| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). 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).
|
65% of the levels
in sham-operated control rats) 8 h postinjection (Fig. 1
). The
increase was observed between 3–4 h after injection and reached a
plateau from 4–8 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.
|
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 Scheffe’s 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.
|
, 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.
|
, 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.
|
-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.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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 3–4 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.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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 1362–1655 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 8–16 plated on 60-mm dishes. When 80–90% 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 manufacturer’s 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 |
|---|
|
FOOTNOTES |
|---|
This work was presented in part at the 3rd International Symposium on Insulin-like Growth Factor Binding Proteins, Tuebingen, Germany, October 6–8, 1995, and at the 10th International Congress of Endocrinology, San Francisco, CA, June 12–15, 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.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-activated sites. J Biol Chem 270:24903–24910-activated sequence-like DNA elements. J
Biol Chem 269:26208–26214This article has been cited by other articles:
 |
|
 |
|
  A. P. R. Sutherland, H. Zhang, Y. Zhang, M. Michaud, Z. Xie, M.-E. Patti, M. J. Grusby, and W. J. Zhang Zinc Finger Protein Zbtb20 Is Essential for Postnatal Survival and Glucose Homeostasis Mol. Cell. Biol., May 15, 2009; 29(10): 2804 - 2815. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. E. Govoni, J. E. Wergedal, R. B. Chadwick, A. K. Srivastava, and S. Mohan Prepubertal OVX increases IGF-I expression and bone accretion in C57BL/6J mice Am J Physiol Endocrinol Metab, November 1, 2008; 295(5): E1172 - E1180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W Kim, R. P Rhoads, N. Segoale, N. B Kristensen, D. E Bauman, and Y. R Boisclair Isolation of the cDNA encoding the acid labile subunit (ALS) of the 150 kDa IGF-binding protein complex in cattle and ALS regulation during the transition from pregnancy to lactation. J. Endocrinol., June 1, 2006; 189(3): 583 - 593. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Huo, R. C. McEachin, T. X. Cui, N. K. Duggal, T. Hai, D. J. States, and J. Schwartz Profiles of Growth Hormone (GH)-regulated Genes Reveal Time-dependent Responses and Identify a Mechanism for Regulation of Activating Transcription Factor 3 By GH J. Biol. Chem., February 17, 2006; 281(7): 4132 - 4141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Chia, M. Ono, J. Woelfle, M. Schlesinger-Massart, H. Jiang, and P. Rotwein Characterization of Distinct Stat5b Binding Sites That Mediate Growth Hormone-stimulated IGF-I Gene Transcription J. Biol. Chem., February 10, 2006; 281(6): 3190 - 3197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S Eleswarapu and H Jiang Growth hormone regulates the expression of hepatocyte nuclear factor-3 gamma and other liver-enriched transcription factors in the bovine liver J. Endocrinol., January 1, 2005; 184(1): 95 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mesotten, P. J. Wouters, R. P. Peeters, K. V. Hardman, J. M. Holly, R. C. Baxter, and G. Van den Berghe Regulation of the Somatotropic Axis by Intensive Insulin Therapy during Protracted Critical Illness J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3105 - 3113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Woelfle and P. Rotwein In vivo regulation of growth hormone-stimulated gene transcription by STAT5b Am J Physiol Endocrinol Metab, March 1, 2004; 286(3): E393 - E401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. Domene, S. V. Bengolea, A. S. Martinez, M. G. Ropelato, P. Pennisi, P. Scaglia, J. J. Heinrich, and H. G. Jasper Deficiency of the Circulating Insulin-like Growth Factor System Associated with Inactivation of the Acid-Labile Subunit Gene N. Engl. J. Med., February 5, 2004; 350(6): 570 - 577. [Full Text] [PDF]
|
|
|
|
|
|
W. H. Daughaday Free Insulin-Like Growth Factor (IGF) in Disorders of IGF Binding Protein 3 Complex Formation J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 3 - 5. [Full Text] [PDF]
|
|
|
|
 |
|
 S.-E. Kong, R. C. Baxter, and P. J. D. Delhanty Age-Dependent Regulation of the Acid-Labile Subunit in Response to Fasting-Refeeding in Rats Endocrinology, December 1, 2002; 143(12): 4505 - 4512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cisternino, M. Draghi, S. Lauriola, D. Scarcella, S. Bernasconi, L. Cavallo, F. De Luca, A. Lomeo, and L. Tato The Acid-Labile Subunit of Human Ternary Insulin-Like Growth Factor-Binding Protein Complex in Girls with Central Precocious Puberty before and during Gonadotropin-Releasing Hormone Analog Therapy J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4629 - 4633. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-E. Kong, S. M. Firth, R. C. Baxter, and P. J. D. Delhanty Regulation of the acid-labile subunit in sustained endotoxemia Am J Physiol Endocrinol Metab, October 1, 2002; 283(4): E692 - E701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. B. O'Brien, J. J. O'Shea, and C. Carter-Su SH2-B Family Members Differentially Regulate JAK Family Tyrosine Kinases J. Biol. Chem., March 1, 2002; 277(10): 8673 - 8681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. Lang, G. J. Nystrom, and R. A. Frost Burn-induced changes in IGF-I and IGF-binding proteins are partially glucocorticoid dependent Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R207 - R215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. V. Silha, Y. Gui, T. Modric, A. Suwanichkul, S. K. Durham, D. R. Powell, and L. J. Murphy Overexpression of the Acid-Labile Subunit of the IGF Ternary Complex in Transgenic Mice Endocrinology, October 1, 2001; 142(10): 4305 - 4313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. W. Davey, T. Xie, M. J. McLachlan, R. J. Wilkins, D. J. Waxman, and D. R. Grattan STAT5b Is Required for GH-Induced Liver Igf-I Gene Expression Endocrinology, September 1, 2001; 142(9): 3836 - 3841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Arosio, S. Garrone, P. Bruzzi, G. Faglia, F. Minuto, and A. Barreca Diagnostic Value of the Acid-Labile Subunit in Acromegaly: Evaluation in Comparison with Insulin-Like Growth Factor (IGF) I, and IGF-Binding Protein-1, -2, and -3 J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1091 - 1098. [Abstract] [Full Text]
|
|
|
|
|
|
C. H. Lang, X. Liu, G. J. Nystrom, and R. A. Frost Acute response of IGF-I and IGF binding proteins induced by thermal injury Am J Physiol Endocrinol Metab, June 1, 2000; 278(6): E1087 - E1096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Rhoads, P. L. Greenwood, A. W. Bell, and Y. R. Boisclair Organization and Regulation of the Gene Encoding the Sheep Acid-Labile Subunit of the 150-Kilodalton Insulin-Like Growth Factor-Binding Protein Complex Endocrinology, April 1, 2000; 141(4): 1425 - 1433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. R. Boisclair, J. Wang, J. Shi, K. R. Hurst, and G. T. Ooi Role of the Suppressor of Cytokine Signaling-3 in Mediating the Inhibitory Effects of Interleukin-1beta on the Growth Hormone-dependent Transcription of the Acid-labile Subunit Gene in Liver Cells J. Biol. Chem., February 11, 2000; 275(6): 3841 - 3847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Suwanichkul, Y. R. Boisclair, R. C. Olney, S. K. Durham, and D. R. Powell Conservation of a Growth Hormone-Responsive Promoter Element in the Human and Mouse Acid-Labile Subunit Genes Endocrinology, February 1, 2000; 141(2): 833 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ji, R. Guan, S. J. Frank, and J. L. Messina Insulin Inhibits Growth Hormone Signaling via the Growth Hormone Receptor/JAK2/STAT5B Pathway J. Biol. Chem., May 7, 1999; 274(19): 13434 - 13442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kanzaki and P. L. Morris Growth Hormone Regulates Steroidogenic Acute Regulatory Protein Expression and Steroidogenesis in Leydig Cell Progenitors Endocrinology, April 1, 1999; 140(4): 1681 - 1686. [Abstract] [Full Text]
|
|
|
|
|
|
H. Olivecrona, A. Hilding, C. Ekström, H. Barle, B. Nyberg, C. Möller, P. J. Delhanty, R. C. Baxter, B. Angelin, T. J. Ekström, et al. Acute and Short-Term Effects of Growth Hormone on Insulin-Like Growth Factors and Their Binding Proteins: Serum Levels and Hepatic Messenger Ribonucleic Acid Responses in Humans J. Clin. Endocrinol. Metab., February 1, 1999; 84(2): 553 - 560. [Abstract] [Full Text]
|
|
|
|
|
|
A. Juul, S. Møller, E. Mosfeldt-Laursen, M. H. Rasmussen, T. Scheike, S. A. Pedersen, K. W. Kastrup, H. Yu, J. Mistry, S. Rasmussen, et al. The Acid-Labile Subunit of Human Ternary Insulin-Like Growth Factor Binding Protein Complex in Serum: Hepatosplanchnic Release, Diurnal Variation, Circulating Concentrations in Healthy Subjects, and Diagnostic Use in Patients with Growth Hormone Deficiency J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4408 - 4415. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. T. Ooi, K. R. Hurst, M. N. Poy, M. M. Rechler, and Y. R. Boisclair Binding of STAT5a and STAT5b to a Single Element Resembling a {gamma}-Interferon-Activated Sequence Mediates the Growth Hormone Induction of the Mouse Acid-Labile Subunit Promoter in Liver Cells Mol. Endocrinol., May 1, 1998; 12(5): 675 - 687. [Abstract] [Full Text]
|
|
|
|
 |
|
 I. Ueki, G. T. Ooi, M. L. Tremblay, K. R. Hurst, L. A. Bach, and Y. R. Boisclair Inactivation of the acid labile subunit gene in mice results in mild retardation of postnatal growth despite profound disruptions in the circulating insulin-like growth factor system PNAS, June 6, 2000; 97(12): 6868 - 6873. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
