Growth Hormone Stimulates Transcription of the Gene Encoding the Acid-Labile Subunit (ALS) of the Circulating Insulin-Like Growth Factor-Binding Protein Complex and ALS Promoter Activity in Rat Liver
Guck T. Ooi,
Fredric J. Cohen1,
Lucy Y.-H. Tseng,
Matthew M. Rechler and
Yves R. Boisclair
Growth and Development Section, Molecular and Cellular
Endocrinology Branch, National Institute of Diabetes and Digestive
and Kidney Diseases, National Institutes of Health (G.T.O., F.J.C.,
L.Y.-H.T., M.M.R.), Bethesda, Maryland 20892,
Department of
Animal Sciences, Cornell University (Y.R.B.), Ithaca, New York
14853
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ABSTRACT
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The growth-promoting activity of GH, the principal
hormonal determinant of body size, is mediated by insulin-like growth
factor I (IGF-I). Most of the IGF-I in plasma circulates in a 150-kDa
complex that contains IGF-binding protein-3 (IGFBP-3) and an
acid-labile subunit (ALS). The 150-kDa complex serves as a reservoir of
IGF-I and determines its bioavailability to the tissues. Formation of
the 150-kDa complex depends upon the synthesis of ALS, which is
synthesized primarily in liver and is regulated by GH. The present
study demonstrates that GH stimulates ALS gene transcription in rat
liver and ALS promoter activity in a rat hepatoma cell line. ALS
messenger RNA (mRNA) and ALS nuclear transcripts were decreased to
similar extents in the livers of GH-deficient hypophysectomized rats.
GH increased hepatic ALS mRNA within 34 h to about 65% of the levels
seen in sham-operated control rats. To confirm that GH stimulated ALS
gene transcription, we transiently transfected an ALS
promoter-luciferase reporter gene construct into H4-II-E rat hepatoma
cells and primary rat hepatocytes. Recombinant human GH (hGH)
stimulated promoter activity about 3-fold. In contrast, basal promoter
activity was lower, and GH stimulation was absent when the ALS reporter
construct was transfected into GH-responsive 3T3-F442A mouse
preadipocyte fibroblasts. GH stimulation of ALS promoter activity in
H4-II-E cells was mediated by functional GH receptors; nonprimate (rat
and bovine) GH gave identical stimulation to hGH, and stimulation by
hGH occurred at physiological concentrations. Reverse transcriptase-PCR
analysis indicated that GH receptor mRNA was present in H4-II-E cells
at approximately 40% of the level seen in rat liver. GH also induced
the expression of the endogenous c-fos gene, indicating
that the signaling pathway necessary for the activation of gene
expression by GH was intact in H4-II-E cells. Thus, H4-II-E cells are a
GH-responsive liver cell line that should provide a useful system in
which to study the molecular mechanism of transcriptional regulation by
GH of ALS and other hepatic genes.
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INTRODUCTION
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GH is the hormone that is primarily responsible for determining
body size (1). In patients with GH insensitivity (2, 3) and in
GH-deficient hypophysectomized (hypox) rats and transgenic mice, growth
can be restored by treatment with insulin-like growth factor I (IGF-I),
the main mediator of the growth-promoting effects of GH. IGF-I, a
polypeptide chemically related to insulin, is expressed in liver and
other tissues and occurs in plasma and tissue fluids complexed to
members of a family of six IGF-binding proteins (IGFBPs) (4). In
plasma, more than 75% of the circulating IGFs are present as a
150-kilodalton (kDa) complex that is composed of IGFBP-3, the
predominant IGFBP in plasma, and an 85-kDa acid-labile subunit (ALS), a
glycoprotein that binds IGFBP-3 but does not bind IGFs directly (4, 5, 6, 7).
Plasma also contains lower molecular mass (
50 kDa) IGFBP:IGF
complexes. Unlike the approximately 50-kDa complexes, which can cross
the endothelial barrier and reach the tissues, 150-kDa complexes are
sequestered in the vascular compartment, allowing IGFs to be stored at
high concentration in plasma without the risk of potentially
deleterious hypoglycemic effects that might result from their intrinsic
insulin-like activity (8, 9, 10, 11, 12).
Formation of the 150-kDa complex critically depends on the synthesis of
ALS, as the 150-kDa complex and ALS are both found almost exclusively
in plasma, whereas IGFBP-3 is distributed ubiquitously (4). The level
of ALS in plasma, like the IGF concentration, is regulated directly by
GH and unaffected by IGF-I (13, 14). GH regulation occurs at the level
of ALS mRNA abundance in liver, the principal site of ALS synthesis
(15, 16). ALS mRNA is decreased in hypox rat liver and is partially
restored after GH treatment (15). GH also increased ALS mRNA in rat
hepatocytes (17) and rapidly induced IGF-I gene transcription in hypox
rat liver (18). Thus, like IGF-I, ALS is an important physiological end
point for GH receptor signaling, whose regulation is likely to occur at
the level of gene expression.
In the present study, we demonstrate that ALS nuclear transcripts and
ALS mRNA are decreased proportionately in hypox rat liver, and that
hepatic ALS mRNA is rapidly increased after GH treatment. Using
isolated primary rat hepatocytes or a GH-responsive rat hepatoma cell
line transfected with reporter constructs whose expression is driven by
the mouse ALS promoter, we demonstrate that GH stimulates ALS promoter
activity, and that this regulation is mediated by functional GH
receptors.
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RESULTS
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Recombinant Human GH (rhGH) Regulates ALS mRNA and ALS Nuclear
Transcripts in Rat Liver
The relative abundance of ALS mRNA in the livers of sham-operated
control rats, hypox rats, and hypox rats at different times after
treatment with rhGH was determined by Northern blotting of total RNA
(Fig. 1
). A complementary DNA (cDNA) probe corresponding
to the 3'-coding region of rat ALS mRNA hybridized to a single band of
approximately 2.2 kilobases (kb), the size of full-length rat ALS mRNA
(19). Hepatic ALS mRNA abundance was decreased by about 90% in rats
made GH deficient by hypophysectomy (Fig. 1
).

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Figure 1. Effects of Hypophysectomy and GH Replacement on
Hepatic Rat ALS mRNA
Total RNA was prepared from the livers of hypox and sham-operated
(control) rats (Charles Rivers Laboratories) and from hypox rats at
different times after a single intraperitoneal injection of rhGH (1.5
mg/kg). Total RNA was analyzed by Northern blotting using a rat ALS
cDNA probe. Top, Autoradiograph of Northern blot showing
hybridization of the rat ALS cDNA probe to 2.2-kb ALS mRNA, the only
positive signal observed. For clarity, a single representative sample
is shown for each experimental condition. Bottom,
Quantification of the abundance of ALS mRNA. Samples from two
experiments are combined. They include 11 control (Cont), nine hypox,
and the indicated number (parentheses) of hypox rats treated with GH
for 1 h (n = 3), 3 h (n = 3), 4 h (n =
4), 6 h (n = 8), 8 h (n = 7), or 24 h (n
= 5). Specific hybridized radioactivity was determined for each sample
by ß-scanning. Results from individual blots in each experiment were
normalized to a set of three reference RNAs from control rat liver run
on each gel. The relative abundance of ALS mRNA is expressed as a
percentage of the sham-operated control values. The mean ±
SEM are shown. *, ALS mRNA levels in livers of hypox rats
4, 6, and 8 h after treatment were significantly different from
the levels in untreated hypox rats and from the levels in hypox rats 1,
3, and 24 h after GH treatment (P < 0.04, by
Scheffes test).
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After a single intraperitoneal injection of hypox rats with rhGH,
hepatic ALS mRNA increased more than 7-fold (to
65% of the levels
in sham-operated control rats) 8 h postinjection (Fig. 1
). The
increase was observed between 34 h after injection and reached a
plateau from 48 h before returning to hypox levels after 24
h.
To determine whether changes in transcription were responsible for the
decrease in steady state abundance of ALS mRNA in hypox rat liver, the
abundance of intron-containing (nascent) ALS transcripts was analyzed
in nuclear RNA prepared from sham-operated and hypox rats. Nascent ALS
transcripts were amplified using the RT-PCR and analyzed by Southern
blotting using a probe specific for the single intron of the rat ALS
gene. An approximately 1.2-kb band corresponding to the nuclear ALS
transcript was readily detected in control rats (Fig. 2
). Its abundance was decreased by about 65% in hypox
rats (Fig. 2
). Northern analysis of total RNA from the livers of the
same animals showed that the steady state abundance of ALS mRNA was
decreased to the same extent in hypox rat liver, suggesting that the
decrease in hepatic ALS mRNA abundance resulted from changes in ALS
gene transcription.

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Figure 2. Rat ALS Nuclear Transcripts Are Decreased in Hypox
Rat Liver
Top, Total RNA was extracted from nuclei prepared from
the livers of three sham-operated rats (lanes 13) and three hypox
rats (lanes 46). After DNase I digestion, the nuclear RNA was reverse
transcribed and amplified using rat ALS primers bracketing the unique
1.1-kb intron. RT-PCR reactions were analyzed by Southern blotting
using a rat ALS intron probe. The predominant, approximately 1.2-kb
product (arrow) agrees with the predicted size (1.1-kb
intron and 0.1-kb cDNA overlap). No hybridization was observed when
AMV-RT was omitted from the RT-PCR reaction (not shown). Bottom
left, Radioactivity hybridized to the approximately 1.2-kb DNA
fragment in the Southern blot (top panel) was
quantitated by ß-scanning. The mean ± SD of the
hybridized counts are plotted. Bottom right, For
comparison, total RNA from matched rat liver samples was analyzed by
Northern blotting using the DNA probe corresponding to nt 13621655 of
the rat ALS cDNA. The mean ± SD of the radioactivity
hybridized to the 2.2-kb rat ALS mRNA are shown. Hypox animals used in
this experiment were obtained from Zivic-Miller and showed only a 65%
decrease in hepatic ALS mRNA compared with sham-operated animals.
Black bars, Sham-operated; hatched bars,
hypox.
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Recombinant hGH Stimulates ALS Promoter Activity in H4-II-E Rat
Hepatoma Cells
To confirm that GH stimulated ALS gene transcription, we next
sought to identify a cell line that could be used to study the
regulation of ALS promoter activity by GH. We compared two established
cell lines [H4-II-E rat hepatoma cells, which synthesize many liver
proteins (20), and GH-responsive 3T3-F442A mouse preadipocyte
fibroblasts (21, 22)] as well as isolated primary rat hepatocytes for
their ability to express a transiently transfected luciferase minigene
driven by a 1953-bp 5'-flanking sequence of the mouse ALS gene (23).
The promoter fragment was ligated in either the sense or reverse
orientation with respect to the luciferase-coding sequence.
In H4-II-E cells transfected with the construct containing the ALS
promoter fragment in the sense orientation and examined in the absence
of GH, promoter activity was about 2-fold higher than that observed in
cells transfected with the reverse orientation construct (Fig. 3A
, left). When cells transfected with the
sense construct were incubated with rhGH (100 ng/ml; 16 h),
promoter activity was increased more than 3-fold (Fig. 3A
, left). Only a small, statistically insignificant
(P > 0.05, by Scheffes test) increase was seen after
rhGH treatment of cells transfected with the reverse orientation
construct, indicating that GH stimulation of rat ALS promoter activity
in H4-II-E cells was orientation dependent. Similar results were
obtained in transfection studies using isolated primary rat
hepatocytes. Promoter activity in cells transfected with the sense
construct was increased more than 4-fold (Fig. 3B
) after rhGH
treatment.

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Figure 3. Recombinant hGH Stimulates ALS Promoter Activity in
Primary Rat Hepatocytes and H4-II-E Rat Hepatoma Cells, but not in
3T3-F442A Mouse Fibroblasts
A, A 1953-bp 5'-flanking sequence from the mouse ALS gene (nt -2001 to
-49) was ligated upstream of a promoterless luciferase gene in plasmid
pGL3 in the sense and reverse orientation. The chimeric plasmids were
transfected into H4-II-E rat hepatoma cells (left panel)
or 3T3-F442A mouse preadipocyte fibroblasts (right
panel) using DEAE-dextran. The transfected cells were
preincubated in serum-containing medium for 1824 h, followed by a
16-h incubation in serum-free medium in either the presence
(black bars) or absence (hatched bars) of
100 ng/ml rhGH. The cells were lysed, and luciferase activity in the
lysates was measured using an automated luminometer. Results are
expressed as relative light units and have been normalized against
ß-galactosidase activity to correct for any differences in
transfection efficiency. The mean ± SD of duplicate
transfections from a single experiment are shown. Similar results were
obtained in four other experiments. B, For comparison, the sense
construct also was transfected into primary rat hepatocytes using
Lipofectin. After 14-h transfection, the cells were refreshed with
serum-free medium in either the presence or absence of 100 ng/ml rhGH.
The medium was replaced every 24 h until the cells were harvested,
and luciferase activity was measured 48 h after transfection. *
and **, Promoter activity of the sense construct after GH treatment was
significantly higher than that in non-GH-treated cells
(P < 0.05, by Scheffes test and t
test, respectively).
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By contrast, when the same constructs were transfected into the
GH-responsive 3T3-F442A mouse fibroblasts, basal promoter activity was
low and did not increase after rhGH treatment (Fig. 3A
, right). The low basal luciferase activity in 3T3-F442A cells
did not result from low transfection efficiency, as ß-galactosidase
activity encoded by the cotransfected plasmid, pSV-ß-galactosidase,
was readily detected in the same cells (results not shown). These
results indicate that H4-II-E cells retain the necessary transcription
factors and signaling pathways of normal liver that account for the
predominant expression of the ALS gene in liver and the stimulation of
hepatic ALS gene transcription by GH in vivo.
Recombinant hGH Stimulates ALS Promoter Activity through GH
Receptors at Physiological Concentrations
Human GH can bind to GH receptors (eliciting a somatogenic
response) and to the related PRL receptors (eliciting a lactogenic
response), unlike nonprimate GH molecules, which only bind to GH
receptors (24). To demonstrate that rhGH activated the ALS promoter by
binding to GH receptors rather than PRL receptors, the ability of
nonprimate GH to stimulate ALS promoter activity was examined. H4-II-E
cells that had been transfected with ALS promoter-luciferase constructs
were incubated with 200 ng/ml of purified rat GH or recombinant bovine
GH (Fig. 4
, left). ALS promoter activity was
induced to the same extent by rat or bovine GH as by rhGH, establishing
that GH stimulated ALS promoter activity via the GH receptor.

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Figure 4. GH Stimulates ALS Promoter Activity in H4-II-E
Cells through GH Receptors in a Dose-Dependent Manner
H4-II-E cells were transiently transfected with the ALS promoter
constructs and treated for 18 h with either 200 ng/ml of GH from
different species (left panel) or varying concentrations
of rhGH (right panel). Luciferase activity was measured
in the cell lysates and normalized against ß-galactosidase activity
to correct for variations in transfection efficiency.
Left, Stimulation of ALS promoter activity by hGH,
recombinant bovine GH (bGH), or rat GH (rGH). The fold stimulation
(with GH/without GH; mean ± SD) of duplicate
transfections is plotted. Right, Dose-dependent
stimulation of ALS promoter activity by different concentrations of
rhGH. Results are expressed as relative light units. The mean ±
SD of quadruplicate transfections are
shown.
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The stimulation of ALS promoter activity by rhGH was dose dependent
(Fig. 4
, right) and occurred at concentrations within the
range of GH levels seen in vivo in the rat (25). Stimulation
was detected at 1 ng/ml, with half-maximal stimulation at 10 ng/ml and
maximal stimulation at 100 ng/ml.
Recombinant hGH Stimulates Expression of the Endogenous
c-fos Gene in H4-II-E Cells
Although rhGH can activate the ALS promoter in transient
transfection assays in H4-II-E cells and primary rat hepatocytes, ALS
mRNA was not detected in H4-II-E cells by Northern analysis (our
unpublished results), suggesting that the endogenous ALS gene might not
be expressed. To establish that the H4-II-E cell line is a valid system
to study the mechanism underlying the activation of transcription by
GH, we determined whether rhGH stimulated expression of the endogenous
c-fos gene. It previously had been shown that GH increased
c-fos transcription and steady state c-fos mRNA
levels in rat liver (26) and in the GH-responsive 3T3-F442A
preadipocyte fibroblast cell line (27, 28). Confluent H4-II-E cells
were incubated with 100 or 400 ng/ml rhGH for 16 h, after which
total RNA was prepared and analyzed by Northern blot hybridization
using a c-fos cDNA probe (Fig. 5
).
Recombinant hGH treatment stimulated c-fos mRNA levels
approximately 4-fold. The stimulation was dose dependent. These results
confirmed that the GH receptors are functional, and that the H4-II-E
cell line contains all of the components of the signal transduction
machinery necessary to mediate GH-dependent activation of the
endogenous c-fos gene.

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Figure 5. Induction of c-fos mRNA in H4-II-E Cells
Treated with rhGH
Confluent H4-II-E cells were incubated in serum-free medium for
1824 h, after which the cells were treated for 16 h with either
100 or 400 ng/ml rhGH. Total RNA was analyzed by Northern blotting
analysis using a c-fos cDNA probe. Radioactivity hybridized
to approximately 2.2-kb c-fos mRNA was quantitated by
densitometric scanning using a NIH Image program. Results plotted are
the mean ± SD of triplicate (control) or
quadruplicate (100 and 400 ng/ml) samples.
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GH Receptor mRNA Is Present in H4-II-E Cells
Few established cell lines contain GH receptors at high enough
concentrations to provide a suitable GH-responsive model for in
vitro studies (29). The abundance of GH receptors in H4-II-E
cells, however, appears to be sufficient for rhGH to activate ALS
promoter activity or stimulate endogenous c-fos mRNA. To
demonstrate this directly, we used a semiquantitative RT-PCR assay to
compare levels of GH receptor mRNA in H4-II-E cells to the levels
present in adult rat liver, one of the tissues with the highest
expression of GH receptor mRNA (30). After reverse transcription of
total RNA (0.1 and 1 µg), a 533-bp DNA fragment [corresponding to
nucleotides (nt) 963 to 1496] of rat GH receptor cDNA was amplified by
PCR and detected by Southern blotting with a nested
-32P-labeled oligonucleotide probe (nt 1368 to 1399 of
the rat GH receptor). The amplified rat GH receptor cDNA fragment was
easily detected in both H4-II-E cells and rat liver (Fig. 6
). This DNA fragment was not detected when AMV-RT was
omitted from the RT reaction (not shown), indicating that it arose by
amplification of GH receptor cDNA rather than contaminating genomic
DNA. The abundance of GH receptor mRNA in H4-II-E cells was estimated
as
40% of that found in rat liver.

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Figure 6. GH Receptor mRNA is Present in H4-II-E Cells and
Rat Liver
Total RNA extracted from H4-II-E cells (lanes 1 and 2) and from normal
adult rat liver (lanes 3 and 4) was treated with DNase I to remove
contaminating DNA, and 0.1 µg (lanes 1 and 3) or 1 µg (lanes 2 and
4) was reverse transcribed and amplified by PCR using primers specific
for the rat GH receptor. Amplified samples were electrophoresed on an
agarose gel and analyzed by Southern blotting using a nested
oligonucleotide probe. Hybridization to the 533-bp hybridized GH
receptor DNA is shown and is quantitated by ß-scanning.
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DISCUSSION
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ALS is the critical component in the formation of the 150-kDa
IGF:IGFBP-3 complex that transports most of the IGF in the circulation
and determines its bioavailability to tissues. Plasma ALS is
principally regulated by GH, and GH regulates ALS mRNA levels in liver,
the major site of ALS synthesis (15, 16). In the present study, we
demonstrate that this regulation is due to the transcriptional
activation of the ALS gene by GH and describe a rat hepatoma cell line
in which GH regulation of the ALS gene can be studied.
After hypophysectomy, ALS mRNA and ALS nuclear transcripts in rat
liver were decreased to similar extents in hypox rat liver, suggesting
that decreased transcription was responsible for the decreased steady
state abundance of ALS mRNA. Treatment of hypox rats with a single
intraperitoneal injection of GH increased ALS mRNA between 34 h to
near-maximal levels, approximately 7-fold greater than that in
untreated hypox rat liver. The kinetics of induction of ALS mRNA in
response to GH are similar to those reported for mRNAs encoded by other
hepatic genes that are regulated by GH, such as IGF-I (18) and serine
protease inhibitor (Spi) 2.1 (31). Systemic administration of GH to
hypox rats increased the levels of hepatic IGF-I and Spi 2.1 mRNAs
within 2 h.
GH regulation of ALS transcription was substantiated by the
demonstration that rhGH stimulated the expression of luciferase
activity in H4-II-E cells transfected with a construct in which a
promoterless luciferase gene was ligated to an approximately 2-kb
5'-flanking sequence of the mouse ALS gene containing the promoter.
GH-stimulated ALS promoter activity was dependent on the orientation of
the promoter relative to the luciferase-coding region, occurring only
when the promoter was in the sense orientation relative to the
luciferase-coding sequence. GH-dependent ALS promoter activation also
was observed in primary hepatocytes. GH stimulation of ALS promoter
activity was cell specific, being observed in H4-II-E cells but not in
3T3-F442A mouse preadipocyte fibroblasts, consistent with the
predominant localization of ALS gene expression to liver observed
in vivo (15). Basal promoter activity also was higher in
H4-II-E cells than in 3T3-F442A fibroblasts. These results indicate
that the approximately 2-kb ALS promoter fragment contains the
necessary cis elements to confer liver-specific and
GH-dependent expression, and that H4-II-E cells express the
necessary transcription factors to mediate these effects. One candidate
liver-specific transcription factor is hepatocyte nuclear factor-3
(HNF-3ß). Putative binding sites for HNF-3 are present in the mouse
ALS promoter (23), and HNF-3ß is present in H4-II-E nuclear extracts
(32).
GH stimulation of ALS promoter activity in H4-II-E rat hepatoma
cells is mediated by functional GH receptors and occurs at GH
concentrations within the physiological range (25). This finding is
important, in that there are currently few established cell lines
available that contain GH receptors at high enough concentrations to
provide a suitable GH-responsive model for in vitro studies
(29). Using RT-PCR, we demonstrated that H4-II-E cells contained
appreciable amounts of mRNA encoding the GH receptor (Fig. 6
). Its
abundance was estimated as about 40% that of GH receptor mRNA in rat
liver, one of the tissues in which GH receptor mRNA is most abundant
(30).
Stred et al. (33) reported that GH can bind to GH receptors
in H4-II-E cells and stimulate receptor phosphorylation, but did not
demonstrate that this GH receptor signaling was linked to a GH-specific
functional end point. The GH receptors in H4-II-E cells are functional,
being capable of signal transduction after GH binding. In transient
transfection assays, nonprimate GH-stimulated ALS promoter activity as
well as rhGH (Fig. 4
), a specificity indicating that GH stimulation was
mediated by GH receptors rather than PRL receptors (24). Although the
endogenous ALS gene does not appear to be expressed in H4-II-E
cells,1 GH activates the endogenous H4-II-E
c-fos gene, a gene whose expression previously was shown to
be GH dependent in 3T3-F442A preadipocytes (27) and rat liver (18, 26, 34). GH induced a dose-dependent increase in c-fos mRNA in
H4-II-E cells, indicating that the GH receptors in H4-II-E cells, when
activated by GH, are capable of downstream signaling events leading to
gene expression.
The availability of the GH-responsive, liver-derived H4-II-E cell
line provides an excellent opportunity to study the
cis-regulatory elements and the transcription factors
binding to them that mediate the GH regulation of genes that are
specifically expressed in liver. The fibroblast-derived 3T3-F442A cell
line, which has been used extensively to study the signaling pathways
for GH (35) and the elements in the c-fos gene that are
responsible for GH regulation (36, 37), does not express liver-specific
genes such as ALS (Fig. 3
) and IGF-I that are important for the
growth-promoting effects of GH. Strategies used by other investigators
to study GH regulation of the expression of hepatic genes have involved
the preparation of isolated primary hepatocytes in culture (38, 39) or
the creation of stable cell lines derived from liver that overexpress
GH receptors (29, 40). Compared with these systems, the H4-II-E cell
line has the advantages that it is readily propagated, and it retains
the ability to synthesize multiple plasma proteins and enzymes that are
produced by normal rat liver (41), indicating that the required
liver-specific transcription factors are present as well as the
components of the signal transduction pathway leading to
transcriptional activation of hepatic genes by GH.
The mechanisms by which GH activates the transcription of genes
that are regulated by GH are poorly understood. In part, this is
because only a limited number of genes, such as c-fos in
3T3-F442A fibroblasts and Spi 2.1 in hepatocytes, have been amenable to
study. For example, although IGF-I is the mediator of the
growth-promoting actions of GH, no continuous cell line has been
identified that expresses IGF-I. After binding of the ligand to the GH
receptor, the receptor dimerizes and binds the cytoplasmic tyrosine
kinase Janus kinase 2 (JAK2), whereupon tyrosine residues on both the
receptor and the kinase are tyrosine phosphorylated. This results in
the tyrosine phosphorylation of signaling molecules such as insulin
receptor substrate-1 and Src homology 2 domain protein, possibly
leading to the activation and nuclear translocation of
mitogen-activated protein kinase (35). In addition, several members of
the STAT (signal transduction and activators of transcription) family
of nuclear proteins also are phosphorylated on tyrosine residues,
leading to their dimerization and translocation to the nucleus
(42).
Studies of the c-fos and Spi 2.1 genes suggest that
transcriptional activation by GH may involve different signaling
pathways and different cis elements. In 3T3-F442A cells, the
serum response element of the c-fos gene is sufficient to
confer GH inducibility to a heterologous promoter (36). This may occur
through activation of the Ras-mitogen-activating protein kinase pathway
by GH, resulting in phosphorylation and activation of Elk-1 (43), a
transcription factor that forms a ternary complex with the serum
response factor and the serum response element (44). GH treatment of
3T3-F442A cells also induces tyrosine phosphorylation, dimerization,
and nuclear translocation of the transcription factors STAT1, -3, and
-5 (42). STAT1 and -3 can bind to a
-interferon activation sequence
(GAS) element (45) in the c-fos promoter (34, 46). This GAS
element is required in conjunction with several other elements for full
induction of the c-fos promoter by GH in Chinese hamster
ovary K1 cells (47). For the Spi 2.1 gene, transfection studies in
primary hepatocytes indicate that GH stimulation requires a 45-bp GH
response element that contains two GAS elements (31, 38). Three copies
of the proximal 3'-GAS element were sufficient to confer GH
responsiveness to a heterologous promoter (48), whereas both GAS
elements were required for full induction of the Spi 2.1 promoter by GH
(39). These GAS elements bind a nuclear factor that is activated after
GH treatment and is immunologically related to STAT5 (39, 49).
Our demonstration of GH-dependent regulation of ALS gene promoter
activity in the H4-II-E rat hepatoma cell line and in isolated primary
rat hepatocytes represents an important addition to the repertoire of
genes available to elucidate the mechanisms by which GH regulates gene
transcription. H4-II-E cells provide a convenient experimental system
to identify the cis elements and transcription factors
involved in GH-dependent expression of the ALS gene, a gene that
encodes a physiologically important end point of GH action, and to
trace the signal transduction pathway leading from activation of the GH
receptor to stimulation of ALS gene transcription.
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MATERIALS AND METHODS
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Materials
Recombinant hGH was provided by Genentech (South San Francisco,
CA); recombinant bovine GH was a gift from Monsanto (St. Louis, MO).
Highly purified rat GH (lot AFP-87401) prepared from frozen whole rat
pituitary glands was obtained from the NIDDK National Hormone and
Pituitary Program (Rockville, MD). It contains less than 0.1% (wt/wt)
contamination by other anterior pituitary hormones. BSA (RIA grade) and
luciferin were purchased from Sigma Chemical Co. (St. Louis, MO);
Lipofectamine, Lipofectin, DMEM, and Williams E medium were obtained
from Life Technologies (Gaithersburg, MD); and diethylaminoethyl
(DEAE)-dextran was purchased from Pharmacia Biotech (Uppsala, Sweden).
Oligonucleotides were synthesized using a model 393 DNA/RNA synthesizer
from Applied Biosystems (Foster City, CA). Radionucleotides were
purchased from Amersham (Arlington Heights, IL). PCR amplifications
were performed using a model 480A DNA Thermal Cycler
(Perkin-Elmer/Cetus, Norwalk, CT).
Experimental Animals
Male Sprague-Dawley rats (150 g, 12 weeks old) were
hypophysectomized or sham-operated by the supplier [Charles River
Laboratories (Wilmington, MA) or Zivic-Miller (Allentown, PA)]. They
were maintained in a temperature-controlled environment on a 12-h
light, 12-h dark cycle, and fed a diet of 1% dextrose in water and rat
chow ad libitum. Rats were considered successfully
hypophysectomized only if they gained less than 20% as much in body
weight as sham-operated animals during the 12-day postoperative
observation period and were used for experiments within the next 2
days.
Twelve days after hypophysectomy, animals were injected
intraperitoneally with a single dose of 1.5 mg/kg rhGH dissolved in
saline in a final volume of 200 µl or with an equal volume of
isotonic saline. The dosage of rhGH used is sufficient to achieve
transiently supraphysiological plasma GH concentrations (18, 25). Rats
were killed by decapitation after carbon dioxide sedation. Their livers
were frozen immediately in liquid nitrogen and stored at -70 C. The
protocols used were approved by the NIDDK animal use committee.
Isolation of Primary Hepatocytes
Primary hepatocytes were aseptically isolated from male
Sprague-Dawley rats (
250 g) by the collagenase perfusion method as
described previously (50).
Northern Blot Hybridization
Total liver RNA was prepared using the acid guanidinium
thiocyanate-phenol-chloroform method (51). The RNA (15 µg/lane) was
electrophoresed on 1.5% agarose-formaldehyde gels in
3-(N-morpholino)propanesulfonic acid buffer and blotted to
nylon membranes [GeneScreen, DuPont-New England Nuclear (Boston, MA)
or Nytran, Schleicher & Schuell (Keene, NH)] as described previously
(52). Ethidium bromide staining confirmed that the ribosomal RNAs were
intact and that sample loading was equal in each lane.
A 294-nt probe corresponding to nt 13621655 of the 3'-coding region
of rat ALS cDNA (GenBank accession no. S46785) was synthesized by
RT-PCR. Total RNA from adult rat liver was reverse transcribed using a
first strand cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA)
and random hexamer priming. A 20-µl aliquot of the reaction mixture
was amplified by PCR using the following primers: sense,
5'-GCATCTCCAGCATCGAAGAACAG-3'; and antisense,
5'-GCAAGGAGTTATTCCTGAGGCTG-3'.
Amplification conditions were 94 C for 1 min (denaturing), 50 C for 1
min (annealing), and 72 C for 1 min (elongation) for 30 cycles. The
resultant product was subcloned into the pCR II plasmid (Clontech) and
verified by EcoRI digestion and partial dideoxy chain
termination sequencing (53).
The rat ALS cDNA probe was labeled with
[
-32P]deoxy-CTP (3000 or 6000 Ci/mmol; Amersham,
Arlington Heights, IL) by random priming and hybridized to RNA as
previously described (52). The hybridization signal was quantitated
using a computer-driven ß-radioactivity scanner (Ambis Scanning
System II, Automated Microbiology Systems, San Diego, CA) operating
within the linear range of its detection ability. Background counts,
determined from an equal area of the blot, were subtracted.
Hybridization signals were normalized to the signals of reference
control RNA samples run on each blot when results from separate blots
were compared.
Quantification of Rat ALS Nuclear RNA
Nuclei were prepared from frozen livers of hypox and
sham-operated control rats as previously described (54, 55). Nuclear
RNA was extracted from the nuclei using the acid guanidinium
thiocyanate-phenol-chloroform method (51) and digested with
deoxyribonuclease I (DNase I) to remove any contaminating DNA. RNA was
quantitated by absorbance at 260 nm after phenol-chloroform extraction
and isopropanol precipitation.
Nascent rat ALS transcripts were amplified by a continuous RT-PCR
procedure (56), using avian myeloblastosis virus reverse transcriptase
(AMV-RT; Boehringer Mannheim, Indianapolis, IN) and the following rat
ALS-specific primer pairs: sense, 5'-CAAGGAACAATGGCCCTGAGGACAG-3'; and
antisense, 5'-CAGAAGCACCACCAGGGCTGG-3'. These primers correspond to nt
-9 to 16 and nt 22 to 42 (with respect to A+1TG) of rat
ALS cDNA, respectively, and bracket the single, approximately 1.1-kb
rat ALS intron that occurs at nt 16 (57). Each reaction tube contained
1 µg nuclear RNA, 310 nmol of each primer, 250 µM
deoxy-NTPs, 10 U AMV-RT, 10 U ribonuclease inhibitor (Boehringer
Mannheim), and 2.5 U Taq polymerase (Perkin-Elmer/Cetus,
Foster City, CA) in 100 µl PCR buffer (10 mM Tris-HCl, pH
8.3; 50 mM KCl; 1.5 mM MgCl2; and
0.01% gelatin). RT-PCR was performed at 65 C for 10 min (annealing),
at 50 C for 15 min (reverse transcription), and at 95 C for 5 min
(denaturation), followed by 36 cycles of 95 C for 1 min, 55 C for 2
min, and 72 C for 2 min. The elongation step of the last cycle was 7
min at 72 C to ensure full extension of DNA fragments. RT-PCR reactions
to which AMV-RT was not added were used as controls.
An aliquot (10 µl) of the amplified samples was electrophoresed on a
1.5% agarose-1 x TBE gel (1 x TBE = 89 mM
Tris-borate and 2 mM EDTA, pH 8.3), blotted, and analyzed
by Southern blotting using a DNA fragment corresponding to the
approximately 1.1-kb probe specific for the single intron of the rat
ALS gene. The cDNA probe was made by RT-PCR of total rat liver mRNA
using the following primer pairs: sense, 5'-
GCTGCCAGCTACAGGCAGTGGGGAAATCCA-3'; and antisense, 5'-
CTCGGCATCTGCCGACGCTCCGGGATCTGTCC-3'. These primers correspond to nt
-101 to -61 and nt 80 to 111 of the rat ALS cDNA, respectively.
Radioactivity hybridized to the amplified approximately 1.2-kb fragment
containing the rat ALS intron was quantitated by ß-scanning.
Plasmids for Transfection
We have cloned the mouse ALS gene and determined its
organization (23). A 1953-bp genomic DNA fragment corresponding to nt
-2001 to -49 (with respect to the A+1TG translation start
site) of the mouse ALS 5'-flanking
region2 was ligated in either the
sense or the reverse orientation relative to the firefly luciferase
reporter gene into plasmid pGL3 (Promega Corp., Madison,
WI), which contains the protein-coding region of the luciferase gene
but lacks a promoter. Unless otherwise specified, sense constructs were
used in all experiments.
Cotransfection with pCMV-SEAP (Tropix, Bedford, MA) encoding secreted
alkaline phosphatase under the control of the cytomegalovirus promoter
or with pSV-ß-galactosidase (Promega Corp.) containing the
galactosidase reporter gene controlled by the simian virus-40 promoter
was used to monitor transfection efficiency.
Cultivation and Transfection of Cells
Stock cultures of H4-II-E rat hepatoma cells (20) were grown in
175-mm2 flasks (Becton Dickinson Co., Lincoln Park, NJ) as
monolayer cultures in RPMI 1640 medium supplemented with 10% FBS (lot
11112405, HyClone Laboratories, Logan, UT) and incubated in 95%
air-5% CO2 at 37 C. Confluent cultures were passaged at a
ratio of 1:5 using 0.05% trypsin-0.53 mM EDTA.
For transfection, H4-II-E cells were adapted to DMEM supplemented with
10% FBS for at least two passages. Transfections were performed on
cells between passages 816 plated on 60-mm dishes. When 8090%
confluent, cells were washed twice with Tris-buffered saline (1 x
TBS = 25 mM Tris-HCl, pH 7.5; 137 mM NaCl;
5 mM KCl; 0.7 mM CaCl2; 0.5
mM MgCl2; and 0.6 mM
Na2HPO4) and exposed for 15 min to
DEAE-dextran:DNA complex \[2 µg luciferase plasmid DNA containing
mouse ALS promoter fragments and 0.05 µg pCMV-SEAP plasmid (or 1 µg
pSV-ß-galactosidase plasmid) complexed to 100 µg DEAE-dextran in a
final volume of 0.2 ml TBS\]. This solution was prepared 15 min before
application to the cells.
After transfection, serum-containing DMEM was added, and the culture
dishes were incubated overnight at 37 C. The medium was then replaced
with fresh serum-containing DMEM and incubated overnight. After
incubation, an aliquot (0.2 ml) of the culture medium was collected for
alkaline phosphatase assay, and the remaining culture medium was
replaced with serum-free DMEM containing 0.1% BSA in either the
presence or absence of GH. After an additional 16-h incubation, cell
lysates were prepared and assayed for luciferase activity as described
previously (58). Chemiluminescence assays were used to measure alkaline
phosphatase secreted in the media or ß-galactosidase in cell lysates
according to the manufacturers instructions (Tropix).
Mouse 3T3-F442A preadipocyte fibroblasts (21) were obtained from Howard
Green (Harvard Medical School, Boston, MA) and cultured in DMEM (4.5
mg/ml glucose, no sodium pyruvate) supplemented with 10% calf serum
(lot 30P1033, Life Technologies). Cells were passaged at a 1:5 ratio
using 0.05% trypsin-0.53 mM EDTA. Only cells maintained
for fewer than 12 passages were used for experiments. Cell were
transfected using DEAE-dextran as outlined above. In some experiments,
3T3-F442A cells were transfected using Lipofectamine (Life
Technologies) as described previously (59).
Isolated primary hepatocytes (3 x 106 cells) grown in
60-mm Falcon Primaria culture dishes (Becton Dickinson Co., Lincoln
Park, NJ) were transfected with 3.5 µg plasmid DNA using Lipofectin
(Life Technologies) as described previously (60). Cells were exposed to
the Lipofectin-DNA complex for 14 h, after which the medium was
replaced with serum-free modified Williams E media (Life Technologies)
in the presence or absence of 100 ng/ml rhGH. The medium was changed
daily for a 48-h period, after which cells were lysed and assayed for
luciferase activity.
Detection of GH Receptor mRNA
Confluent H4-II-E cells grown in 100-mm culture dishes were
incubated in serum-free DMEM containing 0.1% BSA. After 24 h,
total RNA was extracted and treated with DNase I as described above. A
533-bp partial cDNA corresponding to the rat GH receptor was amplified
by continuous RT-PCR using the following primers: sense,
5'-GAGGAGGTGACCACCATCTTGGGC-3'; and antisense,
5'-ACGACCTGCTGGTGTAATGTC-3'. These primers correspond to nt 963 to 986
and nt 1476 to 1496 (with respect to A+1TG) of rat GH
receptor mRNA; their specificity has been verified previously by
in situ PCR of frozen sections of rat pituitary (61). For
comparison, total rat liver RNA was similarly amplified for GH receptor
mRNA. An aliquot (5 µl) of the amplified sample was electrophoresed
on a 1.5% agarose-TBE gel, and the gel was blotted onto a nylon
membrane, which was then hybridized to a 32-base oligonucleotide
(5'-CTGACATTTTGGATACCGATTTCCACACCAGT-3') corresponding to nt 1368 to
1399 of the rat GH receptor. The oligonucleotide probe was labeled with
[
-32P]ATP using T4 polynucleotide kinase. Hybridized
radioactivity on the blot was quantitated by a computer-driven
ß-radioactivity scanner.
Northern Blot Analysis of c-fos mRNA in H4-II-E
Cells
H4-II-E cells were grown to confluence in 100-mm culture dishes
and then incubated for 24 h in serum-free DMEM containing 0.1%
BSA. Cultures were incubated with fresh serum-free DMEM containing
0.1% BSA supplemented with 0, 100, or 400 ng/ml rhGH for an additional
16 h. RNA was extracted as described above and analyzed by
Northern blotting \[50 C, 2 x SSPE (1 x SSPE = 150
mM NaCl, 10 mM NaH2PO4,
and 1 mM EDTA)\] using a 721-bp DNA probe corresponding to
nt 253 to 973 of the rat c-fos cDNA (GenBank accession no.
X06769) labeled as described above. The plasmid containing
c-fos-coding sequences was obtained from V. Baichwal
(Tularik, San Francisco, CA). Quantification of the hybridized signal
was performed by densitometry using the NIH Image program (Division of
Computer Research and Technology, NIH, Rockville, MD).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Lawrence Hirschberger and Martha H. Stipanuk
for providing primary hepatocyte cultures, Matt Poy for technical
assistance, and Dawei Gong for critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Guck T. Ooi, National Institutes of Health, Building 10, Room 8D14, 10 Center Drive, MSC 1758, Bethesda, Maryland 20892-1758.
This work was presented in part at the 3rd International Symposium on
Insulin-like Growth Factor Binding Proteins, Tuebingen, Germany,
October 68, 1995, and at the 10th International Congress of
Endocrinology, San Francisco, CA, June 1215, 1996.
This work was supported in part by a grant (to Y.R.B.) from the
Cornell Center for Advanced Technology in Biotechnology.
1 Present address: Lilly Research Laboratories, Lilly Corporate
Center, Indianapolis, Indiana 46285. 
Received for publication December 2, 1996.
Revision received January 31, 1997.
Accepted for publication March 14, 1997.
 |
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