Abstract
Smith–Lemli–Opitz syndrome (SLOS), desmosterolosis and lathosterolosis are human syndromes caused by defects in the final stages of cholesterol biosynthesis. Many of the developmental malformations in these syndromes occur in tissues and structures whose embryonic patterning depends on signaling by the Hedgehog (Hh) family of secreted proteins. Here we report that response to the Hh signal is compromised in mutant cells from mouse models of SLOS and lathosterolosis and in normal cells pharmacologically depleted of sterols. We show that decreasing levels of cellular sterols correlate with diminishing responsiveness to the Hh signal. This diminished response occurs at sterol levels sufficient for normal autoprocessing of Hh protein, which requires cholesterol as cofactor and covalent adduct. We further find that sterol depletion affects the activity of Smoothened (Smo), an essential component of the Hh signal transduction apparatus.
Similar content being viewed by others
Main
The role of cholesterol in Hh protein biogenesis suggested that impaired Hh autoprocessing might underlie some of the developmental abnormalities in SLOS (Fig. 1 and Table 1; ref. 1). An additional role for cholesterol in Hh signal response was suggested by the observation that cyclopamine and jervine, teratogenic plant alkaloids that block Hh signaling, also inhibit cholesterol transport and synthesis2,3. But cyclopamine has since been shown to specifically inhibit Hh signaling by binding to a pathway component4, and the doses of these alkaloids required to inhibit Hh signaling are lower than those required to block cholesterol transport (ref. 5 and M.K.C., unpublished data).
To determine how cholesterol may affect Hh signaling in embryonic development, we exposed chick embryos to cyclodextrin, a cyclic oligosaccharide that forms non-covalent complexes with sterols6 and can be used to extract and deplete cholesterol from living cells7. Cyclodextrin treatment caused variable loss of the frontonasal process and other midline structures (Fig. 2a), and the spectrum of facial defects was similar to that resulting from exposure to the Hh-pathway antagonist jervine3. The most severely affected embryos developed a proboscis-like structure that phenocopies the nasal rudiments of mouse embryos that are homozygous with respect to mutations in the gene Sonic hedgehog (Shh; ref. 8).
We further investigated Shh signaling in embryonic tissues by exposing chick neural-plate explants to recombinant Shh protein (ShhN, 30 nM) in the presence of cyclodextrin (Fig. 2b). The response to this level of ShhN protein was uniform production of HNF3β (hepatocyte nuclear factor 3β or forkhead box A2; ref. 3), an indicator of floor plate fate. This high-threshold response was blocked in the presence of 400 μM cyclodextrin and replaced by nearly uniform expression of ISL1, an intermediate-threshold response indicative of motor-neuron fate. At 200 μM cyclodextrin, both HNF3β and ISL1 were expressed, suggesting that lower levels of cyclodextrin were less inhibitory. As active ShhN protein was exogenously supplied, this dose-dependent inhibition suggests that sterol deficits affect response to the Hh signal. Furthermore, this effect seemed to be specific, as treatment with 400 μM cyclodextrin alone did not inhibit BMP-induced migration of neural-crest cells9 (Fig. 2c).
Hh protein biogenesis involves internal cleavage and covalent addition of cholesterol through an autoprocessing reaction1. Cyclodextrin treatment of cultured cells has previously been reported to interfere with Shh autoprocessing10, an effect distinct from inhibition of response that we observed. To further investigate whether signal production or signal response is the prevailing inhibitory mechanism in cholesterol synthesis disorders, we established embryonic fibroblast cell lines from mouse models of SLOS11 and lathosterolosis and examined Shh signal biogenesis and response in parallel under identical culture conditions.
Shh protein was efficiently processed in mouse embryonic fibroblasts (MEFs) lacking 7-dehydrocholesterol reductase (Dhcr7) or lathosterol 5-desaturase (Sc5d) enzymes (models of SLOS and lathosterolosis, respectively; see Fig. 1b), with no observable effect of transient cyclodextrin treatment or growth in lipid-depleted culture medium (Fig. 3a). All of the processed N-terminal product (ShhNp) from mutant cultures was cell-associated and had an electrophoretic mobility suggestive of sterol modification (Fig. 3a). The autoprocessing reaction probably proceeds to completion because cholesterol levels are only reduced by roughly 50% under the conditions used for depletion (Fig. 3b) and because 7-dehydrocholesterol and lathosterol both participate efficiently as sterol adducts in the Shh processing reaction3. Likewise, cholesterol levels are reduced but never absent in the serum of individuals with SLOS12.
In contrast with their normal Shh autoprocessing, MEFs with mutations in Dhcr7 and Sc5d had clear deficiencies in their ability to respond to Shh signal when transiently treated with cyclodextrin and grown in lipid-depleted culture medium (Fig. 3b). These results indicate that signal response is more sensitive than is signal biogenesis to perturbations of cholesterol homeostasis. Cells heterozygous with respect to the mutations in Dhcr7 and Sc5d maintained a normal response to ShhNp stimulation under all growth conditions (Fig. 3b), presumably because there was sufficient synthetic activity from the functional allele. In these experiments, the initial transient cyclodextrin treatment is needed to reduce sterol levels to below 40 μg mg−1 protein to affect pathway response (Fig. 3b).
To further investigate Hh signal response in other cultured cells, we tested the ability of pharmacological interventions to mimic the effects of genetic defects in sterol biosynthesis. Continuous treatment with 500 μM cyclodextrin produced about 50% inhibition of Shh signal response in NIH3T3 cells, and 2 mM cyclodextrin produced nearly complete inhibition (Fig. 4a). Transient cyclodextrin treatment for 30 minutes before exposure to ShhNp, however, did not affect response, even at 15 mM cyclodextrin, a concentration 30 times higher than the IC50 for continuous cyclodextrin treatment (Fig. 4b). ShhNp response was also not affected by continuous exposure to compactin, an inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase that blocks sterol synthesis13. The combination of transient cyclodextrin treatment with continuous compactin exposure, however, blocked Shh signal response (Fig. 4b). These data suggest that compactin treatment, when combined with transient cyclodextrin treatment, can mimic a genetic defect in sterol biosynthesis to inhibit Shh signal response. The Shh autoprocessing reaction was not affected by these experimental conditions (data not shown).
Cyclodextrins form complexes with hydrophobic compounds including proteins and phospholipids in addition to sterols6. But the effect of cyclodextrin treatment is probably due to sterol depletion, as transient cyclodextrin treatment only inhibited pathway activity in the presence of sterol biosynthetic mutations or of a statin, and no measurable recovery from the impact of deficits in molecules other than sterols was achieved during prolonged incubation after transient cyclodextrin treatment (Fig. 4c). Furthermore, other sterol-specific perturbations, such as treatment with nystatin or filipin14, also blocked the response of cells to ShhNp protein (data not shown), and the degree of pathway inhibition in sterol biosynthetic mutant and statin-treated fibroblasts correlated inversely with total sterol levels (Fig. 4d). An inhibitory role for 7-dehydro-cholesterol, lathosterol and other cholesterol precursors that accumulate in the Dhcr7 and Sc5d mutant cells seems improbable because synthesis of such precursors in statin-treated wild-type cells is blocked. Indeed, the correlation between responsiveness and overall sterol content (Fig. 4d) suggests that these precursors may contribute to pathway response, although their effectiveness relative to that of cholesterol is unknown.
The ability to pharmacologically mimic sterol biosynthetic defects affords an opportunity to identify the Hh pathway component most directly affected by sterol perturbations. Patched (Ptch) and Smoothened (Smo) stand out as candidate targets because of their integral membrane structure and essential roles in regulating cellular response to the Hh signal. Genetic and biochemical evidence indicates that Ptch suppresses the activity of Smo and that Hh binding to Ptch releases this suppression, allowing Smo to activate a transcriptional response through the Gli family of transcription factors (Fig. 1b). We examined the ability of sterol perturbations to affect the constitutive Hh pathway activity in fibroblasts derived from Ptch−/− embryos15,16 and found a dose-dependent reduction by transient treatment with increasing concentrations of cyclodextrin in combination with compactin (Fig. 5a). Therefore, sterol depletion can block pathway activity independently of Ptch action and may act at a point in the pathway downstream of Ptch.
We next examined the effects of depleting sterols in NIH3T3 cells stably transfected to express wild-type or oncogenic, activated Smo (ref. 16; SmoA1; W539L). We found that sterol depletion could completely block the modest level of pathway activation produced by overexpression of Smo (Fig. 5b), but that it scarcely affected pathway activation by SmoA1 (Fig. 5c). The susceptibility of wild-type Smo but resistance of a mutant activated form to sterol depletion suggests that Smo may be the site of sterol action. Consistent with this conclusion, sterol deprivation did not affect the activation of the pathway due to expression of Gli1, which acts downstream of Smo (data not shown). The retention of normal Hh pathway activity in cells expressing activated Smo indicates that pathway components downstream of Smo function normally under conditions of sterol depletion.
Previous work has suggested that Smo activity is governed by a balance between active and inactive conformations16. The resistance to cholesterol deprivation of activated Smo suggests that Smo conformation may be the target of cholesterol deprivation. This effect could be mediated either through direct interaction of cholesterol with Smo or through an impact on membrane properties, as reported for the function of other seven-transmembrane-domain proteins, such as the oxytocin or the brain cholecystokinin receptors17. One possibility is that sterol depletion could affect a lipid microdomain or raft-mediated process18 required for Smo activity, although we did not observe a change in Smo fractionation with respect to detergent-resistant membranes on sterol depletion (data not shown). Alternatively, the effects of sterol depletion on Smo activity might be indirectly mediated through an as yet uncharacterized interacting component.
Sterol depletion has been previously reported to affect Shh autoprocessing10. This depletion was probably somewhat more severe than that produced by our experimental manipulations. Furthermore, we found that Hh signal response is more sensitive than Hh autoprocessing to inhibition by mutational or pharmacological sterol depletion. We therefore conclude that inhibition of response to Hh protein is a more probable cause of the malformations associated with cholesterol biosynthetic disorders than is inhibition of Hh autoprocessing. Other processes might also be affected by defects in distal cholesterol biosynthesis, as not all of the malformations are necessarily accounted for by impaired Hh signaling. Nevertheless, our findings help explain many developmental malformations associated with a relatively common genetic disorder, SLOS, whose incidence among European Caucasians is 1 in 22,000 (ref. 19). Our findings may also be relevant to other etiologies of abnormal human development, as holoprosencephaly is reported to occur at a frequency of 1 in 250 among aborted fetuses20. The surprising connection between cholesterol synthesis and Hh signal response reported here suggests that signaling pathways involved in developmental patterning must be considered as potential targets of any seemingly simple metabolic defect associated with developmental malformations.
Methods
Chick embryos.
We applied methyl-β-cyclodextrin (200 μl of a 10% w/v solution in L-15 medium (Sigma and Life Technologies)) to windowed, fertile chick eggs (White Leghorn) after 15 h of incubation. For an average egg volume of 50 ml, the final concentration of cyclodextrin was 375 μM. After 4 d of further incubation, we processed the embryos for scanning electron microscopy. We dissected the neural plate and epidermal ectoderm from stage 9–10 chick embryos, cultured them in collagen and induced them with purified ShhN21 as described3. We added methyl-β-cyclodextrin to the chick explant cultures 4 h after induction with ShhN was initiated.
Analysis of Shh protein biogenesis.
We plated MEFs in a 10-cm2 dish (Falcon) and transfected them (Fugene 6, Roche) with a full length Shh expression construct (pRK5-Shh, 5μg) when the cells were roughly 75% confluent. The next day, we changed the culture medium (Dulbecco's modified Eagle medium with 10% fetal bovine serum) to contain either 0.5% fetal bovine serum, 0.5% lipid-depleted serum or lipid-depleted serum after a 30-min treatment with 3.8 mM methyl-β-cyclodextrin (Sigma). After an additional 24 h in culture, we lysed the MEFs in RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 μg ml−1 leupeptin, 1 μg ml aprotinin−1, 0.2 mM phenylmethylsulfonyl fluoride), immunoprecipitated Shh protein from the cell lysates with a monoclonal antibody that recognizes the N-terminal signaling domain (5E1, Developmental Studies Hybridoma Bank) and immunoblotted them with a polyclonal antibody preparation (JH134).
Shh signaling assays.
We carried out Shh signaling assays as described16 in primary fibroblasts lacking functional Dhcr7, Sc5d or Ptch and in NIH3T3 fibroblasts. Dhcr7−/− and Dhcr7+/− MEFs were generated from embryonic day–9 mutant mice and Sc5d−/− and Sc5d+/− MEFs from embryonic day–14.5 mutant mice. We generated a reporter construct with firefly luciferase and Gli (pGL3-8×Gli-luciferase) by cloning eight tandem Gli-binding sites and a lens crystallin promoter from the 8×Gli-BS Luc construct22 into the pGL3-Basic vector (Promega). We determined relative Hh pathway activity in Dhcr7−/−, Dhcr7+/−, Sc5d−/− and Sc5d+/− MEFs from the expression of transiently transfected pGL3-8×Gli-luciferase and control Renilla luciferase (pRL-SV40; Promega) vectors. In Ptch−/− MEFs, we normalized β-galactosidase activity expressed under the control of the Ptch promoter for protein levels to determine relative Hh pathway activity. In the NIH3T3 cell clone Shh-LIGHT Z3, we normalized 8×Gli-BS luciferase activity for β-galactosidase activity from stably transfected vectors (8×Gli-BS Luc22 and pIZ-lacZ). We established clonal sublines of Shh-LIGHT Z3 by cotransfecting either Smo tagged with Myc epitope or SmoA1 with vector encoding G418 resistance (pGT; Invivogen). All transfections were done with Fugene 6. We carried out cyclodextrin treatments for 30 min with methyl-β-cyclodextrin (Sigma) dissolved in Dulbecco's modified Eagle medium, preceded and followed by two washes with phosphate-buffered saline. Fetal bovine serum was depleted of lipids as described23. We purchased compactin as an active sodium salt form of mevastatin (Biomol) and dissolved it in dimethylsulfoxide.
Sterol analysis.
We extracted neutral sterols and analyzed them as described24 from replicate wells of MEFs cultured in parallel with those used for Hh signaling assays.
References
Porter, J.A., Young, K.E. & Beachy, P.A. Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255–259 (1996).
Beachy, P.A. et al. Multiple roles of cholesterol in hedgehog protein biogenesis and signaling. Cold Spring Harb. Symp. Quant. Biol. 62, 191–204 (1997).
Cooper, M.K., Porter, J.A., Young, K.E. & Beachy, P.A. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280, 1603–1607 (1998).
Chen, J.K., Taipale, J., Cooper, M.K. & Beachy, P.A. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, 2743–2748 (2002).
Incardona, J.P. et al. Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport. Dev. Biol. 224, 440–452 (2000).
Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K. & Pitha, J. Differential effects of α-, β- and γ-cyclodextrins on human erythrocytes. Eur. J. Biochem. 186, 17–22 (1989).
Kilsdonk, E.P. et al. Cellular cholesterol efflux mediated by cyclodextrins. J. Biol. Chem. 270, 17250–17256 (1995).
Chiang, C. et al. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407–413 (1996).
Liem, K.F. Jr., Tremml, G., Roelink, H. & Jessell, T.M. Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell 82, 969–979 (1995).
Guy, R.K. Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation. Proc. Natl. Acad. Sci. USA 97, 7307–7312 (2000).
Wassif, C.A. et al. Mutations in the human sterol δ7-reductase gene at 11q12–13 cause Smith–Lemli–Opitz syndrome. Am. J. Hum. Genet. 63, 55–62 (1998).
Kelley, R.I. Inborn errors of cholesterol biosynthesis. Adv. Pediatr. 47, 1–53 (2000).
Brown, M.S., Faust, J.R. & Goldstein, J.L. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts incubated with compactin (ML-236B), a competitive inhibitor of the reductase. J. Biol. Chem. 253, 1121–1128 (1978).
Bittman, R. & Fischkoff, S.A. Fluorescence studies of the binding of the polyene antibiotics filipin 3, amphotericin B, nystatin, and lagosin to cholesterol. Proc. Natl. Acad. Sci. USA 69, 3795–3799 (1972).
Goodrich, L.V., Milenkovic, L., Higgins, K.M. & Scott, M.P. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science 277, 1109–1113 (1997).
Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).
Gimpl, G., Burger, K. & Fahrenholz, F. Cholesterol as modulator of receptor function. Biochemistry 36, 10959–10974 (1997).
Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).
Nowaczyk, M.J., McCaughey, D., Whelan, D.T. & Porter, F.D. Incidence of Smith–Lemli–Opitz syndrome in Ontario, Canada. Am. J. Med. Genet. 102, 18–20 (2001).
Matsunaga, E. & Shiota, K. Holoprosencephaly in human embryos: epidemiologic studies of 150 cases. Teratology 16, 261–272 (1977).
Roelink, H. et al. Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81, 445–455 (1995).
Sasaki, H., Hui, C., Nakafuku, M. & Kondoh, H. A binding site for Gli proteins is essential for HNF-3β floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124, 1313–1322 (1997).
Gibson, K.M. et al. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured fibroblasts from patients with mevalonate kinase deficiency: differential response to lipid supplied by fetal bovine serum in tissue culture medium. J. Lipid Res. 31, 515–521 (1990).
Kelley, R.I. Diagnosis of Smith–Lemli–Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts. Clin. Chim. Acta 236, 45–58 (1995).
Donnai, D., Burn, J. & Hughes, H. Smith–Lemli–Opitz syndromes: do they include the Pallister–Hall syndrome? Am. J. Med. Genet. 28, 741–743 (1987).
Parnes, S., Hunter, A.G., Jimenez, C., Carpenter, B.F. & MacDonald, I. Apparent Smith–Lemli–Opitz syndrome in a child with a previously undescribed form of mucolipidosis not involving the neurons. Am. J. Med. Genet. 35, 397–405 (1990).
Clayton, P., Mills, K., Keeling, J. & FitzPatrick, D. Desmosterolosis: a new inborn error of cholesterol biosynthesis. Lancet 348, 404 (1996).
Ingham, P.W. Hedgehog signaling: a tale of two lipids. Science 294, 1879–1881 (2001).
Wang, B., Fallon, J.F. & Beachy, P.A. Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100, 423–434 (2000).
Chamoun, Z. et al. Skinny hedgehog, an acyltransferase required for palmitoylation and activity of the hedgehog signal. Science 293, 2080–2084 (2001).
Acknowledgements
We thank R.K. Mann and D. Valle for critical review of this manuscript. M.K.C was supported by a career development award from the Burroughs Wellcome Fund and a US National Institutes of Health K08 award from the National Institute of Neurological Disorders and Stroke. This work was supported in part by a grant from the US National Institutes of Health (P.A.B.). P.A.B. is an investigator of the Howard Hughes Medical Institute.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Cooper, M., Wassif, C., Krakowiak, P. et al. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33, 508–513 (2003). https://doi.org/10.1038/ng1134
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ng1134
This article is cited by
-
GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway
Nature Structural & Molecular Biology (2024)
-
Maternal lipid profile in pregnancy and embryonic size: a population-based prospective cohort study
BMC Pregnancy and Childbirth (2022)
-
CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids
Nature Communications (2022)
-
Hedgehog signaling and its molecular perspective with cholesterol: a comprehensive review
Cellular and Molecular Life Sciences (2022)
-
Forebrain Shh overexpression improves cognitive function and locomotor hyperactivity in an aneuploid mouse model of Down syndrome and its euploid littermates
Acta Neuropathologica Communications (2021)