Institute of General Pathology, University of Milan, Via Luigi Mangiagalli 31, 20133 Milan, Italy
1 To whom correspondence should be addressed. Tel: +39 0250315334; Fax: +39 0250315338; Email: a.desiderio{at}unimi.it
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: FBS, fetal bovine serum; HGF, hepatocyte growth factor; HIF-1, hypoxia-inducible factor-1; HO-1, heme oxygenase-1; HRE, hypoxia responsive elements; MEM, minimal essential medium; NF-B, nuclear factor
B; ODC, ornithine decarboxylase; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; ssNF-
B, NF-
B super-repressor; TdT, terminal-dUTP-transferase; 5'-UTR, 5'-untranslated region.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In tumor cells Met activity is often constitutively up-regulated through various molecular mechanisms (6). Breast cancer tissues overexpress the Met receptor, at both the mRNA and protein levels, which is responsive to HGF in a paracrine or autocrine manner (911). Missense point mutations in Met have been identified in breast cancers, childhood hepatocellular carcinomas and renal, gastric and head and neck squamous cell carcinomas. Dysregulated Met signaling consequent to gene amplification is a key event for the metastatic process and, indeed, inappropriate expression of the ligandreceptor pair is often associated with poor prognosis in a variety of tumors, including liver and breast carcinomas (6,12).
In a previous work we showed that HGF-triggered transduction pathways for invasive growth of hepatoma cells lead to the activation of nuclear factor B (NF-
B) and of the transcription factor hypoxia-inducible factor-1 (HIF-1) (13,14). NF-
B is a pleiotropic protein complex, activated from a sequestered cytoplasmic form. It is important for tumor development in that it regulates the expression of genes for cell growth (cyclin D1) as well as pro-metastatic (uPA, IL-6, IL-8 and CXCR4) and anti-apoptotic (cIAP-2 and TRAF-1) genes (15,16). However, NF-
B may render the cells more sensitive to certain pro-apoptotic stimuli on account of its ability to regulate the expression of cellular factors that affect the apoptotic threshold (17).
HIF-1 is a heterodimer composed of and ß subunits, which are helixloophelixPAS domain proteins. HIF-1ß is constitutively expressed, whereas the
subunit is expressed at low levels in most cells under normoxic conditions. HIF-1
is inducible by hypoxia and cytokines, including HGF (14,18). Our data show that HGF stimulates HIF-1 transactivation of genes belonging to the plasminogen activation system, which are important for extracellular matrix degradation and for cell signaling to the nucleus involved in growth/survival (4,19), and HIF-1 may protect hepatoma cells from apoptosis induced by HGF after Met/Fas dissociation (1,20).
To extend our knowledge of the alterations in the cell signaling machinery possibly involved in carcinoma progression we evaluated the expression and regulation of selected sets of genes downstream of HGF-activated NF-B. We studied genes involved in growth and/or apoptosis depending on cell type and intracellular conditions, such as HIF-1
, ornithine decarboxylase (ODC), c-myc, Mcl-1 and heme oxygenase-1 (HO-1) (2125).
ODC is a delayed-early gene involved in control of the cell cycle and is considered a target gene of Myc/Max (22). The polyamines, products of ODC activity, are physiological polycations that help regulate the expression of cyclin D1 and of early response genes (2629). Recent data indicate that HIF-1 is a cancer survival gene (1,30,31). Depletion of polyamines due to degradation of ODC in the proteasome may induce the cells to undergo apoptosis, a process favored by the absence of HIF-1 activity (1,32).
In the present study we compared HepG2 hepatoma cells (our previous model) with MCF-7 (low invasive) and MDA-MB-231 (highly invasive) breast carcinoma cells. These cells differ in their molecular profile. The MDA-MB-231 cell line was also chosen because of a constitutively active phosphatidylinositol 3-kinase (PI3K)/NF-B pathway and elevated Fas levels possibly related to basal apoptosis (3335).
In HepG2 and MCF-7 cells HIF-1 was inducible by HGF through NF-
B/HIF-1 regulation, while in MDA-MB-231 cells ODC and HO-1 were persistently increased after HGF treatment. HIF-1 activity blockade and a decrease in ODC expression reduced cell viability in HGF-treated cells.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell cultures
Human breast carcinoma cells MCF-7 and MDA-MB-231 cultured in RPMI 1640 and human hepatoblastoma HepG2 cells cultured in MEM were from the European Cell Cultures Collection. All cells were routinely maintained in complete medium containing 10% FBS and were starved for 1824 h before treatment with HGF (50200 ng/ml medium without FBS).
Plasmids
Human HIF-1 cDNA was kindly provided by Dr R.D.Thornton (PCOM, Philadelphia, PA), human pODC10/2H by Dr M.Halmekytö (Virtanen-Institute, Kuopio, Finland), the HindIII fragment of 1.8 kb from mouse pMc-myc54 for c-myc by Dr K.B.Marcu (SUNY, New York, NY), human Mcl-1 cDNA by Dr S.W.Edwards (Liverpool, UK) and human cDNA for HO-1 by Dr R.M.Tyrrell (University of Bath, UK). Human pHIF1A(572/+284)Luc and pHIF1A(572/+32)Luc reporter constructs were a generous gift from Dr D.E.Richard (Centre de Recherche, Quebec, Canada, originally prepared by Dr G.L.Semenza's laboratory, Johns Hopkins University, Baltimore, MD). Reporter plasmid p15C was kindly provided by Dr E.Minet (Laboratoire de Biochimie et Biologie Cellulaire, University of Namur, Belgium). The construct in pGL2-enhancer containing pODC(1692/+131)Luc was prepared from pGL2-basic containing pODC(4362/+131)Luc (kindly provided by Dr M.Otieno (Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD, originally prepared by Dr A.K.Verma, Department of Human Oncology, Medical School, University of Wisconsin, Madison, WI). The original plasmid was cut with HindIII to obtain the 1692/+131 fragment, which was sub-cloned into pGL2-enhancer vector, previously cut with HindIII. The pODC(720/+16)Luc fragment was amplified by PCR with Euro-Taq using pODC(1692/+131)Luc as template and the following sense and antisense primers: 5'-CTCGATGTTACAAAGACGAGG-3' and 3'-TTTGTCAGTCCCTCCTGTA-5' (Roche). PCR was done in 50 µl of 1x PCR buffer containing 2 mM MgCl2, 200 µM each dNTP, 2.5 U Euro-Taq, 10% DMSO, 1 µM DNA primers and 100 ng pODC(1692/+131). The sample was heated at 95°C for 180 s, then at 95°C for 60 s, 54.5°C for 90 s and 72°C for 120 s for 35 cycles, then at 72°C for 7 min. The PCR fragment was cloned in pGEM T-Easy and cut with EcoRI, blunted with Klenow polymerase and cloned in pGL2-enhancer previously cut with SmaI. pNF-
BLuc containing five consensus sites for NF-
B was purchased from Stratagene (La Jolla, CA), pGL3PGK6TKp containing six hypoxia responsive elements (HRE) was from Dr P.J.Ratcliffe (Wellcome Trust Center for Human Genetics, Oxford, UK). RSVIkB
MSS super-repressor of NF-
B (ssNF-
B) was kindly provided by Dr N.D.Perkins (University of Dundee, UK). pcDNA3ARNTdelta_b (
ARNT), coding for a dominant negative mutant form of the ARNT subunit, and the void vector pcDNA3 were obtained from Dr M.Schwarz (Institute for Toxicology, University of Tübingen, Germany). pcDNA3-Flag-JNK-1(apf) (
JNK1), coding for a dominant negative mutant form of JNK-1, was from Dr R.J.Davis (Howard Hughes Medical Institute Research Laboratories, University of Massachusetts, MA). Dominant negative SR
(XbaI)
p85 was from Dr P.Raynal (INSERM Unité, Toulouse, France).
Electrophoretic mobility shift assay
For the supergel shift assay, nuclear extracts from HepG2, MCF-7 and MDA-MB-231 cells were first incubated with 1 µg anti-p50 or anti-p65 antibody on ice without the oligonucleotide and then with the labeled oligonucleotide, followed by electrophoresis (13,14). The oligonucleotide sequence containing the NF-B consensus site was 5'-GGATCCTCAACAGAGGGGACTTTCCGAGGCCA-3'. For loading control we used an oligonucleotide containing the Octamer-1 binding site 5'-TGCGAATGCAAATCACTAGAA-3'. These oligonucleotides were synthesized by Primm (Milan, Italy).
Transient transfection assay
For transient transfection experiments MCF-7, MDA-MB-231 and HepG2 cells were seeded in 24-well multiwell plates and transfected at 7080% of confluence; 200 ng reporter plasmid pNF-kBLuc or pGL3GK6TKp and 500 ng reporter plasmid pHIF1A(572/+284)Luc, pHIF1A(572/+32)Luc, p15C, pODC(4362/+131)Luc, pODC(1692/+131)Luc or pODC(-720/+16)Luc was transfected into the cells, with or without 500 ng of ssNF-B or 1 µg
ARNT,
JNK1 or
p85 expression vector. The cells were transfected using Fugene-6, according to the manufacturer's protocol, with pRL-TK for normalization, and after 8 h were starved overnight. Then they were treated for 24 h with HGF (200 ng/ml). Firefly and Renilla luciferase activities were measured with a dual luciferase assay system (4). The void vectors showed practically undetectable luciferase activity. These transfection experiments were carried out on duplicate plates and were repeated at least three times (n = 6).
For total RNA preparation, HepG2 cells (3 x 106) seeded in T25 flasks were transfected with dominant negative ssNF-B (6.25 µg/flask), starved overnight, then treated with HGF (50 ng/ml) for 4 h.
Northern blot and western blot assays
Northern blot analysis of total RNA (30 µg) from HepG2, MCF-7 and MDA-MB-231 cells was done using probes labeled with [-32P]dCTP using a Nick translation kit (14). To confirm that each lane contained equal amounts of total RNA, we checked the rRNA concentration in each lane visually by ethidium bromide staining. For western blotting, total, cytosolic and nuclear protein fractions were prepared (13,14) and protein content was measured by Bio-Rad assay; 100 µg total and cytosolic or 30 µg nuclear proteins were analyzed. The membranes were immunoblotted with anti-Met or anti-HIF-1
antibody. To confirm that each lane contained equal amounts of cytosolic or nuclear extracts, the membranes were hybridized with anti-ß-tubulin or anti-histone deacetylase antibody. After incubation with the appropriate secondary antibody, the signals were detected using an enhanced chemiluminescence kit (ECL-plus; Amersham Biosciences Europe GmbH).
Analysis of the gene promoters
The putative consensus sequences in the gene promoters were investigated with MatInspector analysis software. The GenBank numbers are S71124 for human ODC, AF050115 for HIF-1, AJ315134 for c-myc, AF147742 for Mcl-1 and AF145047 for HO-1.
Cell viability and cytofluorimetric analysis of HGF-treated carcinoma cells
The TOX-1 kit (Sigma) provided the MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], which is reduced to purple formazan crystals by mitochondrial succinate dehydrogenase (EC 1.3.99.1) in viable cells. HepG2, MCF-7 and MDA-MB-231 cells were seeded in 12-well multiwell plates (0.2 x 106/well) and transfected with 1 µg ARNT expression vector per well. Color changes were evaluated in the transfected cells 24 or 48 h after HGF treatment (50 ng/ml). Final values were the result of subtracting readings at 690 nm from those at 570 nm. Cell viability ratios were obtained by relating the values from HGF-treated and untreated control cultures (0.1% FBS) in the presence or absence of
ARNT.
For flow cytometry experiments, HepG2 and MDA-MB-231 cells were seeded in T25 flasks at a density of 1.2 x 106 cells/flask. At various times after HGF treatment the cells were harvested in FBS diluted with phosphate-buffered saline (PBS), centrifuged and fixed using ice-cold 70% ethanol. After permeabilization (0.25% Triton X-100 in PBS for 5 min), the cells were incubated with 50 µl of a solution containing terminal dUTP-transferase (TdT) and FITC-conjugated dUTP deoxynucleotides (1:1) in storage buffer (Boehringer Mannheim, Mannheim, Germany) for 1 h at 37°C in the dark. This method is based on labeling the DNA strand breaks resulting from internucleosomal cleavage by calcium-dependent endonucleases activated during apoptosis. TdT catalyzes the incorporation of FITC-conjugated nucleotides to the 3'-OH free DNA ends in a template-independent manner. After washing in PBS, the cells were analyzed by a flow cytometry system with a laser light source.
Statistical analysis
Densitometric values were analyzed by analysis of variance, with P < 0.05 considered significant. Differences from controls (cells transfected with gene reporter constructs but not exposed to HGF) were evaluated on original experimental data; the controls are assigned an arbitrary value of 1 in the figures.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Gene expression in carcinoma cells after HGF treatment
The pattern of gene expression may be unique to each cell type or cell stimulus depending on the interaction of the transcription factors activated. Many diseases, including neoplasias, ultimately result from disruption of gene expression through inappropriate activation or inhibition of specific transcription factors. We examined a panel of selected target genes with numerous putative NF-B and/or HIF-1 consensus sites in the promoter (Figure 2). Using MatInspector analysis we observed the following consensus sequences in the gene promoters and/or 5'-untranslated region (5'-UTR): for ODC, six HIF-1 and two NF-
B; for c-myc, four HIF-1 and five NF-
B; for Mcl-1, seven HIF-1 and five NF-
B; for HO-1, two HIF-1 and two NF-
B in the promoter, and several NF-
B consensus sequences in the 5'-UTR (36); for HIF-1
, five HIF-1 and four NF-
B.
|
To evaluate the involvement of NF-B activity in the induction of ODC, c-myc, Mcl-1 and HO-1 after HGF, we transfected HepG2 cells with ssNF-
B (Figure 2B). This super-repressor molecule is a highly specific and effective NF-
B inhibitor, with mutations in the conserved serine residues typically targeted for phosphorylation after cellular stimulation (37). The cells were treated with HGF for 4 h, then total RNA was extracted and used for northern blot analysis. NF-
B activity blockade prevented the HGF-induced increases in ODC (50%), c-myc (30%), Mcl-1 (50%) and HO-1 (25%). 18S rRNA was used as a control for RNA loading and for normalizing the densitometric values.
Figure 2C shows the HIF-1 northern blots for the three cell lines treated with HGF, and the densitometric values are reported in the graph as the amount of mRNA in relation to that of starved MCF-7 cells. In HepG2 cells the HIF-1
steady-state mRNA level started to increase 12 h after HGF, peaking at 4 h and decreasing thereafter. Similarly, in MCF-7 cells HIF-1
mRNA level tripled 2 h after HGF and then decreased. In starved MDA-MB-231 cells the HIF-1
steady-state mRNA level, which was 3 times that of the other two cell lines, was unaffected by HGF. To compare the data for the genes examined, we used the same labeled cDNA probes to hybridize filters for the three cell lines, which were then exposed concomitantly to the same X-ray film for an equal period of time. 18S rRNA was used as a control for RNA loading and for normalizing the densitometric values.
Next, we examined the expression of the HIF-1 protein and the transactivating function of HIF-1 in HGF-treated carcinoma cells. Western blots showed HIF-1
protein migrating as a series of bands from 106 to 116 kDa (Figure 2D). In HepG2 and MCF-7 cells HGF raised nuclear HIF-1
protein levels 2.8- and 1.9- to 1.5-fold, respectively, at 46 h. Smaller accumulations of
protein were concomitantly observed in the cytosol, probably indicating protein synthesized but not translocated to the nucleus where the
/ß subunit interaction could occur. In contrast, in MDA-MB-231 cells nuclear
protein of high Mr was strongly elevated under starvation, and a temporary down-regulation was observed 4 h after HGF, followed by partial recovery at 6 h. The
protein pattern was consistent with mRNA inducibility after HGF in HepG2 and MCF-7 cells, while in MDA-MB-231 cells both HIF-1
mRNA and protein were constitutively elevated.
In agreement with HIF-1 protein induction, HIF-1 seemed to be functional in transactivation only in HepG2 and MCF-7 cells (Figure 2E). On transfecting these cells with a luciferase reporter construct under HRE multimer control, HGF increased the activity 8.5- and 2.6-fold. In MDA-MB-231 cells the control luciferase activity of the HRE multimer construct was similar to that of HepG2 and MCF-7 cells and was unaffected by HGF.
Regulation of HIF-1 induction by transient transfection: involvement of transcription factors NF-
B and HIF-1
We investigated the involvement of NF-B and HIF-1 activity in HIF-1
induction after HGF in transient transfection experiments. First, we used MatInspector analysis software to identify putative cis-acting elements within the HIF-1
promoter and in the 5'-UTR (Figure 3A). We found four and five putative binding sites for NF-
B and HIF-1 (HRE), respectively, and only one SP1 consensus sequence in the forward sense. Transient transfections were then done with two luciferase reporter constructs, pHIF1A(572/+284)Luc under the control of the HIF-1
regulatory region, which included the 5'-UTR, and pHIF1A(572/+32)Luc driven only by the HIF-1
promoter (38) (Figure 3B). HGF stimulated the luciferase activity of these two constructs only in HepG2 and MCF-7 cells: the activity of pHIF1A(572/+284)Luc increased 5.6- and 2.8-fold in the two cell lines. The construct lacking the 5'-UTR was noticeably less activated, indicating the essential role of this sequence in HGF-induced HIF-1
translation. In contrast, in MDA-MB-231 cells the activities of both reporter genes were unaffected by HGF (Figure 3B). The basal activity of pHIF1A(572/+32)Luc was 2.5 times higher in MDA-MB-231 than in the other two cell lines (data not shown), consistent with the elevated HIF-1
mRNA level in MDA-MB-231 cells (see Figure 2). ssNF-
B co-transfected in HepG2 and MCF-7 cells prevented the stimulatory effect of HGF on the activities of pHIF1A(572/+284)Luc and pHIF1A(572/+32)Luc, by 7080 and 40%, respectively. A NF-
B super-repressor had no effect on the luciferase activity of the reporter genes in the three cell types, either starved or with 10% FBS, or in HGF-treated MDA-MB-231 cells. We also performed experiments with a dominant negative form of p85, a mutant form that is unable to interact with the p110 subunit, inactivating PI3K. We observed no changes in the luciferase activity of pHIF1A constructs in control MDA-MB-231 cells (data not shown).
|
Regulation of ODC promoter activity in response to HGF in different carcinoma cells
To better define the regulation of human ODC induction by HGF, we analyzed the ODC promoter using MatInspector analysis software (Figure 4A) then prepared reporter genes for transfection experiments. The sequence analyzed was that published by Verma (39) and confirmed by Bac analysis of chromosome 2. The original construct (4362/+131) contained the promoter sequence and part of the first exon. The concentration of consensus sequences was highest in the HindIII fragment 1692/+131, prepared using the restriction enzyme and subcloned in pGL2-enhancer. We also prepared the 720/+16 sequence by RTPCR because it contains six HRE and a carbohydrate response element (Chore) sequence. The E-box (5'-CACGTG-3'), i.e. the consensus core of the Chore, corresponds to the Myc/Max binding site that overlaps the binding site for HIF-1 (CGTG).
|
Effect of HGF-dependent regulation of HIF-1 and ODC on survival of carcinoma cells
Figure 5A shows the viability of HepG2, MCF-7 and MDA-MB-231 cells transiently transfected with ARNT then treated with HGF for 24 or 48 h. Viability was only affected in HepG2 and MCF-7 cells, where overexpression of the dominant negative form in HGF-treated cells reduced viability by 4050% at 24 h, though noticeably less at 48 h. Cell viability was not affected in MDA-MB-231 cells.
ARNT transfection caused only a very small reduction in mitochondrial succinate dehydrogenase activity in untreated HepG2 and MCF-7 cells. HGF-treated HepG2 cells showed steep drops in ODC mRNA and protein levels from 12 until 48 h, ODC protein falling to below control values (Figure 5B). HGF reduced cell viability by
40% at 72 h (Figure 5B), and 25% of the cells underwent apoptosis (Figure 5C). HGF-treated MDA-MB-231 cells did not undergo apoptosis between 24 and 72 h (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study we compared three carcinoma cell lines. HepG2 hepatoma cells are characteristically responsive to HGF, which activates NF-B in an early proliferative phase within 24 h of treatment (21). Then, possibly because of adverse intracellular conditions created by HGF/Met signaling, HepG2 cells undergo apoptosis (43). For example, HGF, like other growth factors, may induce the production of oxygen radicals, although the molecular mechanisms and significance of this phenomenon have not been clarified (44). MCF-7 and MDA-MB-231 cells are, respectively, low and highly invasive breast carcinoma cells. We found similar levels of Met in HepG2 and MDA-MB-231 cells, much higher than in MCF-7 cells. In MDA-MB-231 cells NF-
B activity was constitutively elevated, probably as a consequence of changes in stoichiometry and possibly also of some post-translational modifications of the p65 subunit forming the p50/p65 heterodimer. In MDA-MB-231 cells this was the complex present in largest amount and is known to have transactivation activity (15,42). Phosphorylation can affect several functions of NF-
B, including p65 interaction with the co-activator CREB/p300 and transactivation of Fas (34,42). HGF boosted NF-
B transactivation in the examined cell types to different extents, very likely related to Met levels and constitutive NF-
B activity.
The role of NF-B in cell survival is complex. Through its ability to regulate the expression of cellular factors that affect the apoptotic threshold, NF-
B has been implicated in the regulation of cell death. It seems to act on the upstream pathways of apoptosis, either negatively or positively (41). We evaluated gene expression changes downstream of NF-
B within 24 h of HGF treatment, because this may be the period when cell survival or death is determined. Differences in the genes activated by HGF through NF-
B activity might be related to tumor grade and the apoptotic process already occurring in MDA-MB-231 cells (33,34,41,45). These cells seem to have some peculiarity, because tumor cells acquire resistance to apoptosis during the course of progression.
The genes considered were HIF-1, ODC, c-myc, Mcl-1 and HO-1, because in preliminary experiments we demonstrated the link between HGF-mediated induction and NF-
B activity. HIF-1
codes for the inducible subunit of transcription factor HIF-1 and human tumor biopsies reveal dramatic overexpression of HIF-1
in common cancers. HIF-1
overexpression is also a marker of highly aggressive disease and is associated with an increased risk of mortality in some carcinomas (18). The growth advantage offered by HIF-1 activity is partly related to cell protection from apoptosis (1,30,31). Receptor-mediated signals such as those triggered by the HGF/Met interaction lead to HIF-1
accumulation through pathways and mechanisms separate from hypoxic signaling (46). Under hypoxia there is less degradation of the
subunit in the proteasome due to inhibition of prolyl-4-hydroxylase activity and von Hippel Lindau binding (18).
First, we confirmed that HGF increased HIF-1 mRNA and protein levels in HepG2 hepatoma cells (14) and extended this finding to the low invasive MCF-7 breast cancer cell line. The degree of response might be correlated with Met receptor levels. Here we report new data on the molecular mechanisms involved in HIF-1
induction in HepG2 and MCF-7 cells. We observed a transcriptional/translational control that involved the HGF-inducible NF-
B transactivation activity. This was seen in the transient transfection experiments using two gene reporters, pHIF1A(572/+284) and (572/+32), driven, respectively, by a longer and a shorter regulatory sequence of the HIF-1
gene, containing or not the 5'-UTR, and also on co-transfecting the cells with NF-
B super-repressor. An autoregulatory loop seemed to be set up, because HIF-1 activated through NF-
B probably regulated
gene expression by binding to the HREs in the promoter. This is the first direct demonstration that NF-
B is involved in HIF-1
transcription/translation. Previous papers reported that NF-
B regulates HIF-1
protein stabilization by IL-1 and TNF
(47,48).
Our findings show that the 5'-UTR sequence of HIF-1 mRNA is important in expression of HIF-1
protein after HGF treatment in HepG2 and MCF-7 cells. Involvement of the 5'-UTR in translational control could require HGF-stimulated phosphorylation of the regulatory proteins 4E-BP1, p70S6 kinase and eukaryotic initiation factor 4E, but we cannot exclude induction of these proteins dependent on HGF-activated NF-
B (49). The 5'-UTR sequence also contains an internal ribosome entry site that might permit efficient translation by growth factors (50). The literature indicates that the 5'-UTR of the HIF-1
gene may also play a role in HIF-1
transcription (51). In TATA-less promoters, cis-acting elements are often found downstream of the transcription initiation site. This possibility was supported by the increase (
6-fold) in transcriptional activity of the plasmid p15C (30/+287) transiently transfected in HepG2 cells after HGF treatment (data not shown). p15C contains the entire 5'-UTR sequence and a very short sequence containing at least the initiation site of the HIF-1
gene (51).
In HepG2 and MCF-7 cells HIF-1 seemed to transactivate Mcl-1, an anti-apoptotic Bcl-2 family member known to heterodimerize with Bax and to neutralize its cytotoxic activity (52,53). This would be consistent with the protective role of HIF-1 against HGF-induced apoptosis in hepatoma cells (1,4) and with the transactivation of genes for cancer survival (54). In addition, in both HepG2 and MCF-7 cells the inhibition of HGF-induced HIF-1 activation through ß subunit blockade reduced cell viability.
The precise molecular mechanisms of the switch from an anti-apoptotic to a pro-apoptotic role for NF-B have not been clearly defined. Changes in the transcription factor(s) that functions cooperatively with NF-
B at different promoters would have the effect of switching its activity from one set of genes to another. The possible activation of HIF-1 in the first phase after HGF treatment might be important for carcinoma cell survival, and this was what led us to investigate whether this was a common mechanism.
Second, HIF-1 expression was differently regulated in the three cell types examined. In MDA-MB-231 cells, an aggressive and invasive cancer cell line, HIF-1
was highly expressed under basal conditions through transcription factors probably different from NF-
B, which might be SP1 (one forward and five reverse consensus sequences in the
subunit coding gene) (38). Western blot analysis showed a high nuclear level of the
protein in starved control cells, and slower migrating bands were the most abundant. The very active PI3K/NF-
B pathway of MDA-MB-231 cells might be involved in the stabilization of HIF-1
protein (33,55), but a corresponding basal transactivation activity of HIF-1 was not detectable. Neither
protein transcription/translation nor HIF-1 transactivation activity was stimulated by HGF, as resulted from transfection experiments with the two different pHIF1ALuc gene reporters and the HRE multimer construct. Overexpression of the dominant negative form of HIF-1ß did not affect cell viability. Unlike the other two carcinoma cell types, in MDA-MB-231 cells HGF caused a temporary down-regulation of HIF-1
protein. This might be related to an active proteasome multienzyme system possibly responsible for HIF-1
degradation in the nucleus of these cells. We previously reported that HGF may cause degradation of ODC and p53 proteins in the proteasome in carcinoma cells (1). NF-
B activation in MDA-MB-231 cells may also require I
B
degradation in the proteasome (35).
Finally, MDA-MB-231 cells showed other differences in gene expression after HGF, such as marked and persistent expression of ODC and HO-1. These genes might be important for apoptosis only under particular cellular conditions and stimuli and in some cell types (1,25,43,56,57). ODC plays a key role in cell cycle progression and cell proliferation in various normal and neoplastic conditions (28). This study extends our knowledge on control of ODC expression by growth factors in neoplastic cells. ODC cannot be considered a target gene of HIF-1, but its expression in HGF-treated human carcinoma cells is likely to depend on NF-B. In fact, ODC was strongly induced in MDA-MB-231 cells in which HIF-1 did not respond to HGF and the rise in activity of pODC(1692/+131)Luc after HGF was prevented by a NF-
B super-repressor but not by a dominant negative form of HIF-1ß. A possible explanation for the complete inhibitory effect of a dominant negative form of JNK-1 on pODC (1692/+131)Luc activity might be that NF-
B was activated downstream of the PI3K/JNK-1 cascade, which is stimulated by HGF (4). One pathway for NF-
B activation requires a direct stimulatory action of p38 mitogen-activated protein kinase and JNK on the transcription factor (42). HGF-dependent activation of JNK-1 might also influence the degradation of I
B (58). Myc/Max involvement in HGF-dependent ODC expression is not excluded and might explain the residual activity of the pODC(720/+16)Luc construct. The E-box consensus sequence (at 485 to 480) was that published by Soprano as the functional binding site for Myc/Max (59). A regulatory role of SP1 in pODC(720/+16)Luc activity is under investigation.
Because HIF-1 is non-functional in MDA-MB-231 cells and so cannot perform its anti-apoptotic role, NF-B activity might be crucial in these cells not only for acquisition of the invasive phenotype but also in triggering apoptosis in response to certain stimuli (15,41,60). MDA-MB-231 cells seem to be naturally prone to apoptosis, with a background level of 5% apoptotic cells and elevated levels of Fas and Fas ligand (33,34). However, upon HGF treatment expression of ODC and HO-1 was persistently elevated and might exert a protective role, possibly preventing apoptosis in MDA-MB-231 cells. In contrast, transient ODC mRNA induction without a corresponding increase in enzyme activity in HepG2 cells may be one signal triggering the switch to the apoptotic program (21). Under these conditions ODC is degraded in the proteasome after specific binding to the HGF-induced antizyme (43). Here we show that steep drops in ODC mRNA and protein expression in HGF-treated HepG2 cells were correlated with a loss of cell viability leading to apoptosis at 72 h. The execution phase of apoptosis in HGF-treated hepatoma cells occurs at 4872 h, with the classical changes in pro-apoptotic genes (Bax), cleavage of Bid, release of cytochrome c and activation of caspase 3 (1,43).
The present data might help clarify the molecular events triggered by activation of NF-B in response to the multifunctional cytokine HGF and extend our knowledge of NF-
B function(s) in tumors with different molecular characteristics and origins. The principal significance of identifying alterations in signal transduction pathways involving NF-
B in neoplastic cells is to find targets for therapeutic intervention to sensitize cells to drug-induced apoptosis. Inhibiting HIF-1 activity may help bypass one of the mechanisms of protection against apoptosis of tumor cells.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|