Developmental and Hormonal Regulation of Murine Scavenger Receptor, Class B, Type 1
Guoqing Cao, Liping Zhao, Herbert Stangl, Tomonobu Hasegawa, James A. Richardson, Keith L. Parker, and Helen H. Hobbs
Departments of Molecular Genetics (G.C., H.S., H.H.H.), Internal Medicine (L.Z., K.L.P., T.H., H.H.H.), and Pathology (J.A.R.) University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75235
The scavenger receptor, class B, type I (SR-BI), is the predominant receptor that supplies plasma cholesterol to steroidogenic tissues in rodents. We showed previously that steroidogenic factor-1 (SF-1) binds a sequence in the human SR-BI pro- moter whose integrity is required for high-level SR-BI expression in cultured adrenocortical tumor cells. We now provide in vivo evidence that SF-1 regulates SR-BI. During mouse embryogenesis, SR-BI mRNA was initially expressed in the genital ridge of both sexes and persisted in the developing testes but not ovary. This sexually dimorphic ex- pression profile of SR-BI expression in the gonads mirrors that of SF-1. No SR-BI mRNA was detected in the gonadal ridge of day 11.5 SF-1 knockout embryos. Both SR-BI and SF-1 mRNA were ex- pressed in the cortical cells of the nascent adrenal glands. These studies directly support SF-1 partic- ipating in the regulation of SR-BI in vivo. We exam- ined the effect of cAMP on SR-BI mRNA and pro- tein in mouse adrenocortical (Y1-BS1) and testicular carcinoma Leydig (MA-10) cells. The time courses of induction were strikingly similar to those described for other cAMP- and SF-1-regu- lated genes. Addition of lipoproteins reduced SR-BI expression in Y1-BS1 cells, an effect that was reversed by administration of cAMP analogs. SR-BI mRNA and protein were expressed at high levels in the adrenal glands of knockout mice lack- ing the steroidogenic acute regulatory protein; these mice have extensive lipid deposits in the adrenocortical cells and high circulating levels of ACTH. Taken together, these studies suggest that trophic hormones can override the suppressive ef- fect of cholesterol on SR-BI expression, thus en- suring that steroidogenesis is maintained during stress. (Molecular Endocrinology 13: 1460-1473, 1999
0888-8809/99/$3.00/0 Molecular Endocrinology Copyright @ 1999 by The Endocrine Society
INTRODUCTION
Steroidogenic cells require cholesterol to maintain their plasma membranes and support the synthesis of steroid hormones. Cholesterol for steroidogenesis is preferen- tially obtained from circulating lipoproteins, but also can be acquired from endogenously synthesized cholesterol or hydrolysis of intracellular cholesterol esters (1-3). Both low-density lipoproteins (LDL) and high-density lipopro- teins (HDL) can deliver cholesterol to support steroido- genesis, with the relative contributions of these two li- poproteins differing among species.
More than 20 yr ago, HDL was found to be the major source of cholesterol for steroidogenesis in rodents (4-6). The dominant role of HDL in maintaining cho- lesterol ester stores in steroidogenic tissues is re- flected by the marked lipid depletion seen in the ad- renocortical cells of mice lacking apolipoprotein AI, the major apolipoprotein of HDL (7). The adrenal cortical cells of rats made hypolipidemic by treatment with either high-dose estrogen or 4-aminopyrazolopyrimi- dine are also lipid depleted (5, 8, 9).
Cholesterol delivery to steroidogenic tissues from HDL differs from the well characterized LDL receptor pathway (5, 6). The cholesterol uptake from HDL is selective; lipids are transported into the cell without the concomitant uptake and degradation of the apo- lipoproteins (10). In contrast, after LDL binds to its cell surface receptor, the entire particle is taken up by receptor-mediated endocytosis and delivered to lyso- somes, where the apoproteins are degraded and the cholesterol esters are enzymatically hydrolyzed to re- lease cholesterol (11).
The protein that mediates the selective uptake of lipids from HDL was identified 3 yr ago by Krieger and col- leagues (12) and named scavenger receptor, class B, type I (SR-BI) (12). SR-BI is expressed at highest levels in those tissues and cell types most active in selective uptake in vivo: the liver, the zona fasciculata and zona reticularis of the adrenal glands, the theca cells and corpus luteum of the ovaries, and the Leydig cells of the testes (12-16). Antibodies to the extracellular domain of
SR-BI block HDL-cholesterol ester uptake and HDL- stimulated synthesis of steroids in cultured mouse adre- nocortical cells (17). This finding is consistent with SR-BI playing a major role in supplying steroidogenic cells with cholesterol. Furthermore, SR-BI knockout mice have lip- id-depleted adrenal glands and a 2- to 3-fold increase in plasma levels of HDL-cholesterol (18). Mice with a re- duced amount of SR-BI showed decreased selective uptake of cholesterol esters (19).
Trophic hormones, acting by a cAMP-dependent protein kinase pathway (20), induce the expression of both the LDL receptor and SR-BI (9, 13, 21, 22). Tro- phic hormones fail to increase LDL receptor activity in adrenocortical cells when steroidogenesis is inhibited and the intracellular cholesterol content is maintained by the addition of exogenous lipoproteins (9). These observations are consistent with the model in which trophic hormones deplete intracellular cholesterol stores by stimulating steroidogenesis and thereby in- directly increase LDL receptor activity (23).
The mechanism by which trophic hormones up-regulate SR-BI expression is not known. SR-BI levels are elevated in the adrenal glands of multiple strains of genetically manip- ulated mice that are hypolipidemic, including some apoAl- mice (24) and mice in which the lecithin-choles- terol acyl transferase or the hepatic lipase genes have been inactivated (24, 25). These observations suggest that the levels of SR-BI, like the LDL receptor, may be regulated by the intracellular content of cholesterol.
cAMP acts synergistically with the nuclear hormone receptor steroidogenic factor 1 (SF-1) to activate the genes encoding multiple components of the steroido- genic pathway (26). All cytochrome P450 steroid hy- droxylases and the steroidogenic acute regulatory protein (StAR), which mediates transport of choles- terol from the cytoplasm to the inner mitochondrial membrane, are regulated by SF-1 via SF-1-responsive promoter elements (27, 28). The human CLA-1/SR-BI gene also contains a consensus SF-1 binding motif in its promoter region (29). We previously showed that SF-1 binds to this site in a sequence-dependent man- ner and that this element is required for high-level expression of SR-BI promoter constructs in cultured adrenocortical cells (29).
In this paper, we explore the regulation of SR-BI mRNA expression in steroidogenic tissues of the mouse and examine the relative roles of trophic hor- mones (via cAMP and SF-1) and the intracellular con- centration of cholesterol in regulating the levels of SR-BI in cultured mouse Yl adrenocortical cells.
RESULTS
SR-BI mRNA Is Expressed in the Undifferentiated Urogenital Ridge
To determine the ontogeny of SR-BI expression, in situ mRNA hybridization studies were performed in sec- tions from mouse embryos. The antisense and sense SR-BI probes used in these studies were generated
from a 2.4-kb mouse SR-BI cDNA fragment. The sen- sitivity and specificity of the probes were confirmed with sections of adult mouse adrenal tissue (Fig. 1A). An intense signal was seen in the cortical region of the adrenal gland using the antisense probe, but not the sense probe. In all subsequent in situ mRNA hybrid- ization studies, no signal was apparent using the sense SR-BI probe (data not shown).
To examine the relationship between SR-BI and SF-1 gene expression, in situ mRNA hybridization studies were performed during early embryonic development. During mouse development, the urogenital ridge, which is the an- lage for ovaries, testes, adrenal cortex, and part of the kidney, first appears as a mesenchymal thickening on em- bryonic day 9.5 (E9.5). Shortly thereafter, the genital ridge emerges as a structure distinct from the mesonephros. Serial sagittal sections of male and female mouse embryos were analyzed by in situ mRNA hybridization using anti- sense probes to murine SR-BI and SF-I. Bright-field views of male and female embryos from E10.5 and E11.5 are shown in the left panels of Fig. 1B. Hybridization with the SF-1 antisense probe revealed a discrete linear band of staining in the urogenital ridges of both female and male embryos (middle panels). This is consistent with prior stud- ies showing that SF-1 mRNA can be detected as soon as the urogenital ridge forms (30).
Adjacent sections from E10.5 and E11.5 embryos were hybridized with the SR-BI antisense probe. No staining was seen in the urogenital ridge on E10.5 in either male or female embryos (panels C and F); the only tissue that stains at this early time point is the premordial liver. On E11.5, a signal for SR-BI mRNA was present in the genital ridge of the male embryo (panel I), but only a marginal signal was apparent in the female embryo (panel L). Thus, SR-BI mRNA is first expressed in the genital ridge of the male embryo at E11.5, which is ap- proximately 2 days after the first appearance of SF-1 mRNA and 1 day after the initial appearance of StAR (31) and the cholesterol side chain cleavage enzyme (SCC) (30). The level of SR-BI mRNA expression in the genital ridge at this stage was lower in female than male mice. In contrast to SR-BI, no sex-dependent differences in the expression levels of StAR or SCC mRNA were apparent at this time point (30, 31).
No SR-BI mRNA was detected in the hepatic pri- mordia at day 9.5, which is the first day this structure can be identified (data not shown). In both the male and female embryos, low-level staining was apparent at E10.5 within the septum transversum of the hepatic/ biliary primordia. Significant levels of SR-BI mRNA expression were detected in the liver primordia on E11.5 of the male and female embryos (panels I and L).
Sexually Dimorphic Tissue Expression of SR-BI Mirrors SF-I during Fetal Development
In male mice, the testes become histologically distinct at approximately E12.5 as they organize into round, cord-like structures (the testicular cords), which con- tain both Sertoli cells and primordial germ cells and
A
SR-BI Anti-sense
SR-BI Sense SR-BI
B
SF-1
A
B
C
- Li
E10.5 07
G
D
E
F
E10.5 ㅎ
Li
G
G
Li
H
I
E11.5
07
G
J
~ Li
K
L
E11.5
ㅎ
G
A, Localization of SR-BI mRNA in sections of adult mouse adrenal glands. A bright-field view (left panel) and dark-field microscopy (middle and right panel) of mouse adrenal gland after hybridization with a SR-BI antisense (middle panel) and sense (right panel) probes are shown. Serial sagittal sections of the adrenal gland from an adult mouse were prepared and hybridized with radiolabeled antisense and sense probes derived from the murine SR-BI cDNA as described in Materials and Methods. The sections were exposed to emulsion for 2 weeks before development. B, Localization of SF-1 and SR-BI mRNA in male and female murine embryos on E10.5 and E11.5. Serial sagittal sections of mouse embryos obtained at days 10.5 and 11.5 of pregnancy were prepared as described in Materials and Methods. The sections were incubated with antisense SF-1 or SR-BI radiolabeled RNA probes. Panels A, D, G, and J are bright-field views of the embryos. Panels B, E, H, and K and panels C, F, I, and Lare dark-field views of adjacent sections hybridized with SF-1 or SR-BI antisense RNA probes, respectively. The sections were incubated with emulsion for 3 weeks before development. Green and red fluorescent filters were used for the sections hybridized with the SF-1 and SR-BI probes, respectively. G, Gonadal ridge; Li, liver primordia.
ultimately develop into seminiferous tubules. The Ley- dig cells, which reside between the testicular cords in the interstitial region, synthesize testosterone. High levels of expression of both SF-1 and SR-BI mRNAs are seen within the testes during this time period (Fig. 2A). The patterns of expression of SF-1 and SR-BI mRNAs within the testes were overlapping but not identical (Fig. 2A). SR-BI mRNA has a more restricted and punctate pattern of distribution, with patches of intense signal corresponding to the Leydig cells, thus resembling patterns previously reported for SCC and StAR (30, 31). The signal for SF-1 was more general- ized, consistent with expression in both fetal Sertoli and Leydig cells at this stage of development (30).
In contrast to the testis, the ovary does not produce significant quantities of steroid hormones before pu- berty. SF-1 is expressed in the indifferent gonad of both sexes, and expression persists in the murine ovary at E12.5 (Fig. 2B, panel B). Thereafter, expres- sion levels decline, so that only a trace signal was seen in the E16.5 ovary. Little or no signal for SR-BI was present in the ovaries of the embryos at E12.5, E14.5, and E16.5 (panels C, F, and I). Once again, the pattern of expression of SR-BI in the embryonic ovary resem- bles those described previously for SCC and StAR, as neither SCC nor StAR mRNA is detected in the murine ovary from E12.5 to E16.5 (30, 31).
The expression of SR-BI in the liver was similar in the male and female embryos. SR-BI was expressed in the liver at E12.5 and E14.5 (panels C and F in Fig. 2) but the levels declined by E16.5. In contrast, prior immunocytochemical studies failed to detect SR-BI in the murine fetal liver through E17.5 (32). The apparent discrepancy between the in situ hybridization and im- munocytochemical studies most likely reflects differ- ent sensitivities of the assays. It also is possible that an alternatively spliced form of SR-BI, which is not rec- ognized by the antibody used for the immunocyto- chemical studies, is expressed in the liver at these time points. The SR-BI gene is alternatively spliced at its 3’-end (33). The antibody used for the immunocyto- chemical studies only detects the major form of SR-BI (referred to as SR-BI or SR-BI.1) (12), whereas our antisense probe detects both SR-BI and SR-BII (also called SR-BI.2). More likely, the absence of any im- munodetectable hepatic SR-BI in murine embryos at these time points reflects a level of expression that is too low or too diffuse to detect by immunostaining.
From these studies, we conclude that SR-BI expres- sion in the developing gonads is sexually dimorphic and generally correlates with the expression of SF-I. The pattern and timing of expression of SR-BI in the developing embryo closely resemble those described for the transcripts of two other key participants in the steroid biosynthetic pathway, SCC and StAR (30, 31).
SR-BI is Expressed at High Levels in the Fetal Adrenal Gland
SF-1 transcripts can be detected at E12.5 in the cells that comprise the adrenal primordium (30). Within 24 h, high levels of expression of both SF-1 and SR-BI
transcripts were seen throughout the adrenal gland (Fig. 3, panels A-C). These results correlate well with prior immunocytochemical studies that revealed the first expression of immunodetectable SR-BI in the ad- renal at E14.5 (32). By E16.5 the chromaffin cell pre- cursors have migrated into the central portion of the gland to form the adrenal medulla. SF-1 and SR-BI are not expressed in the adrenal medulla (13), so the stain- ing pattern of the adrenal gland by E16.5 is doughnut shaped.
SR-BI is Not Expressed in the Genital Ridge of SF-1 Knockout Mice, but Is Expressed in the Liver
The expression of SR-BI was also examined in E11.5 male SF-I knockout embryos (Ftz-F1-/-) (34). As shown in Fig. 4, comparable levels of SR-BI mRNA were detected in the developing liver of the Ftz-F1-/- and wild-type embryos, consistent with the fact that SF-1 is not expressed in the liver (30) and thus cannot regulate SR-BI expression in this tissue. A weak SR-BI signal was apparent in the gonadal ridge of the wild- type embryo (bottom panel, left). In contrast, no SR-BI signal was present in the genital ridge of the Ftz-F1-/- embryo (bottom panel, right). To the extent that the developing gonads are still intact at this relatively early stage of development, this finding suggests that SF-I plays an important role in the regulation of SR-BI ex- pression in this tissue in vivo.
Regulation of SR-BI mRNA and Protein Levels by CAMP Analogs in Cultured Murine Steroidogenic Cells
ACTH administration to mice dramatically increases the expression of immunodetectable SR-BI protein in the adrenal cortex, especially in the zonae fasiculata and reticularis (18). Human CG (hCG) administration causes a similar increase in SR-BI expression in the Leydig cells of the rat testes (13). Both ACTH and hCG exert their effects by activating protein kinase A, and their effects can be mimicked by cAMP analogs (20). The time course of the up-regulation of SR-BI protein expression by cAMP was examined in mouse adreno- cortical tumor cells (Y1-BS1) and in a murine testicular Leydig carcinoma cell line (MA-10). Immunoblot anal- ysis was performed using a polyclonal antibody di- rected against the last 14 amino acids of murine SR-BI (12). A 2-fold increase in the level of immunodetect- able SR-BI protein was seen in the Y1-BS1 cells within 4 h, reaching a maximum 5-fold induction at 24 h (Fig. 5A).
Only a trace amount of immunodetectable SR-BI protein was seen in the MA-10 cells before the addi- tion of (Bu)2CAMP (Fig. 5A), which is compatible with the prior observation that MA-10 cells produce almost no steroids unless stimulated with cAMP (35). An in- crease in SR-BI protein levels was detected within 4 h of the addition of the cAMP analog, and by 24 h the
A. MALE
SF-1
SR-BI
A
B
C
E12.5
Li
07
Ts
D
Li
E
F
E14.5
07
Ts
G
Li
H
I
E16.5
07
Ts
B. FEMALE
SF-1
SR-BI
A
·Li
B
C
E12.5
9
Ov
D
Li
E
F
E14.5
ㅎ
Ov
G
H
I
E16.5
?
Ov
The embryos were prepared and incubated with antisense SF-1 and SR-BI probes as described in Fig. 1. The sections were exposed to emulsion for 3 weeks before development. Bright-field (left panel) and dark-field views (middle and right panels) of the sections are shown. Ts, Testis; Li, liver; Ov, ovary.
SF-1
SR-BI
A
B
C
E13.5
A
D
E
F
E14.5
G
H
I
E16.5
&
A
level of SR-BI protein had increased more than 20-fold.
The antibody used in these studies detects only the SR-BI/SR-BI.l transcript, which is the predominant mRNA transcript in steroidogenic cells (36). The other mRNA transcript, SR-BII (or SR-BI.2), differs in se- quence in the last 39 amino acids and thus is not detected by our antibody (33). To determine whether (Bu)2 CAMP increases the levels of both the SR-BI and SR-BII transcripts, we used an RNase protection as- say to assess the relative levels of the two transcripts in the Y1-BS1 and MA-10 cells. When we used this assay to assess the relative proportion of SR-BI and SR-BII in the testes, approximately 75% of the total SR-BI mRNA was SR-BII (data not shown), which is similar to that previously reported (36). At all time points, more than 95% of the total mRNA (SR-BI plus SR-BII) in both Y1 cells and MA-10 cells was SR-BI (Fig. 5B). Moreover, a pronounced increase in the lev- els of SR-BI mRNA was seen in both cell lines, with a time course congruent with that seen for SR-BI protein (Fig. 5A). Thus, there is no evidence for differential
regulation of the two mRNA transcripts in response to (Bu)2 CAMP in the Y1 and MA-10 cells.
SR-BI Levels Are Regulated by Intracellular Cholesterol Levels Independently of Trophic Hormones in YI-BSI Cells at Both a Transcriptional and Posttranscriptional Level
Cholesterol substrate for adrenal steroidogenesis comes from three sources: endogenous synthesis from acetyl CoA, hydrolysis of intracellular cholesterol esters, and uptake from circulating lipoproteins. ACTH dramatically up-regulates expression of SR-BI in the adrenocortical cells of the mouse adrenal gland (18). Is the up-regulation of SR-BI a direct effect of trophic hormones, or the result of depletion of intracellular cholesterol due to the induction of steroidogenesis?
To determine whether SR-BI levels are regulated by changes in the intracellular concentration of choles- terol, we inhibited endogenous cholesterol synthesis with compactin (10 (M), and steroidogenesis with ami- noglutethimide (5 µg/ml). To document that steroid
WT
KO
L
L
L
L
hormone production was effectively inhibited, we measured the level of progesterone in the medium, which fell into the undetectable range (data not shown). Aminoglutethimide treatment without the ad- dition of lipoproteins did not alter the level of SR-BI (data not shown). Addition of 50 µg/ml LDL to the media of aminoglutethimide-treated cells resulted in a 4-fold decrease in the amount of immunodetectable SR-BI protein (Fig. 6A). No further decrease in the level of SR-BI was seen when the concentration of LDL in the media was increased to 100 µg/ml.
To indirectly assess the concentration of cholesterol in the endoplasmic reticulum of the cells, we examined the level of 3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase protein. HMG-CoA reductase is regulated by sterols both at the transcriptional and posttranslational levels (37). The protein was readily detected in the cholesterol-depleted YI-BSI cells. No change in the level of HMG-CoA reductase was seen upon addition of aminoglutethimide in the absence of lipoproteins (data not shown). Addition of LDL to the medium was associated with a marked reduction in the level of immunodetectable HMG-CoA reductase as well as LDL receptor, reflecting an increase in the delivery of cholesterol from the lipoproteins to the endoplasmic reticulum (Fig. 6A).
To determine whether the decrease in SR-BI protein was due to a reduction in the level of SR-BI mRNA, we used our RNase protection assay to assess the effect of aminoglutethimide and LDL on the amount of SR-BI and SR-BII mRNA. The levels of both SR-BI and SR- BII fell by approximately 20% with the addition of LDL (Fig. 6B). Under these conditions, the reduction in the level of SR-BI mRNA was less than that of immuno- detectable SR-BI (Fig. 6A), suggesting posttranscrip- tional regulation of SR-BI. This could be caused by a decrease in the efficiency of mRNA translation or an increase in the degradation of SR-BI protein. Alterna- tively, the epitope that binds the anti-SR-BI antibody may be masked under these conditions.
To differentiate between these models, we as- sessed the half-life of SR-BI by performing a pulse- chase experiment. After a 1-h pulse, the level of im- munoprecipitable SR-BI declined over the 12-h chase at the same rate in the cells incubated in the presence of absence of aminoglutethimide and LDL. The t1/2 in both is 7 h (Fig. 7), which is slightly longer than what was previously reported for recombinant SR-BI that was stably expressed in Chinese hamster ovary cells (38). These results suggest that LDL and aminoglute- thimide treatment does not decrease SR-BI protein by increasing the rate of degradation of SR-BI.
A
B
Time(h)
0246824
Time(h) 0 4 8 24 0 4 8 24
Y1-BS1
SR-BI-
Actin
MA-10
SR-BI
Actin
SR-BII
Y1-BS1
MA-10
CAMP-Mediated Regulation of SR-BI
To determine the hierarchy of regulation of SR-BI ex- pression, we examined the levels of immunodetect- able SR-BI in YI-BSI cells in the presence of both a cAMP analog and lipoproteins. Y1-BS1 cells were in- cubated in newborn calf lipoprotein-deficient serum (NLPPS), 10 µM compactin, and 5 µg/ml of aminoglu- tethimide to block steroidogenesis. Increasing con- centrations of LDL were added to the cells. After 24 h, the levels of immunodetectable SR-BI, HMG-CoA re- ductase, and LDL receptor were assessed by immu- noblotting. As shown in Fig. 8, addition of LDL to the cells progressively decreased the immunodetectable levels of all three proteins.
Treatment with 8-Br-cAMP resulted in 11- and 2-fold increases in SR-BI and HMG-CoA reductase, respectively. No change was seen in the level of LDL receptor under these conditions. Increasing concen- trations of LDL progressively decreased the levels of all three proteins, although the magnitude of the
A
B
LDL (ug /ml)
-
50
100
LDL (ug/ml)
-
50
100
SR-BI-
SR-BI- Actin -
HMG-COA-
SR-BI
-Actin
LDLR
- SR-BII
A, Immunoblot analysis of SR-BI, HMG-CoA reductase, and the LDL receptor in Y1-BS1 cells grown in the presence of LDL and aminoglutethimide (5 µg/ml). Cells were grown according to the protocol outlined in Fig. 5. On day 4, after a 24-h incubation in medium A with 10% NLPPS and 10 µM compactin, 5 µg/ml aminoglutethimide and LDL were added to the cells at the indicated concentrations. After 24 h, the cells were collected, cellular lysates were prepared, and im- munoblot analysis was performed as described in the legend to Fig. 5. B, Total RNA was prepared from the cells and 10 µg RNA were subjected to the RNase protection assays to as- sess the amount of SR-BI mRNA as described in the legend to Fig. 5.
changes in protein levels differed. HMG-CoA reduc- tase and LDL receptor levels fell by 94% and 68%, respectively, when 100 µg/ml of LDL were added to the cells for 24 h. SR-BI levels remained significantly elevated, decreasing by only 20% under these same conditions.
Taken together, these experiments demonstrate that SR-BI is regulated by both cAMP analogs and the intracellular content of cholesterol; the effect of the cAMP analog appears to largely override the suppres- sive effect of the increase in intracellular cholesterol.
SR-BI Expression in the Adrenal Glands of Newborn StAR-/- Knockout (StAR-/-) Mice
StAR-/- mice are unable to efficiently transport cho- lesterol from the cytoplasm across the mitochondria membrane and thus accumulate cholesterol within their steroidogenic cells (39). These mice have ele- vated circulating levels of ACTH due to their adrenal insufficiency (39). In situ hybridization studies were conducted to compare the levels of SR-BI transcripts in newborn StAR-/- and wild-type mice (Fig. 9A). Although the adrenocortical architecture is distorted by the abundant lipid deposits in the StAR-/- adrenals (top panels), the level of SR-BI transcripts is high (bot- tom panels). Thus, the levels of SR-BI mRNA are high in the StAR-/- adrenocortical cells despite their in- creased cellular content of cholesterol, presumably due to the elevated circulating levels of ACTH.
DISCUSSION
Previously we showed that the tissue- and cell-spe- cific distribution of SR-BI protein and mRNA overlaps with that of SF-I in adult rodents and humans (13, 29).
Chase (h)
0 2 4 6 8 12
kDa
Control
- 80
AMG/LDL
- 80
The cells grown in standard conditions until day 2, when the medium was switched to medium A plus 10% NLPPS. After 24 h, the cells were grown in methionine- and cysteine- free medium for 30 min before the addition of 200 uCi/ml of [35S]methionine for 1 h. Then the medium was changed to medium A with 2 mm cold methionine with or without 50 µg/ml LDL, 10 µM compactin, and 5 µg/ml aminoglutethim- ide. The cells were lysed at the indicated time points and immunoprecipitated using a rabbit antimouse SR-BI poly- clonal antibody directed against the last 14 amino acids, as described in Materials and Methods. A total of 35% of the immunoprecipitate was size fractionated on an 8% SDS- polyacrylamide gel. The gel was subjected to Kokak Safety film (Eastman Kodak Co., Rochester, NY) with an intensifying screen for 2 weeks.
We now extend these studies by demonstrating that the temporal and spatial patterns of expression of SF-1 and SR-BI mRNA are similar in the steroidogenic tissues of developing mouse embryos. SR-BI mRNA is expressed in the urogenital ridge shortly after the first appearance of SF-1. SR-BI is expressed in the embry- onic testes, which are active in steroidogenesis, but not in the early developing ovaries, which do not ini- tiate steroidogenesis until late in gestation. This sex- ually dimorphic pattern of SR-BI expression during mouse embryogenesis mirrors that of SF-1. In the adrenal gland, the time course and pattern of expres- sion of SR-BI mRNA and SF-1 were indistinguishable and exhibited no sexual dimorphism.
The timing and cell-specific distribution of expres- sion of SR-BI within the murine embryonic adrenal glands and gonads are similar to those of two other genes that encode proteins in the steroidogenic path- way, StAR and SCC (30, 31). StAR plays a key role in the acute response of steroidogenic cells to trophic hormones by mediating transport of cholesterol from cytoplasm to the inner mitochondrial membrane where SCC catalyzes the first reaction in steroidogenesis (40). The mRNA transcripts for StAR and SCC are first detected in murine embryos 1-2 days after the initial appearance of SF-1 transcripts in urogenital ridge (30, 31). Therefore, the SCC and StAR transcripts are both present in the early developing testes and adrenal glands, but not the ovaries, again resembling the ex- pression pattern of SR-BI. The ontogeny of expression of SR-BI mRNA in the developing embryo, and its similarity to the pattern of expression of two other SF-1-regulated genes (StAR and SCC), strongly sug- gest that SR-BI is part of the coordinated response of steroidogenesis to trophic hormones and plays an important role in embryonic, as well as adult, steroi- dogenic tissues.
LDL(ug/ml)
0
20
50
100
0
20
50
100
8-Br cAMP
-
-
-
-
+
+
+
+
SR-BI
HMG-COA Reductase
LDLR-
1 2 3 4 5 6 7 8
If SF-1 is responsible for activating transcription of SR-BI, then it would be expected that SR-BI expres- sion would not be present in the steroidogenic tissues of mice that do not express SF-1. We could only examine SR-Bl expression in SF-1 knockout mice be- fore E12 because, after this time point, gonads and adrenal primordium undergo apoptosis (30, 35). As shown in Fig. 4, comparable levels of SR-BI transcripts were detected in the developing liver of wild-type and SF-1 knockout embryos at E11.5, consistent with the fact that SF-1 is not expressed in this organ and thus cannot regulate SR-BI. In contrast, SR-BI was ex- pressed in the genital ridge of the wild-type embryo, but not SF-1 knockout mice. To the extent that the developing gonads are still intact at this relatively early developmental stage, this finding suggests that SF-1 plays important roles in SR-B1 expression in vivo.
Although the expression patterns of SF-1 and SR-BI mRNA in the developing rodent are similar, they are not identical. In the testes, SF-1 is expressed in the fetal Sertoli cells as well as Leydig cells. SR-BI has an expression pattern in the testes that is similar to that of SCC and StAR (30, 31); all three transcripts are de- tected only in the steroidogenic cells of the intersti- tium. In other embryonic tissues where SF-1 mRNA is present, like the pituitary and ventromedial hypothal- amus (30), we found no detectable SR-BI transcripts (data not shown). Conversely, SR-BI is expressed at significant levels in some tissues that do not express SF-1, such as the embryonic liver. As discussed in the results, the most likely explanation for the absence of any immunodetectable hepatic SR-BI in murine em- bryos is that the level of expression is either too low or too diffuse to be detected by immunostaining.
To further define the mechanisms that regulate SR-BI in steroidogenic tissues, we examined its ex-
WT
KO
-
pression in cAMP-stimulated cultured murine adreno- cortical (Y1-BS1) and testicular carcinoma (MA-10) cells. SR-BI mRNA and protein levels were dramati- cally increased upon cAMP stimulation in both cell lines. The kinetics of SR-BI induction by cAMP ana- logs in these cell lines differed somewhat from that which was previously reported for StAR (31). The in- crease in SR-BI levels lags behind that of StAR by approximately 2 h. The time course of SR-BI induction in response to cAMP analogs in MA-10 cells is similar to that seen in rat granulosa cells (42). In the unstimu- lated state, these two cell lines are similar in that they synthesize no steroid hormones and express no SR- BI; after cAMP stimulation it takes approximately 6 h
before any detectable increase in SR-BI protein is apparent.
In the absence of any trophic hormones, SR-BI lev- els appear to be regulated by the intracellular content of cholesterol. Our results are compatible with the demonstration that SR-BI expression remains high in desensitized, lipid-depleted, rat luteal cells, which cannot respond to trophic hormones (16); these lipid- depleted cells have high levels of immunodetectable SR-BI protein, as well as HMG CoA reductase and the LDL receptor. In our studies with Y1-BS1 cells, the levels of immunodetectable SR-BI fall into the nonde- tectable range when cells are cultured for 24 h in the presence of LDL (100 µg/ml). The reduction in SR-BI
protein mass was greater than the fall in the level of SR-BI mRNA, which is consistent with a posttranscrip- tional mechanism. We found no evidence that the decrease in SR-BI in the lipoprotein-supplemented Y1-BS1 cells is due to an increased SR-BI degrada- tion. Further studies will be required to define the posttranscriptional mechanism responsible for the ob- served dissociation between the levels of SR-BI mRNA and protein.
Even when Y1-BS1 cells are provided sufficient ex- ogenous LDL to reduce HMG-CoA reductase to trace levels, administration of cAMP resulted in a consider- able increase in SR-BI. These results suggest that trophic hormones up-regulate SR-BI expression di- rectly rather than by depleting intracellular cholesterol stores. These data are consistent with the studies of Dexter et al. (2) who showed that ACTH stimulates the uptake of cholesterol from lipoproteins in the adrenal glands of hypophysectomized rat, even if steroidogen- esis is completely inhibited. The results of our studies in cultured cells are similar to the findings of Gwynne et al. (4), who examined the effect of aminoglutethim- ide on the ACTH-stimulated uptake of radiolabeled cholesterol in rat adrenal slices. They showed that aminoglutethimide did not affect cholesterol uptake despite increasing the cellular cholesterol content by 5-fold. In granulosa cells, like the Y1-BSI cells, high levels of SR-BI were maintained even in the presence of high concentrations of lipoproteins (41).
Further evidence that trophic hormones override the effect of intracellular cholesterol concentrations on SR-BI expression is the finding that SR-BI levels are not decreased in the adrenal glands of StAR knockout mice (39). These mice are unable to efficiently trans- port cholesterol into the mitochondria of steroidogenic tissues and thus fail to synthesize sufficient steroid hormones to suppress pituitary ACTH secretion. As a consequence, the StAR-/- mice accumulate massive amounts of cholesterol in their adrenal glands and have elevated plasma levels of ACTH (39). Despite having cholesterol-laden adrenocortical cells, these mice have normal to elevated levels of SR-BI, which presumably are maintained by the high levels of cir- culating trophic hormones.
Taken together, the results of these studies are con- sistent with SR-BI being part of the repertoire of SF- 1-responsive genes in steroidogenic tissues and the major pathway by which cholesterol is delivered for steroid hormone biosynthesis in the mouse. The phys- iological importance of this regulation may be to en- sure that SR-BI will be up-regulated during times of stress, even if the adrenal gland is replete with cho- lesterol. This regulatory mechanism presumably en- sures that the organism always has a sufficient supply of cholesterol available for maximal steroidogenesis in times of stress. This formulation is consistent with the extensive and careful studies of Reaven et al. (42), who showed that the selective uptake pathway is optimally designed for the dramatic increase in cholesterol de- livery that is required upon stimulation.
Finally, it is important to note that multiple lines of mouse Y1 adrenocortical cells have been developed (20). These cell lines differ in their responsiveness to trophic hormones and are likely to differ in their ex- pression levels of SR-BI. In some of the YI adrenal cell lines, exogenous HDL fails to reduce HMG-CoA re- ductase activity or incorporation of HDL-cholesterol into steroids (23). It is likely that these cell lines have a dysfunctional SR-BI receptor pathway.
MATERIALS AND METHODS
Materials
33P-UTP and 32P-CTP were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Y1-BS1 cells were kindly provided by Bernard Schimmer (University of Toronto, Toronto, Ontario, Canada), and the mouse testicular carci- noma Leydig cells (MA-10) were a kind gift from Mario Ascoli (Department of Pharmacology, University of lowa, lowa City, IA). Cell culture media (DMEM/F-12, and Waymouth’s MB 782/1) were purchased from Life Technologies, Inc. (Gaith- ersburg, MD), FCS, horse serum, (Bu)2cAMP, 8-bromo CAMP, and aminoglutethimide were purchased from Sigma Chemical Co. (St. Louis, MO). The LDL and HDL were pre- pared by ultracentrifugation of human plasma as described (43). A RIA (CT Progesterone Kit) was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, CA) and used according to the recommendations of the manufacturer.
Animals
Timed pregnant NIH Swiss mice were purchased from Harlan Laboratories (Indianapolis, IN). Noon of the day on which the copulatory plug was detected was designated 0.5 day of the gestation (E0.5). Pregnant mice were killed by cervical dislo- cation at the time intervals indicated, and the embryos were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin. Serial sagittal sections of 4 um thickness were pre- pared using a microtome (44). To determine the sex of each embryo, a small aliquot of tissue was taken from the yolk sac and placed in 500 pl of 50 mm KCI, 1.5 mm MgCl2, 10 mm Tris HCI, pH 8.5, 0.01% (wt/vol) gelatin, 0.45% (vol/vol) NP40, 0.45% (vol/vol)Tween 20, and 100 µg/ml proteinase K (Sigma Chemical Co.) at 55 C overnight. PCR was used to amplify a fragment from the mouse SRY gene (45). The amplification reaction included 1 ul of the yolk sac lysate as template and two oligonucleotides (5’-TCATGAGACTGCCAACCACAG-3’ and 5’-CATGACCACCACCACCACCAA-3’). The PCR prod- ucts were size fractionated on a 1% (wt/vol) agarose gel, the gel was stained with ethidium bromide, and the bands were visualized using UV light.
In Situ Hybridization
In situ hybridization was performed as previously described (44). A 2.4-kb fragment containing the mouse SR-BI cDNA (kindly provided by Dr. Monty Krieger, Massachusetts Insti- tute of Technology, Cambridge, MA) was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA). The plasmid was linear- ized using HindIll or Xhol to generate labeled antisense and sense RNA probes, respectively, employing the In Vitro Tran- scription System (Promega Corp., Madison, WI). The probes were partially hydrolyzed by incubating them with 200 mm Na2CO3, pH 10.2, at 60 C for 25 min. Serial sections were deparaffinized and then allowed to hybridize with the probes (1 × 10 (6)cpm/ml) using Hybridization Cocktail (Amresco,
Solon, OH) at 55 C overnight. The sections were washed and the slides were dipped in NTB2 liquid emulsion (Eastman Kodak Co., Rochester,NY) diluted 1:1 in H20. The slides were incubated at 4 C for 23 days and placed in Dektol developing solution (Eastman Kodak Co.) and counterstained with he- matoxylin. Photographs of the slides were taken using a Eclipse E1000M microscope (Nikon, Melville, NY) linked to a video system (Media Cybernetics, Silver Spring, MD). Green and red fluorescent filters was used under dark field illumi- nation for the sections incubated with the SF-1 and SR-BI probe, respectively.
Immunoblot Analysis of SR-BI, LDL Receptor, and HMG-CoA Reductase
The Y1-BS1 cells were maintained in medium A (1:1 mixture of DMEM and Ham’s F-12 medium, plus 100 U/ml penicillin and 100 µg/ml streptomycin sulfate) with 15% horse and 2% FCS. On day 4 the medium was switched to medium A plus 10% NLPPS. After 24 h, the medium was supplemented with 1 mm (Bu)2CAMP. The same protocol was used for MA-10 cells except that the cells were maintained in Waymouths MB 752/1 medium plus 15% horse serum. Cultured cells were washed twice with ice-cold PBS before being collected in 2 ml of PBS. The cells were isolated by centrifugation at 1300 X g for 5 min and resuspended in lysis buffer [1% (vol/vol) Triton, 50 mm Tris, 2 mm CaCl2, 80 mm NaCl, pH 8.2] con- taining protease inhibitors (0.5 mm phenylmethylsulfonylfluo- ride, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, and 2 µg/ml aprotinin). After a 15-min incubation on ice, the samples were centrifuged at 16,000 x g for 10 min. The protein concentra- tion of the lysates was determined using a BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL). Cell lysates for im- munoblots to detect HMG-COA reductase were prepared as described previously (37). Approximately 50 µg of each cell lysate were reduced by the addition of @-mercaptoethanol to 1.5% and then size fractionated on a 6.5% SDS-polyacryl- amide gel. The proteins were transferred to Hybond C Extra Transfer membrane (Amersham Pharmacia Biotech), and im- munoblot analysis was performed using a rabbit antibovine LDL receptor antiserum (1:1000) (46), a rabbit antipeptide polyclonal antibody directed against the last 14 amino acids of mouse SR-BI (10 µg/ml), and IgG-A9, a monoclonal anti- body to HMG-COA reductase (5 µg/ml) (37). Immunoblot analyses were performed using the Enhanced Chemilumines- cence Western Blotting Detection Kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions, and then the filters were exposed to Reflection NEF film (DuPont NEN, Wilmington, DE). The images were scanned into a Power 7500/100 Macintosh computer, and the relative inten- sities of the bands were quantified using NIH Image 1.61 (http://rsb.info.nih.gov/nih-image/download.html).
Immunoprecipitation
On day 0, Y1-BS1 cells were plated at 500,000 cells per well in a 6-well dish and grown for 2 days in medium A plus 15% horse serum and 2% FCS. On day 3 the medium was changed to medium A with 10% NLPPS. After 24 h the cells were incubated in methionine- and cysteine-free DMEM me- dium (ICN Biochemicals, Inc., Costa Mesa, CA) for 30 min and then pulsed with Trans-label methionine-cysteine (200 Ci/ml)(ICN Biochemicals, Inc.) for 1 h. The cells were then chased in medium A plus 10% NLPPS plus 2 mm cold me- thionine with or without 50 µg/ml of LDL, compactin (10 }LM), and 5 µg/ml of aminoglutethimide. Cells were lysed at the indicated time points and SR-BI was immunoprecipitated exactly as described (38) except that the Protein A Sepharose was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
RNase Protection Assay
A 307-bp PCR fragment containing the sequence that en- codes amino acids 397-499 of the mouse SR-BI cDNA was amplified from total murine hepatic RNA using RT-PCR (Stratagene, La Jolla, CA) and two oppositely oriented oligo- nucleotides with the following sequences: 5’-GGGCAAA- CAGGGAAGATCGAGCCA-3’ and 5’-ACCGTGCCCTTG- GCAGCTGGTGAC-3’. The PCR product was subcloned into pGEMT Easy vector (Promega Corp.) and the insert was sequenced. The plasmid was linearized using NcoI, and an in vitro transcription reaction was carried out in the presence of [@-32P]-CTP and SP6 polymerase (Promega Corp.) for 1 h at 37 C using the Riboprobe in vitro Transcription System (Pro- mega Corp.). The reaction product was incubated in 1 U of RQ DNase (Promega Corp.) for 15 min at 37 C to digest the DNA template. The reaction mixture was then diluted with RNase-free water to a final volume of 50 ml, extracted once with 50 ml phenol/chloroform (1:1), and then purified using a G50 spin column (5 Prime→3 Prime, Inc., Boulder, CO). A HybSpeed kit from Ambion, Inc. (Austin, TX) was used for the RNase protection assay. A total of 1 × 105 cpm were mixed with 10 µg of total cellular RNA that was isolated from the cultured cells using RNA STAT (Tel-Test, Friendswood, TX). The RNase protection assay was performed as recom- mended by the manufacturer. The protected fragments were resolved on a 6% denaturing polyacrylamide gel, dried, and exposed to Reflection NEM film for the indicated times. The bands were quantified using a phosphoimager (Fuji Photo Film Co., Ltd., Stamford, CT).
Acknowledgments
We wish to thank Melissa and Tommy Hyatt for their excellent technical assistance. We thank B. Schimmer and M. Ascoli for the kind gift of the Y1-BS1 and MA-10 cells, respectively. The murine SR-BI cDNA was kindly provided by Monty Krieger. We thank David Russell and Hui Tian for helpful discussions.
Received February 1, 1999. Revision received May 17, 1999. Accepted June 3, 1999.
Address requests for reprints to: Helen H. Hobbs, Depart- ment of Molecular Genetics, University of Texas Southwest- ern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235. E-mail: helen.hobbs@email.swmed. edu.
This work was supported by NIH Grants HL-20948 (H.H.H.), DK-54028 (K.L.P.), and the Perot Family Fund.
REFERENCES
1. Morris MD, Chaikoff IL 1959 The origin of cholesterol in liver, small intestine, adrenal gland, and testis of the rat: dietary versus endogenous contributions. J Biol Chem 234:1095-1096
2. Dexter RN, Fishman LM, Ney RL 1970 Stimulation of adrenal cholesterol uptake from plasma by adrenocorti- cotrophin. Endocrinology 87:836-846
3. Borkowski A, Delcroix C, Levin S 1972 Metabolism of adrenal cholesterol in man. J Clin Invest 51:1664-1678
4. Gwynne JT, Mahaffee D, Brewer Jr HB, Ney RL 1976 Adrenal cholesterol uptake from plasma lipoprotein: reg- ulation by corticotropin. Proc Natl Acad Sci USA 73:4329-4333
5. Balasurbramaniam S, Goldstein JL, Faust JR, Brun- schede GY, Brown MS 1977 Lipoprotein-mediated reg-
ulation of 3-hydroxy-3-methylglutaryl coenzyme A reduc- tase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J Biol Chem 252:1771-1779
6. Andersen JM, Dietschy JM 1978 Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J Biol Chem 253:9024-9032
7. Plump AS, Erickson SK, Weng W, Partin JS, Breslow JL, Williams DL 1996 Apolipoprotein A-I is required for cho- lesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production. J Clin Invest 97:2660-2671
8. Andersen JM, Dietschy JM 1976 Cholesterogenesis: de- repression in extrahepatic tissues with 4-aminopyrazolo (3,4-d) pyrimidine. Science 193:903-905
9. Kovanen PT, Goldstein JL, Chappell DA, Brown MS 1980 Regulation of low density lipoprotein receptors by adre- nocorticotropin in the adrenal gland of mice and rats in vivo. J Biol Chem 255:5591-5598
10. Glass C, Pittman RC, Weinstein DB, Steinberg D 1983 Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci USA 80:5435-5439
11. Brown MS, Goldstein JL 1986 A receptor-mediated path- way for cholesterol homeostasis. Science 232:34-47
12. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M 1996 Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518-520
13. Landschulz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH 1996 Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroi- dogenic tissues of the rat. J Clin Invest 98:984-995
14. Mizutani T, Sonoda Y, Minegishi T, Wakabayashi K, Miyamoto K 1997 Cloning, characterization, and cellular distribution of rat scavenger receptor class B type I (SRBI) in the ovary. Biochem Biophys Res Commun 234:499-505
15. Li X, Peegel H, Menon KMJ 1998 In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA during folliculogen- esis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specif- ically in cell types that use cholesterol for steroidogene- sis. Endocrinology 139:3043-3049
16. Reaven E, Nomoto A, Leers-Sucheta S, Temel R, Williams DL, Azhar S 1998 Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipopro- tein receptor) in luteinized and hormone-desensitized rat ovarian models. Endocrinology 139:2847-2856
17. Temel RE, Trigatti B, DeMattos RB, Azhar S, Krieger M 1997 Scavenger receptor class B, type I (SR- BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc Natl Acad Sci USA 94:13600-13605
18. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M 1997 A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its role in HDL metabolism. Proc Natl Acad Sci USA 94:12610-12615
19. Varban ML, Rinninger F, Wang N, Fairchild-Huntress V, Dunmore JH, Fang Q, Gosselin ML, Dixon KL, Deeds JD, Acton SL, Tall AR, Huszar D 1998 Targeted mutation reveals a central role for SR-BI in hepatic selective up- take of high density lipoprotein cholesterol. Proc Natl Acad Sci USA 95:4619-4624
20. Schimmer BP 1985 Isolation of ACTH-resistant Y1 adre- nal tumor cells. Methods Enzymol 109:350-356
21. Rigotti A, Edelman ER, Seifert P, Iqbal SN, Demattos RB, Temel RE, Krieger M, Williams DL 1996 Regulation by
adrenocorticotropic hormone of the in vivo expression of scavenger receptor class B type I (SR-BI), a high density lipoprotein receptor, in steroidogenic cells of the murine adrenal gland. J Biol Chem 271:33545-33549
22. Liu J, Voutilainen R, Heikkila P, Kahri AI 1997 Ribonucleic acid expression of the CLA-1 gene, a human homolog to mouse high density lipoprotein receptor SR-BI, in human adrenal tumors and cultured adrenal cells. J Clin Endo- crinol Metab 82:2522-2527
23. Faust JR, Goldstein JL, Brown MS 1977 Receptor-me- diated uptake of low density lipoprotein and utilization of its cholesterol for steroid synthesis in cultured mouse adrenal cells. J Biol Chem 252:4861-4871
24. Wang N, Weng W, Breslow JL, Tall AR 1996 Scavenger receptor BI (SR-BI) is up-regulated in adrenal gland in apolipoprotein A-I and hepatic lipase knock-out mice as a response to depletion of cholesterol stores. J Biol Chem 271:21001-21004
25. Ng DS, Francone OL, Forte TM, Zhang J, Haghpassand M, Rubin EM 1997 Disruption of the murine lecithin: cholesterol acyltransferase gene causes impairment of adrenal lipid delivery and up-regulation of scavenger re- ceptor class B type I. J Biol Chem 272:15777-15781
26. Parker KL, Schimmer BP 1996 The roles of the nuclear receptor steroidogenic factor 1 in endocrine differentia- tion and development. Trends Encocrinol Metab 7:203-207
27. Morohashi K-i, Honda S-i, Inomata Y, Handa H, Omura T 1992 A common trans-acting factor, Ad4-binding pro- tein, to the promoters of steroidogenic P-450s. J Biol Chem 267:17913-17919
28. Lala DS, Rice DA, Parker KL 1992 Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endo- crinol 6:1249-1258
29. Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH 1997 Human SR- BI/CLA-1 gene structure and localization: evidence for transcriptional control by steroidogenic factor-1. J Biol Chem 272:33068-33076
30. Ikeda Y, Shen W-H, Ingraham HA, Parker KL 1994 De- velopmental expression of mouse steroidogenic fac- tor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654-662
31. Clark BJ, Soo S-C, Caron KM, Ikeda Y, Parker KL, Stocco DM 1995 Hormonal and developmental regula- tion of the steroidogenic acute regulatory protein. Mol Endocrinol 9:1346-1355
32. Hatzopoulos AK, Rigotti A, Rosenberg RD, Krieger M 1998 Temporal and spatial pattern of expression of the HDL receptor SR-BI during murine embryogenesis. J Lipid Res 39:495-508
33. Webb NR, de Villiers WJS, Connell PM, de Beer FC, van der Westhuyzen DR 1997 Alternative forms of the scav- enger receptor BI (SR-BI). J Lipid Res 38:1490-1495
34. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal develop- ment and sexual differentiation. Cell 77:481-490
35. Freeman DA, Ascoli M 1982 Studies on the source of cholesterol used for steroid biosynthesis in cultured Ley- dig tumor cells. J Biol Chem 257:14231-14238
36. Webb NR, Connell PM, Graf GA, Smart EJ, de villiers WJS, de Beer FC, van der Westhuyzen DR 1998 SR-BII, an isoform of the scavenger recptor BI containing an alternate cytoplasmic tail, mediates lipid transfer be- tween high density lipoprotein and cells. J Biol Chem 272:15241-15248
37. Liscum L, Luskey KL, Chin DJ, Ho YK, Goldstein JL, Brown MS 1983 Regulation of 3-hydroxy-3-methylglu- taryl coenzyme A reductase and its mRNA in rat liver as studied with a monoclonal antibody and a cDNA probe. J Biol Chem 258:8450-8455
38. Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RGW, Shangzhe X, Krieger M 1997 Murine SR-BI, a high den-
sity lipoprotein receptor that mediates selective lipid up- take, is N-glycosylated, fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272:13242-13249
39. Caron KM, Soo S-C, Wetsel WC, Stocco DM, Clark BJ, Parker KL 1997 Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein pro- vides insights into congenital lipoid adrenal hyperplasia. Proc Natl Acad Sci USA 94:11540-11545
40. Clark BJ, Stocco DM 1997 Steroidogenic acute regula- tory protein: the StAR still shines brightly. Mol Cell En- docrinol 134:1-8
41. Azhar S, Nomoto A, Leers-Sucheta S, Reaven E 1998 Simultaneous induction of an HDL receptor protein (SR- BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model. J Lipid Res 39:1616-1628
42. Reaven E, Tsai L, Azhar S 1995 Cholesterol uptake by the ‘selective’ pathway of ovarian granulosa cells: early in- tracellular events. J Lipid Res 36:1602-1617
43. Goldstein JL, Basu SK, Brown MS 1983 Receptor-me- diated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol 98:241-260
44. Tian H, Russell DW 1997 Expression and regulation of steroid 5a-reductase in the genital tubercle of the fetal rat. Dev Dynam 209:117-126
45. Carlisle C, Winking H, Weichenhan D, Nagamine CM 1996 Absence of correlation between sry polymorphisms and XY sex reversal caused by the M.m. domesticus Y chromosome. Genomics 33:32-34
46. Herz J, Kowal RC, Ho YK, Brown MS, Goldstein JL 1990 Low density lipoprotein receptor-related protein medi- ates endocytosis of monoclonal antibodies in cultured cells and rabbit liver. J Biol Chem 265:21355-21362