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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 3–4 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 1Go).



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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 Scheffe’s test).

 
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. 1Go). 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. 2Go). Its abundance was decreased by about 65% in hypox rats (Fig. 2Go). 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 1–3) and three hypox rats (lanes 4–6). 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 1362–1655 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.

 
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. 3AGo, 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. 3AGo, 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. 3BGo) 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 18–24 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 Scheffe’s test and t test, respectively).

 
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. 3AGo, 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. 4Go, 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.

 
The stimulation of ALS promoter activity by rhGH was dose dependent (Fig. 4Go, 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. 5Go). 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 18–24 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.

 
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 {gamma}-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. 6Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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. 6Go). 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. 4Go), 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. 3Go) 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 {gamma}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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 [{alpha}-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 [{gamma}-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 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. Back

Received for publication December 2, 1996. Revision received January 31, 1997. Accepted for publication March 14, 1997.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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