7-Dehydrocholesterol–dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome

Smith-Lemli-Opitz/RSH syndrome (SLOS), a relatively common birth-defect mental-retardation syndrome, is caused by mutations in DHCR7, whose product catalyzes an obligate step in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. A null mutation in the murine Dhcr7 causes an identical biochemical defect to that seen in SLOS, including markedly reduced tissue cholesterol and total sterol levels, and 30- to 40-fold elevated concentrations of 7-dehydrocholesterol. Prenatal lethality was not noted, but newborn homozygotes breathed with difficulty, did not suckle, and died soon after birth with immature lungs, enlarged bladders, and, frequently, cleft palates. Despite reduced sterol concentrations in Dhcr7^–/– mice, mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme for sterol biosynthesis, the LDL receptor, and SREBP-2 appeared neither elevated nor repressed. In contrast to mRNA, protein levels and activities of HMG-CoA reductase were markedly reduced. Consistent with this finding, 7-dehydrocholesterol accelerates proteolysis of HMG-CoA reductase while sparing other key proteins. These results demonstrate that in mice without Dhcr7 activity, accumulated 7-dehydrocholesterol suppresses sterol biosynthesis posttranslationally. This effect might exacerbate abnormal development in SLOS by increasing the fetal cholesterol deficiency.

A number of birth defect–malformation syndromes, first characterized by their clinical features, have since proven to be metabolic disorders caused by abnormalities in enzymes essential for cholesterol biosynthesis (1–6). The most common of these, with an estimated frequency of 1 in 10,000 to 40,000 Caucasian births (7–9), is Smith-Lemli-Opitz/RSH syndrome (SLOS; MIM 270400) (10) characterized by a dysmorphic facial appearance; palatal clefting; two-three toe syndactyly; postaxial polydactyly; malformations of heart, kidney, genitalia, and lungs; occasional holoprosencephaly; severe to profound mental retardation; and failure to thrive (10, 11). SLOS is caused by mutations in DHCR7, the gene that codes for 3β-hydroxysteroid Δ⁷-reductase (E.C.1.3.1.21), the enzyme that catalyzes the reduction of the Δ⁷ double bond of 7-dehydrocholesterol (7DHC) (cholesta-5,7-dien-3β-ol) to form cholesterol (cholest-5-en-3β-ol) (12–15). As a result, cholesterol biosynthesis is hindered, leading to reduced plasma and tissue cholesterol concentrations and the accumulation of 7DHC and its epimer 8DHC (cholesta-5,8-dien-3β-ol) in all tissues (12, 16–18).

Cholesterol plays an essential role in development because it is needed to activate the morphogenic sonic hedgehog (SHH) signal pathway during early gestation (19), and adverse effects of cholesterol deprivation in the prenatal period are amply illustrated in SLOS by the strong inverse dependence of symptom severity and perinatal mortality on plasma cholesterol levels (8, 20). In contrast to cholesterol, 7DHC fails to stimulate the SHH pathway (21). 7DHC also appears to be an especially potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (22), the rate-limiting enzyme for cholesterol biosynthesis, explaining why plasma total sterol and mevalonic acid concentrations (16, 23, 24) and rates of sterol biosynthesis, as measured by sterol balance (25), are reduced by an average of 40–50% in SLOS.

To explore the mechanisms by which 7DHC inhibits sterol biosynthesis, the mouse Dhcr7 gene was mutated by homologous recombination to create an animal lacking measurable Dhcr7 activity. Except for low 8DHC/7DHC ratios, the biochemical phenotype in the mice was indistinguishable from that in SLOS, and several physical and metabolic abnormalities found in newborn Dhcr7^–/– mice have their counterparts in affected children. As in SLOS, sterol biosynthesis was highly abnormal in homozygous mice. HMG-CoA reductase, LDL receptor, and sterol response element–binding protein-2 (SREBP-2) mRNA levels were found to be similar to levels measured in heterozygous and wild-type littermates, yet tissue total sterol concentrations and HMG-CoA reductase protein levels and activities were markedly reduced. Ancillary in vitro studies provided the explanation for this seemingly paradoxical result, demonstrating that 7DHC accelerates proteolysis of HMG-CoA reductase enzyme several fold. Thus, in the Dhcr7-null mouse, as in children with SLOS, elevated concentrations of 7DHC suppress cholesterol synthesis, exacerbating the sterol deficiency and further contributing to abnormal development.

Targeted mutation of the Dhcr7 locus eliminates enzyme activity. The most frequently encountered DHCR7 mutation in SLOS patients (IVS8-1G→C) causes aberrant splicing of the last exon and leads to a frameshift and truncation of the gene product (15). The few SLOS patients homozygous for this defect are among the most severely affected SLOS patients, and the mutation appears to be a null allele (18). To completely inactivate Dhcr7 we created a mutation analogous to IVS8-1G→C by deleting the last exon (exon 8) and its flanking splice acceptor site in the mouse gene (Figure 1a), thereby removing the sequence for amino acid residues 318–471 (Figure 1b) that code for approximately one-third of the protein. This deletion prevents transcription of two putative transmembrane segments that contain part of a predicted sterol-sensing domain and an extended hydrophilic sequence thought to form a large cytosolic loop (39). Analysis of genomic DNA (Figure 2) verified the engineered genomic alterations shown in Figure 1a. As expected from the nature of the targeted Dhcr7 mutation, RT-PCR could not amplify the deleted 3′-coding region of Dhcr7 mRNA in liver (Figure 1c) or brain (not shown) from pups homozygous for the Dhcr7^delEx8 allele while Dhcr7-specific Ab’s failed to detect the Dhcr7 protein in Dhcr7^–/– mice (Figure 1d). In contrast, 5′-exonic regions of Dhcr7, which were not deleted by the mutation, were amplified successfully (data not shown), indicating that the targeted gene was transcribed. Dhcr7 activities from homozygous newborns were virtually undetectable (1 ± 1 pmol/mg protein/min), while enzyme activities in heterozygotes were close to half of that noted in wild-type mice (Figure 3).

Figure 2

Genotyping of Dhcr7–/–, Dhcr7+/–, and Dhcr7+/+ newborn mice from heterozygous matings by PCR using primers specific for mouse exons 7 and 8 as described in Methods and in Figure 1a.

Figure 3

Activity of Dhcr7 (picomoles per milligram of protein per minute) as measured by the conversion of [³H]-7DHC to cholesterol in liver homogenates from newborn Dhcr7^–/–, Dhcr7+/–, and Dhcr7+/+ mice.

Dhcr7^ΔEx8 mice display developmental abnormalities and die perinatally. Heterozygous matings resulted in litters that averaged 6–7 animals (range 2–12) yielding 61 homozygotes, 130 heterozygotes, and 60 wild-type animals in 251 consecutive newborns (approximately half male and half female) examined within 14 hours of birth. As this result closely approximates the expected Mendelian ratio, homozygotes do not die prenatally. Pups lacking Dhcr7 activity, however, were easily identified shortly after birth by their labored breathing, blue coloration, and lack of movement, while heterozygous and wild-type littermates appeared to be identical in physical appearance and size to each other. None of the Dhcr7^–/– pups suckled, and they weighed significantly less (P < 0.001) than their Dhcr7^+/– and wild-type littermates (1.14 ± 0.10 g, n = 15, vs. 1.41 ± 0.15 g, n = 38, and 1.42 ± 0.13 g, n = 17, respectively). Blood glucose levels (92 ± 11, 94 ± 17, and 96 ± 21 mg/dl, respectively; P = NS) were similar in all animals. All homozygotes died within 18 hours, presumably from respiratory failure or dehydration. Histological analysis showed no gross abnormalities of brain, heart, intestine, adrenal gland, or kidney. However, microscopic examination of fixed and stained sections of lung sections from Dhcr7^–/– mice demonstrated compact lungs with sparse, unconnected air spaces (Figure 4) that were similar in appearance to normal 15- to 16-gestational day mice (40). These results suggest that lungs in newborn Dhcr7^–/– mice may be immature and would explain their breathing difficulties and early death. Although not seen in any Dhcr7^+/– or Dhcr7^+/+ littermates, isolated cleft palates were noted in 12% of Dhcr7^–/– pups (Figures 5, a and b), while more than 90% of Dhcr7^–/– newborns but no Dhcr7^+/– or Dhcr7^+/+ mice exhibited greatly distended urinary bladders (Figures 5, c and d).

Figure 4

Palates were unremarkable in newborn Dhcr7+/–and Dhcr7+/+ mice (a) but were frequently cleft in Dhcr7–/– newborns (b), while bladders were of normal size in controls (c) but were nearly always distended (indicated by arrows in the opened abdomen) in Dhcr7–/– mice (d). ×10.

Figure 5

Histological sections of control mouse lung, (a) ×10 and (b) ×40, and Dhcr7–/– mouse lung, (c) ×10 and (d) ×40, illustrating lung hypoplasia in the latter. Especially noteworthy is the excessive cellular density, the failure of the lungs to fill the pleural cavity (*), and their immature appearance.

Sterol abnormalities in Dhcr7^–/– mice and humans with SLOS are similar. Cholesterol and total sterol (cholesterol plus dehydrocholesterol) concentrations were significantly reduced, while levels of 7DHC and 8DHC were increased 30- to 40-fold in isolated liver microsomes, in whole liver, and in brain homogenates from Dhcr7^–/– newborn mice (Table 1). Except for 8DHC/7DHC ratios that were lower than those noted in humans (16, 20), sterol compositions in Dhcr7^–/– mice closely resembled that in SLOS. Cholesterol deficits in homozygous mice were most profound in the brain with dehydrocholesterols making up 80% of total sterols. Furthermore, although desmosterol (cholesta-5,24-dien-3β-ol) constituted nearly 20% of neutral sterols in the Dhcr7^+/– and Dhcr7^+/+ newborn mouse brain, this cholesterol precursor was not detected in Dhcr7^–/– mice, which instead accumulated 7-dehydrodesmosterol (cholesta-5,7,24-trien-3β-ol).

Table 1

Sterol concentrations in whole brain, whole liver, and liver microsomes from newborn Dhcr7–/– compared with pooled Dhcr7+/– and wild-type mice

Plasma cholesterol levels, 111 ± 27 mg/dl and 82 ± 16 in heterozygous breeders and wild-type adult mice, respectively (n = 6, P = NS), were similar. Liver cholesterol concentrations in 23-day-old Dhcr7^+/– and Dhcr7^+/+ pups were identical (2.7 ± 0.4 mg/g vs. 2.8 ± 0.6 mg/g, respectively; n = 4) and were similar in composition to newborn Dhcr7^+/– and Dhcr7^+/+ livers. Unlike liver, however, cholesterol levels in Dhcr7^+/– and Dhcr7^+/+ mouse brains increased threefold to fourfold in the first 3 weeks of life, while desmosterol concentrations fell nearly tenfold (Table 2). Except for subtly elevated 7DHC levels, heterozygous animals appeared otherwise normal, so there was only a minor biochemical phenotype associated with the effect of haploinsufficiency on Dhcr7 activity.

Table 2

Brain sterols in 23-day-old Dhcr7+/– and Dhcr7+/+ mice

HMG-CoA reductase activities and enzyme protein, but not mRNA levels, were markedly reduced in Dhcr7^–/– newborns. Consistent with low total sterol concentrations in Dhcr7^–/– mice, HMG-CoA reductase activities were reduced sixfold in liver and 2.7-fold in brain microsomes (Table 3), while levels of HMG-CoA reductase protein were reduced commensurately (Figure 6a) compared with Dhcr7^+/– and Dhcr7^+/+ littermates. Generalized protein degradation, however, was not a feature of the homozygous mice because levels of LDLR (Figure 6b) and β-actin concentrations appeared to be similar in all animals. In contrast to HMG-CoA reductase activity, mRNA levels for HMG-CoA reductase as well as for HMG-CoA synthase, squalene synthase, LDLR, SREBP-1, and SREBP-2 were similar in Dhcr7^–/–, Dhcr7^+/–, and Dhcr7^+/+ newborns (Figure 7).

Figure 6

Immunoblot analysis of 50 μg of liver protein for (a) HMG-CoA reductase (HMGR) and (b) LDLR in Dhcr7+/+, Dhcr7+/–, and Dhcr7–/– newborn mice demonstrating significantly reduced levels of HMG-CoA but not LDLR protein in homozygous animals. Relative intensities of each band normalized to β-actin are listed below the blots. Levels of β-actin present in each lane were nearly identical, indicating equal loading and transfer (not shown).

Figure 7

Northern blot analysis of (a) hepatic HMG-CoA reductase, HMG-CoA synthase, squalene synthase, LDLR, SREBP-1, and SREBP-2, and (b) RT-PCR–amplified brain HMG-CoA reductase and LDLR mRNA extracted from Dhcr7–/–, Dhcr7+/–, and Dhcr7+/+ newborn mice, demonstrating that expression of all of the genes is the same in the three genotypes. Intensities in both a and b were normalized to GAPDH mRNA. These are representative samples of blots carried out in three Dhcr7–/– mice.

Table 3

HMG-CoA reductase activity in liver and brain microsomes from Dhcr7–/– compared with Dhcr7+/+ newborn mice

7DHC accelerates catabolism of HMG-CoA-reductase protein. 7DHC, but not cholesterol, reduced levels of steady state β-galactosidase activity in a dose-dependent manner in CHO cells transfected with the HMGal construct (Figure 8). Incubating these cells for 24 hours with 10–20 μg/ml 7DHC raised their 7DHC/cholesterol ratio to levels similar to those measured in the livers of Dhcr7^–/–mice while reducing activity to 40–50% of pretreatment values. After a 24-hour incubation following the addition of 1 μg/ml and 10 μg/ml 7DHC to the culture medium (Figure 9), HMG-CoA reductase half-lives were reduced to 3.8 ± 0.2 and 2.5 ± 0.4 hours, respectively, compared with 9.2 ± 2.3 hours in cells grown without exogenous precursor sterol (P < 0.05). Similarly, HMGal half-life, 5.0 ± 0.7 hours in control cells, was shortened to 2.7 ± 0.2 hours and 2.0 ± 0.4 hours (P < 0.005) in cells incubated with 1 and 10 μg/ml 7DHC, respectively (Figure 10).

Figure 8

β-galactosidase activity in CHO cells transfected with the HMGal construct and driven by the galactosidase promoter. Cells were incubated for 24 hours with increasing concentrations of either 7DHC or cholesterol before determining activity. Because HMG CoA reductase activity is known to be proportional to HMGal activity in this system, these results demonstrate that 7DHC, but not cholesterol, causes excessive destruction of HMG CoA reductase enzyme protein.

Figure 9

The percentage of [³⁵S]-HMG-CoA reductase remaining 1.5, 3.5, and 5.5 hours after a chase with unlabeled methionine in CHO cells incubated for 24 hours in the presence or absence of 1 and 10 μg/ml 7DHC, demonstrating a dose-dependent acceleration of enzyme catabolism by 7DHC.

Figure 10

The percentage of [³⁵S]-HMGal remaining 1.5, 3.5, and 5.5 hours after a chase with unlabeled methionine in CHO cells incubated for 24 hours in the presence or absence of 1 and 10 μg/ml 7DHC, demonstrating a dose-dependent acceleration of HMG CoA reductase enzyme protein catabolism by 7DHC.

Suppressing hydroxylation of the sterol side chain had no effect on the stability of HMGal. Half-lives measured in cells incubated for 24 hours in the presence and absence of 30 μM ketoconazole were 4.1 ± 0.4 and 5.0 ± 0.7, respectively (P = NS), while half-lives in cells grown under identical conditions but after adding 10 μg/ml 7DHC were 2.6 ± 0.1 and 2.0 ± 0.4 hours, respectively (P = NS).

In tissues from Dhcr7^–/– mice, despite the application of a very sensitive and specific technique for the amplification and detection of the transcript for the 3′-end of Dhcr7, none could be found, no Dhcr7 immunoreactive protein was observed, and Dhcr7 enzyme activity could not be detected. Furthermore, Dhcr7 mRNA levels and Dhcr7 activities in Dhcr7^+/– mice were reduced by approximately 50% compared with their Dhcr7^+/+ littermates (Figures 1 and 3). Thus, gene targeting was successful, and Dhcr7^ΔEx8 is a null allele unable to reduce 7DHC to cholesterol. The targeted mouse recapitulated most of the biochemical defects described in SLOS with 7DHC levels elevated 30- to 40-fold, and cholesterol and total sterol concentrations were markedly reduced. In addition, because the biosynthesis of desmosterol requires the reduction of a Δ⁷ double bond, 7-dehydrodesmosterol, a compound that retains double bonds at both C-7,8 and C-24,25 accumulated in the brains of Dhcr7^–/– pups (Table 1). Unlike SLOS, however, in which 8DHC and 7DHC concentrations are nearly equal (16, 20), 8DHC/7DHC ratios in newborn Dhcr7^–/– mice were closer to 0.1 (Table 1).

The cholesterol/7DHC ratios in livers of newborn homozygous mice were considerably higher than ratios noted in the most severely affected children with SLOS, that is in those individuals predicted to be without DHCR7 activity (8, 18, 20). This result suggests that the fetal mouse receives a greater proportion of its cholesterol from its mother than does the human fetus, in agreement with numerous observations that maternal cholesterol is obligate for murine (41–43) but not for human (44) development and that considerable amounts of maternal cholesterol are probably transferred to the fetus early in rodent gestation (45, 46). The gross structural features of most organs are set within the first 7–10 gestational days in rats and mice. Thus, using atherogenic diets to raise plasma cholesterol levels in pregnant rats being treated with DHCR7 inhibitors probably enabled the mothers to transfer enough extra cholesterol to the fetuses to abolish most abnormal development (47). Because mice normally have much higher plasma cholesterol levels than rats, one has difficulty in generating fetal mice with severe abnormalities using these same inhibitors (48). Elevated cholesterol transfer from mother to fetus early in gestation is also likely to be the reason that the Dhcr7^–/– mice did not develop more of the SLOS-like craniofacial and limb abnormalities.

Although embryonic lethality was not noted in homozygous mice, some developmental abnormalities including incomplete lung development, distended bladders, and cleft palates were seen and all Dhcr7^–/– mice died within 18 hours of birth from “failure to thrive” with respiratory failure. While palatal clefting and lung abnormalities are found in nearly half of all SLOS patients, the latter usually consist of lobular defects while cellular immaturity has not as yet been reported (49). The exact molecular pathways by which alterations in sterol composition perturb development are not entirely certain but two possibilities seem relevant to this study. Development of lung and bladder, and a number of other organ systems, depends on the sonic hedgehog/patched/smoothened/Gli protein family pathway (50, 51) and inappropriate signaling resulting from Shh gene modification leads to abnormal epithelial/mesenchymal interactions (50) and defective lung development in mice (52). Because proper functioning of Shh requires cholesterol (53) and 7DHC does not permit Shh to be activated (21, 54), it was not surprising to find abnormal development in organs that use this signal pathway. Unlike lung and bladder, palatal clefting, noted in 50% of children with SLOS (8) and 12% of Dhcr7^–/– mice, may be somewhat dependent on abnormal folate metabolism (55). The folate receptor is a glycosylphosphatidylinositol-bound protein complex probably sorted to caveolae or other regions of the cell membrane having a high sterol content and that might be specially sensitive to lipid composition (56). For instance, SLOS fibroblasts grown in a cholesterol-free medium so as to increase their 7DHC concentration develop membrane-specific defects including increased fluidity and reduced hydrolysis of membrane phosphoinositol (57), all of which might contribute to palatal clefting.

Sterol biosynthesis is suppressed in the average child with SLOS (16, 22, 23, 25). Biosynthesis is also reduced by similar amounts in Dhrc7^–/– mice as percentage of reductions in liver total sterols, HMG-CoA reductase protein levels, and HMG-CoA reductase activities (68%, 64%, and 83%, respectively) are comparable. Similar differences were also noted in the mouse brain, with the exception that the magnitude of HMG-CoA reductase activities in the brains of all mice were several-fold higher than those noted in the liver. This greater activity reflects the increased need for cholesterol by the CNS soon after birth, illustrated by the fourfold rise in brain, but not liver, cholesterol in 23-day-old compared with newborn mice (Tables 1 and 2). Markedly elevated HMG-CoA reductase activity also explain why, compared with other organs, cholesterol and total sterol deficits are much larger in the brains of both Dhcr7^–/– mice and severely affected human fetuses (16).

In contrast to activities and protein concentrations, mRNA levels for HMG-CoA reductase, and for number of other genes critical for cholesterol synthesis and metabolism, appeared to be similar in all of the newborn mice (Figure 7). Cholesterol homeostasis is normally maintained by a tightly controlled coordinate regulation of a number of genes modulated, for the most part, by the microsomal protein SREBP-2. When intracellular cholesterol levels are sensed as being too low, SREBP-2 is cleaved from the endoplasmic reticulum (ER), releasing a nuclear transcription factor that upregulates expression of HMG-CoA reductase, HMG-CoA synthase, squalene synthase, LDLR, and SREBP-2 (58). Thus, for example, treating rats with the squalene synthase inhibitor zaragozic acid and reducing hepatic cholesterol levels by 40% induces a fourfold to fivefold increase in HMG-CoA reductase and LDLR transcription rates, mRNA levels, and protein concentrations, and in HMG-CoA reductase activities (59, 60). Yet, in Dhcr7^–/– mice, despite similar reductions in total sterol concentrations and much greater reductions in tissue cholesterol levels, the SREBP-2–modulated pathway seems not to be activated (Figure 7). It may be that 7DHC perturbs (57, 61) the ER membrane, making cleavage of the active nuclear transcription factor more difficult, or changes membrane properties so as to prevent the cycling of SCAP to the Golgi apparatus so that it can be modified and activated (62). However, because LDLR mRNA and protein levels were similar in both affected, wild-type, and heterozygous mice, high levels of 7DHC seem not to interfere with translation.

Unchanged mRNA levels would not, under any circumstances, explain why HMG-CoA reductase protein levels are depressed in homozygous mice or why de novo sterol synthesis is inhibited. But, the combination of normal HMG-CoA reductase and LDLR mRNA and reduced protein levels for HMG-CoA reductase, but not for LDLR or β-actin, suggested that 7DHC might be accelerating the degradation of HMG-CoA reductase itself. We assayed the effect of 7DHC on the catabolism of HMG-CoA reductase using a well-established in vitro model that employs CHO cells transfected with the HMGal construct, which consists of the transmembrane region of HMG-CoA reductase fused to the reporter β-galactosidase gene and its promoter. This portion of the HMG-CoA reductase protein is sufficient to initiate the HMG-CoA reductase proteolytic cascade, which, at the same time, also degrades β-galactosidase. Thus, in this system, HMG-CoA reductase activity and protein levels are both proportional to β-galactosidase activity (34, 38). Furthermore, because expression is driven by the galactosidase, not the HMG-CoA reductase, promoter assays are carried out under conditions of constant gene expression unrelated to either cholesterol or isoprenoid concentrations.

The addition of sufficient 7DHC to raise the 7DHC/cholesterol ratio to a level nearly equal to that in the livers of Dhcr7^–/– mice suppressed steady-state β-galactosidase activity by about 50% (Figure 8), a value similar to the reduction in HMG-CoA reductase activity and protein noted in these animals. In contrast to 7DHC, cholesterol had no effect on activity. These results, taken together, suggest that 7DHC probably destabilizes HMG-CoA reductase enzyme. We were able to prove that this is indeed the mechanism that causes reduced sterol biosynthesis in Dhcr7^–/– mice and in patients with SLOS by incubating CHO cells with 7DHC and finding that the half-lives of HMG-CoA reductase (Figure 9) and HMGal (Figure 10) both decreased threefold when cellular 7DHC increased to about 30% of total sterols.

HMG-CoA reductase is an ER protein that is degraded within its transmembrane region by a membrane-bound cysteine protease (35, 63). It normally has a half-life of only a few hours, and its stability is reduced by Ca²⁺ depletion (38), perhaps involving a specific Ca transporter (64), and by the addition of mevalonate or mevalonate-derived sterols (65, 66). Nevertheless, in the Dhcr7^–/– mouse and in SLOS, despite reduced tissue total sterol concentrations and plasma mevalonate levels (24), HMG-CoA reductase protein appears to be degraded at a highly accelerated rate. This result, in turn, suggests that 7DHC has an effect within the ER membrane contrary to that of cholesterol or isoprenoid cholesterol precursors, and in some manner activates the proteolytic process. Although it has not been well studied, replacing cholesterol with 7DHC has a number of effects on membranes, including increasing their thickness and fluidity as well as their permeability to Ca ions (57). In contrast to 7DHC, ketoconazole, an inhibitor of sterol side-chain hydroxylation that mitigates the suppression of HMG-CoA reductase activity by 7DHC in fibroblast cultures (22), did not affect the HMGal half-life. This latter result suggests that, while 27-hydroxylated sterols may well be important inhibitors of HMG-CoA reductase expression (67), they probably have little effect on enzyme stability in physiologic concentrations.

Thus, the 7DHC that accumulates in patients with SLOS accelerates proteolysis of HMG-CoA reductase enzyme protein, thereby depriving the developing organism of needed sterols. Although this effect might be protective, forestalling even more massive accumulations of a possibly toxic precursor such as 7DHC, it is just as likely to be deleterious. This would be especially disadvantageous for children with mutations that retain some DHCR7 activity (18) by suppressing the production of 7DHC and further restricting the amount of cholesterol available prenatally (68). The newborn Dhcr7-null mouse is, for many purposes, a good model for SLOS. However, because it does not demonstrate many of the more common abnormalities noted in SLOS, it will have to be further manipulated (48) to create a system to study abnormal development. Nevertheless, this mouse has been especially helpful because it has permitted identification of one of the major mechanisms that suppresses sterol synthesis in children with SLOS.

Note added in proof. During preparation of this manuscript Wassif et al. published a description of a SLOS/RSH mouse (69). Using a different targeting strategy, they created an animal without apparent Dhcr7 activity but with impaired glutamate uptake by frontal cortex neurons that demonstrated nearly identical sterol concentrations and phenotype as the Dhcr7^ΔEx8 mice. Both models have been described before in abstract form (70–72).

This work was supported by a grants from the Department of Veterans Administration Research Service (to G.S. Tint and to G. Salen), grant 9950855V from the American Heart Association, Florida/Puerto Rico Affiliate (to G.C. Ness), NIH grant HL-4263 (to N. Maeda), training grant T32NS07431 (to H. Waage-Baudet), grants from Fonds zur Förderung der wissenschaftlichen Forschung (P14112-MOB to H. Glossmann) and the Jubiläumsfond der Österreichischen Nationalbank (to H. Glossmann). The authors also wish to thank Bibiana Pcolinsky for her excellent technical assistance.

Barbara U. Fitzky and Fabian F. Moebius contributed equally to this work.

1. Hoffmann, G, et al. Mevalonic aciduria: an inborn error of cholesterol and nonsterol isoprene biosynthesis. N Engl J Med 1986. 314:1610-1614.
   View this article via: PubMed Google Scholar
2. Clayton, P, Mills, K, Keeling, J, FitzPatrick, D. Desmosterolosis: a new inborn error of cholesterol biosynthesis. Lancet 1996. 348:404.
   View this article via: PubMed Google Scholar
3. Kelley, RI, et al. Abnormal sterol metabolism in patients with Conradi-Hunermann-Happle syndrome and sporadic lethal chondrodysplasia punctata. Am J Med Genet 1999. 83:213-219.
   View this article via: PubMed CrossRef Google Scholar
4. Braverman, N, et al. Mutations in the gene encoding 3 beta-hydroxysteroid-Δ8,Δ7-isomerase cause X-linked dominant Conradi-Hunermann syndrome. Nat Genet 1999. 22:291-294.
   View this article via: PubMed CrossRef Google Scholar
5. König, A, Happle, R, Bornholdt, D, Engel, H, Grzeschik, K-H. Mutations in the NSDHL gene, encoding a 3β-hydroxysteroid dehydrogenase, cause CHILD syndrome. Am J Med Genet 2000. 90:339-346.
   View this article via: PubMed CrossRef Google Scholar
6. Grange, DK, Kratz, LE, Braverman, NE, Kelley, RI. CHILD syndrome caused by deficiency of 3β-hydroxysteroid-Δ8, Δ7-isomerase. Am J Med Genet 2000. 90:328-335.
   View this article via: PubMed CrossRef Google Scholar
7. Lowry, RB, Yong, S-L. Borderline intelligence in the Smith-Lemli-Opitz syndrome. Am J Med Genet 1980. 5:137-143.
   View this article via: PubMed CrossRef Google Scholar
8. Cuniff, C, Kratz, LE, Moser, A, Natowicz, MR, Kelley, RI. Clinical and biochemical spectrum of patients with RSH/Smith-Lemli-Opitz syndrome and abnormal cholesterol metabolism. Am J Med Genet 1997. 68:263-269.
   View this article via: PubMed CrossRef Google Scholar
9. Witsch-Raumgartner, M, et al. Frequency gradients of DHCR7 mutations in patients with Smith-Lemli-Opitz syndrome in Europe: evidence for different origins of common mutations. Eur J Hum Genet 2001. 9:45-50.
   View this article via: PubMed CrossRef Google Scholar
10. Smith, DW, Lemli, L, Opitz, JM. A newly recognized syndrome of multiple congenital anomalies. J Pediatr 1964. 64:210-217.
    View this article via: PubMed CrossRef Google Scholar
11. Curry, CJR, et al. Smith-Lemli-Opitz syndrome-type II: multiple congenital anomalies with male pseudohermaphroditism and frequent early lethality. Am J Med Genet 1987. 26:45-57.
    View this article via: PubMed CrossRef Google Scholar
12. Tint, GS, et al. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J Med 1994. 330:107-113.
    View this article via: PubMed CrossRef Google Scholar
13. Shefer, S, et al. Markedly inhibited 7-dehydrocholesterol-Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes. J Clin Invest 1995. 96:1779-1785.
    View this article via: JCI PubMed CrossRef Google Scholar
14. Moebius, FF, Fitzky, BU, Lee, JN, Paik, Y-K, Glossmann, H. Molecular cloning and expression of the human Δ7-sterol reductase. Proc Natl Acad Sci USA 1998. 95:1899-1902.
    View this article via: PubMed CrossRef Google Scholar
15. Fitzky, BU, et al. Mutations in the Δ7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome. Proc Natl Acad Sci USA 1998. 95:8181-8186.
    View this article via: PubMed CrossRef Google Scholar
16. Tint, GS, et al. Markedly increased concentrations of 7-dehydrocholesterol combined with low levels of cholesterol are characteristic of the Smith-Lemli-Opitz syndrome. J Lipid Res 1995. 36:89-95.
    View this article via: PubMed Google Scholar
17. Kelley, RI. Diagnosis of Smith-Lemli-Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts. Clin Chem Acta 1995. 236:45-58.
    View this article via: CrossRef Google Scholar
18. Witsch-Baumgartner, M, et al. Mutational spectrum in the Δ7-sterol reductase gene and genotype-phenotype correlation in 84 patients with the Smith-Lemli-Opitz syndrome. Am J Hum Genet 2000. 66:402-412.
    View this article via: PubMed CrossRef Google Scholar
19. Porter, JA, Young, KE, Beachy, PA. Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996. 274:255-259.
    View this article via: PubMed CrossRef Google Scholar
20. Tint, GS, et al. Correlation of severity and outcome correlate with plasma sterol levels in variants of the Smith-Lemli-Opitz syndrome. J Pediatr 1995. 127:82-87.
    View this article via: PubMed CrossRef Google Scholar
21. Incardona, JP, Gaffield, W, Kapur, RJ, Roelink, H. The teratogenic Veratrum alkaloid cyclopamine inhibits Sonic hedgehog signal transduction. Development 1998. 125:3553-3562.
    View this article via: PubMed Google Scholar
22. Honda, M, et al. 7-Dehydrocholesterol down-regulates cholesterol biosynthesis in cultured Smith-Lemli-Opitz syndrome skin fibroblasts. J Lipid Res 1998. 39:647-657.
    View this article via: PubMed Google Scholar
23. Tint, G.S., et al. 1997. The Smith-Lemli-Opitz syndrome: a potentially fatal birth defect caused by a block in the last enzymatic step in cholesterol biosynthesis. In Subcellular biochemistry: cholesterol: its metabolism and functions in biology and medicine. Volume 28. R. Bittman, editor. Plenum Press. New York, New York, USA. 117–143.
    View this article via: PubMed Google Scholar
24. Honda, M, et al. Regulation of cholesterol biosynthetic pathway in patients with the Smith-Lemli-Opitz syndrome. J Inherit Metab Dis 2000. 23:464-474.
    View this article via: PubMed CrossRef Google Scholar
25. Steiner, RD, Linck, LM, Flavell, DP, Lin, DS, Connor, WE. Sterol balance in the Smith-Lemli-Opitz syndrome: reduction in whole body cholesterol synthesis and normal bile acid production. J Lipid Res 2000. 41:1437-1447.
    View this article via: PubMed Google Scholar
26. Shehee, WR, Oliver, P, Smithies, O. Lethal thalassemia after insertional disruption of the mouse major adult beta-globin gene. Proc Natl Acad Sci USA 1993. 90:3177-3181.
    View this article via: PubMed CrossRef Google Scholar
27. Batta, AK, Tint, GS, Abuelo, D, Shefer, S, Salen, G. Identification of 8-dehydrocholesterol (cholesta-5,8-dien-3β-ol) in patients with Smith-Lemli-Opitz syndrome. J Lipid Res 1995. 36:705-713.
    View this article via: PubMed Google Scholar
28. Honda, M, et al. Measurement of 3β-hydroxysteroid Δ7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome. J Lipid Res 1996. 37:2433-2438.
    View this article via: PubMed Google Scholar
29. Nguyen, LB, et al. A molecular defect in hepatic cholesterol biosynthesis in sitosterolemia with xanthomatosis. J Clin Invest 1990. 86:923-931.
    View this article via: JCI PubMed CrossRef Google Scholar
30. Wu, J, Zhu, YH, Patel, SB. Cyclosporin-induced dyslipoproteinemia is associated with selective activation of SREBP-2. Am J Physiol 1999. 277:E1087-E1094.
    View this article via: PubMed Google Scholar
31. Chambers, CM, Ness, GC. Dietary cholesterol regulates hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in rats primarily at the level of translation. Arch Biochem Biophys 1998. 354:317-322.
    View this article via: PubMed CrossRef Google Scholar
32. Ness, GC, Sample, CE, Smith, M, Pendleton, LC, Eichler, DC. Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Biochem J 1986. 233:167-172.
    View this article via: PubMed Google Scholar
33. Ness, GC, Zhao, Z. Thyroid hormone rapidly induces hepatic LDL receptor mRNA levels in hypophysectomized rats. Arch Biochem Biophys 1994. 315:199-202.
    View this article via: PubMed CrossRef Google Scholar
34. Skalnik, DG, Narita, H, Kent, C, Simoni, RD. The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol regulated degradation onto β-galactosidase. J Biol Chem 1988. 263:6836-6841.
    View this article via: PubMed Google Scholar
35. Jingami, H, Brown, MS, Goldstein, JL, Anderson, RGW, Luskey, KL. Partial deletion of membrane-bound domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase eliminates sterol-enhanced degradation and prevents formation of crystalloid endoplasmic reticulum. J Cell Biol 1987. 104:1693-1704.
    View this article via: PubMed CrossRef Google Scholar
36. Rothblat, GH, Arbogast, LY, Ouellette, L, Howard, BV. Preparation of delipidized serum protein for use in cell culture systems. In Vitro 1976. 12:554-557.
    View this article via: PubMed CrossRef Google Scholar
37. Lowry, OH, Rosebrough, NJ, Farr, AL, Randall, RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 1951. 193:265-275.
    View this article via: PubMed Google Scholar
38. Roitelman, J, Bar-Nun, S, Inoue, S, Simoni, RD. Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase. J Biol Chem 1991. 266:16085-16091.
    View this article via: PubMed Google Scholar
39. Bae, S-H, Lee, JN, Fitzky, BU, Seong, J, Paik, Y-K. Cholesterol synthesis from lanosterol. Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein. J Biol Chem 1999. 274:14624-14631.
    View this article via: PubMed CrossRef Google Scholar
40. Talbot, CL, et al. Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung. Am J Respir Cell Mol Biol 1999. 20:398-406.
    View this article via: PubMed Google Scholar
41. Farese (Jr), RV, Ruland, SL, Flynn, LM, Stokowski, RP, Young, SG. Knockout of the mouse apolipoprotein B genes results in embryonic lethality in homozygotes and protection against diet-induced hypercholesterolemia in heterozygotes. Proc Natl Acad Sci USA 1995. 92:1774-1778.
    View this article via: PubMed CrossRef Google Scholar
42. Raabe, M, et al. Knockout of the abetalipoproteinemia gene in mice - reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci USA 1998. 95:8686-8691.
    View this article via: PubMed CrossRef Google Scholar
43. Herz, J, Farese, RV. The LDL receptor gene family, apolipoprotein B and cholesterol in embryonic development. J Nutr 1999. 129:473S-475S.
    View this article via: PubMed Google Scholar
44. Linton, MF, Farnese (Jr), RV, Young, SG. Familial hypobetalipoproteinemia. J Lipid Res 1993. 34:521-541.
    View this article via: PubMed Google Scholar
45. Chevallier, F. Transferts et synthèse du cholesterol chez le rat au course de sa croissance. Biochim Biophys Acta 1964. 84:316-339.
    View this article via: PubMed Google Scholar
46. McConihay, JA, Honkomp, AM, Granholm, NA, Woollett, LA. Maternal high density lipoproteins affect fetal mass and extra-embryonic fetal tissue sterol metabolism in the mouse. J Lipid Res 2000. 41:424-432.
    View this article via: PubMed Google Scholar
47. Roux, C, Dupuis, R, Horvath, C, Talbot, J-N. Teratogenic effect of an inhibitor of cholesterol synthesis (AY 9944) in rats: correlation with maternal cholesterolemia. J Nutr 1980. 110:2310-2312.
    View this article via: PubMed Google Scholar
48. Lanoue, L, et al. Limb, genital, CNS and facial malformations result from gene/enviroment-induced cholesterol deficiency: further evidence for a link to Sonic Hedgehog. Am J Med Genet 1997. 73:24-31.
    View this article via: PubMed CrossRef Google Scholar
49. Kelley, RI, Hennekam, RCM. The Smith-Lemli-Opitz syndrome. J Med Genet 2000. 37:321-335.
    View this article via: PubMed CrossRef Google Scholar
50. Bitgood, MJ, McMahon, AP. Hedgehog and Bmp genes are co-expressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev Biol 1995. 172:126-138.
    View this article via: PubMed CrossRef Google Scholar
51. Hogan, BL, et al. Branching morphogenesis of the lung: new models for a classical problem. Cold Spring Harb Symp Quant Biol 1997. 62:249-256.
    View this article via: PubMed Google Scholar
52. Pepicelli, CV, Lewis, PM, McMahon, AP. Sonic hedgehog regulates branching morphogenesis in the mammalian lung. Curr Biol 1998. 8:1083-1086.
    View this article via: PubMed CrossRef Google Scholar
53. Cooper, MK, Porter, JA, Young, KE, Beachy, PA. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 1998. 280:1603-1607.
    View this article via: PubMed CrossRef Google Scholar
54. Gofflot, F, Gaoua, W, Bourguignon, L, Roux, C, Picard, JJ. Expression of Sonic Hedgehog downstream genes is modified in rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis. Develop Dynam 2001. 220:99-111.
    View this article via: CrossRef Google Scholar
55. Heil, SG, van der Put, NMJ, Trijbels, FJM, Gabreels, FJM, Blom, HJ. Molecular genetic analysis of human folate receptors in neural tube defects. Eur J Hum Genet 1999. 7:393-396.
    View this article via: PubMed CrossRef Google Scholar
56. Luhrs, CA, Slomiany, BL. A human membrane-associated folate binding protein is anchored by a glycosylphosphatidylinositol tail. J Biol Chem 1989. 264:21446-21449.
    View this article via: PubMed Google Scholar
57. Tulenko, TN, LaBelle, E, Boesze-Battaglia, K, Mason, RP, Tint, GS. A membrane bilayer defect in the Smith-Lemli-Opitz syndrome. FASEB J 1998. 12:A827. (Abstr.)
58. Brown, MS, Goldstein, JL. A proteolytic pathway that controls the cholesterol content of membranes, cells and blood. Proc Natl Acad Sci USA 1999. 96:11041-11048.
    View this article via: PubMed CrossRef Google Scholar
59. Ness, GC, Zhao, ZH, Keller, RK. Effect of squalene synthase inhibition in the expression of hepatic cholesterol biosynthetic-enzymes, LDL receptor, and cholesterol 7α hydroxylase. Arch Biochem Biophys 1994. 311:277-285.
    View this article via: PubMed CrossRef Google Scholar
60. Lopez, D, Chambers, CM, Keller, RK, Ness, GC. Compensatory responses to inhibition of hepatic squalene synthase. Arch Biochem Biophys 1998. 351:159-166.
    View this article via: PubMed CrossRef Google Scholar
61. Bruckerdorfer, KR, Demel, RA, De Gier, J, Van Deenen, LLM. The effect of partial replacements of membrane cholesterol by other steroids on the osmotic fragility and glycerol permeability of erythrocytes. Biochim Biophys Acta 1969. 183:334-345.
    View this article via: PubMed CrossRef Google Scholar
62. Nohturfft, A, De Bose-Boyd, RA, Scheek, S, Goldstein, JL, Brown, MS. Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci USA 1999. 96:11235-11240.
    View this article via: PubMed CrossRef Google Scholar
63. Moriyama, T, Sather, SK, McGee, TP, Simoni, RD. Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease. J Biol Chem 1998. 273:22037-22043.
    View this article via: PubMed CrossRef Google Scholar
64. Cronin, SR, Khoury, A, Ferry, DK, Hampton, RY. Regulation of HMG-CoA reductase degradation requires the P-type ATPase Cod1p/Spf1p. J Cell Biol 2000. 148:915-924.
    View this article via: PubMed CrossRef Google Scholar
65. Roitelman, J, Simoni, RD. Distinct sterol and non-sterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase. J Biol Chem 1992. 267:25264-25273.
    View this article via: PubMed Google Scholar
66. Bradfute, DL, Simoni, RD. Non-sterol compounds that regulate cholesterogenesis. Analogues of farnesyl pyrophosphate reduce 3-hydroxy-3-methylglutaryl-coenzyme A reductase levels. J Biol Chem 1994. 269:6645-6650.
    View this article via: PubMed Google Scholar
67. Axelson, M, Larsson, O. Low density lipoprotein (LDL) cholesterol is converted to 27-hydroxycholesterol in human fibroblasts. J Biol Chem 1995. 270:15102-15110.
    View this article via: PubMed Google Scholar
68. Gaoua, W, Wolf, C, Chevy, F, Ilien, F, Roux, C. Cholesterol deficit but not accumulation of aberrant sterols is the major cause of the teratogenic activity in the Smith-Lemli-Opitz syndrome. J Lipid Res 2000. 41:637-646.
    View this article via: PubMed Google Scholar
69. Wassif, CA, et al. Biochemical, phenotypic and neurophysiogical characterization of a genetic mouse model of RSH/Smith-Lemli-Opitz syndrome. Hum Mol Gen 2001. 10:555-564.
    View this article via: PubMed CrossRef Google Scholar
70. Tint, GS, et al. A mouse model for the Smith-Lemli-Opitz syndrome. Am J Hum Genet 1999. 65:A96. (Abstr.)
71. Tint, GS, et al. Cholesterol (CH) metabolism is highly abnormal in the mouse model of Smith-Lemli-Opitz syndrome. Hepatology 2000. 32:387A. (Abstr.)
72. Porter, FD, et al. Phenotypic, biochemical, and neurophysiological characterization of a SLOS mouse model. Am J Hum Genet 2000. 67:280.. (Abstr.)