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Original Studies |
Departments of Gynecology and Obstetrics (J.S., S.I.T., L.F.-S., L.C.G.) and Radiation Oncology (F.K., A.J.G.), Stanford University Medical School, Stanford, California 94305-5317; and Department of Pediatrics (D.R.P.), Baylor College of Medicine, Houston, Texas 77030
Address correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., Department of Gynecology and Obstetrics, Stanford University Medical Center, Room HH-333, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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10-fold and 20-fold, respectively. In normoxia,
8-Br-cAMP stimulated IGFBP-1 protein and mRNA levels in a
dose-dependent manner (7-fold and 10-fold at 1 mM). Hypoxia
and 8-Br-cAMP showed additive stimulatory effects on IGFBP-1 protein
and mRNA levels (35-fold and 50-fold at 1 mM) that were
time and dose dependent. Primary transcripts of IGFBP-1 mRNA were
increased concordantly with IGFBP-1 mRNA. The half-life of the
IGFBP-1 mRNA was markedly increased (6-fold) by hypoxia, and cAMP
minimally enhanced this effect. These results demonstrate that hypoxia
and compounds that increase intracellular cAMP additively regulate
IGFBP-1 gene expression by transcriptional and posttranscriptional
mechanisms. Regulation of IGFBP-1 mRNA and protein by cAMP and hypoxia
may be important for understanding the physiologic and pathophysiologic
roles of IGFBP-1. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current study, we have examined the effects of hypoxia and a cAMP analog, 8Br-cAMP, on IGFBP-1 protein and mRNA expression, primary transcripts of IGFBP-1, and IGFBP-1 mRNA stability in HepG2 cells. We report additive effects of hypoxia and 8Br-cAMP on IGFBP-1 protein and mRNA expression in HepG2 cells, suggesting that cAMP and hypoxia may operate through independent pathways for IGFBP-1 gene activation
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HepG2 cells, a human hepatocellular carcinoma cell line (13) known to produce IGFBP-1 (14), were purchased from the American Type Culture Collection (Manassas, VA). HepG2 cells were grown at 37 C in 95% air-5% CO2 in 175-mm2 cell culture dishes and fed every 3–4 days with MEM (Life Technologies, Inc., Grand Island, NY) plus 10% FBS and 50 µg/mL gentamicin (Gemini Bio Products, Inc., Calabasas, CA), 0.1 mM nonessential amino acids (Life Technologies, Inc.), and 1 mM sodium pyruvate (Life Technologies, Inc.). Cultures were passaged weekly when confluent at a ratio of 1:4 using trypsin-EDTA (Life Technologies, Inc.).
Materials
8-Bromoadenosine-3',5'-cyclic monophosphate (8Br-cAMP; Sigma, St. Louis, MO) was dissolved in H2O to a stock concentration of 100 mM. Actinomycin D (Sigma) was dissolved in dimethyl sulfoxide (600 µg/mL), and 5,6-dichloro-1-D-ribofuranosyl benzimidazole (DRB; Sigma) was dissolved in ethanol (10 mM) and stored at -20 C. [125I] IGF-I (µCi/µg) and [125I] IGF-II (µCi/µg) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ).
Hypoxia treatment and hormonal supplementation
Cells were plated in duplicate in custom-made 60-mm glass plates, with notched sides to allow gas change (15), at 2–3 x 106 cells, and stabilized in serum-free RPMI 1640 (Life Technologies, Inc.) for 24 h before the following experiments. To examine the effect of different concentrations of 8Br-cAMP under hypoxic or normoxic conditions, dishes were placed in specially designed aluminum hypoxia chambers that were prewarmed to 37 C, sealed, and subjected to successive rounds of evacuation, followed by flushing with 95% N2/5% CO2 while being slowly agitated. The final oxygen concentration in the media after one cycle was reduced to 2% measured with a Clark-type electrode (Controls Katharobic, Edmonton, Alberta, Canada). The chambers were then placed in a 37 C incubator for 24 h in the absence or presence of varying concentrations of 8Br-cAMP (0.1, 0.5, and 1 mM) in serum-free RPMI 1640. Controls were cells cultured in normoxia for 24 h in the presence or absence of 0.1, 0.5, or 1 mM 8Br-cAMP. Kinetic studies were conducted in which cells were treated in normoxia or hypoxia for 2, 6, 12, or 24 h with or without 8Br-cAMP (1 mM). Five-milliter aliquots of conditioned media were collected, centrifuged, and stored at -20 C for subsequent analysis. Total cellular RNA was isolated, as described below. All conditions were examined in duplicate in three separate experiments.
Western ligand blotting
Western ligand blotting was performed according to the method of Hossenlopp et al. (16). Seventy-five microliters of conditioned media were electrophoresed on 12% SDS-PAGE with a 5% stack at 50 V. The size-fractionated proteins were electroblotted onto nitrocellulose at 160 mA for 1 h and 20 min. Blocking was with 1% BSA in saline for 2 h. The filter-immobilized proteins were incubated with 1.5 x 106 cpm/mL [125I] IGF-I and [125I] IGF-II overnight at 4 C, washed, air-dried, and exposed to autoradiography film (NEN Life Science Products, Boston, MA) at -80C. The intensity of the blots was quantitated by laser scanning densitometry, as described (3).
IGFBP-1 immunoradiometric assay (IRMA)
IGFBP-1 levels in conditioned medium from duplicate cultures were analyzed by IRMA, according to the manufacturer’s instructions (Diagnostics Systems Laboratories, Inc., Webster, TX). The sensitivity was 0.01 ng/mL, and the intra- and interassay coefficients of variation were 4.7% and 2.3%, respectively. IGFBP-1 levels were normalized by the total protein in conditioned medium by the method of Bradford (17).
Northern blot hybridization
Total RNA was isolated with a modification of Chomczynski
and Sacchi (18), using TRIzol (Life Technologies, Inc.)
according to the manufacturer’s instructions. Ten micrograms of total
RNA were loaded per lane for 1.2% agarose-formaldehyde
electrophoresis. Gels were then stained with ethidium bromide to
confirm even loading of total RNA in each lane. RNA was transferred
onto nitrocellulose membrane by capillary transfer, and the filters
were irradiated in a Stratagene ultraviolet cross-linking apparatus
(Stratagene, La Jolla, CA). The following cDNA probes were
used: a 938-bp EcoRI fragment of human IGFBP-1
complementary DNA (cDNA) and PstI/SphI
1260-bp intron-1 fragment of the human IGFBP-1 gene (19). The probes
were labeled with deoxycytidine
5'
-phosphate32-labeled triphosphate (NEN Life Science Products) using a random priming kit
(Amersham Pharmacia Biotech). Membranes were blocked at 42
C in 50% formamide, 5x SSC, 0.3% SDS, 5x Denhart’s solution, and
100 µg/mL salmon sperm DNA and then probed overnight at 42 C using
1 x 106 cpm/ml of the appropriate
random-primed probe. Filters were washed twice at room temperature in
5x SSC and 0.5% SDS, twice at 37 C in 1x SSC and 0.5% SDS, and
twice at 65 C in 0.1x SSC and 1.0% SDS. Films were exposed at -70 C
using DuPont enhancing screens. Molecular sizes were confirmed using an
RNA marker (Promega Corp., Madison, WI) and ribosomal RNA
(18S and 28S) as standards. The hybridization signals on the blots were
analyzed quantitatively on a laser scanning densitometer
(LKB, Bromma, Sweden). The intensity on the blots was
normalized with 18S ribosomal RNA on ethidium bromide staining, and the
relative intensity was calculated as a fold of the mean RNA value
compared to controls.
Measurements of IGFBP-1 mRNA half-life
To investigate IGFBP-1 mRNA stability, HepG2 cells were treated with either one of two transcription blockers, actinomycin D (3 µg/mL) (20) or DRB (100 µM) (21). The blocking agents were added 15 min before treatment (zero time point), and cultured in hypoxic or normoxic conditions for 2, 6, 12, or 24 h in the presence or absence of 1 mM 8Br-cAMP. Cells were collected, and total cellular RNA was extracted at the appropriate time point and processed for Northern blot analysis, as described above.
Statistical analysis
Results were presented as the mean ± SEM. Differences between groups were examined by one-way ANOVA, followed by Scheffe’s F-test, and significance was assigned at P less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HepG2 cells were used as a model system to investigate IGFBP-1
gene regulation under hypoxic conditions and by cAMP. HepG2 cells in
serum-free medium were incubated in normoxic (20%
O2) and hypoxic (2% O2)
conditions for 24 h. IGFBP-1 protein in conditioned media was
analyzed by Western ligand blotting (Fig. 1A
), which revealed a complement of
IGFBPs with molecular weights of 34, 28, and 24 kDa, previously
identified as IGFBP-2, IGFBP-1, and IGFBP-4, respectively (3). In
normoxia and hypoxia, IGFBP-1 levels were markedly greater than other
IGFBPs and increased in a dose-dependent manner after the incubation
with 8Br-cAMP at 1 mM by 3-fold and 7-fold, respectively
(Fig. 1B). Other IGFBPs were not affected by 8Br-cAMP and hypoxia
treatment, detected by this method.
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) of conditioned
medium revealed that under hypoxic, compared with normoxic, conditions
IGFBP-1 protein levels increased by an average of 10-fold. Under
normoxic conditions, the cAMP analog 8Br-cAMP (0.1, 0.5, and 1
mM) stimulated IGFBP-1 protein levels in a dose-dependent
manner (7-fold at 1 mM), compared with the normoxic
control.
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), identical in size to
the IGFBP-1 mRNA described previously (22), was observed. IGFBP-1 mRNA
levels were increased by an average of 20-fold in hypoxic, compared
with normoxic, conditions (Fig. 2C). Under normoxic conditions,
8Br-cAMP (0.1, 0.5, and 1 mM) stimulated IGFBP-1 mRNA
levels in a dose-dependent manner (10-fold at 1 mM,
compared to control). Effects of hypoxia and 8Br-cAMP on IGFBP-1 expression: time course response
IRMA analysis (Fig. 3A) revealed that
with 8Br-cAMP and under normoxic conditions IGFBP-1 in the conditioned
medium reached a maximum (9-fold compared to without cAMP) at 12
h. Under hypoxic conditions with 8Br-cAMP, IGFBP-1 increased rapidly
and reached maximum stimulation (33-fold, compared to normoxia control)
at 12 h.
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50% of
maximum stimulation (Fig. 3, B and C). Under hypoxic conditions without
8Br-cAMP, IGFBP-1 mRNA increased rapidly and reached maximum
stimulation at a 6-h incubation (Fig. 3). By the addition of 8Br-cAMP
in hypoxia, IGFBP-1 mRNA was synergistically increased after a 6-h
incubation (20-fold, compared to normoxic control) and was maintained
throughout 24 h of culture. Under hypoxic conditions, increases in
IGFBP-1 in conditioned medium and IGFBP-1 mRNA were about the same by
24 h (Fig. 3, A and C). Effects of hypoxia and 8Br-cAMP on the abundance of IGFBP-1 primary transcripts
Total cellular RNA was extracted from cultured HepG2 cells treated
with 0, 0.1, 0.5, and 1 mM 8Br-cAMP in hypoxic or normoxic
conditions for 24 h (Fig. 4A).
Northern hybridization using an IGFBP-1 intron-1 fragment identified
several high molecular weight RNA species, which are primary
transcripts and shorter splicing intermediates containing intron-1 (8).
By sequence analysis (22), these species comprise the full-length
transcript (FLT; 5.2 kb), FLT missing intron-3 (4.3 kb), FLT missing
intron-2 (4.0 kb), FLT missing intron-2 and 3 (3.1 kb), and free
intron-1 (1.5 kb), respectively (Fig. 4A). Under normoxic conditions,
by the addition of 8Br-cAMP, the IGFBP-1 primary transcript and
splicing intermediates are changed in the same manner as IGFBP-1 mRNA,
consistent with a previous report that cAMP regulates IGFBP-1 primarily
at the transcriptional level (8). Under hypoxic, compared with
normoxic, conditions, those species are markedly increased by hypoxia,
and 8Br-cAMP enhances this effect in a dose-dependent manner. Kinetic
analysis (Fig. 4B) revealed that the primary transcripts rapidly
increased by the addition of 1 mM 8Br-cAMP and that hypoxia
enhances this effect. Densitometric analysis of the full-length primary
transcripts revealed that 3-fold hypoxic induction was observed and
that 8Br-cAMP further stimulates this induction (3-fold at 1
mM). This induction is considerably less than the increase
in IGFBP-1 mRNA (20-fold without 8Br-cAMP, 50-fold at 1 mM
8Br-cAMP), suggesting that the increase of IGFBP-1 mRNA by hypoxia is
composed primarily of a posttranscriptional and, in part, by a
transcriptional component.
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Increase in the steady-state levels of IGFBP-1 mRNA can be caused
by changing the rate of transcription, by stabilizing the mRNA
(i.e. decreasing the rate of mRNA degradation), or by both
mechanisms. To determine the effect of 8Br-cAMP and hypoxia on IGFBP-1
mRNA stability, HepG2 cells were treated with either one of two
transcription blockers, actinomycin D (3 µg/mL) or DRB (100
µM). These blockers were added 15 min before
treatment (zero time point), and cells were then cultured in hypoxic or
normoxic conditions for 2, 6, 12, or 24 h without (Fig. 5A) or with (Fig. 5B) 1
mM 8Br-cAMP. After inhibiting RNA synthesis, the
remaining IGFBP-1 RNA was quantitated by Northern hybridization, as
described above. In the absence of cAMP, IGFBP-1 mRNA levels during
normoxia decreased to 50% in 2 h, after the addition of actinomycin
D or DRB (Fig. 5A). Under hypoxic conditions, IGFBP-1 mRNA was markedly
stabilized, and the half-life of IGFBP-1 mRNA was 12 h. With 1
mM 8Br-cAMP (Fig. 5B), the half-life of IGFBP-1
mRNA was not affected under normoxic conditions, whereas under hypoxic
conditions IGFBP-1 did not decline to 50% of the initial value by
24 h. These results indicate that hypoxia increases IGFBP-1 mRNA
stability and that 8Br-cAMP contributes (minimally) to this
stabilization.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The oxygen-regulated system controlling induction of a variety of genes by hypoxia is present in most cells and involves induction of a nuclear transcription factor, HIF-1, which binds to the HRE and, with contributions from a variety of enhancers, results in gene transcription (23, 24, 25). IGFBP-1 mRNA and protein are synthesized by HepG2 cells and are stimulated in these cells by hypoxia via mechanisms that seem to involve HIF-1 (3). Recently, a role for cAMP has been reported in the hypoxia response of other genes, including LDH-A (12) and EPO (26). LDH-A mRNA and protein are inducible by hypoxia as well as by cAMP. Mutational analyses of the LDH promoter revealed three domains in the 5' flanking region that are functionally important. These include a HIF-1-binding site in between an enhancer and a sequence characteristic of a CRE. Functional cooperativity was observed between these sites, and maximal hypoxia inducibility was observed with all three sites (12). Of note, the HIF-1-binding element alone was unable to support inducible expression. With regard to EPO, it is not induced by cAMP, but is induced by cAMP under hypoxic conditions (26). cAMP is not crucial for the hypoxia-activated signal transduction pathway per se, although it may act with other signaling pathways to enhance the response to hypoxia. Hypoxia inducibility of IGFBP-1 gene expression has several differences, compared to the LDH-A and EPO. For example, the HREs in the IGFBP-1 gene are in an intron (3) and not in the promoter that contains the CRE (8). Also, one of the HREs (5'-714-721-3') is functional in a reporter construct with a heterologous promoter and without a CRE (3). These observations suggest that the IGFBP-1 response to cAMP and to hypoxia are independent and further support the effects observed in the current study. Data on the EPO 3' enhancer, for instance, suggest that the HIF-1 DNA recognition site is cAMP responsive in hypoxic conditions (27). Whether this is so for IGFBP-1 remains to be determined. Experiments to define whether HIF-1 is the hypoxia-sensitive factor in HepG2 cells mediating the observed IGFBP-1 induction and, if so, whether the HIF-1 DNA recognition site in the IGFBP-1 gene is cAMP responsive, are planned in future studies in our laboratory.
Northern blots using IGFBP-1 intron 1 identified several high molecular weight RNA species containing intron 1. Sequence analysis determined that the RNAs are the FLT, splicing intermediates, and free intron 1. In normoxia, the primary transcript of IGFBP-1 mRNA is increased by 8Br-cAMP in a dose-dependent manner, concordant with the changes of steady-state levels of IGFBP-1 mRNA, consistent with the previous reports that cAMP regulates IGFBP-1 at the transcriptional level (8). Under hypoxic, compared with normoxic, conditions, those species are remarkably increased, and 8Br-cAMP enhances this effect. By kinetic analysis, the primary transcripts are rapidly increased by 8Br-cAMP, and hypoxia further stimulated their induction. These results indicate that IGFBP-1 gene expression is stimulated by hypoxia at the transcriptional level that is concordant with our previous results (3). However, hypoxic induction of primary transcripts cannot fully account for the increase in steady-state IGFBP-1 mRNA, suggesting that the increase of IGFBP-1 mRNA by hypoxia is due to posttranscriptional and, in part, by transcriptional regulatory mechanisms. It has been reported that other hypoxia-inducible genes such as EPO (27, 28), vascular endothelial growth factor (29), and tyrosine hydroxylase (30) are posttranscriptionally regulated by hypoxia (i.e. hypoxia stabilizes those mRNAs). The data reported herein suggest that hypoxia regulates IGFBP-1 gene expression by transcriptional control as well as by stabilization of IGFBP-1 mRNA. Furthermore, overall, the data suggest independent pathways of hypoxia and cAMP regulation of IGFBP-1 in HepG2 cells by either independent mechanisms for IGFBP-1 transcription or by one acting primarily through posttranscriptional mechanisms.
By Western ligand blotting, in normoxia and hypoxia, IGFBP-1 levels were greater than other species (IGFBP-2 and IGFBP-4) and increased in a dose-dependent manner after incubation with 8Br-cAMP. These results may reflect tissue-specific expression of IGFBPs and will require further investigation.
IGFBP-1 is synthesized primarily in the liver, pregnancy decidua, and luteinizing granulosa cells and is elevated in physiologic conditions such as pregnancy, in the maternal circulation. It is also elevated in pathophysiologic conditions, including renal failure, insulin-dependent diabetes mellitus, GH receptor deficiency, and in the fetal circulation of pregnancies complicated by uteroplacental insufficiency and in utero hypoxia (1, 2). Regulation of IGFBP-1 by hypoxia, cAMP, and other known regulators, including glucocorticoids, progesterone, and insulin, has been observed in a variety of cell types (1). HepG2 cells have provided a useful model to investigate regulation of IGFBP-1 by these effector molecules, although extrapolation of regulation of IGFBP-1 by hypoxia and cAMP in HepG2 cells to pathophysiologic conditions requires cautious interpretation.
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Footnotes |
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Received December 2, 1999.
Revised April 12, 2000.
Revised June 8, 2000.
Accepted July 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or
hypoxic conditions (filled symbols) with (
) or
without (•) 8Br-cAMP 1 mM for 2, 6, 12,and 24 h.
IGFBP-1 levels in conditioned medium were quantitated by IRMA and
normalized by total protein concentrations. The values shown are the
mean ± SEM of three different experiments. B, HepG2
cells were preincubated in serum-free medium for 24 h, and at time
zero (lane a), the medium was replaced with fresh serum-free medium
containing 1 mM 8Br-cAMP. Total RNA was
extracted after 2, 6, 12, and 24 h of exposure of HepG2 cells in
normoxia (lanes b-e) or hypoxia (lanes f-i). A 1.5-kb steady-state
IGFBP-1 mRNA was detected using a 938-bp fragment of IGFBP-1 cDNA. C,
Densitometric analysis of Northern blots. Data are shown as a fold over
normoxic control (lane a). The intensity on the blots was normalized
with 18S ribosomal RNA on ethidium bromide staining, and the relative
intensity was calculated as a fold of the mean RNA value compared to
controls. Results shown are the mean ± SEM of three
different experiments.
)
conditions for 2, 6, 12, or 24 h in the absence (A) or the
presence (B) of 1 mM 8Br-cAMP. The intensity on the blots
was normalized with 18S ribosomal RNA on ethidium bromide staining, and
the relative intensity was calculated as a fold of the mean RNA value
compared to controls at zero time points. The results shown are the
mean ± SEM from three different experiments. *,
P < 0.05.