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

Abstract

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.

Figure 1

(a) Disruption of the Dhcr7 gene. The coding sequence of exon 8 and the 5′ flanking splice acceptor site and part of the 3′ flanking untranslated region (indicated in gray) of the wild-type gene were replaced with a neomycin resistance gene in opposite orientation to Dhcr7 transcription. Restriction sites used for subcloning of short and long Dhcr7 gene segments are shown. The targeting construct was linearized with ClaI (arrow). Primers employed for PCR genotyping are labeled a, b, and neo (see Methods). (b) Tentative topology model of Dhcr7. The induced Dhcr7 mutation is expected to delete one-third of the Dhcr7 protein (the residues shown as filled circles). The box indicates the putative sterol-binding site REF. (c) Total RNA extracted from the livers of Dhcr7–/–, Dhcr7+/–, and wild-type newborn mice was reverse transcribed and amplified using specific primers for the 3′-end of Dhcr7 mRNA and GADPH. (d) Immunoblot analysis of Dhcr7. Upper panel: Loading decreasing amounts of total hepatic protein results in corresponding reductions in Dhcr7 Ab response. Lower panel: Microsomal protein from yeast (WA0) and human tsA-201 cells (tsA) heterologously expressing DHCR7 with an N-terminal myc tag (15) and from livers of newborn wild-type mice demonstrated immunoreactive Dhcr7 protein. Bands corresponding to myc-tagged (myc) and wild-type (WT) DHCR7/Dhcr7 proteins, respectively, are indicated by arrowheads. In contrast, no immunoreactive band corresponding to wild-type Dhcr7 was detected in 40 μg of microsomal protein from either homozygous mice (last two lanes) or mock-transfected yeast (MOCK).

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.

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.

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.

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.