Selective reconstitution of liver cholesterol biosynthesis promotes lung maturation but does not prevent neonatal lethality in Dhcr7 null mice
© Yu et al; licensee BioMed Central Ltd. 2007
Received: 21 November 2006
Accepted: 04 April 2007
Published: 04 April 2007
Targeted disruption of the murine 3β-hydroxysterol-Δ7-reductase gene (Dhcr7), an animal model of Smith-Lemli-Opitz syndrome, leads to loss of cholesterol synthesis and neonatal death that can be partially rescued by transgenic replacement of DHCR7 expression in brain during embryogenesis. To gain further insight into the role of non-brain tissue cholesterol deficiency in the pathophysiology, we tested whether the lethal phenotype could be abrogated by selective transgenic complementation with DHCR7 expression in the liver.
We generated mice that carried a liver-specific human DHCR7 transgene whose expression was driven by the human apolipoprotein E (ApoE) promoter and its associated liver-specific enhancer. These mice were then crossed with Dhcr7+/- mutants to generate Dhcr7-/- mice bearing a human DHCR7 transgene. Robust hepatic transgene expression resulted in significant improvement of cholesterol homeostasis with cholesterol concentrations increasing to 80~90 % of normal levels in liver and lung. Significantly, cholesterol deficiency in brain was not altered. Although late gestational lung sacculation defect reported previously was significantly improved, there was no parallel increase in postnatal survival in the transgenic mutant mice.
The reconstitution of DHCR7 function selectively in liver induced a significant improvement of cholesterol homeostasis in non-brain tissues, but failed to rescue the neonatal lethality of Dhcr7 null mice. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity.
The role of cholesterol in embryonic development is an important question in biology, with significant ramifications for human disease [1, 2]. Defects in post-squalene cholesterol biosynthesis, such as in Smith-Lemli-Opitz syndrome (SLOS, MIM 270400) or desmosterolosis (MIM 603398), disrupt the synthesis of cholesterol and cause a variety of severe developmental abnormalities [3–8]. SLOS is a complex inborn error of cholesterol biosynthesis caused by mutations of the 3β-hydroxysterol-Δ7 reductase gene (DHCR7) [9–11]. The lack of Dhcr7 expression in mouse mimics the early postnatal lethality observed in severely affected individuals (those with the condition formerly referred to as SLOS type II). The biological changes in development caused by disruption of normal cholesterol biosynthesis that result in this early postnatal lethality remain obscure. As a genetic model for understanding of the SLOS in human, mice lacking Dhcr7 provide a useful model to determine which affected tissues are critically responsible for the lethality and which ones may contribute to the morbidity.
One cause of early postnatal death in the Dhcr7 null animals appears to be associated with anoxia due to diffuse atelectasis of the late gestational lungs [12–14], which may cause immature formation of gas-exchange unit and subsequent respiratory insufficiency after birth [2, 14]. Therefore, lung involvement seems to be a cause of neonatal death in Dhcr7 null mouse model. However, there is still uncertainty regarding the mechanism(s) responsible for the late gestational lung hypoplasia caused by the loss of endogenous cholesterol biosynthesis in Dhcr7-/- mice. As with any biological aspect of a generalized cholesterol deficiency, the pathophysiology is likely to be complex and multi-factorial. It remains unclear whether the delayed lung maturation is caused by a developmental defect intrinsic to lung or whether it represents a systemic abnormality, especially of the central nervous system (CNS), because of impaired cholesterol homeostasis during embryogenesis. A different variety of developmental abnormalities in brain have also been noted in both human patients and, to some extent, in Dhcr7-deficient animal models. In this regard, we have also recently demonstrated that Dhcr7-/- mice can be partially rescued from neonatal death by a low level restoration of DHCR7 expression in brain, indicating that neuropathophysiology is one cause of their neonatal lethality .
In attempting to rescue Dhcr7-deficient mice, we felt that it was necessary to consider the liver because that organ is a major source of cholesterol for most developing organs, except for brain [4, 16–19]. Thus, we hypothesized that restoration of normal cholesterol synthesis in the liver might be able to abrogate the lethality. In the current study, we asked whether the specific reconstitution of DHCR7 expression in liver would alleviate the cholesterol deficiency and afford protection from early postnatal death in Dhcr7 null mice. Our results suggest that, although expression of DHCR7 in liver alone during development induced a significant improvement in cholesterol homeostasis in non-brain tissues and promoted lung maturation, it failed to rescue the neonatal lethality. These results provided further evidence that CNS defects caused by Dhcr7 null likely play a major role in the lethal pathogenesis of Dhcr7-/- mice, with the peripheral organs contributing the morbidity.
Characterization of TgDHCR7 lines
Sterol metabolic profile of TgDHCR7/Dhcr7-/-mice
Collectively, these data indicated that selective reconstitution of DHCR7 expression in liver improved significantly cholesterol homeostasis in liver, lung and circulation of Dhcr7 null animals during embryogenesis, but did not affect metabolism in the brain.
Late gestational lung development in Dhcr7-/-Tg+ mice
Postnatal survivability of Dhcr7-/-Tg+ mice
Survival beyond 24 h of birth in transgenic and non transgenic Dhcr7-/pups
Survival rate (%)
TgDHCR7 line 2
TgDHCR7 line 3
Association of cholesterol and 7/8DHC with lipid membranes
Severe cholesterol deficiency, caused by the intentional genetic ablation of Dhcr7 in the mouse, has proven to be incompatible with perinatal life and is associated with respiratory insufficiency after birth [12–14]. However, Dhcr7 null pups also exhibit other defects that indicate other organs/systems are also disrupted by absence of normal cholesterol synthesis. A key question is therefore which of these organs (whose lack of normal development) leads to the neonatal lethality. The hypoxia and abnormal lung phenotype would suggest this could be a major defect, though it is also possible that a failure to integrate respiration at the CNS level could be equally important. In our preliminary effort to restore DHCR7 activity in the CNS, we reported a stochastic rescue of pups up to 3 weeks beyond birth, despite almost no increase in brain cholesterol, or fall in precursor sterol concentrations . At the same time, we had also embarked upon restoration of cholesterol synthesis in peripheral (non-brain) tissues to see if this could abrogate the lethality. We report herein the effects of restoration of cholesterol synthesis in the developing liver, an organ that can circulate lipoproteins and cholesterol at a very embryonic age.
Selective reconstitution of liver cholesterol biosynthesis resulted in significant improvement of cholesterol homeostasis in all of the tissues examined in Dhcr7 null mice with the exception of the brain, confirming the embryonic liver's ability to supply cholesterol to peripheral tissues and the inability of cholesterol in the circulation to cross the blood-brain barrier. Cholesterol was detectable in liver and lung of non-transgenic Dhcr7-null term pups at ~40% of normal levels, which represents maternal transfer of this cholesterol and confirmed previous findings . Cholesterol levels in both liver and lungs of transgenic Dhcr7-null term pups increased to ~80% of WT level (and statistically not different from WT levels), indicating robust transgenic liver expression of DHCR7 led to adequate delivery of endogenously synthesized cholesterol to the lungs (presumably via lipoprotein-mediated transfer). The alleviation of the non-brain tissue cholesterol deficit in the transgenic Dhc7-null embryos led to improvement in the lung development, but not complete restoration of normal development.
Although our data support the conclusion that the lung defects may not be the major reason for the neonatal mortality, some caveats need to be mentioned. Despite significantly increased cholesterol in the lung, the sterol precursors were reduced by ~40% and not completely suppressed. Sterol precursors have been demonstrated to be part of plasma membranes and their presence may have significant implications in altering membrane signaling, by altering raft formation and functions [28–33]. In this study, membrane sterol distributions were further investigated and compared between transgenic and non-transgenic Dhcr7-/- lungs. Adapting an established method, lipid-rafts and non-rafts were prepared by sucrose gradient fractionation of Triton X100 extracted tissue homogenates . Bimodal profiles of cholesterol and 7/8DHC in sucrose gradient fractions were found, with first peak enriched in "raft" fractions and the second peak found in non-raft fractions. Thus, the bimodal profile may represent sterol portions distributed in lipid-raft microdomains and subcellular membranes [23, 25]. As expected, although significant restoration of cholesterol level, a considerable amount of 7/8DHC was still accumulated in the rafts of transgenic Dhcr7-/- lungs. Thus one mechanistic possibility is that raft-mediated signaling may be important for the terminal differentiation of Type 1 AECs (and capillary endothelial cells), and that this defect was not fully complemented by increasing the uptake of cholesterol from exogenous sources and thus the lung-associated mortality was not altered. The defect in cholesterol biosynthesis in the lungs will continue to lead to generation of precursor sterols and since these sterols are readily incorporated into plasma membranes, restoration of normal raft function may require direct restoration of Dhcr7 activity [30–34].
Another possibility is that pulmonary surfactant alteration may also play a role in the Dhcr7-deficient lung phenotype, since pulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lung, has been described to function as lipid microdomains. However, we have reported that expression of surfactant proteins, both at the mRNA and at the immunohistochemical level, was indistinguishable between Dhcr7-null and wild-type embryos . Cholesterol concentration, a critical parameter in modulating the lateral structure of pulmonary surfactant membranes as evidenced by in-vitro studies, may provide a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition . A difference in the condensing ability of cholesterol and 7DHC in monolayer films of dipalmitoyl phosphatidylcholine (DPPC), a major surfactant phospholipid, and their different abilities to form lateral domains with DPPC has also been demonstrated . Sterols can induce upregulation of phosphatidylcholine synthesis in cultured fibroblasts and this process is affected by the double-bond position in the sterol tetracyclic ring structure . Therefore, markedly reduced cholesterol content and the massive accumulation of precursor sterols, such as 7/8DHC, may affect lung developmental at late gestational stages by the modulation of surfactant properties, even if surfactant protein expression is normal. However, restoration of DHCR7 activity in the lung using a transgene that expressed DHCR7 activity did not rescue this lethality (Yu and Patel, unpublished observations). Additionally, our previous brain-specific DHCR7 transgenic expression studies showed that some rescue was possible, even though lung sterol profiles were not altered . Thus, while lung defects remain a contributory factor for this lethality, we contend that a defect in the CNS is the major reason why SLOS pups die soon after birth.
Some positive aspects should also be highlighted. Since the only cells that could synthesize cholesterol were the hepatocytes in the developing liver, yet there was robust restoration of cholesterol in the peripheral organs, this further confirms previous studies on the importance of lipoprotein-mediated liver-derived cholesterol delivery to these organs . Additionally, this study also confirms the now well-established physiological observation that the blood-brain barrier is an absolute barrier for entry of pre-formed cholesterol into the brain and it is operational as early as E10 in the mouse .
One curious finding confounding the above interpretations is that, although cholesterol levels in the transgenic Dhcr7-null livers increased to almost WT levels, the precursor levels in the liver were reduced by only 70 %, despite an almost 5-fold increase in the relative expression of the transgene in the liver. Our explanation for this is that the fetal and neonatal mouse liver contains more than hepatocytes, with a significant amount of hematopoetic activity that persists beyond birth. The ApoE promoter chosen will only result in robust hepatic-specific expression. Since total dissected organ tissue sterols are measured, we presume that the precursor accumulations are therefore primarily in these non-hepatic cells and not hepatocytes. If so, it also suggests that close proximity of the hepatocytes and hematopoetic stem cells does not result in complementation or clearance of the precursor sterols from the latter. Thus, while cholesterol can be efficiently delivered to the various tissues readily via the lipoprotein-mediated pathways, removal of the precursor sterols back to the liver may not be as efficient.
Restoration of liver cholesterol synthesis is an efficient method to restore peripheral tissue cholesterol in Dhcr7-null embryos, and this can lead to an improvement in the lung developmental defects reported previously and a minimal improvement in the neonatal lethality was also observed. However, this strategy does not affect the sterol defect in the CNS and this organ may be critically responsible for the neonatal lethality.
Generation of transgenic mice expressing human DHCR7specifically in liver
To generate transgenic mice expressing DHCR7 in the liver, we used a pLIV-LE6 vector that contains the constitutive human Apo E gene promoter and its hepatic control region (a gift from John Taylor, J. David Gladstone Institutes, San Francisco). N-terminal 3 hemagglutinin (HA) epitope tagged human DHCR7 cDNA with intron 5 (3HA-DHCR7in5) was generated by routine PCR insertion method. The transgenic plasmid (pLiv-11-3HA-DHCR7in5) was generated by cloning 3HA-DHCR7in5 fragment encoding the open reading frame of human DHCR7 into Mlu I-Cla I sites of pLIV-LE6. The 6-kb Not I-Spe I fragment of pLiv-11-3HA-DHCR7in5 (Fig. 1) was then isolated and injected into fertilized eggs (C57Bl × FVB/N) to generate transgenic mice by Medical University of South Carolina Transgenic Core facility. Transgene-specific PCR with the forward primer 5'-ATGGAGAGGAGGGGGCTGAGA-3' and the reverse primer 5'-TGGATTTTGCCAATAATGTCCAGC-3' was used for routine genotyping for transgene status. Tail DNA of transgenic (TgDHCR7) mice produced a PCR product of 530 bp (data not shown). Southern blots of tail DNAs were performed to identify three founder mice that harbored the integrated transgene. For Southern analysis, 10 μg of DNA was digested with Bam HI. The digested DNA was separated on a 0.8% agarose gel, transferred to a Hybond-N membrane, and hybridized with the 32P-labeled 1.5 kb human DHCR7 cDNA. Expression levels of the human DHCR7 transgene were determined by RT-PCR and Western blot analyses, on tissues of offspring from positive founders bred with C57BL/6J mice. Quantitative analyses of transgene expression were performed by real-time PCR (see below). Mice with high levels of transgene expression in the liver were bred to C57BL/6J mice, and two integrated transgenic lines, TgDHCR7-2 and TgDHCR7-3, were established. Both lines showed almost identical patterns of transgene expression and were used interchangeably in all crosses. Dhcr7+/- mice (N>12 on C57Bl/6J background), described previously [14, 27], were crossed with TgDHCR7 lines to generate Dhcr7+/- Tg+ animals. These were subsequently intercrossed to obtain Dhcr7-/-Tg+ mice. The transgenic mice were maintained as hemizygotes by breeding with C57BL/6J mice (Jackson Laboratories). All mice were housed in colony cages with a 12-hour light/12-hour dark cycle and fed Teklad Mouse/Rat Diet 7002 from Harlan Teklad (Madison, Wisconsin, USA). For animal experiments, non-transgenic littermates were used as controls for transgenic mice. All animal experiments were performed with the approval of the Institutional Animal Care and Research Advisory Committee at Medical College of Wisconsin.
Tissue expression of TgDHCR7
Total RNA was extracted from multiple tissues from postnatal 10-day-old offspring or livers of embryos at E11.5, E13.5, E16.5 from TgDHCR7 lines, as described previously [14, 15]. Random hexamers were used as primers for reverse transcription of 1 μg of total RNA, and cDNA first-strand synthesis carried out using Thermoscript (Invitrogen) according to manufacturer's protocol. Specific mRNA for TgDHCR7 was amplified using human DHCR7 cDNA primers (forward: 5'-GGACTGGTTTTCACTGGCGAGCG-3' and reverse: 5'-CCAGAGCAGGTGCGTGAGGAG-3'). The endogenous murine Dhcr7 and β-actin were amplified using Dhcr7 cDNA primers (forward: 5'-CCAAAGTCAAGAGTCCCAACGG-3' and reverse: 5'-ACCAGAGGATGTGGGTAATGAGC-3') and the β-actin gene primers, as described previously . PCR amplification was carried out as follows: denaturation for 3 min at 94°C and then a succession of 30 cycles by 30 sec at 94°C, 30 sec at 58°C, 1 min at 72°C, and a final extension at 72°C for 10 min. TgDHCR7 RT-PCR products (350 bp) were gel-purified and sequenced to confirm it contained correct spliced products using an automated capillary sequencer (Beckman Coulter CEQ 8000, CA), as previously described . SYBR-Green real-time PCR quantification of transgene transcripts was performed with ABI 7300 Real-time PCR system, using human DHCR7 cDNA primers (forward: 5'-CCCAGCTCTATACCTTGTGG-3' and reverse: 5'-CCAGAGCAGGTGCGTGAGGAG-3'), murine Dhcr7 cDNA primers (forward: GCCAAGACACCACCTGTGACAG-3' and reverse: 5'-TGGACGCCTCCCACATAACC-3') and β-actin primers (forward: ACCCTGTGCTGCTCACCGAG-3'and reverse: 5'-TGCCTGTGGTACGACCAGAGG-3'). Quantification of TgDHCR7 mRNA levels in transgenic livers on E16.5, relative to endogenous murine Dhcr7 mRNA, was performed using quantitative real-time PCR (qRT-PCR), with normalization performed to the β-actin mRNA abundance from the same sample.
For immunoblotting analysis of TgDHCR7, tissue membrane fractions were extracted using ProteinExtract native membrane protein extraction kit (Calbiochem, San Diego, CA) according to manufacturer's protocol. Briefly, approximately 50 mg of frozen tissues from brain, lung, liver, spleen, kidney, or skeletal muscle was homogenized in 2 ml of ice cold homogenate buffer added with cocktail of protease inhibitors, using a tissue homogenizer (PRO Scientific Inc., Oxford, CT) by three pulses of 10 seconds each and then subjected to Dounce homogenizer, pestle A, by 20 strokes. The homogenates were centrifuged at 16,000 g for 15 minutes at 4°C and supernatants referred as to cytosolic fractions. The pellets were resuspended in lysis buffer and gentled shaken for 30 minutes at 4°C, followed by centrifugation at 16,000 g for 15 minutes at 4°C. The supernatants were collected and referred as to membrane fractions. Protein concentrations in each cytosolic and membrane fractions were determined using Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) and 40 μg of protein from each sample was separated by 8% SDS-PAGE, transferred onto nitrocellulose membranes, incubated with HA monoclonal antibody (1:400, Santa Cruz Biotechnology, Santa Cruz, CA), Calnexin polyclonal antibody (1:200, Santa Cruz Biotechnology), or caveolin-1 (1:400, Santa Cruz Biotechnology) for 2 hours, followed by HRP second antibodies (1:4000, Santa Cruz Biotechnology), and visualized by chemiluminescent detection according to manufacturer's protocols (Perkin-Elmer Life Sciences, Boston, MA).
Sterol composition in tissues and plasma (pooled plasma collected by decapitation from 9 animals, pooling performed by combining plasma from 3 pups) were identified and quantitated by gas-chromatography-mass spectrometry (GC-MS) as described previously .
Histology and immunohistochemistry
Timed pregnant females at embryonic day (E) 19.5 were sacrificed. E19.5 fetuses were dissected free from the uteri and thoracic body parts harvested, fixed in 10% neutral buffered formalin, and embedded in paraffin. Tissues from non-transgenic Dhcr7+/+ (WT), Dhcr7-/- Tg- and Dhcr7-/- Tg+ animals were cut into 5-μm sections and stained with hematoxylin-eosin for routine histological examination. Immunohistochemistry (IHC) to determine T1-α, SP-C, caveolin-1 (cav-1) and PECAM-1 expression in tissue sections was performed in standard IHC procedures as described previously . Immunofluorescence staining was performed to determine TgDHCR7 expression in livers by use of a monoclonal anti-HA probe conjugated with TRITC (1:200, Santa Cruz Biotechnology).
Isolation of detergent-resistant lipid rafts
A modification of the method of Mukherjee et al.  was used to isolate lipid rafts from neonatal lung tissues. Briefly, frozen lung tissues were homogenized in an ice-cold lysis buffer containing 5% glycerol in buffer A (50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 0.15 M NaCl, 20 mM NaF, 1 mM Na3VO4, 5 mM β-mercaptoethanol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF). Tissue debris and nuclei were removed by centrifugation at 1,000 g for 5 minutes. Protein concentration of the postnuclear supernatants (PNS) was measured using Protein Reagent (Bio-Rad) and adjusted to 2 mg/ml. 10 % Triton X-100 (TX) was added to 2 ml of PNS to a final concentration of 0.5 % TX followed by a 30 min incubation on ice. The samples were mixed with equal volume of 80 % (w/v) sucrose in buffer A, and then overlaid with 2.0 ml each of 35, 30, 25, and 5 % (w/v) sucrose (all in buffer A). Sucrose gradient was spun at 38,000 rpm in a Sorval 90 ultracentrifuge using TH-641 rotor for 15 hr at 4°C. Twelve fractions of each 1.0 ml were collected from the top to bottom. Sterol composition in each 12 fractions and pooled lipid-raft and non-raft fractions was quantitatively determined as described above and normalized to organic-phosphate as determined by a phosphate assay . Based on cholesterol profile, as well as cellular organelle protein markers and sphingolipid profile (data not shown), lipid rafts or detergent resistant membranes (DRMs) were defined as the membrane materials that floated at the interface of 5% and 25% sucrose in the density range 1.055~1.115 g/ml (fraction 2~4); non-raft materials were collected in the density range 1.130~1.180 g/ml (fraction 8~11).
The authors would like to thank Dr. John Taylor for providing human apoE gene promoter plasmid, to Abigail Maciolek, Yanhong Cai and Bibi Pcolinsky for technical assistance. This research was supported by a Biomedical Research Grant RG-11311-M from the American Lung Association (HY), by grants from the Office of Research and Development, Department of Veterans Affairs (GST and GX); and by PHS grant HL68660 from the National Heart, Blood and Lung Institute, NIH (SBP).
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