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Endocrinology Vol. 145, No. 12 5540-5547
Copyright © 2004 by The Endocrine Society

Insulin-Like Growth Factor-I Regulation of Hepatic Scavenger Receptor Class BI

Wen M. Cao, Koji Murao, Hitomi Imachi, Xiao Yu, Hiroaki Dobashi, Kazuya Yoshida, Tomie Muraoka, Noriko Kotsuna, Sachi Nagao, Norman C. W. Wong and Toshihiko Ishida

First Department of Internal Medicine (W.M.C., K.M., H.I., X.Y., H.D., K.Y., T.M., N.K., S.N., T.I.), Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan; and Departments of Medicine and Biochemistry and Molecular Biology (N.C.W.W.), Faculty of Medicine, University of Calgary, Health Sciences Center, Calgary, Alberta, Canada T2N 4N1

Address all correspondence and requests for reprints to: Koji Murao, M.D., Ph.D., First Department of Internal Medicine, Faculty of Medicine, Kagawa University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. E-mail: mkoji{at}kms.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High-density lipoprotein mediates a normal physiological process called reverse cholesterol transport. This process enables the transfer of cholesterol from peripheral tissues to the liver for further metabolism and eventual secretion in the form of bile. The scavenger receptor of the B class (SR-BI), human homolog of SR-BI, and CD36 and LIMPII analogous-1 (CLA-1) are different names for the same receptor that facilitates hepatocellular uptake of cholesterol from high-density lipoprotein. The pivotal role of this receptor in enterohepatic circulation of cholesterol and bile salts underlies our interest to study the regulation of hepatic SR-BI gene in response to the actions of IGF-I. The results of our studies showed that endogenous expression of SR-BI/CLA-1 was suppressed by exposure to GH or IGF-I in cultured HepG2 cells. This observation extended to a whole animal model of rats continuously infused with IGF-I. IGF-I decreased transcriptional activity of the SR-BI promoter. However, the inhibitory effect of IGF-I on SR-BI/CLA-1 promoter activity was abrogated by wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3-K). Exposure of HepG2 cells to IGF-I elicited a rapid phosphorylation of Akt. We also demonstrated that the constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the human SR-BI/CLA-1 promoter. Furthermore, the dominant-negative mutant of Akt abolished the ability of IGF-I to suppress activity of the SR-BI/CLA-1 promoter. In conclusion, PI3-K/Akt pathways participate in IGF-I-suppression of SR-BI/CLA-1 expression, which suggests that the activation of Akt plays an important role in cholesterol metabolism in liver.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE LIVER IS a key organ in regulating the metabolism of cholesterol. It controls the synthesis, plasma clearance, and excretion of cholesterol from the body (1, 2). Cholesterol homeostasis in the adult is comprised of a balance between mechanisms that increase body cholesterol, such as intestinal absorption plus endogenous synthesis, and the process that eliminates free cholesterol or bile acids from the body. Whereas transporters necessary for enterohepatic cycling of bile salts have been identified (3), many of the genes that control biliary cholesterol secretion and intestinal cholesterol absorption have not been identified.

Although detailed mechanisms remain uncertain, it has been proposed that high-density lipoprotein (HDL) promotes reverse cholesterol transport by facilitating the transfer of cholesterol from peripheral tissues to the liver for secretion into bile (4, 5). Early studies using HDL and low-density lipoprotein with radiolabeled free cholesterol have shown that free cholesterol in HDL is the preferred source for biliary cholesterol (6, 7). Recent work with plant sterols provides evidence that HDL is the preferred carrier for cholesterol, which is metabolized into bile salts (8). Based on the observations that HDL free cholesterol is preferentially used for biliary secretion and that, in tissue culture studies, HDL, but not low-density lipoprotein, selectively binds free cholesterol (9), Schwartz et al. (6) predicted that a cell-surface receptor might be involved in the hepatic uptake of HDL free cholesterol. The mouse scavenger receptor of class BI (SR-BI) fulfills this role and mediates selective uptake of HDL cholesterol ester into transfected Chinese hamster ovary cells. This finding provides an important link between a specific cell surface receptor and a pathway for the uptake of HDL (10).

Our previous report shows that human SR-BI [hSR-BI/CD36 and LIMPII analogous-1 (CLA-1)], like mouse SR-BI, functions as a receptor for HDL (11, 12, 13, 14, 15, 16). hSR-BI/CLA-1 is also similar to the mouse homolog because it also mediates selective uptake of cholesterol ester and is expressed in liver plus steroidogenic tissues. These features suggest that hSR-BI/CLA-1 is functionally related to mouse SR-BI. Overexpression of SR-BI in the mouse liver dramatically decreased plasma HDL (17, 18, 19) and increased hepatic and gallbladder biliary cholesterol concentrations (17, 20). In contrast, SR-BI-deficient mice have increased plasma HDL cholesterol (21, 22) and significantly lower levels of biliary cholesterol in the gallbladder (23, 24).

The average life expectancy in patients with active acromegaly is reduced by approximately 10 yr, with cardiovascular disease as the principal cause of premature death (25). Chronic excess of GH and IGF-I are suspected of causing atherosclerosis because a reduction of the mean serum GH leading to normalization of serum IGF-I restores life expectancy to normal (26). The high incidence of cardiovascular diseases in patients with acromegaly may in part be attributable to lipoprotein abnormalities. To determine whether hormonal defects in acromegaly affects HDL metabolism and thus enhances the risk of cardiovascular disease, we have examined hormonal regulation of hepatic hSR-BI/CLA-1 expression, especially in connection with IGF-I. We found that IGF-I inhibited the hepatic hSR-BI/CLA-1 expression via the phosphoinositide 3-kinase (PI3-K)/Akt pathway. The finding may underlie lipoprotein abnormalities in patients with acromegaly.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Wortmannin, PD98059, JNK (c-Jun amino terminal kinase) inhibitor-1, and SB203580 were purchased from Calbiochem (La Jolla, CA). Osmotic mini pumps (model 2001, delivering 1 ml solvent per hour) were from Alza Corp. (Palo Alto, CA). Recombinant human GH (Genotropin) and recombinant IGF-I were purchased from Fujisawa Phamaco Co. Ltd (Osaka, Japan). An anti-IGF-I antibody was obtained from American Research Products Inc. (Belmont, MA).

Plasmid preparation
An expression vector encoding a constitutively active Akt and a dominant-negative mutant of Akt was described previously (27). An expression vector encoding a constitutively active p110 subunit was a gift from J. Downward (Imperial Cancer research Foundation, London, UK). An expression vector encoding a phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was a gift from J.E. Dixon (University of Michigan, Arbor, MI).

Cell culture
HepG2 cells (obtained from RIKEN CELL BANK, Ibaragi, Japan) were grown in DMEM (Life Technologies, Tokyo, Japan) supplemented with 10% fetal calf serum.

Antibodies
To create an antibody directed against the extracellular domain (residues 185–300) of CLA-1 (28), the corresponding cDNA fragment was amplified from human monocyte-derived THP-1 cells (ATCC, Manassas, VA) cDNA using PCR. The product of this reaction was inserted into the vector pGEX-2T (Pharmacia, Piscataway, NJ). The nucleotide sequence was verified and the peptide was expressed in Escherichia coli. The resulting fusion peptide fused to glutathione-S-transferase was isolated using glutathione-Sepharose 4B beads (Pharmacia). The bound material was used to inject guinea pigs and generate antisera against the protein. Western blot analysis of proteins extracted from the cells stably expressing CLA-1 showed that the antibody was directed against an extracellular portion of the protein. The antibody recognized a single protein band with an estimated molecular mass of 83 kDa as previously described (11).

Western blot analysis
Cells were washed in PBS and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.1% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 0.15 M NaCl; 1 mM EDTA; and 10 µg/ml aprotinin). The proteins were resuspended under reducing conditions, and 15 µg was fractionated by size on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes for immunoblotting (29). The membranes were blocked overnight at room temperature with 0.1% Tween 20 in PBS (PBS-T) containing anti-hSR-BI/CLA-1 antibody (1:3000 dilution from whole antiserum) (11) or antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (dilution 1:1000; Biomol Research, Plymouth Meeting, PA). These membranes were washed with PBS-T, incubated for 1 h at room temperature in PBS-T containing horseradish peroxidase-linked anti-guinea pig IgG (dilution 1:3000), and rinsed in PBS-T, and antibody binding was visualized by chemiluminescence detection (ECL; Amersham Corp., Arlington Heights, IL).

Northern blot analysis
A full-length cDNA of hSR-BI/CLA-1 was synthesized by PCR using reverse-transcribed RNA from HepG2 cells and labeled with [32P]-deoxycytidine triphosphate (3000 Ci/mmol) by the random priming method (Takara, Tokyo, Japan). Electrophoresis and hybridization were performed as described (30). Blots were also probed with human GAPDH to ensure equal loading of the RNA samples (31). After autoradiography at room temperature for 24 h, hybridization signals were detected using a Bioimaging Analyzer (BAS 1000; Fuji Photo Film, Tokyo, Japan).

Transfection of HepG2 cells and luciferase reporter gene assay
The reporter construct contained the hSR-BI/CLA-1 gene sequence spanning the region from –1200 to +2 as determined from the published sequence (32). The segment of interest was amplified using PCR and cloned into the luciferase reporter gene (pCLA-LUC). Purified reporter plasmid was transfected into HepG2 cells (at 60% confluence) using a conventional cationic liposome transfection method (Lipofectamine; Life Technologies, Gaithersburg, MD). All assays were corrected for ß-galactosidase activity, and the total amount of protein in each reaction was identical. Twenty-microliter aliquots were taken for the luciferase assay, which was performed according to the manufacturer’s instructions (ToyoInk, Tokyo, Japan).

Immunoblotting of Akt
Cells were lysed for 10 min in ice-cold buffer A (50 mM Tris-HCl, pH 7.5; 1 mM EDTA; 1 mM EGTA; 0.5 mM Na3VO4; 0.1% 2-mercaptoethanol; 1% Triton X-100; 50 mM NaF; 5 mM sodium pyrophosphate; 10 mM Na-glycerophosphate; 0.1 mM phenylmethylsulfonylfluoride; 1 µM microcystin; and 1 µg/ml each of pepstatin, aprotinin, and leupeptin). The lysates were centrifuged, and the supernatants were collected. The supernatants containing protein concentrations of 20 µg/ml were used for immunoblotting according to standard procedures. Akt phosphorylated at Ser473 or Thr308 was detected using a phosphospecific Akt polyclonal antibody, and total Akt was detected by using phosphorylation-independent antibodies (Upstate Biotechnology, Lake Placid, NY) as described previously (33). The protein bands were visualized by chemiluminescence.

In vivo experiment
Eight-week-old male Wistar rats were purchased from Clea Japan Inc. (Osaka, Japan), and rats were maintained in controlled light conditions (14 h light, 10 h dark). The protocol used in this experiment was reviewed and approved by the Kagawa University Institutional Animal Care and Use Committee (Kagawa, Japan). Hormonal stimulation was given as continuous infusions using osmotic mini pumps that were implanted surgically under light ether anesthesia (34). Animals were treated with IGF-I (2.4 mg/kg body weight·d) or vehicle for 7 d. Plasma was extracted with acid-ethanol to remove binding proteins, and the IGF-I concentration in the extracted plasma was measured using RIA.

Statistical analysis
Statistical comparisons were made possible through the use of one-way ANOVA and the Student’s t test, with P < 0.05 considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH and IGF-I decrease hSR-BI/CLA-1 expression in HepG2 cells
To analyze the effects of GH and IGF-I on hSR-BI/CLA-1 expression, we measured the levels of endogenous hSR-BI/CLA-1 expression in the human hepatoma cell line, HepG2, using Western blot analysis. Exposure of these cells to GH or IGF-I for 24 h decreased the abundance of endogenous hSR-BI/CLA-1 protein to roughly one half compared with the amount in cells maintained in control media (Fig. 1AGo).



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  		FIG. 1. Effect of IGF-I on hSR-BI/CLA-1 expression in HepG2 cells. A, IGF-I and GH decrease hSR-BI/CLA-1 protein expression. HepG2 cells were exposed to IGF-I and GH for 24 h, and hSR-BI/CLA-1 protein in total cell lysate was detected using Western blot analysis probed with an anti-hSR-BI/CLA-1 antibody. Abundance of GAPDH served as a control and is shown on the bottom of each lane. C, Control; GH, 10 µg/ml GH; IGF-I, 10 nM IGF-I. A graph showing the mean ± SEM of three experiments for each treatment group is shown on the right. The asterisk denotes a significant difference (P < 0.01). B, hSR-BI/CLA-1 expression after inhibition by GH in the presence and absence of an anti-IGF-I neutralization antibody. Western blot analysis of total cell protein extracted from HepG2 cells 24 h after treatment with control media (C), media containing GH, and GH plus an anti-IGF-I antibody (anti-IGF) probed with hSR-BI/CLA-1-specific antibody is shown. Abundance of GAPDH served as a control and is shown on the bottom of each lane. A graph showing the mean ± SEM of three experiments for each treatment group is shown on the right. The asterisk denotes a significant difference (P < 0.01). NS, Nonsignificant difference. C, IGF-I decreases hSR-BI/CLA-1 mRNA expression. The abundance of hSR-BI/CLA-1 and GAPDH mRNA levels in 20 µg of total RNA were measured using Northern blot hybridization and an imaging analyzer. Lane 1, no treatment; lane 2, 10 nM IGF-I. A graph showing the mean ± SEM of three experiments for each treatment group is shown on the right. The asterisk denotes a significant difference (P < 0.01).

 
Exposure of HepG2 cells to GH has previously been shown to stimulate IGF-I secretion (35). To determine whether the GH effect on hSR-BI/CLA-1 expression was direct or via IGF-I, we treated HepG2 cells exposed to GH with a neutralizing antibody against IGF-I. Results (Fig. 1BGo) showed that GH inhibition of hSR-BI/CLA-1 expression was not evident in the presence of the IGF-I antibody. Northern blot analysis of total RNA showed that the abundance of hSR-BI/CLA-1 mRNA decreased after treatment with IGF-1 (Fig. 1CGo), but expression of GAPDH, the control, did not change. These results clearly indicate that IGF-I inhibits the expression of hSR-BI/CLA-1 in HepG2 cells.

Effect of IGF-I on hSR-BI/CLA-1 promoter activity
The preceding observations prompted us to measure transcriptional activity of the hSR-BI/CLA-1 promoter in the HepG2 cells. Therefore, activity of pCLA-LUC to either 10 µg/ml GH or 10 nM IGF-I was tested in HepG2 cells (Fig. 2AGo). Consistent with the observed changes in the levels of hSR-BI/CLA-1 protein and mRNA, both GH and IGF-I inhibited activity of the promoter.



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  		FIG. 2. Effect of IGF-I on hSR-BI/CLA-1 transcriptional activity and in HepG2 cells. A, IGF-I decreases hSR-BI/CLA-1 gene transcription. HepG2 cells were transfected with 1 µg of pCLA-LUC and treated with 10 µg/ml GH or 10 nM IGF-I for 24 h before cell harvest. All assays were corrected for ß-galactosidase activity, and the total amounts of protein per reaction were identical. The results were expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± SEM of four separate transfections that were performed on separate days. The asterisk denotes a significant (P < 0.01). Cont, Control. B, A PI3-K inhibitor blocks the actions of IGF-I. Effects of PI3-K inhibitor wortmannin (WM), MAPK kinase 1 inhibitor PD98059 (PD), JNK inhibitor I (JNKI-I), or protein kinase C inhibitor Bisindolylmaleimide I (BIS; Calbiochem) on IGF-I (10 nM) inhibited hSR-BI/CLA-1 transcriptional activity in HepG2 cells. Each data point shows the mean ± SEM of three separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01). NS, Nonsignificant difference.

 
Next we tested whether protein kinases were involved in IGF-I-mediated inhibition of hSR-BI/CLA-1 promoter activity. To address this question, we studied the effect of pharmacological inhibitors on hSR-BI/CLA-1 promoter activity. In this study, IGF-I stimulation (10 nM) was added in combination with inhibitors, such as PI3-K (10 µM wortmannin), a mitogen-activated ERK (10 µM PD98059), a JNK (1 µM JNK inhibitor-I), or a p38 MAPK (1 µM SB203580), to the HepG2 cells. Results (Fig. 2BGo) showed that the inhibitory effect of IGF-I on hSR-BI/CLA-1 promoter activity was not sensitive to inhibitors of ERK, JNK, and p38 MAPK, but it was sensitive to wortmannin, an inhibitor of PI3-K. This finding suggests that the actions of IGF-I appear to be mediated by PI3-K.

Time course of Akt phosphorylation by IGF-I
The preceding studies show that PI3-K may be required for the inhibitory effects of IGF-I on hSR-BI/CLA-1 expression. Because Akt is a potential target of PI3-K, we wondered whether IGF-I activated the Akt kinase. This possibility led us to examine the kinetics of Akt activation by measuring its ability to phosphorylate residues Thr308 and Ser473 of the protein. These modifications are a prerequisite for catalytic activity of Akt. The results (Fig. 3Go) showed that Akt phosphorylation was evident within 5 min after exposure of the HepG2 cells to IGF-I, and this activity reached a peak at 15 min. These findings indicate that IGF-I activates the Akt kinase and that IGF-I induction of Akt phosphorylation is rapid and attains maximal values at 15 min.



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  		FIG. 3. IGF-I stimulates the phosphorylation of Akt. HepG2 cells were exposed to 10 nM IGF-I for 0, 5, 15, 60, and 120 min. Abundance of phosphorylated Akt was detected by Western blot analysis of total cell protein using a phosphospecific Akt antibody (Akt-P, top row). To show equal loading of protein in each lane, the same blot was probed a second time with an Akt-specific antibody. An identical experiment independently performed gave similar results.

 
Akt regulates hSR-BI/CLA-1 promoter activity
Because Akt phosphorylation participates in IGF-I inhibition of hSR-BI/CLA-1 expression, we wondered whether these events occurred at the level of the promoter. Therefore, we asked whether IGF-I affected hSR-BI/CLA-1 promoter activity. If so, then the actions of constitutively active Akt, p110 (a subunit of PI3-K), and PTEN (a natural inhibitor of PI3-K) should affect activity of the promoter. As predicted, the results (Fig. 4AGo) showed that constitutively active Akt and p110 suppressed hSR-BI/CLA-1 promoter activity in HepG2 cells. In contrast, the cotransfection of PTEN expression vector and pCLA-LUC activated the promoter activity.



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  		FIG. 4. Role of PI3-K/Akt signal transduction pathway on hSR-BI/CLA-1 promoter activity by IGF-I. A, Effects of PI3-K components on hSR-BI/CLA-1 promoter activity. HepG2 cells were transfected with pCLA-LUC and empty vector (Cont), empty vector plus IGF-I-treatment (IGF-I), constitutively active Akt expression vector (Akt), p110 expression vector (p110), and PTEN expression vector (PTEN) 24 h before cell harvest. All assays were corrected for ß-galactosidase activity, and the total amounts of protein per reaction were identical. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± SEM of four separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01). B, Dominant-negative Akt (Akt-DN) blocks IGF-I inhibition of hSR-BI/CLA-1 transcription. HepG2 cells were transfected with pCLA-LUC and empty vector or Akt-DN and then treated with IGF-I for 24 h before cell harvest. The results are expressed as relative luciferase activity compared with control cells arbitrarily set at 100. Each data point shows the mean ± SEM of four separate transfections that were performed on separate days. The asterisk denotes a significant difference (P < 0.01).

 
Although PI3-K has many potential downstream targets, we focused our attention on Akt because of several reports showing the importance of this kinase on the effects of IGF-I. We asked whether a dominant-negative mutant of Akt had an effect on hSR-BI/CLA-1 promoter activity (Fig. 4BGo). Consistent with proceeding studies, IGF-I inhibited the activity of the hSR-BI/CLA-1 promoter. As expected, the expression of a dominant-negative mutant of Akt inhibited the actions of IGF-I on hSR-BI/CLA-1 promoter activity. Together, these findings support the idea that the PI3-K/Akt pathway is required for IGF-I inhibition of hSR-BI/CLA-1 promoter activity in HepG2 cells.

Effect of IGF-I on hSR-BI/CLA-1 expression in vivo
Although our in vitro experiments in cultured cells have helped to elucidate the action of IGF-I, whether IGF-I has the same effect in an in vivo model remains unanswered. To test whether the findings in vitro extended to an in vivo model, we infused rats with IGF-I for 7 d, thus giving the animals high circulating levels of IGF-I (43.5 ± 4.2 nmol/liter) compared with the levels in the control rats (33.2 ± 2.4 nmol/liter). IGF-I treatment of rats should suppress hSR-BI/CLA-1 mRNA and protein in these animals. Consistent with our findings in vitro, hSR-BI/CLA-1 mRNA decreased to one half when compared with control (Fig. 5Go). These findings show that the IGF-I effects on hSR-BI/CLA-1 gene expression in rats are similar to the effects in the cultured HepG2 cells.



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  		FIG. 5. Decreased level of hepatic SR-BI mRNA in IGF-I-treated rats. This graph shows the level of rat SR-BI mRNA in IGF-I-treated animals compared with vehicle-treated animals. The abundance of SR-BI and GAPDH mRNA levels in 20 µg of total RNA were measured using Northern blot hybridization and an imaging analyzer, and the ratio of SR-BI mRNA to GAPDH mRNA is shown as the percentage of control in the figure. Numbers in parentheses indicate numbers of observations in each group. Control, Vehicle-treated animals; IGF-I, IGF-I-treated animals. The asterisk denotes a significant difference (P < 0.01) between treated and control rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have summarized the results of studies showing that IGF-I regulated hSR-BI/CLA-1 gene expression. Furthermore, we have explored the signaling pathways by which the actions of IGF-I inhibit hSR-BI/CLA-1 expression in HepG2 cells. This effect appears to be mediated by PI3-K because a specific inhibitor of this kinase, wortmannin, prevents IGF-I suppression of hSR-BI/CLA-1 expression (Fig. 2BGo). To further define the actions of IGF-I, we used a transient transfection assay to show that growth factor inhibits activity of the hSR-BI/CLA-1 promoter. Furthermore, p110, the constitutively active form of the PI3-K catalytic subunit, reduces hSR-BI/CLA-1 transcriptional activity. Finally, in vivo experiments were performed in which rats were infused with IGF-I for 7 d, followed by Northern blot analysis of hSR-BI/CLA-1 mRNA in liver. IGF-I treatment of rats suppressed hSR-BI/CLA-1 mRNA. However, the mechanism of the IGF-I effects on hSR-BI/CLA-1 in vivo remains to be explored.

Because Akt/protein kinase B (PKB) is one of the downstream components of intracellular signaling triggered by IGF-I, it is not surprising that Akt/PKB was phosphorylated after treatment of HepG2 cells with IGF-I. Akt/PKB phosphorylation is negatively regulated by PTEN/MMAC1/TEP1, a tumor-suppressor gene product. This protein is a phosphatase that dephosphorylates the 3' position to reverse the reactions catalyzed by PI3-K (35). Results in Fig. 4BGo showed that overexpression of PTEN increased hSR-BI/CLA-1 promoter activity.

To connect the actions of Akt with hSR-BI/CLA-1 expression, we assessed the effects of both a constitutively active and a dominant-negative form of Akt on hSR-BI/CLA-1 promoter activity (Fig. 4BGo). In agreement with our hypothesis, constitutively activated Akt mimics the inhibitory action of IGF-1 on hSR-BI/CLA-1 promoter activity, and the dominant-negative mutant blocks this effect. However, we cannot discard the possibility that the long-term effect of IGF-I on hSR-BI/CLA-1 expression is an indirect one. Further studies of the role of Akt in IGF-I-mediated hSR-BI/CLA-1 inhibition will be necessary to define the pathway by which IGF-I affects nuclear transcription.

In this study, we showed that the exposure of HepG2 cells to GH decreased the expression of hSR-BI/CLA-1. Furthermore, the addition of a neutralizing antibody against IGF-I to GH-treated HepG2 cells blocked GH stimulation of hSR-BI/CLA-1 expression. Although this finding strongly suggests that GH acted via IGF-I to stimulate hSR-BI/CLA-1 expression, it does not exclude the possibility of a direct effect of GH. The HepG2 cells in our studies were treated with relatively high concentrations of GH to illicit IGF-I secretion, and this likely reflects the phenotype of these cancer cells. Further studies of the effects of GH on hSR-BI/CLA-1 expression using cells that more closely resemble the normal liver cell will be needed.

The current belief is that the protective effect of HDL comes from its participation in reverse cholesterol transport. This is a normal physiological process whereby cholesterol from cells in the arterial wall are transported onto HDL particles and then shuttled to the liver for further metabolism and disposal (36, 37). Efflux of cellular free cholesterol from peripheral cells to the acceptor HDL particles is the first step in reverse cholesterol transport and requires the binding of HDL to the hepatocyte membrane. Several reports have suggested that rodent SR-BI is functionally related to hSR-BI/CLA-1, and this protein is likely the receptor that selectively takes up HDL cholesterol ester (10, 17, 21). SR-BI is believed to act as a docking receptor for HDL in connection with selective uptake of cholesterol esters (38, 39). Hepatic overexpression of SR-BI causes increased biliary secretion of cholesterol without a concomitant increase in phospholipids or bile salt secretion (20). SR-BI enhances the exchange of cholesterol between the surface of HDL and the cell.

The net flux and direction of cholesterol flow largely depends on the gradient of cholesterol between the two surfaces. SR-BI may be expressed at both sinusoidal and canalicular membranes of hepatocytes (17). On the sinusoidal side of hepatocytes, SR-BI facilitates the net transfer of free cholesterol from HDL to the plasma membrane, whereas on the canalicular side, SR-BI might promote the release of free cholesterol from the membrane to such acceptors as phospholipid vesicles. The excretion of HDL cholesterol into bile is facilitated by the biliary secretion of phospholipid at the canalicular membrane (40). Although these data point to an important role for SR-BI in HDL and cholesterol metabolism, the deficiency of this protein did not alter hepatic cholesterol (ester) content. Additionally, SR-BI deficiency did not affect the expression of the key regulators of hepatic cholesterol homeostasis, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the low-density lipoprotein receptor, and cholesterol 7{alpha}-hydroxylase (41). But the deficiency did reduce the cholesterol content in the bile by approximately 40% (41). Rigotti et al. (21) generated mice with a targeted null mutation in the SR-BI gene and indicated that the plasma cholesterol concentrations were increased because of the formation of large, apolipoprotein A-I-containing particles in heterozygous and homozygous mutants relative to wild-type controls. The plasma concentration of apolipoprotein A-I, the major protein in HDL, was unchanged in the mutants. This data, in conjunction with the increased lipoprotein size, suggested that the higher level of plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results indicate that SR-BI may stimulate the hepatic uptake of HDL free cholesterol and its transport into bile.

Acromegaly is associated with premature cardiovascular mortality. Reduction of mean serum GH and normalization of serum IGF-I restores life expectancy to normal (25). The harmful effect of GH/IGF-I excess in acromegaly, which gives rise to atherosclerosis, may help explain the shortened life expectancy, but the mechanism(s) underlying this defect remain undefined. A potential source of useful information comes from mice that carry a liver-specific or generalized knockout of the IGF-I and IGF-I receptor genes (42). Unfortunately, little is known about lipid metabolism in such animals, except that the liver in IGF-I-deficient mice exhibits a 2-fold increase in serum triglycerides (43). Further studies will be needed to clarify the lipid metabolism in these animals.

We are interested in the potential role of SR-BI-mediated hepatic uptake of HDL cholesterol in controlling hepatic cholesterol content, bile acid metabolism, and biliary lipid secretion. The ability to regulate this process may lead to beneficial ways to control cholesterol and thus impact on the risk of cardiovascular disease. Our findings show the suppressive effect of IGF-I on hSR-BI/CLA-1 expression, raising the possibility that decreased hSR-BI/CLA-1 may be one of the risk factors for atherosclerosis in patients with acromegaly.

The expression of SR-BI in steroidogenic cells in vivo and in vitro is regulated by trophic hormones. This regulation is mediated by cAMP/protein kinase, a signal transduction pathway that involves transcription factors such as CCAAT/enhancer-binding proteins and steroidogenic factor-1 (44, 45). For example, in vivo adrenocortical SR-BI expression and steroidogenesis are stimulated by the systemic administration of adrenocorticotropic hormone and inhibited by dexamethasone. In this study, we have examined the potential mechanism for the actions of IGF-I by studying the promoter of the hSR-BI/CLA-1 gene. The promoter contains several cis-acting regulatory elements located within the 5'-flanking region. In the SR-BI promoter, there are a number of consensus-binding motifs for transcription factors (44, 45), including CCAAT/enhancer-binding proteins, steroidogenic factor-1, and sterol regulatory element binding protein-1. Recent studies (46, 47, 48, 49) suggest cross-talk between cAMP and PI3-K in regulating bile acid secretion across the canalicular membrane. It is possible that cAMP activates PI3-K in a cell type-specific manner, and this activation is required for bile acid secretion. Our results and previous reports indicate that both the protein kinase A and PI3-K/Akt pathways regulate the expression of hSR-BI/CLA-1. In support of this possibility, the sequence of the hSR-BI/CLA-1 promoter contains binding sites for transcription factors shown to participate in cross-talk between signal transcription and gene regulation (31). Further studies will be needed to determine the detailed regulatory mechanisms on hSR-BI/CLA-1 gene.

In summary, the results in this study show that IGF-I inhibits the expression of the endogenous hSR-BI in HepG2 cells. This inhibitory effect of IGF-I on hSR-BI/CLA-1 promoter is mediated by the PI3-K/Akt signal transduction pathway. These findings raise the possibility that IGF-I may affect reverse cholesterol transport by controlling hSR-BI/CLA-1 expression.


   Footnotes
 
This work was supported in part by Grant-in-Aid for Scientific Research 14770601 (to K.M.) and Grant-in-Aid for Scientific Research 15590944 (to T.I. and K.M.).

Abbreviations: CLA-1, CD36 and LIMPII analogous-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; JNK, c-Jun amino terminal kinase; PI3-K, phosphoinositide 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SR-BI, scavenger receptor of class BI.

Received March 15, 2004.

Accepted for publication August 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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