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Department of Pediatrics, Oregon Health Sciences University School of Medicine, Portland, Oregon 97201-3042
Address all correspondence and requests for reprints to: Dr. Zoran S. Gucev, Department of Pediatrics, NRC5, Oregon Health Sciences University School of Medicine, Portland, Oregon 97201-3042.
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Abstract |
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Introduction |
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IGFBP-3 is the major IGFBP in human adult serum, forming a 150-kDa complex with an acid-labile subunit (ALS) and IGF. IGF-I and IGFBP-3 are positively correlated with GH secretion; increased serum concentrations for all three proteins are observed in patients, with acromegaly and decreased concentrations are found in patients with GH deficiency (GHD) (3, 4). Administration of GH to patients with GHD corrects serum IGF-I and IGFBP-3 concentrations (5). Surprisingly, IGF-I treatment failed to increase serum concentrations of IGFBP-3 in patients with GH insensitivity (GHI) (6, 7), suggesting that GH directly regulates IGFBP-3. Kanety et al. (8), on the other hand, reported that chronic IGF-I treatment of GHI results in increased serum levels of IGFBP-3. Several in vivo studies in rats also suggested that IGF-I administration can increase serum IGFBP-3 concentrations (9, 10, 11).
To investigate further the issue of IGFBP-3 regulation, we used a cultured human liver adenocarcinoma cell line, SKHEP-1. Our results provide evidence that supports an IGF-I-independent mechanism for GH regulation of IGFBP-3 while demonstrating that IGF-I can also itself directly stimulate IGFBP-3 synthesis.
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Materials and Methods |
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IGFBP-3g1 was raised against
glycosylated recombinant IGFBP-3 (12).
5,6-Dichloro-1ß-D-ribofuranosylbenzimidazole (DRB) was
purchased from Sigma Chemical Co. (St. Louis, MO). Iodination of IGF-I,
IGF-II, IGFBP-3, and IGFBP-4 was performed by a modification of the
chloramine-T technique, to a specific activity between 350–500
µCi/µg (IGF-I and IGF-II) or of 100 µCi/µg (IGFBP-3 and
IGFBP-4).
Cell culture
SKHEP-1 human liver adenocarcinoma cells (American Type Culture
Collection, Rockville, MD) were maintained in MEM supplemented with
Earle’s salts, L-glutamine, and 10% FBS. Cells were grown
until 80% confluent in medium containing 10% FBS, and then switched
for 12 h to serum-free MEM. Medium was aspirated, and cells were
cultured in serum-free MEM with or without various hormones, as
described in the text. Conditioned media (CM) were collected and
centrifuged at 1000 x g for 10 min to remove cell
debris. The harvested CM from triplicate wells within each experiment
were pooled and stored at -70 C until assay.
RIA for IGF-I
Aliquots of CM were concentrated and subjected to acidic
size-exclusion chromatography, followed by a specific and sensitive
double antibody RIA (13). All samples were measured in duplicate. The
intraassay coefficient of variation was less than 10%, and the
interassay coefficient of variation was 14%. The lower limit of
detection was 0.1 ng/ml in CM.
Western ligand blots
IGFBPs in SKHEP-1 CM were analyzed by ligand blotting, as
described by Hossenlopp et al. (14). Samples were diluted
with nonreducing SDS-dissociation buffer (0.5 M Tris, pH
6.8; 69% glycerol; and 4% SDS), loaded onto a 1-mm discontinuous
SDS-polyacrylamide gel, and electrophoresed through a 12% gel
overnight. Electrotransfer of the proteins from the gels to 0.45-µm
pore nitrocellulose (Schleicher and Schuell, Keene, NH) was performed
using a Hoefer Semi-Dry Transphor unit (San Francisco, CA).
Nitrocellulose filters were blocked with 1% BSA for 2 h and
incubated with a combination of 125I-radiolabeled IGF-I and
IGF-II (1 x 106 cpm each) in Tris-buffered saline
overnight at 4 C. Nitrocellulose filters were exposed to x-ray film
(Kodak X-Omat AR, Eastman Kodak Co., Rochester, NY) in the presence of
Cornex Hi-Plus Intensifying Screens (DuPont, Wilmington, DE) for 3–7
days at -70 C. The density of the bands was assessed by an LKB
densitometer (LKB, Rockville, MD).
IGFBP-3 and IGFBP-4 protease assay
IGFBP-3 and IGFBP-4 protease activities were measured by
incubating 30,000 cpm [125I]IGFBP-3E.
coli or [125I]IGFBP-4yeast
with 100 µl CM (15). As protease controls, 2 µl pooled pregnant or
nonpregnant human sera or 100 µg/ml PSA were also tested. Samples
were subjected to 10% SDS-PAGE, and the gels were vacuum-dried and
exposed for autoradiography for 3–7 days.
Glycosylation studies
Proteins were deglycosylated with endoglycosidase F (Endo F;
Calbiochem-Novabiochem International, La Jolla, CA), as previously
described (16). For each sample, 100 µl CM or 2 µl human serum were
heated at 95 C for 2 min. After cooling to 22 C, 300 mU Endo F were
added, and the pH was adjusted to 5.0. Samples were incubated at 37 C
for 5 h. Reactions were terminated by the addition of
electrophoresis buffer, followed by boiling for 2 min. Western ligand
blotting of the samples was then performed, as described above.
IGFBP-3 immunoblots
Immunoblots were performed, using Amersham’s enhanced
chemiluminescence system (Little Chalfont, UK) and IGFBP-3 antibody
(12). Proteins were separated by 12% SDS-PAGE. After transfer,
nitrocellulose membranes were blocked by 2% BSA (RIA grade) overnight
at 4 C. First antibody was added (1:500 dilution) and incubated
overnight. After washing twice for 10 min each time in 0.1% Tween-20,
the second antibody, a goat antirabbit IgG-horseradish peroxidase
conjugate (Bio-Rad, Richmond, CA; dilution, 1:5000) was incubated for
2 h and subsequently exposed to Amersham enhanced
chemiluminescence reagents according to the manufacturer’s
protocol.
Northern blots
A modification of the guanidinium isothiocyanate precipitation
method (17) was used to isolate total RNA from cells. RNA samples were
size-fractionated on 1.2% agarose-formaldehyde gels, transferred to
nitrocellulose, and hybridized with specific probes. The following cDNA
clones were prepared for use as [32P]deoxy-CTP-labeled
(Prime-It, Stratagene, La Jolla, CA) cDNA probes for Northern blot
analysis: a 1082-bp EcoRI-PvuII fragment of the
human IGFBP-3 cDNA, a 440-bp SmaI fragment of the human
IGFBP-4 cDNA, a 376-bp Sau3A-EcoRI fragment of the rat IGF-I
cDNA, and a 570-bp PstI-BamHI human IGF-II
restriction fragment as a probe for IGF-II. An 18S ribosomal RNA probe
(Arrbion, Austin, TX) was used on each Northern blot as an internal
control. Bands were visualized by autoradiography after exposure to XAR
film with intensifying screens at -70 C. RNA ladder size markers (BRL,
Grand Island, NY) were used to obtain size estimates of the specific
transcripts. Band densities were analyzed using the area under the
curve, as calculated by LKB densitometer. The relative densities of the
bands were expressed as absorption per mm.
Effects of GH and IGF-I on IGFBP-3 messenger RNA (mRNA) decay (DRB
treatment)
The RNA polymerase II inhibitor, DRB (18), was used for
estimation of the half-life of IGFBP-3 mRNA. At time zero, cells were
extensively washed, and the culture medium was replaced with fresh
serum-free medium containing DRB (25 µg/ml) in the presence or
absence of 10 ng/ml GH or 100 ng/ml IGF-I. RNA isolated from control
and GH- and IGF-I-treated cultures at the indicated times was subjected
to Northern blot analysis and hybridized with a 32P-labeled
human IGFBP-3 cDNA probe, visualized by autoradiography, and
quantitated by densitometry, as described above.
Reverse transcriptase-PCR (RT-PCR)
The reverse transcription system (Promega, Madison, WI) was used
for reverse transcribing 1 µg total RNA to 20 µg cDNA. For each
gene, 2.5 µg cDNA were amplified for 35 cycles with the appropriate
set of primers (Table 1
) using Tag polymerase (Promega)
according to the manufacturer’s protocol. Products were visualized on
a 2% ethidium bromide-stained agarose gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The predominant 39-
to 41-kDa band was recognized on immunoblot by an IGFBP-3 antibody
(data not shown), and additionally, deglycosylation with Endo F yielded
a 29-kDa protein, consistent with IGFBP-3. The 32-kDa was proved to be
IGFBP-2 by immunoblotting with IGFBP-2 antibody, HEC-1 (data not
shown); RT-PCR demonstrated that SKHEP-1 cells express IGFBP-1, -2, -3,
-4, and -5, mRNAs (Fig. 2). In the present study,
regulation of the predominant binding protein (IGFBP-3) by GH and IGF-I
was investigated further.
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IGFBP-3 protein levels in CM were increased, in a dose-dependent
manner, by more than 3-fold. Increases in IGFBP-3 concentrations were
observed at GH concentrations as low as 0.1 ng/ml. Treatment with IGF-I
or Des(1, 2, 3)-IGF-I also increased basal IGFBP-3 protein levels in CM in
a dose-dependent manner up to more than 3-fold (Fig. 1, B and C).
Interestingly, all three agents concurrently decreased the 24-kDa
IGFBP-4 band in a dose-dependent manner. Higher concentrations [10
ng/ml GH or 100 ng/ml IGF-I or Des(1, 2, 3)-IGF-I] resulted in complete
loss of IGFBP-4 from CM. Furthermore, the 32-kDa IGFBP-2 increased in
intensity with treatment with 1 ng/ml GH or 10 ng/ml IGF-I or
Des(1, 2, 3)-IGF-I, whereas the 28-kDa IGFBP species significantly
decreased in intensity with treatment with either IGF-I or
Des(1, 2, 3)-IGF-I, but not with GH treatment. As it has been demonstrated that specific proteases are important regulators of IGFBP-3 and IGFBP-4 levels (15, 19, 20), CM from SKHEP-1 cells were tested for IGFBP-3 and IGFBP-4 protease activities (15). In this cell system, no IGFBP-3 or IGFBP-4 protease activity was detectable in CM from control cells or cells treated with GH, IGF-I, or Des(1, 2, 3)-IGF-I (data not shown).
A time-course (days 1–5) experiment was performed for untreated as
well as GH- or IGF-I-treated cells (Fig. 3). The GH (10
ng/ml)-induced increase in IGFBP-3 concentrations in CM was more
pronounced on the first day than the increase produced with IGF-I
treatment (100 ng/ml; P < 0.05 in the densitometric
analysis of the three experiments performed). Both GH and IGF-I
increased IGFBP-3 concentrations on days 3 and 5.
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; P < 0.05 in the densitometric
analysis of the three experiments performed), whereas IGF-I increased
IGFBP-3 mRNA after 24 h (Fig. 4B). GH and IGF-I treatment resulted
in no change in IGFBP-4 mRNA at 6 h, although a significant
decrease in IGFBP-4 mRNA was found with either treatment at 24 and
48 h. The pattern of changes was similar at 24 and 48 h
(Fig. 4C).
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14–18 h. Moreover, it
was not changed by treatment with either 10 ng/ml GH or 100 ng/ml IGF-I
(Fig. 5). The DRB experiment thus demonstrates that both
GH and IGF-I transcriptionally regulate IGFBP-3 mRNA, raising the
question of whether GH’s effect is IGF mediated or IGF
independent.
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To further test our hypothesis that GH is acting in an IGF-independent
manner, we investigated whether GH stimulates IGF-I synthesis in
SKHEP-1 cells by 1) RIA, 2) Northern blotting, and 3) RT-PCR. Using a
specific RIA to measure IGF-I concentrations in SKHEP-1 CM after
removal of IGFBPs by acid chromatography, no immunoreactive IGF-I was
found in the CM of control or GH-treated cells. Furthermore, on
Northern blot analysis, no IGF-I mRNA was detectable in control or
GH-treated SKHEP-1 cells (data not shown). In addition, IGF-I mRNA
expression could not be detected using RT-PCR (Fig. 6).
Although IGF-II mRNA was detected by RT-PCR, IGF-II mRNA did not change
under GH-stimulated conditions on Northern blots (data not shown),
making the possibility that IGF-II mediates the GH effect on IGFBP-3
unlikely. As no immunoreactive IGF-I was present (by RIA), and no IGF-I
mRNA was detected by Northern blots or RT-PCR in SKHEP-1 cells, it
appears that GH regulates IGFBP-3 at least partly by IGF-I-independent
mechanisms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I and/or GH have been well documented to increase IGFBP-3 levels in vitro (21, 22, 23, 24, 25, 26, 27, 28, 29). Interestingly, using cultured osteoblastic cells, Ernst and Rodan (30) found that IGFBP-3 was markedly stimulated by GH and only minimally stimulated by IGF-I. The stimulation of IGFBP-3 by GH, but not by IGF-I, in that study as well as GH stimulation of IGFBP-3 in the absence of detectable IGF-I in human osteoblasts (31) are consistent with our results, in which GH stimulates IGFBP-3 mRNA and protein, whereas IGF-I expression by SKHEP-1 cells was absent. Furthermore, in support of our hypothesis of an IGF-independent GH regulation of IGFBP-3, the GH signal transduction pathway has been found to be not exclusively coupled to IGF-I gene expression; in human IM-9 lymphocyte cells, treatment with 200 ng/ml GH induced tyrosine phosphorylation of 93-, 120-, and 134-kDa proteins, but did not alter IGF-I gene expression (32).
On the other hand, several animal studies have suggested that IGF-I, rather than GH, is the primary regulator of IGFBP-3 in the rat. Serum IGFBP-3 concentrations are decreased in hypophysectomized (9, 10, 11) and GH-deficient transgenic mice (33) and are restored after GH replacement or IGF-I infusion (9, 10, 11). Additionally, overexpression of IGF-I in GH-deficient transgenic mice (33) restored IGFBP-3 levels. In the neonatal hypophysectomized rat, administration of IGF-I, but not GH, increased serum concentrations of IGFBP-3, whereas IGFs, in contrast to GH, resulted in only modest body weight gain (11). In a spontaneous dwarf rat model that lacks only GH and not other pituitary hormones, continuous GH administration induced all three components of the 150-kDa complex, whereas administration of IGF-I restored only IGFBP-3, suggesting that ALS and IGF-I are directly GH dependent, whereas IGFBP-3 is regulated by IGF-I (34).
However, several animal studies also point toward direct, IGF-independent IGFBP-3 regulation by GH. Albiston and Herington (35) reported that hypophysectomy decreased IGFBP-3 mRNA levels in liver by 50%, suggesting that hepatic IGFBP-3 mRNA was partially GH dependent. Moreover, in hypophysectomized rats, GH induced only the ß-subunit (IGFBP-3) and not the high mol wt complex (9), suggesting that GH may regulate IGFBP-3 specifically. In addition, hepatic IGFBP-3 mRNA levels were found to be reduced in hypophysectomized rats, even after IGF-I treatment (36).
In clinical studies, evidence has emerged that IGFBP-3 concentrations are markedly age dependent (37, 38), influenced by nutrition (39), and firmly linked to GH status (decreased in GHD and increased in acromegaly). Nevertheless, careful clinical studies indicate IGF-independent GH regulation of IGFBP-3. In normal human subjects, small increases in the 150-kDa complex are induced after GH infusion and in the 50-kDa region after IGF-I infusion, suggesting that ALS is not induced by IGF-I (40). Moreover, IGFBP-3 serum levels declined after IGF-I administration in normal subjects, presumably reflecting IGF-I negative feedback on pituitary GH secretion (41).
The strongest clinical evidence for direct GH regulation of IGFBP-3 has been found in studies of IGF-I treatment of GHI patients (42). Treatment of GHI with IGF-I failed to increase radioimmunoassayable IGFBP-3 levels (43, 44). Several mechanisms to explain this failure were proposed: 1) GH itself directly regulates IGFBP-3 synthesis; 2) GH stimulation of the ALS is necessary to maintain normal serum concentrations of IGF-induced IGFBP-3; and finally, 3) GH stimulates IGF-I production through an autocrine or a paracrine mechanism, which is not reflected by an increase in serum IGF levels, and local production of IGF-I then leads to IGFBP-3 synthesis.
The data from the SKHEP-1 cell line are best explained by an ability of both IGF-I and GH to independently regulate IGFBP-3 gene transcription. Time-course studies at both the mRNA and protein levels are consistent with an IGF-independent regulation by GH, and these findings are further supported by the ability of picomolar concentrations of GH to stimulate IGFBP-3 production. IGF-I gene expression in SKHEP-1 cells was undetectable by both Northern analysis and RT-PCR, and radioimmunoassayable IGF-I was below the limits of detection. In a cell system where GH regulates neither IGF-I nor ALS, it is of interest that it should directly modulate IGFBP-3. Further studies of the IGFBP-3 promoter should lead to a better understanding of the regulation of this critical binding protein and help explain why dual control of IGFBP-3 gene expression is physiologically important.
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Footnotes |
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2 Supported by a scholarship from Fondo de Investigacion Sanitaria
(94/5373, Spain). Present address: Unidad de Endocrinologia, Hospital
Infantil Miguel Servet, Avda. Isabel la Catolica 1–3, Zaragoza 50009,
Spain.
Received September 12, 1996.
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