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Endocrinology Vol. 139, No. 1 260-265
Copyright © 1998 by The Endocrine Society

ARTICLES

The Regulation of Acid-Labile Subunit Gene Expression and Secretion by Cyclic Adenosine 3',5'-Monophosphate1

Patric J. D. Delhanty and Robert C. Baxter

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Dr. Patric J. D. Delhanty, Kolling Institute of Medical Research, St. Leonards, New South Wales 2065, Australia. E-mail: delhanty{at}med.usyd.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Circulating acid-labile subunit (ALS) is mainly hepatocyte derived and is GH dependent. ALS buffers the metabolic effects of the insulin-like growth factors by sequestering them in a ternary complex with insulin-like growth factor-binding protein-3. Nutritional regulation of ALS may be mediated by cAMP and changes in circulating GH levels or tissue GH sensitivity. Therefore, we examined the regulation by cAMP of ALS steady state messenger RNA (mRNA) levels and secretion in isolated hepatocytes under basal and GH-induced conditions. Increasing intracellular cAMP in primary hepatocytes produced a dose-dependent suppression of ALS mRNA levels and secretion. This effect was not related to a reduction in mRNA stability. In the presence of GH there was a parallel suppression of mRNA levels and secretion. However, under basal conditions cAMP had less effect on ALS mRNA levels than on secretion. Thus, in the absence of GH, expression of ALS may be predominantly posttranscriptionally regulated by cAMP. Our study suggests that cAMP affects ALS gene transcription, perhaps by interrupting the GH signaling pathway, and also inhibits posttranscriptional events in ALS expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factors (IGF-I and -II) are related in structure to proinsulin and have developmental and growth stimulatory effects as well as insulin-like metabolic actions (1). In the circulation the IGFs are stabilized in a ternary complex with IGF-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS) (2, 3). In comparison with other IGF-IGFBP complexes, this ternary complex is thought to cross the capillary barrier relatively poorly. This suggests a significant role for ALS in regulating the release of IGF from the circulation into the extracellular tissue compartment, thereby modulating their metabolic and other biological activities. The liver is the principal source of circulating ALS, which is synthesized by hepatocytes (4, 5, 6, 7).

Under conditions of starvation or severe protein deprivation, circulating levels of IGF-I are decreased, which can be partially attributed to reduced gene expression (8). However, another possible cause may be the reduction in levels of the circulating ternary complex due to a decrease in either circulating IGFBP-3 or ALS, which would effectively reduce the IGF-holding capacity of the blood. Such changes might be expected to contribute to the increased clearance rate of IGF-I observed in protein-restricted rats (9). Previous studies have demonstrated that both acutely fasted and chronically malnourished rats had significantly decreased levels of serum ALS (10, 11). In the liver, a major mediator of the response to starvation is cAMP, which modulates the transcription of a number of genes involved in this response. For example, cAMP stimulates gluconeogenesis by rapidly stimulating phosphoenolpyruvate carboxykinase gene transcription (12, 13, 14). To determine whether nutritional deprivation might regulate ALS levels via changes in cAMP production, we investigated the roles of cAMP and its interaction with GH in the regulation of ALS messenger RNA (mRNA) levels and secretion in primary cultures of rat hepatocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
N6,2'-O-Dibutyryl cAMP [sodium salt; (Bu)2cAMP], 8-(4-chlorophenylthio)-cAMP (sodium salt), forskolin, theophylline, cholera toxin, diethylpyrocarbonate, dichloro-1ß-D-ribofuranosyl benzimidazole (DRB), BSA (fraction V, RIA grade), H22358, and Williams’ E medium were obtained from Sigma Chemical Co. (St. Louis, MO). Collagenase was obtained from Boehringer Mannheim (Sydney, Australia). Tissue culture plates were purchased from Corning (Trace Biosciences, Sydney, Australia). Zeta-Probe GT nylon membranes were obtained from Bio-Rad (Richmond, CA). Na125I was obtained from ANSTO (Sydney, Australia). Recombinant human GH (rhGH) was provided by Kabi Peptide Hormones (Stockholm, Sweden).

Preparation of rat hepatocytes
Hepatocytes were prepared from 10-week-old (~250-g) female Wistar rats by in situ perfusion of livers with collagenase and were plated at 2 x 106 cells/60-mm plate in 2 ml Williams’ E medium containing 300 nM insulin, 10% FCS, and antibiotics (0.1 µg/ml streptomycin and 0.06 µg/ml penicillin). The protocol was approved in advance by the institutional animal care and ethics committee. The DNA content of representative plates of cells from each hepatocyte preparation was measured by fluorometry using dye H22358 (15). The mean value was 189 ± 11 µg DNA/plate. Medium was removed after 5 h, and cells were maintained serum free in fresh Williams’ E medium containing 0.2% BSA, 300 nM insulin, and 0.06 µg/ml penicillin. Generally, additions were made 24 h after the initial plating, and cells were then maintained for up to 48 h beyond this time. (Bu)2cAMP, theophylline, and cholera toxin were dissolved in Williams’ E medium. Forskolin was dissolved in dimethylsulfoxide at a stock concentration of 10 mM. In this case control plates were treated with dimethylsulfoxide at a concentration equivalent to the highest concentration of forskolin used. The highest concentrations of reagents used had no apparent toxic effect, and during the period of incubation with the various reagents, the appearance and attachment of the cells to the plates remained unchanged.

RNA extraction and Northern analysis
Total RNA was extracted from duplicate plates of hepatocytes by the guanidine isothiocyanate/acid-phenol technique (16). Total RNA samples (20 µg) were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde. The integrity of the ethidium bromide-stained RNA samples was confirmed on a UV light box. The RNA was then transferred by capillary blotting to Zeta-Probe GT membranes and cross-linked by baking at 80 C in a gel-drying apparatus. RNA was isolated from duplicate plates, and total RNA from three sets of experiments was analyzed on separate blots to avoid interblot variation.

A 350-bp rat ALS DNA probe was generated by PCR from a genomic DNA construct containing exon 2 of the rat ALS gene, using oligodeoxynucleotides described previously (10). The rat IGFBP-1 comple-mentary DNA (cDNA) probe was provided by S. Shimasaki (Scripps Research Institute, La Jolla, CA). These cDNAs were labeled using a Ready-to-GO random priming kit (AMRAD-Pharmacia, Melbourne, Australia) and [{alpha}-32P]deoxy-CTP (AMRAD-NEN, Melbourne, Australia). Filters were prehybridized and hybridized (2 x 106 cpm/ml) as described previously, then washed using 0.1 x SSC (standard saline citrate) at 42 C. Filters were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The equality of RNA loading was determined by stripping the blots in 0.01 x SSC-0.5% SDS at 80 C, then rescreening with an 18S ribosomal RNA (rRNA) cDNA probe (Dr. D. Denhardt, Rutgers University, Piscataway, NJ). Data were normalized by expressing the ratio of ALS mRNA to 18S rRNA. Results are expressed as the percentage of the maximum ALS/18S ratio observed in each experiment on a particular blot. This usually corresponded to cells treated with 30 ng/ml rhGH alone.

RIAs
Conditioned media were collected and stored at -20 C. These media were then thawed once and assayed using a specific rat ALS RIA, as previously described (10). Standard curves were constructed with purified rat serum ALS resuspended in Williams’ E medium.

Effect of cAMP on the rate of ALS mRNA decay
Duplicate plates of hepatocytes in three separate experiments were maintained in serum-free Williams’ E medium, supplemented with 0.2% BSA and 300 nM insulin, for 20 h. Subsequently, the cells were incubated in serum-free medium supplemented with 30 ng/ml rhGH. Sixteen hours after initiation of rhGH treatment, (Bu)2cAMP was added to half the cells to give a final concentration of 100 µM. Four hours later, DRB, a specific RNA polymerase II inhibitor (17), was added to a final concentration of 75 µM. Total RNA was then extracted from the cells 0, 1, 6, 12, and 24 h after the addition of DRB and screened for ALS mRNA and 18S rRNA by Northern analysis, which were quantified using a PhosphorImager. The ability to measure ALS mRNA in cells maximally inhibited with 100 µM (Bu)2cAMP in the absence of GH was limited by the lack of sensitivity of the Northern analyses.

Statistics
The data for experiments involving (Bu)2cAMP and forskolin represent the mean ± SEM of results from experiments performed with quadruplicate plates of hepatocytes from at least three independent liver perfusions. The experiments with cholera toxin are derived from quadruplicate plates of cells from two independent liver perfusions. The data for the mRNA decay experiments were from duplicate plates of cells from three independent liver perfusions. Results were analyzed by ANOVA, with P values calculated using Fisher’s protected least significant differences test (StatView 4.02 for Apple Macintosh, Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of (Bu)2cAMP on basal and GH-stimulated ALS mRNA abundance and ALS secretion
Hepatocytes were treated with (Bu)2cAMP for 48 h in the presence or absence of rhGH (30 ng/ml). We have previously shown that this GH concentration maximally stimulates hepatocyte ALS production (18). In the presence of rhGH, (Bu)2cAMP produced a significant suppression of steady state ALS mRNA levels to 49.2 ± 8% of the maximum GH-induced level at 200 µM (Fig. 1AGo). Similarly, ALS secretion was significantly reduced to 37.6 ± 7% of the maximum GH-induced level by 200 µM (Bu)2cAMP (Fig. 1BGo). However, under basal conditions (i.e. minus rhGH), ALS mRNA levels were suppressed to only 63.7 ± 5.7% of maximum basal levels (untreated controls) (Fig. 1AGo), although a significant suppression of ALS secretion to 36.3 ± 4.4% (P < 0.05) of the untreated control value was observed at 200 µM (Fig. 1BGo). Another cAMP analog, 8-(4-chlorophenylthio)-cAMP, gave similar results when used over the same concentration range (data not shown). In GH-stimulated states, the pattern of suppression of ALS mRNA paralleled that of ALS secretion over the range of (Bu)2cAMP doses used. However, cAMP had a less potent effect on ALS mRNA relative to ALS secretion in cells not exposed to rhGH.



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  		Figure 1. (Bu)2cAMP suppresses ALS steady state mRNA levels and secretion in primary hepatocytes. A, Dose response effect of (Bu)2cAMP on GH-stimulated and basal ALS mRNA levels. *, P < 0.05 relative to GH-treated cells without (Bu)2cAMP. B, Dose response effect of (Bu)2cAMP on GH-stimulated and basal ALS secretion. **, P < 0.05 relative to untreated cells. C, Representative Northern blot of total RNA from primary hepatocytes treated with (Bu)2cAMP screened sequentially with rat ALS, rat IGFBP-1, and 18S cDNA probes. The expression of IGFBP-1 mRNA is up-regulated by (Bu)2cAMP, demonstrating that the cells are still responding normally and that (Bu)2cAMP up to 200 µM is not toxic to the cells.

 
The highest concentration of (Bu)2cAMP did not have a toxic effect on the cells, and their morphology and attachment to the plates remained normal. In addition, IGFBP-1 gene expression was stimulated by (Bu)2cAMP, as demonstrated by others (19), even in the presence of 300 nM insulin and 30 ng/ml GH (Fig. 1CGo).

Effect of theophylline on basal and GH-stimulated ALS mRNA abundance and ALS secretion
Hepatocytes treated with 1 mM theophylline, a phosphodiesterase inhibitor, showed an approximately 50% reduction (P < 0.05) in both ALS mRNA levels and secretion under GH-stimulated conditions relative to those in cells cultured in the absence of theophylline (Fig. 2Go, A and B, compared to Fig. 1Go, A and B). However, this reduction was not statistically significant under basal conditions. This additive effect of theophylline was consistent across the (Bu)2cAMP dose response.



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  		Figure 2. Theophyline (1 mM) suppresses GH-stimulated and basal ALS mRNA levels and secretion in primary hepatocytes and has additive effects on exogenous cAMP. A, Dose response effect of (Bu)2cAMP in the presence of theophylline on GH-stimulated and basal ALS mRNA levels. *, P < 0.05 relative to GH and 1 mM theophylline-treated cells without (Bu)2cAMP; {dagger}, P < 0.05 relative to GH-treated cells alone (see Fig. 1AGo). B, Dose response effect of (Bu)2cAMP in the presence of theophylline on GH-stimulated and basal ALS secretion. *, P < 0.05 relative to GH and 1 mM theophylline-treated cells without (Bu)2cAMP; {dagger}, P < 0.05 relative to GH-treated cells alone (see Fig. 1BGo). C, A representative Northern blot screened sequentially with rat ALS, rat IGFBP-1, and 18S cDNA probes. The expression of IGFBP-1 mRNA is up-regulated normally by (Bu)2cAMP, suggesting that the cells are still responding normally, and that (Bu)2cAMP up to 200 µM is not toxic to the cells.

 
In the presence of 1 mM theophylline, hepatocytes induced by rhGH exhibited the same parallel dose-dependent suppression of ALS mRNA and secretion by (Bu)2cAMP as that observed in the absence of theophylline [200 µM (Bu)2cAMP suppressed mRNA levels to 45.6 ± 6% and secretion to 55 ± 4.3% of the maximum levels observed in cells with combined GH and 1 mM theophylline treatment but without (Bu)2cAMP]. This contrasted with the reduced effect of cAMP on ALS mRNA (68.5 ± 9.8% of the maximum basal levels in the presence of 1 mM theophylline) relative to that on ALS secretion (43.3 ± 3.3%) in theophylline-treated cells not exposed to rhGH. ALS mRNA levels in cells treated with 200 µM (Bu)2cAMP in the presence of rhGH were not significantly different from those under basal conditions (basal levels were 83.6 ± 6.3% of GH-induced levels; Fig. 2AGo), whereas ALS secretion by the same experimental groups of cells was suppressed (basal levels were 43.7 ± 6.1% of GH-induced levels; Fig. 2BGo).

In the presence of theophylline, the cells retained normal morphology and attachment, and there was a dose-dependent stimulation of IGFBP-1 by (Bu)2cAMP up to 200 µM (Fig. 2CGo).

Cholera toxin and forskolin suppress basal and GH-stimulated ALS mRNA abundance and ALS secretion
Cholera toxin, which causes constitutive activation of the Gs subunit of the G protein complex, had a marked effect on GH-stimulated hepatocytes, causing significant suppression of ALS mRNA levels to approximately 40% of maximum GH-induced levels at 0.1 µg/ml and down to approximately 20% at 10 µg/ml (Fig. 3AGo). However, no significant change in mRNA levels was observed under basal conditions. Cholera toxin caused a dose-dependent suppression of ALS secretion in both GH-treated and untreated cells (Fig. 3BGo), with significant suppression at the lowest dose used (0.1 µg/ml; P < 0.05).



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  		Figure 3. The effects of cholera toxin on ALS gene expression and secretion. A, Cholera toxin significantly suppresses ALS mRNA levels in GH-stimulated cells, but has no significant effect on basal levels. *, P < 0.05 relative to GH-treated cells without cholera toxin. B, Cholera toxin significantly suppresses ALS secretion under both GH-stimulated and basal conditions. *, P < 0.05 relative to GH-treated cells without cholera toxin; **, P < 0.05 relative to untreated cells.

 
GH-stimulated hepatocytes treated for 48 h with 0.1–25 µM forskolin, which activates the catalytic subunit of adenylate cyclase, showed a significant suppression of ALS secretion (P < 0.025) relative to that in the vehicle-treated control. However, although there was a trend toward suppression of mRNA levels, no significant effect was observed over this range of concentrations (Fig. 4Go, A and B). Although demonstrating a similar trend toward suppression, the effects of forskolin on mRNA levels and secretion were almost completely abrogated under basal conditions.



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  		Figure 4. The effects of forskolin on ALS expression. A, Forskolin had no significant effect on ALS mRNA levels either under GH-induced or basal conditions. B, Forskolin at 25 µM significantly suppressed ALS secretion under GH-stimulated conditions, although this effect was not observed in untreated cells. *, P < 0.025 relative to GH-treated cells without forskolin.

 
cAMP does not affect the stability of ALS mRNA in GH-treated cells
Our results suggest that the sensitivity of regulation of ALS mRNA levels by cAMP is increased in cells treated with GH. To determine whether this was due to an increased susceptibility of ALS mRNA to degradation, we examined the effects of cAMP on 75 µM DRB-treated cells that had been preincubated with GH. Figure 5Go shows the results of three experiments, which were run using duplicate plates of cells. We found that there was no difference in the rate of ALS mRNA decay (t1/2 ~20 h) in either the presence or absence of 100 µM (Bu)2cAMP.



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  		Figure 5. cAMP does not affect the rate of ALS mRNA decay (t1/2 ~20 h). Primary hepatocytes were treated for 4 h with 100 µM (Bu)2cAMP in the presence of 30 ng/ml rhGH. DRB was then added to the medium to a concentration of 75 µM, and total RNA was prepared from these cells at 0, 1, 6, 12, and 24 h. Northern blots of this RNA were screened sequentially with rat ALS and 18S cDNA probes, and quantitated using a PhosphorImager. The results represent the mean ± SEM of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have examined the regulation by cAMP of ALS gene expression and secretion over 48 h in isolated hepatocytes, either stimulated by or in the absence of rhGH (18). In this cell system, ALS production has previously been shown to be linear over 48 h in both the presence and absence of rhGH. Treatment of primary hepatocytes with reagents that mimic or cause an increase in intracellular cAMP gave a dose-dependent suppression of basal and GH-induced ALS secretion. However, a similar dose-dependent effect on ALS mRNA levels was only observed in GH-treated cells. Under basal conditions ALS mRNA levels appeared less responsive to cAMP treatment. The effect of cAMP on steady state ALS mRNA levels in GH-treated hepatocytes was unrelated to a decrease in ALS mRNA stability, suggesting that the suppression observed was a transcriptional effect, although this remains to be confirmed by other techniques. The dose-dependent relationship between ALS mRNA levels and secretion in GH-stimulated hepatocytes suggests pretranslational regulation of ALS expression by cAMP. However, when GH is limiting or absent, this dose-dependent relationship is not so apparent, suggesting that a translational or posttranslational effect of cAMP may also be important under these conditions.

Although (Bu)2cAMP, theophylline, and cholera toxin all markedly inhibited ALS secretion and reduced mRNA levels, the effect of forskolin was not so pronounced. Significant (~30%) inhibition of ALS secretion by hepatocytes occurred only in the presence of 25 µM forskolin with GH stimulation despite only a small change in mRNA levels. Under basal conditions there was a slight, but statistically insignificant, inhibition of ALS secretion. The range of concentrations we used has been shown by others to significantly elevate intracellular cAMP levels in primary hepatocytes, albeit only transiently for 60–90 min. Forskolin has been demonstrated to have a variety of cAMP-independent effects, such as glucose transporter and P-glycoprotein binding, and steroid-like activities, which may be confounding its specific role in activating adenylyl cyclase (20). Another possibility is that the transient forskolin-induced accumulation of cAMP (peaking at 30–60 min, then declining to basal levels between 4–24 h) that has been reported by others (20) is not sufficient to produce the sustained suppression of ALS mRNA levels and secretion over 48 h seen with cAMP analogs and cholera toxin. We found that it was not possible to examine the acute (1–6 h) effects of cAMP on ALS mRNA and secretion because of the lack of sensitivity of the assays available. However, it is evident that ALS expression is markedly sensitive to intracellular cAMP.

During fasting, serum GH levels are depressed in the rat, and a number of organs, including the liver, have reduced sensitivity to GH (9). In addition, fasting modulates the expression of a number of important metabolic enzymes, such as phosphoenolpyruvate carboxykinase, whose gene expression is directly mediated by cAMP (12). In relation to our finding with isolated primary hepatocytes, we have previously shown that although fasting significantly suppresses serum ALS levels, there is no associated suppression of hepatic mRNA levels (10). This suggests that fasting primarily affects posttranscriptional events in ALS processing in the liver that may be linked to conditions where GH signaling is limiting due to low hormone levels or decreased GH sensitivity.

Translational initiation in eukaryotes is mediated by the eukaryotic initiation factor-4F (eIF-4F) complex (21). The rate-limiting component of this large complex appears to be eIF-4E, which binds to the mRNA 7'-methylguanylic acid cap. The activity of eIF-4E is modulated by a 22-kDa binding protein, termed PHAS-1 in rats. cAMP decreases the phosphorylation of PHAS-1, which stimulates its association with eIF-4E, thereby suppressing translational initiation (22). We hypothesize that under basal conditions or in cases of GH insensitivity, the effects of cAMP on ALS translation become dissociated from those acting on transcription. This observation fits with our finding that although serum ALS levels are suppressed in fasted rats that are relatively GH insensitive, as estimated by their lowered serum GH-binding protein levels (11) or direct measurement of GH receptor (23), hepatic ALS mRNA levels are unaffected.

Unlike hepatocytes under basal conditions, those treated with GH show similar levels of suppression of both ALS mRNA and secretion by cAMP. Although cAMP has been shown to have direct suppressive effects on the transcription of a number of genes in hepatocytes (24), cAMP may have indirect effects on GH signaling and subsequent ALS transcriptional regulation. GH stimulation of its receptor leads to activation of the GH receptor-associated cytoplasmic tyrosine kinase, JAK2 (25). In turn, JAK2 promotes the stimulation of a number of signaling pathways that mediate GH-induced gene expression. These include activation of the signal transducers and activators of transcription (STAT) family of transcription factors, in particular STAT1, -3, and -5 (26), and the mitogen-activated protein kinase (MAPK) pathway (27). A number of studies demonstrate that cAMP can block growth factor-stimulated MAPK activity in a variety of cells types (28, 29, 30). This effect is probably mediated by protein kinase A, which has been shown to inhibit MAPK activity in a cell-free system (31). There is recent evidence that cAMP can also inhibit interferon-{gamma}-stimulated activity of JAK1, STAT1, and STAT3 in mononuclear cells (32, 33), although it remains to be determined whether cAMP retains these specific effects on GH-induced JAK2 activation and ALS gene expression in primary hepatocytes.

Our data show that cAMP suppresses ALS expression by primary hepatocytes at both the pre- and posttranscriptional levels, and that the specific site(s) of action may depend on the GH status of the system. Under basal conditions, ALS expression appears to be controlled predominantly at the posttranscriptional level, whereas in the presence of GH, ALS mRNA levels are particularly sensitive to cAMP. Because ALS mRNA stability appears to be unaffected by cAMP, we hypothesize that the effect of cAMP on hepatocytes in the presence of GH may be amplified by its suppression of GH signaling events. This has implications for the regulation of hepatic ALS gene expression and secretion in conditions of GH insensitivity.


   Acknowledgments
 
We gratefully acknowledge Jin Dai for initiating the work on regulation of ALS by cAMP, and Vanessa Baxendale for excellent technical assistance.


   Footnotes
 
1 This work was supported by a National Health and Medical Research Council Project Grant. Back

Received June 9, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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