Comparison of expression and regulation of the high-density lipoprotein receptor SR-BI and the low-density lipoprotein receptor in human adrenocortical carcinoma NCI-H295 cells
Geneviève Martin1, Antoine Pilon1, Caroline Albert2, Michel Valle2, Dean W. Hum2, Jean-Charles Fruchart1, Jamila Najib1, Véronique Clavey1 and Bart Staels1
1U.325 INSERM, Département d’Athérosclérose, Institut Pasteur de Lille, and Faculté de Pharmacie, Université Lille II, France; 2 Laboratoire d’Endocrinologie Moléculaire, Le Centre Hospitalier de l’Université Laval, Laurier, Québec, Canada
In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high-density lipoproteins (HDL) via the HDL receptor, scavenger receptor-BI (SR-BI). In humans cholesterol for steroidogenesis is considered to be derived from the low-density lipoprotein (LDL) receptor pathway, and the contribution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription-PCR amplification and sequence analysis demonstrated the presence of SR-BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR-BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI-H295 cells, indicating its presence in the steroid-producing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR-BI and LDL receptor mRNA levels in a time- and dose-dependent manner. The induction of SR-BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR-BI protein in NCI-H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMP- stimulated cells. Scatchard analysis under basal conditions indicated that NCI-H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (Ka; in nM) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.
Keywords: adrenal gland; cholesterol metabolism; lipoproteins; receptor; steroidogenesis.
Correspondence to B. Staels, U.325 INSERM, Département
d’Athérosclérose, Institut Pasteur, 1 Rue Calmette, 59019 Lille, France.
Fax: + 33 3 20 87 73 60. Tel .: + 33 3 20 87 73 88. E-mail: Bart.Staels@pasteur-lille.fr
Abbreviations: ActD, actinomycin D; ACTH, adrenocorticotropic hormone; apo, apolipoprotein; CHX, cycloheximide; GAPDH, glyceraldehyde-3- phosphate dehydrogenase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LPL, lipoprotein lipase; MIX, 3-isobutyl 1-methylxanthine; PKA, protein kinase A; P450scc, cytochrome P450 side chain cleavage enzyme; RT, reverse transcription; SR-BI, scavenger receptor class B type I; HMG-COA, 3-hydroxy 3-methylglutaryl-coenzyme A; db-cAMP, dibutyryl- cAMP; CLA-1, CD36 and LIMPII Analogous-1; HDL3, HDL subfraction 3; CT, cholera toxin; FBS, fetal bovine serum.
(Received 28 August 1998, revised 4 January 1999, accepted 1 February 1999)
The adrenal gland is the principal site for the biosynthesis of steroid hormones, such as glucocorticoids and mineralocorti- coids [1]. In the adrenal cortex, steroidogenesis is controlled by specific tropic peptide hormones [2]. Glucocorticoid synthesis in the zonae fasciculata and reticularis is principally stimulated by the anterior pituitary-derived adrenocorticotropic hormone (ACTH), whereas mineralocorticoid synthesis is principally induced by angiotensin-II in the zona glomerulosa. After bind- ing to specific cell-surface receptors, ACTH and angiotensin-II, respectively, activate the protein kinase A (PKA) and protein kinase C pathways of intracellular second messengers. Short- term, immediate regulation of adrenal steroidogenesis is deter- mined by the rate of cholesterol uptake in the mitochondria principally through the action of the steroidogenic acute regu- latory protein [3,4]. Long-term regulation of steroidogenesis by these tropic hormones occurs mainly via alterations in the expression level of the different mitochondrial enzymes involved in the conversion of cholesterol into steroids, of which the first
step is catalysed by the cytochrome P450 side chain cleavage enzyme (P450scc) [5-7]. However, both short- and long-term induction of steroidogenesis depend critically on the availability of cholesterol substrate. Indeed, depletion of adrenal cholesterol pools, as occurs in mice genetically modified to be deficient in adrenal cholesterol delivery pathways, results in impaired basal and stress-stimulated corticosteroid synthesis [8].
Cholesterol for steroidogenesis can be derived either from endogenous de novo synthesis, of which the rate-limiting step is catalysed by the enzyme 3-hydroxy 3-methylglutaryl-coenzyme A (HMG-COA) reductase, or via receptor-mediated uptake from circulating lipoproteins. Humans, and other animal species carry- ing their plasma cholesterol mainly in low density lipoprotein (LDL), are believed to derive the cholesterol necessary for steroid hormone synthesis mainly from LDL via receptor-mediated endocytosis through the LDL receptor [9-13]. In contrast in animal species which carry the majority of their cholesterol in HDL (e.g. rodents) cholesterol for steroidogenesis appears to be derived principally from high density lipoprotein (HDL) via a nonendocytotic, apolipoprotein-dependent receptor-mediated cholesteryl ester uptake pathway [12,14-18]. Although the contribution of the latter pathway to steroidogenesis in humans is unknown, the absence of major adrenal dysfunction in patients with defects in the LDL receptor pathway and treated with HMG-COA reductase inhibitors implicates the existence of functional back-up pathways of cholesterol delivery to the adrenal gland for steroid synthesis, possibly via mechanisms similar to those of rodents [19-23].
Studies in rodents have identified the scavenger receptor-BI (SR-BI) as the principal receptor mediating the selective uptake of cholesteryl esters from HDL by the adrenal gland in these species [24]. SR-BI is a membrane receptor, which belongs to the family of the class B type I scavenger receptors. So far hamster and mouse SR-BI cDNAs [24-26], as well as their human homologue CLA-1 (CD36 and LIMPII Analogous-1 [27]), have been cloned and characterized. In contrast with the class A scavenger receptors, class B scavenger receptors display a more restricted ligand specificity: SR-BI binds native and modified LDL, anionic phospholipids and HDL, but not poly- anions [25,28]. Recent studies indicated that HDL binding to murine SR-BI is mediated via its apolipoproteins, apoA-I, apoA-II and apoC-III [18]. Interestingly, adenovirus-mediated overexpression of SR-BI in mouse liver resulted in a significant decrease of plasma HDL concomitant with an increase in biliary cholesterol, indicating the functional involvement of SR-BI in the reverse cholesterol transport pathway in this species [29]. SR-BI mRNA and protein are expressed at high levels in steroidogenic tissues (adrenal gland and ovary) and to a lesser extent in lung and liver [26,28,30-33]. Immunohistochemical analysis demonstrated a high level of SR-BI expression in the zonae fasciculata and reticularis and a lower level of expression in the zona glomerulosa of the adrenal gland in rats [31]. SR-BI expression is under control of stimuli that change sterol meta- bolism, such as stress and ACTH in adrenal tissue [30,32,34], and human chorionic gonadotropin in testicular steroidogenic Leydig cells [31]. Furthermore, animals rendered deficient (by homologous recombination) in apoA-I and hepatic lipase, two genes involved in the delivery of cholesterol from HDL to peripheral cells, respond to depletion of the adrenal cholesterol stores by up-regulating adrenal SR-BI expression; this indicates that in rodents, SR-BI is a functional HDL receptor under feedback control [30]. Recently, it was demonstrated that SR-BI is the major route for the delivery of cholesteryl esters from HDL to the steroidogenic pathway in cultured mouse adrenal cells [35]. Together with the restricted ligand-specificity, the
tissue distribution pattern and regulation by steroidogenic stimuli suggests a general role for receptors of this class in lipid metabolism and more specifically in adrenal gland cholesterol metabolism. Nevertheless, functional data have so far been provided only in rodents and receptor binding and cholesterol uptake studies have not yet been performed on human adrenocortical cells.
In view of the involvement of SR-BI in rodent adrenal cholesterol metabolism [30,31,34], we decided to compare the expression and regulation of human SR-BI to the LDL receptor in human adrenal cortex cells. Furthermore, in an attempt to evaluate the relative importance of the HDL receptor pathway to adrenal cholesterol metabolism, we compared the efficacy of HDL in delivering cholesterol to human adrenocortical cells to that of LDL, the classical vehicle for cholesterol delivery to the human adrenal gland. Our results show that SR-BI is expressed at high levels in the steroid-producing cells of the human foetal and adult adrenal cortex as well as in NCI-H295 adrenocortical carcinoma cells. In addition, we show that SR-BI and LDL receptor expression are coordinately regulated by activators of glucocorticoid synthesis in NCI-H295 cells. However, uptake of cholesteryl esters from HDL by the adrenal cells appears quantitatively less important than from LDL, suggesting that the HDL receptor SR-BI is likely to be of lesser importance in providing cholesterol to human adrenocortical cells under normal physiological conditions.
MATERIALS AND METHODS
Materials
Cycloheximide (CHX), actinomycin D (ActD), transferrin, selenium and insulin were from Boehringer Mannheim. ACTH, cholera toxin (CT), 3-isobutyl-1-methylxanthine (MIX), forskolin, dibutyryl-cAMP (db-cAMP) and 8Br-cAMP were from Sigma. Superscript reverse transcriptase and cell culture media were from Life Technologies. [a-32P]dCTP and [Q-32PJUTP were from NEN and [3H]-cholesteryl oleyl ether was from Amersham.
Cell culture
Human adrenal tissue was obtained from renal transplant donors through a collaboration with the Quebec Transplant Association. Primary foetal adrenal and adult zonae glomerulosa/fasciculata cells were isolated as described [36]. Human HepG2 hepato- blastoma, THP-1 monocyte, Caco2 colon carcinoma and adreno- cortical carcinoma NCI-H295 and NCI-H295R cells were obtained from the ATCC. THP-1 monocytes were differentiated and THP-1, Caco2, NCI-H295 and Hep-G2 cell cultures were maintained exactly as described previously [37,38]. NCI-H295 cells were maintained in RPMI supplemented with 2% fetal bovine serum (FBS) and transferred to RPMI with 10% FBS before treatment. db-cAMP, 8Br-cAMP, CT and CHX, dissolved in sterile H2O, ActD and forskolin, dissolved in ethanol, and MIX, dissolved in dimethylsulphoxide were added to the medium at the concentrations and periods of time indicated. Control cells received vehicle only. Each experiment was performed at least twice with similar results.
Tissue and cell RNA extraction and analysis
Total cellular RNA was prepared by the guanidinium isothio- cyanate/caesium chloride procedure (tissues) or by the acid guanidinium thiocyanate/phenol/chloroform method (cell cul- tures) [39,40]. Poly(A+)RNA was subsequently prepared using
Poly(A)Quik push columns (Stratagene). For analysis of SR-BI and LDL receptor expression in adult and foetal adrenal, primary adrenal fasciculata-glomerulosa cells, NCI-H295 and HepG2 cells, total RNA (50 ng) was reverse transcribed using random hexamer primers and Superscript reverse transcriptase. SR-BI and LDL receptor mRNA was subsequently amplified by PCR (35 cycles of 30 s 94 ℃, 30 s 60 ℃, 30 s 72 ℃) using, respectively, as primers the sense oligonucleotides 5’-CCT CGG AAA ACA ATG GAG TGA GCA-3’ and 5’-GAA CCT GGA GGG TGG CTA CAA GTG CCA GTG TGA G-3’ and the antisense oligonucleotides 5’-CAC AGG TTT GCC CCA GGG TCC A-3’ and 5’-TGT CCC TGC TGA TGA CGG TGT CAT AGG A-3’, yielding fragments of the expected size of 318 (nucleotides 2151-2468 of the SR-BI/CLA-1 cDNA) and 311 (nucleotides 1107-1417 of the LDL receptor cDNA) bp for SR-BI and LDL receptor, respectively [27,41]. Identity of the amplified frag- ments was confirmed further by restriction enzyme analysis using Sau3AI yielding fragments of 238 and 79 bp for SR-BI and 245 and 66 bp for LDL receptor [27,41]. The resulting products were separated on a 2% agarose gel and undigested reverse transcription (RT)-PCR fragments were subsequently subcloned into the pBluescript plasmid vector. Sequence analy- sis revealed complete identity to the human SR-BI/CLA-1 cDNA sequence reported previously [27].
Northern and dot blot hybridizations of total cellular RNA and poly(A+)RNA were performed as described previously [42] using the human LDL receptor and SR-BI/CLA-1 cDNA clones [27,43]. A GAPDH probe was used as a control probe in all experiments [44]. All cDNA probes were labelled by random primed labelling (Boehringer Mannheim). Filters were hybrid- ized to 1.5 x 10° cpm.mL-1 of each probe as described [42]. They were washed once in 0.5 x NaCl/Cit and 0.1% SDS for 10 min at room temperature and twice for 30 min at 65 ℃ and subsequently exposed to X-ray film (X-OMAT-AR, Kodak). Autoradiograms were analysed by quantitative scanning densito- metry (Biorad GS670 Densitometer) and results normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels as described [42].
Isolation of nuclei and transcriptional rate assay
Nuclei were prepared from NCI-H295 cells treated either with 8Br-cAMP (300 µM) or vehicle for the times indicated and transcription run-on assays were performed as described by Nevins [45]. Equivalent counts of nuclear RNA labelled with [Q-32P]UTP (3000 Ci.mmol-1) were hybridized for 36 h at 42 ℃ to 5 µg of SR-BI, LDL receptor, P450scc [46], GAPDH and vector DNA (pBluescript) immobilized on Hybond-C Extra filters (Amersham). After hybridization, filters were washed at room temperature for 10 min in 0.5X NaCl/Cit and 0.1% SDS and twice for 30 min at 65 ℃ and subsequently exposed to X-ray film (X-OMAT-AR, Kodak). Quantitative analysis was performed by scanning densitometry (BioRad GS670 densitometer).
Lipoprotein isolation and labelling
LDL and HDL subfraction 3 (HDL3) were isolated from human plasma by ultracentrifugation at densities of 1.030-1.063 and 1.12-1.21 g.mL-1, respectively [47]. Protein content was measured by the method of Peterson [48]. The only major proteins in HDL3 were apoA-I and apoA-II, whereas apoE was undetectable. Lipids were measured by enzymatic methods and HDL3 and LDL composition were in agreement with values reported previously [49]. LDL and HDL3 apolipoproteins were
radiolabelled with [125]] as described by Bilheimer et al. [50]. The specific radioactivity usually obtained was 300-600 c.p.m .. ng protein. Labelling of LDL and HDL3 with [3H]-cholesteryl oleyl ether as tracer was performed as described elsewhere [51]. [3H]-cholesteryl oleyl ethers were incorporated into donor liposomes [52] and incubated overnight at 37 ℃ with lipo- proteins and a plasma fraction enriched with cholesterol ester transfer protein (density > 1.21 g.mL-1). Donor liposomes were removed by flotation at density 1.006 g.mL-1 and labelled LDL and HDL3 were reisolated, respectively, at density 1.063 and 1.21 g.mL-1. The specific activity usually obtained was 5000 and 40 000 d.p.m .. µg-1 cholesterol ester in HDL or LDL.
Binding of [125]] HDL3 and [1251] LDL to NCI-H295R cells
NCI-H295R cells, which grow as monolayers and respond similarly to cAMP treatment as the parental NCI-H295 cells (data not shown, [53]), were treated with 8Br-cAMP (300 µM) or vehicle for 24 h. To determine specific binding of [125I]lipoproteins, cells (6 x 10° cells/well) were washed and incubated for 1 h at 37 ℃ with serum-free culture medium. Cells were subsequently incubated for 2 h at 4 ℃ with serum-free medium containing different concentrations of [125I]lipoproteins with or without a 50-fold excess of unlabelled lipoproteins. Dishes were washed twice with ice-cold 2% (w/v) albumin/Tris buffer (10 mM Tris/HCI, pH 7.4, containing 0.150 M NaCl) and washed twice again with cold Tris buffer. Cells were then lysed in 0.1 M NaOH, and the [125I] radio- activity was determined with a Beckman gamma scintillation counter. An aliquot of each sample was assayed for protein content [48]. Specifically bound [125I]lipoprotein is defined as high affinity binding calculated by subtracting the binding of [125I]lipoproteins in the absence and presence of an excess of unlabelled lipoprotein. Results are the mean of triplicate assays. Results were analysed by the Scatchard method either in ug apolipoprotein.mL-1 or mol lipoprotein.L-1. Equilibrium associ- ation constants between lipoproteins and cells were calculated based on molecular mass and lipoprotein compositions reported by Kostner and Laggner [49] (HDL3: 200 kDa; LDL: 2.5 MDa). Bmax (maximal binding capacity) is expressed in pmol apolipo- protein.mg- cell protein- and Ka is expressed in nmol apolipo- protein.L-1.
Selective uptake of cholesteryl esters from HDL and LDL by NCI-H295R cells
Cellular uptake of [125]] was determined using the same procedure as above, but incubations were performed for 4 h at 37 ℃. After treatment with 8Br-cAMP (300 µM) or vehicle for 48 h, NCI-H295R cells were washed and incubated for 1 h at 37 ℃ with serum-free culture medium. Cells were then incubated for 30 min or 5 h at 37 ℃ with serum-free medium containing the indicated concentrations of [3H]cholesteryl oleyl ether-labelled lipoproteins. The nonspecific uptake obtained with a 50-fold excess of unlabelled lipoprotein was < 10%. Cellular lipids from each well were extracted twice with 1 mL hexane/isopropyl alcohol (3 : 2 v/v) for 30 min, cellular [3H]-cholesteryl oleyl ether radioactivity was counted [54] and total and esterified cholesterol was measured using an enzymatic assay (Boehringer Mannheim). The delipidated cells were dissolved in 1 mL 1 M NaOH and an aliquot was used for protein determination [48]. Results are the mean of triplicate assays. Results are expressed either in absolute values (ng cholesteryl ester incorporated h-1.mg-cell protein-1) or in relative values as a percentage of the untreated control.
Membrane preparation, ligand and immunoblot assays
Cells were treated with 8Br-cAMP (300 µM) or vehicle for 48 h and cell membranes were prepared according to Basu et al. [55] with few modifications. Briefly, cell monolayers were scraped from dishes in buffer containing 50 mM Tris/HCI (pH 7.4), 50 mM NaCl, 300 mM saccharose and a mixture of protease inhibitors (1 mM benzamidine, 1 mM iodoacetamide, 1 mM phenantroline, 1 µM pepstatine, 1 mM phenylmethanesulfonyl fluoride). Cells were pelleted by centrifugation at 200 g for 5 min, resuspended in the same buffer, homogenized with two 5-s pulses at 80% and one 5-s pulse at 100% level using a Bioblock 375-W ultrasonic homogenizer, and then centrifuged at 800 g for 10 min at 4 ℃. The supernatant was centrifuged at 100 000 g for 60 min at 4 ℃, and the pellet was resuspended in the buffer indicated above. The membranes were either analysed immediately or stored frozen.
Membrane proteins (100 µg) were separated under nonreduc- ing conditions by SDS/PAGE using 4-15% gradient gels as described by Laemmli [56] and blotted onto nitrocellulose filters by electrotransfer (10 mA.cm-2). The protein patterns were revealed by Ponceau S (Sigma) staining. The blots were first incubated for 2 h at room temperature in blocking buffer (10 mM phosphate buffer, pH 7.4, 150 mm NaCl, 10% (w/v) nonfat dried milk) and then for 2 h with buffer containing 5% (w/v) nonfat dried milk and 30 µg of HDL3 protein.mL-1. Subsequently, the blots were washed five times for 10 min at room temperature with phosphate buffer and incubated over- night at 4 ℃ with 0.5 µg.mL-1 of an equimolar mixture of peroxidase-labelled mouse monoclonal antibodies raised against human apoA-I (A03, A51, A17, A05) [57] in phosphate buffer containing 5% (w/v) nonfat dried milk. For LDL receptor immunoblots, the blots were incubated for 2 h at room tem- perature with a mouse monoclonal antibody against LDL receptor (0.5 µg-mL-1; IGC7-Amersham) then overnight at 4 ℃ with 0.5 µg.mL-1 of peroxidase-labelled goat anti-mouse
antibodies. The blots were washed as above and revealed by enhanced chemiluminescence (Amersham) followed by auto- radiography. For SR-BI immunoblots, the blots were incubated as indicated for LDL receptor with a rabbit polyclonal antipeptide antiserum (diluted 1 : 1000) raised against amino acids 470-509 of human CLA-1/SR-BI [27] coupled at Cys470 to keyhole limpet haemocyanin. The apparent molecular mass of each protein was determined using molecular mass standards (BioRad).
RESULTS
SR-BI is expressed in the steroid-producing cells of the human adrenal cortex
In order to determine whether the SR-BI gene is expressed in the human adrenal gland, RT-PCR amplification and Northern blot analysis was performed on human foetal adrenal and adult adrenal cortex RNA. RT-PCR amplification using SR-BI- specific primers [27] yielded a DNA fragment of the expected size of 318 bp in both tissues (Fig. 1A). SR-BI was coexpressed with LDL receptor mRNA, which yielded a fragment of 313 bp by RT-PCR amplification using LDL receptor-specific primers [41] (Fig. 1A). Identity of the amplified DNA fragments was further confirmed by restriction enzyme digestion with Sau3AI, which yielded the expected restriction fragments of 239/79 bp and 250/63 bp for SR-BI and LDL receptor, respectively [27,41] (Fig. 1A). Furthermore, both the SR-BI and LDL receptor genes are expressed in the steroid-producing cells of the human adrenal cortex, as both SR-BI and LDL receptor mRNA are detected by RT-PCR analysis of primary cultures of human fasciculata-glomerulosa cells as well as in the human adreno- cortical carcinoma cell line NCI-H295, which actively synthe- sizes the major adrenal steroids, including gluco- and mineralocorticoids [38] (Fig. 1A). As a control, GAPDH mRNA was also detected in all cell lines and tissues examined
A
Marker
Mix only
Adult adr
Fetal adr
Prim. adr
NCI-H295
HepG2
C
Fetal adr
NCI-H295
Sau3AI
-+ - + - + - + - +
506
396
344
LDL-R
298
220
-28S
506
396
344
298
SR-BI
220
B
HepG2
THP-1
THP-1diff.
CaCo2
NCI-H295
Prim.hep.
-18S
LDL-R
SR-BI
(data not shown). The resulting SR-BI PCR products from human fetal adrenal and NCI-H295 cells were cloned into pBluescript and sequence analysis revealed complete identity to the previously published human SR-BI/CLA-1 cDNA sequence (data not shown) [27]. Quantitative Northern blot analysis comparing the mRNA levels of SR-BI and LDL receptor in different human cell lines demonstrated high levels of expres- sion of both SR-BI and LDL receptor mRNA in NCI-H295 cells, whereas all other cell lines examined, including human hepatoma and primary hepatocyte cells, THP-1 monocytes and colon carcinoma Caco2 cells, expressed both genes, but to a much lower extent (Fig. 1B). Northern blot analysis comparing SR-BI expression levels between human foetal adrenal and NCI- H295 cells indicated comparable, high levels of expression of SR-BI (Fig. 1C). Altogether these results indicate that the SR-BI and LDL receptor genes are coexpressed both in human primary adrenal as well as in NCI-H295 cells.
SR-BI and LDL receptor mRNA levels are coordinately regulated by activators of adrenal glucocorticoid synthesis
To determine whether, as in primary adrenal cells [32], SR-BI gene expression is under the control of activators of adrenal glucocorticoid synthesis in NCI-H295 cells, the cells were treated for 24 h with various activators of the cAMP/PKA signal transduction pathway which mediate the adrenal response to ACTH. Addition of the cAMP analogues db-cAMP and 8Br-cAMP resulted in an increase of SR-BI mRNA levels (Fig. 2). Stimulation of endogenous cAMP production by forskolin, CT (a stimulator of adenylyl cyclase activity), or CT with MIX (an inhibitor of phosphodiesterase) also resulted in increases of SR-BI mRNA levels (Fig. 2). As for SR-BI, activators of the cAMP/PKA signal transduction pathway increased LDL receptor mRNA levels, but this induction was slightly more pronounced than the increase of SR-BI mRNA levels (Fig. 2).
The kinetics and dose-dependency of response to cAMP in NCI-H295 cells were then examined. Treatment with 8Br-cAMP
Control
Forskolin
MIX + CT
CT
db-cAMP
8Br-CAMP
Control
+LDL-R
-SR-BI
GAPDH
A
LDL-R
B
C
SR-BI
500
O- - O GAPDH
600
LDL-R
% of control
LDL-R
400
% of control
500
SR-BI
O- - O GAPDH
400
300
+SR-BI
300
200
200
4 GAPDH
100
100
0 36 12
24
48
0
10
30
100
300 1000
0 10 30 100 300
Time (hr)
[db-cAMP]
LDL-R mRNA (% induction)
A
Control
B
700
CAMP
CHX
SR-BI mRNA (% induction)
600
CHX+CAMP
400
500
300
400
300
200
200
8
100
100
:
0
3
6
12
24
0
3
6
12
24
Time (hr)
resulted in a gradual increase of SR-BI mRNA levels, which attained a maximum 24 h after treatment (Fig. 3A). By contrast, LDL receptor mRNA levels increased much more rapidly, already reaching maximum induction 3 h after treatment (Fig. 3A). Treatment of NCI-H295 cells for 24 h, the optimal time-point for cAMP induction of SR-BI gene expression (Fig. 3A), with different doses of 8Br-cAMP resulted in dose- dependent increases of both SR-BI and LDL receptor mRNA levels, which were already maximal at 100 µM of cAMP (Fig. 3B). Similar results were obtained by Northern blot analysis of RNA from an independent experiment (Fig. 3C). Interestingly, in all of these independent experiments the induction of LDL receptor mRNA levels was consistently higher than those of SR-BI (Fig. 3).
The induction of SR-BI and LDL receptor gene expression by CAMP is independent of ongoing protein synthesis and occurs at the transcriptional level
To determine whether the induction of SR-BI and LDL receptor gene expression by cAMP is dependent upon ongoing protein synthesis, the influence of simultaneous treatment with the protein synthesis inhibitor CHX on the cAMP-mediated induc- tion of SR-BI and LDL receptor mRNA levels was determined in NCI-H295 cells. Treatment with db-cAMP resulted in a rapid induction of LDL receptor mRNA levels, whereas SR-BI mRNA increased more gradually (Fig. 4), thereby confirming our pre- vious observations (Fig. 3A). Simultaneous addition of CHX did not change the induction of SR-BI or LDL receptor mRNA
levels (Fig. 4), indicating that the effects of cAMP are inde- pendent of ongoing protein synthesis. Interestingly, LDL recep- tor mRNA levels increased ~ threefold 6 h after CHX addition (Fig. 4A). This increase was transient and independent of the presence of cAMP, suggesting that basal LDL receptor gene expression in NCI-H295 cells is under negative control by a short-lived protein.
Next, the induction of SR-BI and LDL receptor gene expression by cAMP was examined at the transcriptional level. In a first set of experiments, NCI-H295 cells were treated with cAMP in the presence or absence of the RNA polymerase II inhibitor ActD. Pretreatment for 90 min of NCI-H295 cells with ActD completely prevented the induction of both SR-BI and LDL receptor mRNA levels at all time-points examined, suggesting that cAMP induces both genes at the transcriptional level (Fig. 5).
The results from the ActD transcription inhibition experi- ments were further confirmed by nuclear run-on experiments performed at optimal time points according to the kinetics of SR-BI (12 h) and LDL receptor (2 and 6 h) induction by cAMP (Figs 3A, 4 and 5). Both 2 and 6 h after 8Br-cAMP (300 µM) treatment the LDL receptor gene transcription rate increased > fourfold (Fig. 6). Similarly, SR-BI gene transcription increased ~2.5-fold 12 h after cAMP treatment (Fig. 6). As a control, gene transcription of P450scc, the first enzyme in the steroidogenic pathway, increased after 8Br-cAMP treatment at all time points analysed (Fig. 6). These data unequivocally demonstrate that cAMP increases both LDL receptor and SR-BI expression at the transcriptional level.
LDL-R mRNA (% induction)
A
B
500
SR-BI mRNA (% induction)
-200
400
Control
Control
CAMP
CAMP
150
300
ActD
ActD
ActD+CAMP
ActD+CAMP
-100
200
100
50
0
3
6
12
24
0
3
6
12
24
Time (hr)
A
Control
B
C
500
CAMP
250
% of control
Control
CAMP
Control
CAMP
400
200
LDL-R
P450scc
300
150
P450scc
BSK
200
100
BSK
SR-BI
100
50
GAPDH
GAPDH
Time (hr)
2
6
2
6
12
12
6
12
LDL-R
P450scc
SR-BI P450scc
cAMP treatment increases the expression of SR-BI and LDL receptor protein in NCI-H295 cells
When HDL3 ligand blot analysis was performed on NCI-H295 cells, binding of HDL3 localized principally to a protein doublet of 82 and 84 kDa, a molecular mass similar to that described for murine SR-BI (about 82 kDa) and for human CLA-1 (about 85 kDa) [58]; a minor HDL3-binding protein of ~120 kDa was also detected (Fig. 7A). Furthermore, a protein of the same molecular weight was detected by antibodies raised against the 40 C-terminal amino acids of CLA-1. These results indicate that SR-B1 is the major HDL-binding protein in NCI-H295 cells. Immunoblot analysis indicated that the expression of SR-B1 is ~ twofold stimulated by cAMP treatment (Fig. 7B). Analysis of LDL receptor protein by Western blotting under nonreducing conditions indicated the presence of a 130-kDa protein corre- sponding to the LDL receptor in NCI-H295 cells (Fig. 7C). Stimulation of NCI-H295 cells with cAMP resulted in a more pronounced increase of LDL receptor protein levels (Fig. 7C).
Basal and cAMP-stimulated lipoprotein binding and uptake of cholesteryl esters is much lower from HDL than from LDL
As SR-BI has been shown to be a HDL receptor mediating the binding of HDL apolipoproteins and uptake of cholesteryl esters from HDL in rodent tissues [18,31,35], we next determined the binding of and cholesteryl ester uptake from HDL and compared it with LDL both in basal and cAMP-stimulated NCI-H295 cells. NCI-H295 cells were pretreated for 24 h with cAMP or vehicle and subsequently incubated at 4 ℃ with different amounts of human LDL or HDL3, which was devoid of the LDL
receptor ligand apoE and therefore could not bind to the LDL receptor (data not shown). Compared to control cells, treatment with cAMP resulted in a similar increase of both LDL and HDL binding to the surface of the NCI-H295 cells (Fig. 8A and B. NB. Note that the axes scales are different). Interestingly, when expressed in protein mass, absolute binding levels of LDL were higher than that of HDL, under both basal and stimulated conditions (Fig. 8A and B). Scatchard analysis of data of two independent experiments with independently prepared LDL and HDL batches resulted in Bmax and Ka values, respectively of 107 ± 5.6 ng.mg- cell protein and 19.9 ± 2.1 µg protein. mL-1 for HDL and 912 ± 236 ng.mg-1 cell protein and 15 ± 5 µg protein.mL-1 for LDL. The Ka values for HDL3 binding to NCI-H295 cells reported in this study are similar to those reported recently by Murao et al. (35 µg.mL-1) [33] and Xu et al. (20 µg.mL-1) [18], which were obtained using CLA-1 overexpressing cells in which CLA-1 is likely to be the major HDL binding protein. By contrast, in CLA-1 transfected insect SF9 cells Calvo et al. [58] obtained a higher binding affinity for HDL (Ka = 2 µg.mL-1). When expressed in mol lipopro- tein.L-1 Bmax values were 0.747 ± 0.004 pmol·mg cell pro- tein-1 for HDL and 1.625 ± 0.389 pmol.mg cell protein-1 for LDL. Therefore, the number of HDL and LDL binding sites are within the same order of magnitude; there are approximately twice as many LDL than HDL binding sites. However, calcu- lation of Ka values indicated that affinity (which is proportional to 1/Ka) for LDL (Ka = 28.3 ± 9.5 nm) is higher than that for HDL (Ka = 138.1 ± 14.8 nM).
When uptake of [125I]HDL was measured at 37 ℃, no significant degradation of HDL apolipoprotein was observed (data not shown), but [125I]LDL uptake (Fig. 9A) and degra- dation (data not shown) was important and stimulated to 224%
A
B
+ CAMP
-
kDa
kDa
82
- CLA-I/SR-BI
121-
82
C
kDa
+ CAMP
-
165
130 -
-LDL-R
A
O-O Control
B
0-O Control
CAMP
CAMP
80
600
LDL binding (ng/mg cell protein)
Q
1
60
(ng/mg cell protein)
HDL binding
400
40
200
20
0
0
02 5 10
25
50
02 5 10
50
LDL (ug/ml medium)
HDL(ug/ml medium)
25
of control by cAMP. The basal uptake of [125]]LDL corresponds to an incorporation rate of ~1100 ng cholesteryl ester·h-1 mg cell protein-1.
In order to compare the relative contribution of HDL and LDL to deliver cholesterol to the human adrenal cortex, the cholesteryl ester uptake from HDL and LDL was measured in basal and cAMP-stimulated NCI-H295 cells. NCI-H295 cells were pretreated for 48 h with cAMP or vehicle and subsequently incubated at 37 ℃ with human HDL or LDL labelled in vitro with a [3H]cholesteryl ether, which cannot be metabolized intracellularly into steroid hormones and therefore allows accurate measurement of cholesteryl ester uptake; intracellular radioactivity was measured 0.5 or 5 h after incubation (Fig. 9). Results from two independent experiments using different HDL3 and LDL preparations demonstrated that, compared with vehicle treated control cells, cAMP treatment for 48 h significantly increased cholesteryl ester uptake both from HDL3 (+55%) or from LDL (+67%). At the same protein concentration (25 µg-mL-1), in basal conditions (in the absence of cAMP stimulation) the cholesteryl ester uptake from LDL (423 ± 115 ng cholesteryl ester-mg cell protein-1.h-1) was about 20 times more efficient than from HDL (20.7 ± 2.3 ng cholesteryl ester·mg cell protein-1·h-1). Interestingly, under cAMP stimulation intracellular free and esterified cholesterol pools varied, respectively, from 22.9 ± 1.8 and 21.8 ± 2.6 µg-mg cell protein-1 in untreated control cells to 20.1 ± 1.7 and 14.1 ± 1.7 µg.mg cell protein-1 after cAMP treatment, indi- cating that cAMP treatment results in an increased utilization of
cholesterol from the esterified cholesterol pool for steroido- genesis without changing the free cholesterol concentrations.
DISCUSSION
The results from this study show that the human SR-BI/CLA-1 gene is expressed in the fetal and adult adrenal cortex. Comparison of SR-BI expression between different human tissues and cell lines indicates that, of all tissues and cell lines examined, SR-BI is expressed at highest levels in cells from the adrenal cortex. Moreover, SR-BI expression is detected in human primary adrenocortical cells as well as in the human adrenocortical carcinoma cell line NCI-H295, indicating that in man SR-BI is actually produced in the steroid-synthesizing cells of the adrenal cortex. These observations in human adreno- cortical NCI-H295 cells are in line with other reports which appeared while this work was in progress [26,32,33]. Therefore, the expression pattern of human SR-BI is similar to the one in rodents, in which the highest expression of SR-BI mRNA and protein are observed in steroidogenic tissues, such as the adrenal cortex and ovary [30,31].
Both in primary adrenocortical [32] as well as in NCI-H295 cells (this study), SR-BI expression is induced to a comparable level by activation of intracellular second messenger systems, which mediate the effects of specific tropic peptide hormones on adrenal steroidogenesis. In the adrenal cortex, ACTH via cAMP activates steroidogenesis resulting in an increased production of glucocorticoid hormones by the zonae fasciculata and
2000
A
O-O Control CAMP
1
B
Control CAMP
200
LDL uptake (ng/mg cell protein)
(% of control)
Cholesteryl ether uptake
1500
150
1000
Ỗ
100
500
50
0
012
5
10
25
HDL
LDL
LDL (ug/ml)
reticularis. In primary cultures of human adrenocortical cells, ACTH treatment resulted in a significant induction of SR-BI mRNA levels [32]. A similar induction by ACTH has been observed previously in vivo in mice [34] as well as in vitro in murine Y1 adrenal cells [30], indicating conservation between species of SR-BI regulation by the ACTH signalling pathway. Our results show that in NCI-H295 cells SR-BI gene expression is induced by different activators of intracellular cAMP and PKA, the ACTH second messenger signalling pathway. This induction of SR-BI mRNA levels by cAMP parallels the induction of most steroidogenic enzymes, such as P450scc, P450c17 and P450c21 [38].
Both the induction of SR-BI and LDL receptor gene expression by cAMP occur independent of ongoing protein synthesis. Similarly, the induction of P450scc and P450c17 by cAMP are independent of ongoing protein synthesis in NCI-H295 cells, whereas the induction of P450c21 and lipo- protein lipase gene expression by cAMP have been shown to be, respectively, inhibited and superinduced by CHX treatment in NCI-H295 cells [38,59]. Interestingly, as observed for lipopro- tein lipase expression in NCI-H295 cells [59], basal LDL receptor, but not SR-BI mRNA levels increase transiently after addition of the protein synthesis inhibitor, CHX, suggesting that LDL receptor gene expression is negatively controlled by a labile protein in the adrenal gland. A similar induction by CHX has been observed previously in human hepatoma HepG2 and monocytic leukaemia THP-1 cells [60].
Results from nuclear run-on and ActD transcription inhibition experiments show that, similar to the major steroidogenic enzymes, P450scc, P450c17 and P450c21 [38,59], cAMP treatment induces both SR-BI and LDL receptor expression at the transcriptional level. This transcriptional induction of the LDL-R and SR-BI genes may occur via cooperation between cAMP/PKA-activated transcription factors of the CREB/CREM (cAMP response element-binding protein(s)/cAMP response element modulator) family [61-65], different specific adrenal- enriched transcription factors and ubiquitous transcription factors, such as Sp1, binding to cAMP-responsive elements and to adjacent sites [66]. Specific adrenal-enriched transcription factors, such as NGFI-B/nur77, SF-1/Ad4BP and DAX-1 [67-70], have been shown to enhance the cAMP responsiveness of various genes involved in steroidogenesis [63,71-73]. Interestingly SF-1 has been shown to regulate transcription from the SR-BI gene promoter [26]. Whether these adrenal- enriched transcription factors participate in the basal and cAMP- mediated expression of the SR-BI and LDL receptor genes remains to be determined.
Quantitative differences are observed between LDL receptor and SR-BI expression and activity in human adrenocortical cells. First, in comparison with SR-BI, LDL receptor gene expression is much more rapidly induced already reaching a maximum after 3 h, whereas SR-BI expression increases more gradually and reaches a maximum only after 24 h of cAMP treatment. In addition, in all experiments, the level of induction of SR-BI expression was consistently lower compared with the induction of the LDL receptor. Second and most important, binding of HDL and, especially, cholesteryl ester uptake from HDL are quantitatively less important than these functions from LDL. Although the number and affinity of the binding sites for LDL are higher than for HDL, this higher efficiency of LDL to deliver cholesteryl esters to the adrenal cells is determined mostly by the high cholesteryl ester level in LDL (41% of the LDL mass) compared with the lower level in HDL (15% of the HDL3 mass) as well as to the lower molecular mass of HDL particles. Although binding and cholesterol uptake from HDL
reflects the contribution of the entire HDL pathway and the relative contribution of the SR-BI vs. other, as yet unidentified, HDL receptors cannot be assessed, our ligand blot data indicate that a protein of similar size to SR-BI represents the major HDL-binding protein present in NCI-H295 cells and immuno- blot experiments confirm that this protein is SR-BI. These observations suggest that both the total HDL as well as the SR-BI specific pathways are lesser contributors to adrenal cholesterol delivery under normal physiological conditions. Together with the obviously limited intracellular cholesteryl ester pools, the LDL receptor pathway would then be the principal pathway providing cholesterol for steroidogenesis during an acute stress response. Therefore, and in contrast with rodents [8], our data indicate that the SR-BI pathway may not be the predominant pathway under normal physiological conditions in humans [10].
Nevertheless, as the induction of SR-BI expression is associated with both increased binding of HDL and uptake of HDL-derived cholesteryl esters by the adrenal cell, the SR-BI pathway may be part of a back-up system providing cholesterol for steroidogenesis to the human adrenal gland under certain pathological conditions. The existence of such alternative pathways to the LDL receptor for cholesterol delivery to the adrenal gland in humans has been suggested by the observation that patients homozygous for familial hypercholesterolemia, who are deficient in LDL receptors, show no major abnormal- ities of adrenal steroid synthesis, except for a slightly lowered response to ACTH stimulation [19-21]. Similarly, patients suffering from abetalipoproteinema, and therefore lacking LDL in plasma, also have normal basal corticosteroid levels, but display a slightly reduced ACTH response [22,23,74]. Our results suggest that these slight abnormalities may be, at least in part, due to the much slower response of SR-BI expression compared with the LDL receptor upon stimulation with ACTH. These observations corroborate the existence of functional, back-up systems for cholesterol delivery to the adrenal gland in humans, probably involving SR-BI. However, as SR-BI has been shown to bind both native LDL and HDL, both lipoprotein classes may contribute cholesterol for steroidogenesis through this pathway. Altogether, these data suggest that the LDL receptor responds immediately to stimulation of adrenal steroidogenesis, whereas the HDL receptor may provide a slower inducible back-up system.
In conclusion, these results demonstrate the presence of SR-BI mRNA in the steroid-synthesizing cells of the foetal and adult adrenal cortex as well as in adrenocortical carcinoma NCI-H295 cells. In primary adrenal and NCI-H295 cells SR-BI and LDL receptor expression are regulated coordinately by ACTH and activators of the PKA second messenger pathway, in a manner similar to that of the steroidogenic enzymes. However, our data indicate that under normal physiological conditions the HDL pathway may be of lesser importance than the LDL pathway in providing cholesterol to human adrenal cortex cells.
ACKNOWLEDGEMENTS
This research was sponsored by grants from INSERM, the Région Nord- Pas de Calais and the Medical Research Council of Canada (#MT-12901). We are grateful to B. Derudas and C. Copin for expert technical assistance.
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