Downregulation of Lactoferrin by PPAR{alpha} Ligands: Role in Perturbation of Hepatocyte Proliferation and Apoptosis

Susan Hasmall, George Orphanides, Neil James, William Pennie, Kathryn Hedley, Anthony Soames, Ian Kimber and Ruth Roberts,1

Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, SK10 4TJ, England, United Kingdom

Received January 25, 2002; accepted March 22, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPAR{alpha} (peroxisome proliferator activated receptor {alpha}) is a transcription factor that mediates the rodent liver tumorigenic responses to peroxisome proliferators via regulation of genes that remain to be identified. Using microarray gene expression profiling of mRNA from wild type versus PPAR{alpha} null mice, we detected a 3- to 7-fold downregulation of hepatic lactoferrin (LF) in response to the PP, diethylhexylphthalate (DEHP; 1150 mg/kg). Northern blot analyses confirmed a significant downregulation of LF mRNA by DEHP in wild type mouse liver. Since LF has been reported to repress tumor necrosis factor-{alpha} (TNF-{alpha}), LF downregulation by PPs may permit TNF-{alpha} levels to rise, enhancing hepatocyte survival and proliferation. To test this hypothesis, we asked if exogenous LF could prevent the perturbation of hepatocyte growth by PPs but not by TNF-{alpha}. In vitro, the PPs monoethylhexylphthalate (MEHP; 500 µM, the active metabolite of DEHP) and another PP, nafenopin (50 µM) or exogenous TNF-{alpha} (5000 U/ml) induced hepatocyte proliferation and suppressed apoptosis. LF (200 µM) blocked the growth but not the peroxisome proliferation response to PPs but could not block the growth response to TNF-{alpha}. Immunocytochemistry using specific antibodies to LF but also to transferrin (TF), a related gene previously shown to contain a PP response element (PPRE), demonstrated that both LF and TF are expressed in murine liver. Furthermore, both were downregulated by DEHP in both wild type and PPAR{alpha} null mouse liver. These data suggest that the regulation of iron binding proteins by PPAR{alpha} ligands plays a role in PP-mediated liver growth, but not in peroxisome proliferation.

Key Words: nongenotoxic carcinogenesis; lactoferrin; peroxisome proliferators; PPAR{alpha}; hypolipidaemic drugs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PPs are a class of nongenotoxic rodent hepatocarcinogens that includes industrial plasticizers such as diethylhexyl phthalate and fibrate hypolipidaemic drugs such as nafenopin (reviewed in Ashby et al., 1994Go; Reddy et al., 1980Go). In mice and rats, treatment with PPs results in hepatic peroxisome proliferation, increased hepatocyte DNA synthesis, suppression of hepatocyte apoptosis, liver enlargement, and hepatocarcinogenesis (reviewed in Ashby et al., 1994Go; Bayly et al., 1994Go). The peroxisome proliferator activated receptor alpha (PPAR{alpha}), cloned originally from mouse liver (Issemann and Green, 1990Go), was shown to mediate the pleiotropic effects of PPs in rodents such as enzyme induction, peroxisome proliferation, and hepatocarcinogenesis (Aldridge et al., 1995Go; Gonzalez, 1997Go; Lee et al., 1995Go; Peters et al., 1997Go; Schoonjans et al., 1996Go; Tugwood et al., 1992Go). PPAR{alpha} is activated by artificial PPs and by natural ligands such as fatty acids and eicosanoids (Auwerx, 1992Go; Forman et al., 1997Go). Activated PPAR{alpha} binds to DNA as a heterodimer with the retinoid x receptor (RXR). This heterodimer binds to DR1 elements that comprise 2 degenerate direct AGGTCA repeats spaced by 1 base pair, termed PPAR{alpha} response elements (PPRE, Issemann et al., 1993Go; Kliewer et al., 1992Go). PPREs have been identified in the promoter regions of a number of genes coding for enzymes that are transcriptionally regulated by PPs (Bardot et al., 1993Go; Hertz et al., 1995Go; Kliewer et al., 1992Go; Schoonjans et al., 1996Go; Tugwood et al., 1992Go).

The observation that deletion of the transcription factor, PPAR{alpha}, renders hepatocytes refractory to PP-induced growth perturbation suggests that PPAR{alpha} mediated gene transcription is causative in this response. With this in mind, much effort has been invested in determining the identity of PP-regulated genes involved in maintaining hepatocyte survival and proliferation. Recent data support a role for cytokines such as tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin 1 {alpha}/ß (IL1{alpha}/ß) in mediating the response to PPs via engagement of nuclear factor {kappa}B (NF{kappa}B; Cosulich et al., 2000; Rusyn et al., 1998Go; West et al., 1999Go) and mitogen activated protein (MAP) kinase (Cosulich et al., 2000; Eder, 1997Go) survival pathways. However, the evidence on transcriptional regulation of cytokines by PPs is conflicting (Holden et al., 2000Go; Ledda-Columbano et al., 1998Go; Rose et al., 2000Go). In addition to the approach of investigating expression of genes for cytokines already implicated in the response to PPs, recent articles have described the use of gene microarray analysis to identify PP-regulated genes (Cherkaoui-Malki et al., 2001Go; Yamazaki et al., 2001Go). Although potentially of interest, the new leads identified were not investigated further. Here, we have applied gene microarray analysis to identify genes regulated by PPs in rodent liver then taken a hypothesis-based approach to investigate the role of the regulated genes in a robust in vitro model of PP responsiveness. DEHP was selected for administration at the carcinogenic dose (NTP, 1982Go) since we have shown previously that this regime produces a strong induction of hepatic proliferation and suppression of apoptosis (James et al., 1998Go). Subsequent in vitro studies used monoethylhexylphthalate (MEHP), the principal metabolite of DEHP and the proximal peroxisome proliferator (Mitchell et al., 1985Go) or nafenopin, included to extend the observations made for MEHP to other PPs. The data presented here suggest that iron binding proteins such as lactoferrin (LF) and possibly transferrin (TF) may play a role in the perturbation of hepatic growth control leading to carcinogenesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Nafenopin was a gift from Ciba-Geigy (Basel, Switzerland) and MEHP was a gift from Gerhart Gans, BASF, Germany. Diethylhexylphthalate (DEHP) and TF were purchased from Sigma-Aldrich Chemical Company, Dorset, UK. Recombinant human LF was produced in Aspergillus awamori as described previously and was a gift from Dr. D. R. Headon, Agennix Inc., Houston, TX (Ward et al., 1995Go, Ward et al., 1997Go). ToxBlot nylon filter cDNA arrays were generated as described previously (Pennie, 2000Go; Pennie et al., 2000Go).

Animals and animal procedures.
PPAR{alpha} null and wild type (SV129) mice were a gift from Frank Gonzalez, NIH. Male mice (3 per group) received 2 doses of DEHP (1150 mg/kg/day) or were left untreated. In the second experiment, groups of 3 mice received 2 doses of DEHP (1150 mg/kg/day) or vehicle (corn oil, 5 ml/kg/day). All animals were killed by terminal anesthesia 48 h later (24 h after the second dose). Sections of each of the 3 main lobes (left lateral, right median, and caudate) were evaluated for peroxisome proliferation and the remaining liver tissue used for preparation of RNA, protein, and for immunocytochemistry.

Microarray gene expression profiling.
Total liver RNA and mRNA were isolated using Total RNA Isolation Reagent (Advanced Biotechnologies) and mMACS mRNA Isolation Kit (Miltenyi Biotech), respectively. Radiolabelled 1st strand cDNA probes were prepared using 200 U Superscript II (Gibco BRL) according to the manufacturer’s instructions. Reactions contained 500 ng oligo dT12-18 (Gibco BRL), 200–400 ng mRNA, 0.5 mM dATP, dGTP, and dTTP, 5 mM dCTP, and 30 mCi 33P dCTP (Amersham). Probes were purified using G50 Sephadex spin columns (Boehringer Mannheim). ToxBlot (Pennie, 2000Go; Pennie et al., 2000Go) nylon filter cDNA arrays were incubated with 10 ml hybridization solution (0.5 M sodium phosphate [pH 7.2], 7% SDS, 1 mM EDTA) for 1 h, hybridized with 10 ml hybridization solution containing 1,000,000 cpm/ml labeled probe for 16 h and washed with 20 ml 40 mM sodium phosphate (pH 7.2), 1% SDS for 3 times for 20 min. All ToxBlot incubations were performed at 65°C. Filters were exposed to phosphorimager screens for 48 h and scanned using a Molecular Dynamics SI phosphorimager. ArrayVision software (Molecular Dynamics) was used to detect hybridization spots, calculate relative hybridization intensities and subtract background hybridization values. Relative hybridization values were normalized between filters before calculation of expression ratios.

Peroxisome proliferation in vivo by volume fraction.
The effects of DEHP on liver peroxisomal volume fraction was determined as described previously (James and Roberts, 1996Go). Briefly, peroxisomes were highlighted by staining for catalase and micrographs taken at random and analyzed for peroxisomal volume fractions using a point-counting technique.

Hepatocyte preparation and treatments.
Hepatocytes were prepared from male wild type SV129 mice using a 2-stage collagenase perfusion technique as described previously (James and Roberts, 1996Go). Hepatocytes (1 x 106) were inoculated onto 12.5 cm2 falcon Biocoat treated tissue culture flasks and maintained at 37°C as described previously (James and Roberts, 1996Go). MEHP (750 µM final concentration) or nafenopin (50 µM final concentration) was added to the medium of hepatocytes from a 400X stock solution in dimethylformamide (DMF). TGFß1 (5 ng/ml) was added to the medium from a 2 mg/ml stock solution in 4 mM HCl, 0.1% BSA. LF or TF (100 µM) was added from a 40 mg/ml stock in saline.

Measurement of hepatocyte DNA synthesis, apoptosis, or peroxisome proliferation in vitro.
To assess the ability of LF or TF to block PP-stimulated proliferation, suppression of apoptosis or peroxisome proliferation, fresh medium with or without LF or TF was added to the hepatocyte monolayers 22 h after plating. MEHP, nafenopin, or DMF was added after a further 2 h (at 24 h after plating). To evaluate proliferation, BrdU was added 48 h after plating and the cultures maintained for a further 16 h prior to evaluation of replicative DNA synthesis as described previously (James and Roberts, 1996Go). To evaluate apoptosis, TGFß1 was added 24 h later in the continued presence or absence of ligand and cultures stained with Hoechst 33258 to evaluate apoptosis as described previously (James and Roberts, 1996Go). For peroxisomal ß-oxidation, hepatocytes were harvested at 72 h and the oxidation of a palmitoyl-CoA substrate was determined as described previously (Lazarow and De Duve, 1976Go).

Northern blots.
Total RNA was harvested from liver using Total RNA Isolation Reagent (Advanced Biotechnologies, UK) at 48 h after initial treatment with DEHP. Samples of RNA (20 or 30 mg) from liver were separated by electrophoresis on 1% agarose gels containing formaldehyde (Ausubel et al., 1995Go) and transferred to Hybond nitrocellulose membranes (Amersham). CYP4A1, LF, and acidic ribosomal phosphoprotein (ARPP) probes were prepared using either PCR-amplified DNA fragments or gel purified restriction fragments. Probes were 32P dCTP radiolabelled using Megaprime (Amersham) or HighPrime (Boehringer Mannheim) following manufacturer’s instructions. Probes were hybridized to membranes in RapidHyb buffer (Amersham) overnight at 65°C, then washed twice in 2% SSC/0.1% SDS at 65°C. Following washing, filters were exposed to film or a phosphorimager screen overnight and the relative abundance of RNA transcripts was determined using a Bio-Rad GS-670 scanning densitometer or a Molecular Dynamics SI phosphorimager, respectively.

Western blots.
Protein samples (5 µg) or standards (200 ng LF or 100 ng TF) were mixed with NuPage sample buffer (Novex, San Diego, CA) and loaded onto a 10% Bis-Tris polyacrylamide gel (Invitrogen). Gels were run in MOPS running buffer (MOPS 1M, Tris base 1M, SDS 69.3mM, and EDTA 20.5mM at pH 7.7) at 200 V for approx. 60 min. Proteins were blotted onto a PVDF filter (Immobilon-P, Millipore, UK) using Tris-glycine transfer buffer (Tris base 12 mM, glycine 96 mM, methanol (20% final vol.). Equal protein loading was verified using Ponceau S staining. The PVDF filter was then blocked with 10% nonfat dried milk in TBS Tween 20 (0.5%). LF and TF were detected using primary antibodies (1:1000; both gifts from Pauline Ward, Baylor College of Medicine, Houston, TX) and horseradish peroxidase linked secondary antibody (1:5000) diluted in TBS Tween 20 (0.5%). Detection was via enhanced chemiluminescence and Hyperfilm ECL (Amersham Life Science, UK).

Immunocytochemistry.
Liver tissues were fixed in Bouins fixative, embedded in paraffin wax, and 5 mm sections cut. Sections were then dewaxed and rehydrated prior to being submerged in peroxidase quenching solution (1:9 30% hydrogen peroxidase:100% methanol) for 30 min to block endogenous peroxidase activity. Sections were then incubated in 10% normal goat serum diluted in PBS for 1 h at room temperature before addition of primary antibody. Primary LF or TF antibody was diluted 1:1000 in 10% normal goat serum, and sections incubated for 18 h at 4°C. Rabbit serum diluted in 10% goat serum was used in place of primary antibody as a negative control for nonspecific staining. Sections were then washed 3 times in PBS before addition of horseradish peroxidase-linked streptavidin-biotin complex (Strept ABC Complex/HRP linked duet kit; Dako Ltd., Denmark). Peroxidase activity was visualised using a 3,3'-diaminobenzidine (DAB) substrate (Sigma, UK) and sections were counterstained with Mayer’s hematoxylin, before dehydration and mounting.

Statistics.
Differences between treatment group means and the appropriate control were compared using Student’s t-tests. Data are expressed as mean ± SD and statistical significance at p <= 0.01 or p <= 0.05 is indicated by ** or *, respectively.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genes Identified by Microarray as Being Regulated by DEHP in Vivo
Probes derived from control and DEHP-treated livers were hybridized to custom-made cDNA arrays (ToxBlot) containing sequences representing approximately 600 genes relevant to mechanisms of toxicity (Pennie, 2000Go; Pennie et al., 2000Go). Figure 1Go shows that 12 genes were either up- or downregulated by greater than 1.5-fold in all 3 wild-type mice compared with the average level of their expression in the untreated wild-type group. The regulation of these genes by DEHP was absent or reduced in the PPAR{alpha}-/-mice. Of those genes found to be regulated by DEHP, the biggest changes were an upregulation of CYP4A1 and a downregulation of LF mRNA.



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FIG. 1. Bar histogram of the mean changes in spot intensities between naïve and DEHP-treated mice. Twelve genes were up- or downregulated by greater than 1.5-fold in the livers of all 3 wild type animals compared with the average level of expression in the control wild type group.

 
Confirmation of the Repression of LF mRNA Expression by DEHP
To confirm the downregulation of LF mRNA identified by microarray, a second in vivo experiment was performed using DEHP versus vehicle (corn oil) control; the livers were isolated for analysis of LF expression by Northern blot analysis of both this and the previous experiment. In addition, peroxisome proliferation was analyzed as a positive control for the hepatic response to PPs. DEHP caused an increase in peroxisomal volume fraction in all 3 wild type mice but not in the PPAR{alpha} null mice (Table 1Go). Northern blot analysis of the first (Fig. 2AGo) and the second independent experiment (Fig. 2BGo) confirmed significant (Fig. 2A,Gop = 0.03; Fig. 2B,Gop = 0.001) downregulation of LF mRNA by DEHP in all wild type mice, and a slight but not statistically significant (Fig. 2A,Gop = 0.08; Fig. 2B,Gop = 0.09) downregulation of LF in PPAR{alpha} null mouse livers.


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TABLE 1 Induction of Peroxisome Proliferation by DEHP in Wild Type SV129 but Not in PPAR{alpha} Null Mice
 


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FIG. 2. Northern blot analysis of LF expression in liver in 2 independent in vivo experiments comparing DEHP treatment with naïve controls (A) or corn oil (vehicle) control (B). The bar histograms of mean LF band intensities relative to ARPP control (n = 3) show significant downregulation of LF in DEHP-treated wild type mice (A, p = 0.03; B, p = 0.001) but not in PPAR{alpha} null mice (A, p = 0.08; B, p = 0.09). *Significant at the 0.05 level. **Significant at the 0.01 level.

 
LF Prevents the Growth Response, But Not the Peroxisome Proliferation Response to PPs
Having established that LF mRNA is downregulated in mouse liver in vivo by the PP DEHP, we wanted to determine whether LF may play a role in PP induced hepatocyte growth perturbation. Previously, we have shown that both PPs and the hepatic mitogen epidermal growth factor (EGF) can suppress apoptosis and induce DNA synthesis in cultured mouse hepatocytes. Additionally, PPs induce peroxisome proliferation. The biological response to DEHP in vivo requires metabolism of DEHP to the proximate peroxisome proliferator MEHP (Mitchell et al., 1985Go); for this reason MEHP was used for in vitro analyses. Figures 3A and 3BGo show that exogenous LF inhibited the induction of DNA synthesis and suppression of apoptosis seen in response to the PP, MEHP. However, exogenous LF did not block the induction of peroxisomal ß-oxidation by MEHP nor did it block the growth response to the hepatic mitogen EGF (Fig. 3CGo).



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FIG. 3. Inhibition by LF (100 µM) of the effects of MEHP (500 µM) but not EGF (25 ng/ml) on SV129 mouse hepatocyte S-phase (A) and apoptosis (B) in vitro. Endogenous LF did not block the induction of peroxisomal ß-oxidation by MEHP (C). *Significant at the 0.05 level. **Significant at the 0.01 level. Data are normalized to levels of S-phase and apoptosis in controls, which averaged 2.4 and 1.1, respectively.

 
LF Does Not Prevent the Growth Response to TNF-{alpha}
LF is implicated in regulating the production of cytokines such as TNF-{alpha} that in turn are implicated in mediating the growth but not the peroxisome proliferation response to PPs (Roberts and Kimber, 1999Go). Thus, we examined the ability of exogenous LF to block the response to TNF-{alpha} using a second PP, nafenopin, as a positive control. Nafenopin is a hypolipidaemic PP shown previously to suppress apoptosis and induce proliferation in vitro (James et al., 1998Go). Figures 4A and 4BGo show that in vitro LF inhibited the induction of DNA synthesis and suppression of apoptosis by nafenopin (Figs. 4A and 4BGo). However, LF was unable to prevent the induction of ß-oxidation (Fig. 4CGo). Furthermore, LF was unable to prevent the growth changes seen in response to exogenous TNF-{alpha} (Figs. 4A and 4BGo) suggesting that TNF-{alpha} may act secondary to the downregulation of LF. The related iron binding protein TF exhibited some ability to reduce the growth response to PPs (data not shown). However, unlike the consistent response to LF, the effects of TF were equivocal.



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FIG. 4. Inhibition by LF (100 µM) of the effects of nafenopin (50 µM) but not TNF{alpha} (5000 U/ml) on SV129 mouse hepatocyte S-phase (A) and apoptosis (B) but not peroxisomal ß-oxidation (C) in vitro. *Significant at the 0.05 level. **Significant at the 0.01 level. Data are normalized to levels of S-phase and apoptosis in controls, which averaged 2.2 and 1.2, respectively.

 
DEHP Downregulates Both LF and TF in Vivo
Since the gene sequences for LF and TF are sufficiently homologous for potential cross-hybridization, we used specific anti-TF and anti-LF antibodies to determine whether either or both proteins could be regulated by PPs. Figure 5Go shows that the LF antibody recognizes LF but not TF and that the TF antibody recognizes TF and not LF. In addition, Figure 5Go shows that TF is expressed in mouse liver whereas the level of LF protein was below the limit of detection by Western blot analysis, at least for whole liver. Nonetheless, these data did not rule out the possibility of detectable expression in a subpopulation of nonparenchymal liver cells such as those of the hepatic sinusoids. To address this, we analyzed TF and LF expression by immunocytochemistry in section of liver from DEHP or control (vehicle) treated mice. In the livers of control wild type and PPAR{alpha} null mice, LF could be detected in the nonparenchymal sinusoidal cells and also in discrete pools within the cytoplasm of some cells that were probably hepatocytes of the midzone (the region between but distant from the central or portal veins; Fig. 6Go). Following administration of DEHP, LF was largely lost from midzonal cytoplasmic pools and was retained in the sinusoidal cells (Figs. 6C and 6DGo). TF, which could be detected by Western blot, was prominent in discrete cells in the midzone in the livers of control wild type and control PPAR{alpha} null mice (Figs. 6E and 6FGo). TF was also detectable in the sinusoids but fewer sinusiodal cells contained TF than LF (Figs. 6E and 6FGo). Following administration of DEHP, TF was largely lost from the midzonal pools but was retained in the few sinusoidal cells (Figs. 6G and 6HGo). In contrast to LF, TF was distributed throughout the cytoplasm of a number of hepatocytes and the sinusoidal membranes of most cells contained some TF; this was not affected by treatment with DEHP.



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FIG. 5. Western blotting for TF and LF in control or DEHP treated mouse liver. TF or LF were included as standards on both blots.

 


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FIG. 6. Immunocytochemical detection of LF and TF in wild type (left-hand panel) and PPAR{alpha} null (right-hand panel) mouse liver. In control wild type (A) and PPAR{alpha} null (B) mice LF could be detected in the nonparenchymal sinusoidal cells (S) and also in discrete pools within the cytoplasm of some cells (H) located between the central (CV) and portal veins (PV). Following 2 daily doses of DEHP to wild type (C) and PPAR{alpha} null (D) mice, LF was largely lost from the cytoplasmic pools (H) and was retained in the sinusoidal cells (S). TF was prominent in discrete pools in the midzonal cells (H) between the veins (CV) in the livers of control wild type (E) and control PPAR{alpha} null mice (F). TF was also detectable in the sinusoids, but fewer sinusiodal cells (S) contained TF than LF. Following 2 daily doses of DEHP to wild type (G) and PPAR{alpha} null (H) mice, TF was largely lost from the cytoplasmic pools but was retained in the few sinusoidal cells (S).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms through which PPs regulate hepatocyte proliferation and apoptosis remain to be identified. Here, we present data to suggest that the downregulation of iron binding proteins such as LF by PPs may play a role in initiating this growth response. The observation that LF could not block the peroxisome proliferation response suggests that the effects of LF on PP-induced growth are not caused by nonspecific interaction of LF with MEHP. In addition, LF could not prevent the growth response to the hepatic mitogen EGF, suggesting that LF has a specific effect on the induction of growth by PPs rather than a nonspecific inhibition on cell replication.

LF is an iron binding glycoprotein that is present in milk, tears, saliva, and other biological fluids (reviewed in Lonnerdal and Iyer, 1995Go; Teng, 1999Go). LF is known to have antibacterial properties, to influence epithelial cell proliferation, and to regulate immune and inflammatory responses (reviewed in Britigan et al., 1994Go; Lonnerdal and Iyer, 1995Go; Teng, 1999Go). LF is expressed in many tissues including uterus, breast, liver, spleen, and lung (Shigeta et al., 1996Go). Recent data have shown that the expression of LF by human endometrial carcinoma cells plays a role in their resistance to tamoxifen (Albright and Kaufman, 2001Go). In addition, LF expression is decreased or absent in most cancer cells, suggesting that LF gene expression is impaired during tumorigenesis (Teng et al., 2000Go). Of potential significance to the findings presented herein, the liver has been implicated in LF synthesis and clearance (McAbee, 1995Go).

Previous data have suggested that proflammatory cytokines such as TNF-{alpha} and IL-1, mediate some or all of the changes induced during exposure of hepatocytes to PPs (reviewed in Roberts and Kimber, 1999Go). Thus, for instance, it has been shown in vitro that recombinant TNF-{alpha} is able to induce hepatocyte S phase and suppress apoptosis (Rolfe et al., 1997Go). Moreover, anti-TNF-{alpha} blocking antibodies prevent the induction of liver proliferation by PPs in vivo (Bojes et al., 1997Go) and in vitro (West et al., 1999Go). Transgenic mice where the TNF-{alpha} receptor 1 (TNFR1) has been disabled are compromised in their ability to mount a hepatic regenerative response after partial hepatectomy (Yamada and Fausto, 1998Go). In addition, inactivation of TNFR1 with a blocking antibody has been shown to inhibit significantly the response of hepatocytes to nafenopin in vitro (West et al., 1999Go). Conversely, evidence from TNF-{alpha} and TNF-{alpha} receptor null transgenic mice challenges the role of TNF-{alpha} in the proliferation response to PPs since PPs can still induce hepatic DNA synthesis in these mice (Givler et al., 2000Go; Lawrence et al., 2001Go), although in this case compensatory mechanisms may operate. Interestingly, we have shown that although growth responses to nafenopin were inhibited almost completely by LF, LF did not affect the stimulation of S-phase or suppression of apoptosis induced by addition of exogenous TNF-{alpha}. Collectively these data suggest that LF may be able to regulate liver expression of TNF-{alpha}, and possibly other proinflammatory cytokines. The argument is that following exposure to MEHP or nafenopin, the downregulation of LF expression would result in increased levels of TNF-{alpha} that in turn would mediate some or all of the growth changes associated with PPs. It seems likely that this increase in TNF-{alpha} levels occurs by release of pre-existing cytokine since no increase in expression of TNF-{alpha} mRNA was detected either in these experiments or in those previously reported (Holden et al., 2000Go).

There exist precedents in the literature for the regulation by LF of TNF-{alpha} and other cytokines (Machnicki et al., 1993Go). Moreover, the role we hypothesize for LF in the liver bears some striking similarities to what is known of the ability of LF to influence cutaneous immune and inflammatory reactions. It has been demonstrated in both mice and humans that topical exposure to homologous recombinant LF inhibits substantially the migration of epidermal Langerhans cells normally induced by skin sensitization with a chemical allergen (Cumberbatch et al., 2000Go). Of importance is the fact that allergen-induced LC migration is dependent upon the availability of TNF-{alpha} (Kimber et al., 2000Go). Investigations have shown that although LF is able to compromise allergen-induced LC migration, for which the de novo production of TNF-{alpha} is required, LF was without influence on migration induced by the intradermal injection of TNF-{alpha} itself (reviewed in Kimber et al., 2000Go). Collectively, these data support the view that LF provides a mechanism for the homeostatic regulation of constitutive and inducible TNF-{alpha} expression. The implication from the results reported here is that the ability of PPs to downregulate LF mRNA will result in the upregulation, or at least uncontrolled, expression of TNF-{alpha} that may provoke perturbation of hepatocyte growth regulation and the development of tumors.

Hertz et al. (1996) have reported PPARß-dependent specific downregulation of another iron binding protein, TF, by PPs. Since the genes for LF and TF are sufficiently homologous for potential cross-hybridization, this raises the possibility that the mRNA identified as being regulated by PPs could either be LF, TF, or both. Indeed, immunocytochemical analyses revealed that the hepatic expression pattern of both proteins is altered after DEHP exposure. The murine LF gene promoter was cloned in 1991 (Liu and Teng, 1991Go) and shown subsequently to contain elements responsive to estrogen and also to mitogens such as epidermal growth factor (EGF; Teng, 1999Go). The promoter contains an estrogen response element (ERE) at –351/327 and a GC rich mitogen responsive element at –10/–103. The ERE contains overlapping elements for the chicken ovalbumin upstream promoter transcription factor (COUP-TF) and for estrogen receptor (ER; Liu and Teng, 1992Go). In reproductive tissues, LF mRNA and protein is strongly induced by estrogens (Shigeta et al., 1996Go) and ER and COUP-TF appear to play opposing roles in estrogen responsiveness (Shigeta et al., 1996Go). Other transcription factors such as IKLF (Shi and Teng, 1994Go), Sp1 (Khanna-Gupta et al., 2000Go), C/EBP (Khanna-Gupta et al., 2000Go) and LF itself have also been implicated in regulating LF gene expression (Furmanski et al., 1997Go). Thus, the regulation of the LF gene is highly complex and differs in different tissues, possibly due to tissue specificity in expression of transcription factors (Teng et al., 1998Go). Additionally, although the human and mouse LF promoters both contain imperfect EREs, the molecular mechanisms that governs the estrogen responsiveness differ between human and mouse (Teng, 1994Go).

The data presented herein suggest that LF expression is constitutive in liver, possibly under the control of endogenous ER and estrogens. It remains to be determined how PPs downregulate LF gene expression since there was some downregulation of mRNA in the PPAR{alpha} null mouse and LF protein was clearly downregulated in null mouse liver in response to DEHP. Interestingly, TF protein was also downregulated in the PPAR{alpha} null mouse liver in response to DEHP despite previous data showing that the TF gene expression is switched off by displacement of HNF4 by nonproductive binding of PPAR{alpha} (Hertz et al., 1996Go). Thus, it is possible that both the LF and TF promoters could be regulated by PPAR{alpha} via displacement of positive regulatory factors such as ER or via engagement of silencer proteins that can bind to the LF promoter such as CDP/cut (Khanna-Gupta et al., 1997Go). However, it is clear that the expression of both proteins is altered by PPs in the livers of both wild type and PPAR{alpha} null mice. In summary, these data suggest that downregulation or redistribution of iron binding proteins such as LF plays a role in the response of hepatocytes to PPs, possibly via permitting upregulation of cytokines such as TNF-{alpha} that can recruit NF{kappa}B and MAP kinases to promote survival and proliferation (Cosulich et al., 2000aGo,bGo; Eder, 1997Go). Of significance is the recent finding that LF is decreased or absent in most cancer cells, suggesting that LF gene expression is impaired during tumorigenesis (Teng et al., 2000Go). These data provide new insights into the molecular mechanisms of PP-induced rodent hepatocyte growth perturbation that may be key to nongenotoxic carcinogenesis.


    NOTES
 
1 To whom correspondence should be addressed at present address: Aventis Pharma, Centre de Recherche de Vitry-Alfortville, 13 Quai Jules Guesde, F94400 Vitry-sur-Seine, France. Fax: 3 3158 938164. E-mail: ruth.roberts{at}aventis.com. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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