transplanted hepatocytes at 4 weeks with glycogen content similar to that of adjacent periportal hepatocytes of the host.

The regulation of phenobarbitone-inducible CYPIIB1 activity in the suckling liver was instructive. When hepatocytes were isolated from a donor with extensive P450 induction following 7 days of treatment with phenobarbitone, 80-90% cells showed immunostaining for CYPIIB1 activity. Upon transplantation of these cells into the suckling rat liver, CYPIIB1 expression was present in transplanted cells at 1 h of transplantation (Fig. 5). However, all transplanted cells rapidly lost CYPIIB1 activity in the suckling rat liver subsequently (p < 0.001, Chi-square) when animals were studied at 1 week after cell transplantation, despite phenobarbitone administration to pups for these 7 days. Moreover, P450 activity did not return in transplanted hepatocytes at 4 weeks, although perivenous cells in the suckling liver were faintly stained with anti-CYPIIB1 at this time following 7 days of phenobarbitone treatment. The findings indicated that in response to signals emanating from the nonpermissive host hepatic microenvironment, P450 activation was lost in transplanted hepatocytes.


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Fig. 5.   Phenobarbitone-inducible CYPIIB1 activity in the suckling liver. A, donor rat liver showing extensive immunoreactivity with an antibody against CYPIIB1. The animal was treated with phenobarbitone for 7 days prior to isolation of cells. B, the suckling liver from pups treated with phenobarbitone for 7 days did not react with the antibody. Control tissues from an uninduced normal adult rat liver or when the primary antibody was omitted also showed no reaction product. C, showing transplanted hepatocytes staining with CYPIIB1 antibody in portal areas and hepatic sinusoids of a suckling pup within 1 h of intrasplenic injection. D, at 7 days after cell transplantation, CYPIIB1 immunostaining was detected in neither transplanted hepatocytes (arrows) nor host liver.

Regulation of Gene Expression in Hepatocytes Isolated from Different Positions of the Liver Lobule-- We reasoned that if external signals were important determinants in hepatic gene regulation, it should be possible to test whether specific function could be induced in hepatocytes isolated from permissive and nonpermissive areas of the liver lobule. For this purpose, we utilized Percoll gradients to isolate hepatocytes enriched in periportal or perivenous areas of the same liver. In parallel experiments, cell fractions were characterized by a variety of parameters, including differences in their ploidy states and phenotypic analysis to ascertain the lobular origin (18). The hepatocytes were isolated from a normal F344 rat. Perivenous hepatocytes exhibited greater abundance of tetraploid nuclei on flow cytometry, greater glycogen content and P450 activity, and lesser Glu-6-P activity. None of the isolated hepatocytes expressed gamma -glutamyl transpeptidase, indicating an absence of contamination with biliary cells. When these isolated hepatocytes were cultured with phenobarbitone and 3-methylcholanthrene, CYP450 activity using resorufin conversion as a measure was not apparent at 24 h of cell culture. Subsequently, although basal P450 activity was greater in cultured perivenous hepatocytes (H2 fraction) (up to 3-fold greater, p < 0.001), periportal hepatocytes (H4 fraction) rapidly acquired this facility in culture conditions. Indeed, the relative magnitude of P450 induction in both cell fractions was comparable after 2, 3, and 5 days in culture, although P450 inducibility declined in cultured hepatocytes with time (Fig. 6). In some experiments, 3-methylcholanthrene-inducible P450 activity was greater in H4 cell fraction compared with H2 cell fraction (p < 0.001, t test) at 2 days in culture, but then became similar in both cell fractions.


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Fig. 6.   Induction of P450 expression in periportal hepatocytes nonpermissive in vivo. Hepatocytes isolated from the same rat liver were cultured as described under "Experimental Procedures." Utilization of 7-ethoxyresorufin is shown in H2 cells (perivenous hepatocyte fraction) and H4 cells (periportal hepatocyte fraction) following culture with phenobarbitone. The magnitude of resorufin conversion in the two fractions was similar with no difference upon statistical analysis (t tests, p = not significant). None of the cell fractions showed significant resorufin conversion at 24 h. Results of experiments with 3-methylcholanthrene induction of cells were essentially similar. The data were representative of at least six independent experiments performed.


    DISCUSSION

These findings establish that signals emanating from the microenvironment play critical roles in directing liver gene expression. Our findings are novel in respect with the use of transplanted hepatocytes as reporters to analyze physiological mechanisms in the liver. Also, the results demonstrate unequivocally that hepatocytes can both acquire and lose specialized functions depending upon the permissiveness or otherwise of the microenvironment. All three of our approaches, namely shifting the position of reporter hepatocytes in the adult liver, switching hepatocytes from a permissive environment to a nonpermissive one, and analysis of hepatocytes from perivenous and periportal areas in the neutral territory of identical culture conditions and substrate availability provided mutually compatible results.

Our studies were greatly facilitated by the recent development of novel cell transplantation systems and insights into mechanisms of liver repopulation with transplanted hepatocytes (14). A most critical feature concerned the ability of transplanted hepatocytes to enter hepatic cords in the adult liver, which was demonstrated previously (16), as well as in the neonatal liver, as shown here. While transplanted hepatocytes can proliferate extensively in the diseased liver with constant depletion of host hepatocytes (22, 23), in the setting of self-limited liver injury, such as acute CCl4 toxicity, proliferation of transplanted hepatocytes was tightly regulated (17). Although it could potentially be argued that the progeny of transplanted hepatocytes could have shown differences in gene regulation mechanisms, such was not the case in our experience. Despite significantly increased sizes of hepatocyte clusters in animals treated repeatedly with CCl4, gene expression in transplanted hepatocytes resembled that in adjacent host cells. During extensive repopulation of the mouse liver, transplanted adult hepatocytes have also been shown to display gene expression patterns consistent with their position in the liver lobule (23).

A multitude of studies established previously that periportal and perivenous hepatocytes differ in their biochemical capacities (18, 24-29). Our findings of differences in the abundance of Glu-6-P and glycogen content were compatible with models of metabolic zonation in the liver (30, 31). It was interesting that the periportal preference of Glu-6-P activity in the suckling rat liver was similar to the adult liver, whereas glycogen stores were less pronounced and showed no zonal predilection in very young suckling pups. Metabolic activities localized preferentially in periportal areas are broadly classified to be involved in glucose and amino acid utilization and urea, cholesterol and bile synthesis, etc., whereas glycogen, fatty acid and glutamine synthesis, and xenobiotic disposal are preferentially localized in perivenous areas (30, 31). Our findings concerning restriction of P450 activity in perivenous areas of the intact adult liver are compatible with metabolic zonation of gene expression. This peculiar zonal selection of gene expression has been correlated with structural features of cells, e.g. the occurrence of greater hepatocellular ploidy in perivenous areas of the liver lobule (32). Similarly, post-translationally regulated distributions of gene products in cellular compartments and plasma membrane domains have been interpreted as unique position-specific features of some hepatocytes (2). On the other hand, our findings concerning transplantation of P450-expressing hepatocytes in suckling pups indicated that this activity was reversible. The findings suggested regulation at a functional rather than structural level. Moreover, we found that hepatocytes isolated from periportal areas of the liver were able to rapidly acquire P450 inducibility in the presence of appropriate substrates. The findings were compatible with the previous demonstrations of phenobarbitone-inducible CYPIIB1 mRNA expression in the developing rat liver (33). Studies with in situ mRNA hybridization showed that CYPIIB1 mRNAs were distributed across the hepatic lobule in this situation, except for periportal cells, as in our studies of adult rats with immunostainable CYPIIB1 protein following phenobarbitone induction. The lack of immunostainable CYPIIB1 in our suckling rats is in agreement with post-transcriptional regulation if previous mRNA expression data are extrapolated (33). Nonetheless, our findings should help understand previously unreconciled results concerning regulation of hepatic alcohol dehydrogenase activity. In vivo studies showed that alcohol dehydrogenase localized principally in perivenous areas of the adult liver (1). In contrast, studies using periportal and perivenous hepatocytes isolated by enzymatic digestion of the liver showed that the rates of ethanol metabolism were similar in both cell fractions (34).

The studies suggest that position-specific gene expression must reflect the consequence of complex interactions among cellular transcription factors, signals from adjacent cells, extracellular matrix components, hormones, intermediary metabolites, etc. Regulation of position-specific gene expression in the liver by manipulating ambient oxygen or hormone concentrations has been documented (35, 36). Recent studies showed changes in the distribution of specific cellular transcription factors, as well as abundance of cytokines in various zones of the liver lobule (37, 38). Similarly, gradients of extracellular matrix components are recognized in the liver and may have important influences upon hepatic gene expression (39). Cell-cell interactions have also been shown to regulate hepatic gene expression, e.g. in cocultures of hepatic stellate cells and hepatocytes, differentiated hepatocyte functions are retained in a superior fashion (40). Our findings indicate that hepatocytes do not possess irrevocable mechanisms to account for position-specific gene expression patterns and are in agreement more with a major role of external influences, albeit not necessarily exclusive ones.

The results have implications upon further analysis of liver gene regulation and characterization of specific liver cell subpopulations. For instance, there is burgeoning interest in searching for hepatic stem cells. Cell lines capable of differentiating into hepatocytes have been obtained from progenitor liver cells (41). One goal of these general efforts is to establish mechanisms for bio-artificial liver devices to support the failing liver, which would benefit from suitable devices that can reproduce the hepatic microenvironment and offer greater preservation of desired functions in immobilized cells. Other investigations are aimed at using progenitor cells for cell and gene therapy approaches. In undertaking characterization of progenitor liver cells with analysis of gene regulation, our findings concerning the effects of microenvironment on cell differentiation should be especially germane.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1 DK46952 (to S. G.), P33 DK41296 (Marion Bessin Liver Research Center, P. I., D. A. Shafritz), and P30-CA13330 (Einstein Cancer Research Center, P. I., D. I. Goldman), and by an award from the Irma T. Hirschl Trust (to S. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Ullmann 625, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2098; Fax: 718-430-8975.

The abbreviation used is: PBS, phosphate-buffered saline.
    REFERENCES
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Abstract
Introduction
References

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