Disruption of Dhcr7 and Insig1/2 in cholesterol metabolism causes defects in bone formation and homeostasis through primary cilium formation

Abstract

Human linkage studies suggest that craniofacial deformities result from either genetic mutations related to cholesterol metabolism or high-cholesterol maternal diets. However, little is known about the precise roles of intracellular cholesterol metabolism in the development of craniofacial bones, the majority of which are formed through intramembranous ossification. Here, we show that an altered cholesterol metabolic status results in abnormal osteogenesis through dysregulation of primary cilium formation during bone formation. We found that cholesterol metabolic aberrations, induced through disruption of either Dhcr7 (which encodes an enzyme involved in cholesterol synthesis) or Insig1 and Insig2 (which provide a negative feedback mechanism for cholesterol biosynthesis), result in osteoblast differentiation abnormalities. Notably, the primary cilia responsible for sensing extracellular cues were altered in number and length through dysregulated ciliary vesicle fusion in Dhcr7 and Insig1/2 mutant osteoblasts. As a consequence, WNT/β-catenin and hedgehog signaling activities were altered through dysregulated primary cilium formation. Strikingly, the normalization of defective cholesterol metabolism by simvastatin, a drug used in the treatment of cholesterol metabolic aberrations, rescued the abnormalities in both ciliogenesis and osteogenesis in vitro and in vivo. Thus, our results indicate that proper intracellular cholesterol status is crucial for primary cilium formation during skull formation and homeostasis.

References

[1] Porter FD, Herman GE. Malformation syndromes caused by disorders of cholesterol synthesis. J. Lipid Res., 2011, 52:6-34

[2] Iwata J et al. Modulation of lipid metabolic defects rescues cleft palate in Tgfbr2 mutant mice. Hum. Mol. Genet., 2014, 23:182-193

[3] Engelking LJ et al. Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin. J. Clin. Invest., 2006, 116:2356-2365

[4] Rossi M et al. Clinical phenotype of lathosterolosis. Am. J. Med. Genet. A, 2007, 143A:2371-2381

[5] Porter FD. Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management. Eur. J. Hum. Genet., 2008, 16:535-541

[6] Edison RJ et al. Adverse birth outcome among mothers with low serum cholesterol. Pediatrics, 2007, 120:723-733

[7] Maymunah AO, Kehinde O, Abidoye G, Oluwatosin A. Hypercholesterolaemia in pregnancy as a predictor of adverse pregnancy outcome. Afr. Health Sci., 2014, 14:967-973

[8] Battaile KP, Steiner RD. Smith-Lemli-Opitz syndrome: the first malformation syndrome associated with defective cholesterol synthesis. Mol. Genet. Metab., 2000, 71:154-162

[9] Fitzky BU et al. Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome. Proc. Natl Acad. Sci. USA, 1998, 95:8181-8186

[10] Porter FD. RSH/Smith-Lemli-Opitz syndrome: a multiple congenital anomaly/mental retardation syndrome due to an inborn error of cholesterol biosynthesis. Mol. Genet. Metab., 2000, 71:163-174

[11] Fitzky BU et al. 7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith-Lemli-Opitz/RSH syndrome. J. Clin. Invest., 2001, 108:905-915

[12] Engelking LJ et al. Schoenheimer effect explained-feedback regulation of cholesterol synthesis in mice mediated by Insig proteins. J. Clin. Invest., 2005, 115:2489-2498

[13] Dong XY, Tang SQ, Chen JD. Dual functions of Insig proteins in cholesterol homeostasis. Lipids Health Dis., 2012, 11:173

[14] Emmer BT, Maric D, Engman DM. Molecular mechanisms of protein and lipid targeting to ciliary membranes. J. Cell Sci., 2010, 123:529-536

[15] Senin II et al. Recoverin and rhodopsin kinase activity in detergent-resistant membrane rafts from rod outer segments. J. Biol. Chem., 2004, 279:48647-48653

[16] Iomini C, Li L, Mo W, Dutcher SK, Piperno G. Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas. Curr. Biol.: CB, 2006, 16:1147-1153

[17] Travis AJ et al. Expression and localization of caveolin-1, and the presence of membrane rafts, in mouse and Guinea pig spermatozoa. Developmental Biol., 2001, 240:599-610

[18] Tull D et al. SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania. Mol. Biol. Cell, 2004, 15:4775-4786

[19] Chang CF, Schock EN, Attia AC, Stottmann RW, Brugmann SA. The ciliary baton: orchestrating neural crest cell development. Curr. Top. Dev. Biol., 2015, 111:97-134

[20] Nachury MV, Seeley ES, Jin H. Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier? Annu Rev. Cell Dev. Biol., 2010, 26:59-87

[21] Goetz SC, Anderson KV. The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet., 2010, 11:331-344

[22] Lewis AE, Vasudevan HN, O’Neill AK, Soriano P, Bush JO. The widely used Wnt1-Cre transgene causes developmental phenotypes by ectopic activation of Wnt signaling. Dev. Biol., 2013, 379:229-234

[23] Chai Y et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development, 2000, 127:1671-1679

[24] Wilkie AO. Craniosynostosis: genes and mechanisms. Hum. Mol. Genet., 1997, 6:1647-1656

[25] Knodler A et al. Coordination of Rab8 and Rab11 in primary ciliogenesis. Proc. Natl Acad. Sci. USA, 2010, 107:6346-6351

[26] Westlake CJ et al. Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proc. Natl Acad. Sci. USA, 2011, 108:2759-2764

[27] Pedersen LB, Veland IR, Schroder JM, Christensen ST. Assembly of primary cilia. Dev. Dyn., 2008, 237:1993-2006

[28] Arquint C, Sonnen KF, Stierhof YD, Nigg EA. Cell-cycle-regulated expression of STIL controls centriole number in human cells. J. Cell Sci., 2012, 125:1342-1352

[29] Ko MA et al. Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis. Nat. Genet., 2005, 37:883-888

[30] Kong D et al. Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles. J. Cell Biol., 2014, 206:855-865

[31] Pearson CG, Winey M. Plk4/SAK/ZYG-1 in the regulation of centriole duplication. F1000 Biol. Rep., 2010, 2:58

[32] Peel N, Stevens NR, Basto R, Raff JW. Overexpressing centriole-replication proteins in vivo induces centriole overduplication and de novo formation. Curr. Biol., 2007, 17:834-843

[33] Strnad P et al. Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle. Dev. Cell, 2007, 13:203-213

[34] Tsou MF et al. Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Dev. Cell, 2009, 17:344-354

[35] Vulprecht J et al. STIL is required for centriole duplication in human cells. J. Cell Sci., 2012, 125:1353-1362

[36] Day TF, Yang Y. Wnt and hedgehog signaling pathways in bone development. J. Bone Jt. Surg. Am., 2008, 90 Suppl 1 19-24

[37] Gaur T et al. Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression. J. Biol. Chem., 2005, 280:33132-33140

[38] Yuan X, Yang S. Primary cilia and intraflagellar transport proteins in bone and cartilage. J. Dent. Res., 2016, 95:1341-1349

[39] Bai CB, Auerbach W, Lee JS, Stephen D, Joyner AL. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development, 2002, 129:4753-4761

[40] Goodrich LV, Johnson RL, Milenkovic L, McMahon JA, Scott MP. Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev., 1996, 10:301-312

[41] Ingham PW. Localized hedgehog activity controls spatial limits of wingless transcription in the Drosophila embryo. Nature, 1993, 366:560-562

[42] Marigo V, Johnson RL, Vortkamp A, Tabin CJ. Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol., 1996, 180:273-283

[43] Platt KA, Michaud J, Joyner AL. Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice. Mech. Dev., 1997, 62:121-135

[44] Aza-Blanc P, Ramirez-Weber FA, Laget MP, Schwartz C, Kornberg TB. Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell, 1997, 89:1043-1053

[45] Aza-Blanc P, Lin HY, Ruiz i Altaba ,A, Kornberg ,TB. Expression of the vertebrate Gli proteins in Drosophila reveals a distribution of activator and repressor activities. Development, 2000, 127:4293-4301

[46] Hammond N.L., Brookes K.J., Dixon M.J.. Ectopic Hedgehog Signaling Causes Cleft Palate and Defective Osteogenesis. Journal of Dental Research, 2018, 97 13 1485-1493

[47] St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev., 1999, 13:2072-2086

[48] Lenton K et al. Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling. Genesis, 2011, 49:784-796

[49] McMurray RJ, Wann AK, Thompson CL, Connelly JT, Knight MM. Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells. Sci. Rep., 2013, 3

[50] Yu HM et al. The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development, 2005, 132:1995-2005

[51] Lancaster MA, Schroth J, Gleeson JG. Subcellular spatial regulation of canonical Wnt signalling at the primary cilium. Nat. Cell Biol., 2011, 13:700-707

[52] Baron R, Kneissel M. WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat. Med., 2013, 19:179-192

[53] Ali SA et al. Regulation of cholesterol homeostasis by hedgehog signaling in osteoarthritic cartilage. Arthritis Rheumatol., 2016, 68:127-137

[54] Tsushima Hidetoshi, Tang Yuning J., Puviindran Vijitha, Hsu Shu-Hsuan Claire, Nadesan Puviindran, Yu Chunying, Zhang Hongyuan, Mirando Anthony J., Hilton Matthew J., Alman Benjamin A.. Intracellular biosynthesis of lipids and cholesterol by Scap and Insig in mesenchymal cells regulates long bone growth and chondrocyte homeostasis. Development, 2018, 145 13 dev162396

[55] Holick MF. Vitamin D: a millenium perspective. J. Cell Biochem., 2003, 88:296-307

[56] Morris JG, Earle KE, Anderson PA. Plasma 25-hydroxyvitamin D in growing kittens is related to dietary intake of cholecalciferol. J. Nutr., 1999, 129:909-912

[57] Prabhu AV, Luu W, Sharpe LJ, Brown AJ. Cholesterol-mediated degradation of 7-dehydrocholesterol reductase switches the balance from cholesterol to vitamin D synthesis. J. Biol. Chem., 2016, 291:8363-8373

[58] Bonjour JP et al. The increase in skin 7-dehydrocholesterol induced by an hypocholesterolemic agent is associated with elevated 25-hydroxyvitamin D3 plasma level. Pflug. Arch., 1987, 410:165-168

[59] Movassaghi M, Bianconi S, Feinn R, Wassif CA, Porter FD. Vitamin D levels in Smith-Lemli-Opitz syndrome. Am. J. Med Genet. A, 2017, 173:2577-2583

[60] Novakovic B et al. Maternal vitamin D predominates over genetic factors in determining neonatal circulating vitamin D concentrations. Am. J. Clin. Nutr., 2012, 96:188-195

[61] Stordal K et al. Fetal and maternal genetic variants influencing neonatal vitamin D status. J. Clin. Endocrinol. Metab., 2017, 102:4072-4079

[62] Kelley RI. Inborn errors of cholesterol biosynthesis. Adv. Pediatr., 2000, 47:1-53

[63] Lindegaard ML et al. Characterization of placental cholesterol transport: ABCA1 is a potential target for in utero therapy of Smith-Lemli-Opitz syndrome. Hum. Mol. Genet., 2008, 17:3806-3813

[64] Tint GS, Yu H, Shang Q, Xu G, Patel SB. The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols. J. Lipid Res., 2006, 47:1535-1541

[65] Hoey DA, Chen JC, Jacobs CR. The primary cilium as a novel extracellular sensor in bone. Front. Endocrinol., 2012, 3:75

[66] Anderson CT et al. Primary cilia: cellular sensors for the skeleton. Anat. Rec., 2008, 291:1074-1078

[67] Qiu N et al. Disruption of Kif3a in osteoblasts results in defective bone formation and osteopenia. J. Cell Sci., 2012, 125:1945-1957

[68] Lehti MS et al. Cilia-related protein SPEF2 regulates osteoblast differentiation. Sci. Rep., 2018, 8

[69] Cooper MK et al. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat. Genet., 2003, 33:508-513

[70] Blassberg R, Macrae JI, Briscoe J, Jacob J. Reduced cholesterol levels impair Smoothened activation in Smith-Lemli-Opitz syndrome. Hum. Mol. Genet., 2016, 25:693-705

[71] Blassberg R, Jacob J. Lipid metabolism fattens up hedgehog signaling. BMC Biol., 2017, 15

[72] Abzhanov A, Rodda SJ, McMahon AP, Tabin CJ. Regulation of skeletogenic differentiation in cranial dermal bone. Development, 2007, 134:3133-3144

[73] Jacob S, Wu C, Freeman TA, Koyama E, Kirschner RE. Expression of Indian Hedgehog, BMP-4 and Noggin in craniosynostosis induced by fetal constraint. Ann. Plast. Surg., 2007, 58:215-221

[74] Veistinen LK et al. Regulation of calvarial osteogenesis by concomitant de-repression of GLI3 and activation of IHH targets. Front Physiol., 2017, 8:1036

[75] Jeong J, Mao J, Tenzen T, Kottmann AH, McMahon AP. Hedgehog signaling in the neural crest cells regulates the patterning and growth of facial primordia. Genes Dev., 2004, 18:937-951

[76] Lan Y, Jiang R. Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth. Development, 2009, 136:1387-1396

[77] Rice R et al. Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. J. Clin. Invest., 2004, 113:1692-1700

[78] Cobourne MT et al. Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome. Dev. Biol., 2009, 331:38-49

[79] Tu X et al. Osteocytes mediate the anabolic actions of canonical Wnt/beta-catenin signaling in bone. Proc. Natl Acad. Sci. USA, 2015, 112:E478-486

[80] Chen S et al. Adverse effects of osteocytic constitutive activation of ss-catenin on bone strength and bone growth. J. Bone Miner. Res.: Off. J. Am. Soc. Bone Miner. Res., 2015, 30:1184-1194

[81] Jia M et al. Effects of constitutive beta-catenin activation on vertebral bone growth and remodeling at different postnatal stages in mice. PLoS ONE, 2013, 8

[82] Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell, 2005, 8:739-750

[83] Corbit KC et al. Kif3a constrains beta-catenin-dependent Wnt signalling through dual ciliary and non-ciliary mechanisms. Nat. Cell Biol., 2008, 10:70-76

[84] Brugmann SA et al. A primary cilia-dependent etiology for midline facial disorders. Hum. Mol. Genet., 2010, 19:1577-1592

[85] Correa-Cerro LS et al. Development and characterization of a hypomorphic Smith-Lemli-Opitz syndrome mouse model and efficacy of simvastatin therapy. Hum. Mol. Genet., 2006, 15:839-851

[86] DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development, 1999, 126:4557-4568

[87] Lustig B et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol., 2002, 22:1184-1193

[88] Iwata J et al. Modulation of noncanonical TGF-beta signaling prevents cleft palate in Tgfbr2 mutant mice. J. Clin. Invest., 2012, 122:873-885

[89] Iwata J et al. Transforming growth factor-beta regulates basal transcriptional regulatory machinery to control cell proliferation and differentiation in cranial neural crest-derived osteoprogenitor cells. J. Biol. Chem., 2010, 285:4975-4982

[90] Suzuki A, Pelikan RC, Iwata J. WNT/beta-catenin signaling regulates multiple steps of myogenesis by regulating step-specific targets. Mol. Cell. Biol., 2015, 35:1763-1776

[91] Kinzler KW, Vogelstein B. The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol. Cell. Biol., 1990, 10:634-642

[92] Winklmayr M et al. Non-consensus GLI binding sites in Hedgehog target gene regulation. BMC Mol. Biol., 2010, 11

[93] Shimano H. Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog. Lipid Res., 2001, 40:439-452

[94] Edwards PA, Tabor D, Kast HR, Venkateswaran A. Regulation of gene expression by SREBP and SCAP. Biochimica et. Biophysica Acta, 2000, 1529:103-113

Funding

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)(DE024759)

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)

U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)