Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-α signalling in endochondral bone growth
Anita Woods
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorClaudine G. James
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorGuoyan Wang
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorHolly Dupuis
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorCorresponding Author
Frank Beier
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Correspondence to: Frank BEIER, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.Tel.: (519) 661–2111-85344Fax: (519) 661–3827E-mail: [email protected]Search for more papers by this authorAnita Woods
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorClaudine G. James
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorGuoyan Wang
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorHolly Dupuis
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Search for more papers by this authorCorresponding Author
Frank Beier
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada
Correspondence to: Frank BEIER, Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario N6A 5C1, Canada.Tel.: (519) 661–2111-85344Fax: (519) 661–3827E-mail: [email protected]Search for more papers by this authorAbstract
Elucidating the signalling pathways that regulate chondrocyte differentiation, such as the actin cytoskeleton and Rho GTPases, during development is essential for understanding of pathological conditions of cartilage, such as chondrodysplasias and osteoarthritis. Manipulation of actin dynamics in tibia organ cultures isolated from E15.5 mice results in pronounced enhancement of endochondral bone growth and specific changes in growth plate architecture. Global changes in gene expression were examined of primary chondrocytes isolated from embryonic tibia, treated with the compounds cytochalasin D, jasplakinolide (actin modifiers) and the ROCK inhibitor Y27632. Cytochalasin D elicited the most pronounced response and induced many features of hypertrophic chondrocyte differentiation. Bioinformatics analyses of microarray data and expression validation by real-time PCR and immunohistochemistry resulted in the identification of the nuclear receptor retinoid related orphan receptor-α (Ror-α) as a novel putative regulator of chondrocyte hypertrophy. Expression of Ror-α target genes, (Lpl, fatty acid binding protein 4 [Fabp4], Cd36 and kruppel-like factor 5 [Klf15]) were induced during chondrocyte hypertrophy and by cytochalasin D and are cholesterol dependent. Stimulation of Ror-α by cholesterol results in increased bone growth and enlarged, rounded cells, a phenotype similar to chondrocyte hypertrophy and to the changes induced by cytochalasin D, while inhibition of cholesterol synthesis by lovastatin inhibits cytochalasin D induced bone growth. Additionally, we show that in a mouse model of cartilage specific (Col2-Cre) Rac1, inactivation results in increased Hif-1α (a regulator of Rora gene expression) and Ror-α+ cells within hypertrophic growth plates. We provide evidence that cholesterol signalling through increased Ror-α expression stimulates chondrocyte hypertrophy and partially mediates responses of cartilage to actin dynamics.
Supporting Information
Table S1 Probe sets regulated by Y27632
Table S2 Probe sets regulated by cytochalasin D
Table S3 Probe sets regulated by jasplakinolide
Fig. S1 Gene ontology and Kegg pathways in response to Y27632 treatment (A) Microarray gene sets from primary chondrocytes treated with 10 μM Y27632 for a period of 24 hrs were assessed according to GO annotations categorized by Fatigo+. (B) Kegg annotations were used to determine common gene changes within different signalling pathways.
Fig. S2 Gene ontology and Kegg pathways in response to jasplakinolide treatment. (A) Microarray gene sets from primary chondrocytes treated with 50 nM jasplakinolide for a period of 24 hrs were assessed according to GO annotations categorized by Fatigo+. (B) Kegg annotations were used to determine common gene changes within different signalling pathways.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
| Filename | Description |
|---|---|
| JCMM_684_sm_Table1.pdf111.9 KB | Supporting info item |
| JCMM_684_sm_Table2.pdf342.7 KB | Supporting info item |
| JCMM_684_sm_Table3.pdf86.2 KB | Supporting info item |
| JCMM_684_sm_fig1.pdf127.5 KB | Supporting info item |
| JCMM_684_sm_fig2.pdf123.5 KB | Supporting info item |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1 Hunziker EB. Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Tech. 1994; 28: 505–19.
- 2 Noonan KJ, Hunziker EB, Nessler J, et al . Changes in cell, matrix compartment, and fibrillar collagen volumes between growth-plate zones. J Orthop Res. 1998; 16: 500–8.
- 3 Abad V, Meyers JL, Weise M, et al . The role of the resting zone in growth plate chondrogenesis. Endocrinology. 2002; 143: 1851–7.
- 4 Hunziker EB, Schenk RK, Cruz-Orive LM. Quantitation of chondrocyte performance in growth-plate cartilage during longitudinal bone growth. J Bone Joint Surg Am. 1987; 69: 162–73.
- 5 Wilsman NJ, Farnum CE, Leiferman EM, et al . Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics. J Orthop Res. 1996; 14: 927–36.
- 6 Beier F. Cell-cycle control and the cartilage growth plate. J Cell Physiol. 2005; 202: 1–8.
- 7 Breur GJ, VanEnkevort BA, Farnum CE, et al . Linear relationship between the volume of hypertrophic chondrocytes and the rate of longitudinal bone growth in growth plates. J Orthop Res. 1991; 9: 348–59.
- 8 Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell. 2002; 2: 389–406.
- 9 Schwartz NB, Domowicz M. Chondrodysplasias due to proteoglycan defects. Glycobiology. 2002; 12: 57–68.
- 10 Spranger J, Winterpacht A, Zabel B. The type II collagenopathies: a spectrum of chondrodysplasias. Eur J Pediatr. 1994; 153: 56–65.
- 11 Drissi H, Zuscik M, Rosier R, et al . Transcriptional regulation of chondrocyte maturation: potential involvement of transcription factors in OA pathogenesis. Mol Aspects Med. 2005; 26: 169–79.
- 12 Aigner T, Gerwin N. Growth plate cartilage as developmental model in osteoarthritis reserach–potentials and limitations. Curr Drug Targets. 2007; 8: 377–85.
- 13 James CG, Appleton CT, Ulici V, et al . Microarray analyses of gene expression during chondrocyte differentiation identifies novel regulators of hypertrophy. Mol Biol Cell. 2005; 16: 5316–33.
- 14 Woods A, Wang G, Beier F. RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. J Biol Chem. 2005; 280: 11626–34.
- 15 Woods A, Beier F. RhoA/ROCK signaling regulates chondrogenesis in a context-dependent manner. J Biol Chem. 2006; 281: 13134–40.
- 16 Wang G, Woods A, Sabari S, et al . RhoA/ROCK signaling suppresses hypertrophic chondrocyte differentiation. J Biol Chem. 2004; 279: 13205–14.
- 17 Benya PD, Brown PD, Padilla SR. Microfilament modification by dihydrocytochalasin B causes retinoid acid-modulated chondrocytes to re-express the differentiated collagen phenotype without a change in shape. J Cell Biol. 1988; 106: 161–70.
- 18 Brown PD, Benya PD. Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression. J Cell Biol. 1988; 106: 171–9.
- 19 Benya PD. Modulation and re-expression of the chondrocyte phenotype; mediation by cell shape and microfilament modification. Pathol Immunopathol Res. 1988; 7: 51–4.
- 20 Newman SA, Watt FM. Influence of cytochalasin D induced changes in cell shape on proteoglycan sythesis by cultured articular chondrocytes. Exp Cell Res. 1988; 178: 199–210.
- 21 Loty S, Forest N, Boulekbache H, et al . Cytochalasin D induces changes in cell shape and promotes in vitro chondrogenesis: a morphological study. Biol Cell. 1995; 83: 149–61.
- 22 Agoston H, Khan S, James CG, et al . C-type natriuretic peptide regulates endochondral bone growth through p38 MAP kinase-dependent and independent pathways. BMC Devl Biol. 2007; 7: 18.
- 23 Ulici V, Hoenselaar KD, Gillespie JR, et al . The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation. BMC Dev Biol. 2008; 11: 40.
- 24 Wang G, Woods A, Agoston H, et al . Genetic ablation of Rac1 in cartilage results in chondrodysplasia. Dev Biol. 2007; 306: 612–23.
- 25 James CG, Ulici V, Tuckermann J, et al . Expression profiling of dexamethasone treated primary chondrocyte identifies targets of glucocorticoid signaling in endochondral ossification development. BMC Genomics. 2007; 8: 205.
- 26 Kang HS, Angers M, Beak JY, et al . Gene expression profiling reveals a regulatory role for ROR alpha and ROR gamma in phase I and phase II metabolism. Physiol Genomics. 2007; 31: 281–94.
- 27 James CG, Woods A, Underhill TM, et al . The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription. BMC Mol Biol. 2006; 7: 30.
- 28 Bursell L, Woods A, James CG, et al . Src kinase inhibition promotes the chondrocyte phenotype. Arthritis Res Ther. 2007; 9: R105.
- 29 Bubb M, Senderowicz A, Sausville E, et al . Jasplakinolide, a cytotoxic natural product, induces actin polyerization and competitively inhibits the binding of phalloidin to F-actin. J Biol Chem. 1994; 269: 14869–71.
- 30 Goddette DW, Frieden C. The kinetics of cytochalasin D binding to monomeric actin. J Biol Chem. 1986; 261: 15970–3.
- 31 Miralles F, Posern G, Zaromytidou A-I, et al . Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell. 2003; 113: 329–42.
- 32 Al-Shahrour F, Carbonell J, Minquez P, et al . Babelomics: advanced functional profiling of transcriptomics, proteomics and genomics experiments. Nucleic Acids Res. 2008; 36: W341–6.
- 33 Shao YY, Wang L, Hicks DG, et al . Expression and activation of peroxisome proliferator-activated receptors in growth plate chondrocytes. J Orthop Res. 2005; 23: 1139–45.
- 34 Hill TP, Spater D, Taketo MM, et al . Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell. 2005; 8: 727–38.
- 35 Li TF, O’Keefe RJ, Chen D. TGF-beta signaling in chondrocytes. Front Biosci. 2005; 1: 681–8.
- 36 Stanton LA, Beier F. PPARgamma2 expression in growth plate chondrocytes is regulated by p38 and GSK-3. J Cell Mol Med. DOI: 10.1111/j.1582-4934.2008.00396.x
- 37 Enomoto-Iwamoto M, Kitagawa J, Koyama E, et al . The Wnt antagonist Frzb-1 regulates chondrocyte maturation and long bone development during limb skeletogenesis. Dev Biol. 2002; 251: 142–56.
- 38 Coleman CM, Tuan RS. Functional role of growth/differentiation factor 5 in chondrogenesis of limb mesenchymal cells. Mech Dev. 2003; 120: 823–36.
- 39 Cunningham NS, Jenkins NA, Gilbert DJ, et al . Growth/differentiation factor-10: a new member of the transforming growth factor-beta superfamily related to bone morphogenetic protein-3. Growth Factors. 1995; 12: 99–109.
- 40 Schipani E, Ryan HE, Didrickson S, et al . Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival. Genes Dev. 2001; 15: 2865–76.
- 41 Chauvet C, Bois-Joyeux B, Berra E, et al . The gene encoding human retinoic acid-receptor-related orphan receptor alpha is a target for hypoxia-inducible factor 1. Biochem J. 2004; 384: 79–85.
- 42 Kallen JA, Schlaeppi JM, Bitsch F, et al . X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Structure. 2002; 10: 1697–707.
- 43 Wiesenberg I, Missbach M, Kahlen JP, et al . Transcriptional activation of the nuclear receptor RZR alpha by the pineal gland hormone melatonin and identification of CGP 52608 as a synthetic ligand. Nucleic Acids Res. 1995; 23: 327–33.
- 44 Alberts AW, Chen J, Kuron G, et al . Mevinolin: A highly potent competitve inhibitor of hydroxymethylglutaryl coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA. 1980; 77: 3957–61.
- 45 Lau P, Nixon SJ, Parton RG, et al . RORalpha regulates the expression of genes involved in lipid homeostasis in skeletal muscle cells: caveolin-3 and CPT-1 are direct targets of ROR. J Biol Chem. 2004; 279: 36828–40.
- 46 Pfander D, Cramer T, Deuerling D, et al . Expression of thrombospondin-1 and its receptor CD36 in human osteoarthritic cartilage. Ann Rheum Dis. 2000; 59: 448–54.
- 47 Mic FA, Molotkiv A, Fan X, et al . RALDH3, a retinaldehyde dehydrogenase that generates retinoic acid, is expressed in the ventral retina, otic vesical and olfactory pit during mouse development. Mech Dev. 2000; 97: 227–30.
- 48 Gray S, Wang B, Orihuela Y, et al . Regulation of gluconeogenesis by Kruppel-like factor 15. Cell Metab. 2007; 5: 305–12.
- 49 Schnabel M, Marlovits S, Eckhoff G, et al . Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis Cartilage. 2002; 10: 62–70.
- 50 Mallein-Gerin F, Garrone R, van der Rest M. Proteoglycan and collagen synthesis are correlated with actin organization in dedifferentiating chondrocytes. Eur J Cell Biol. 1991; 56: 364–73.
- 51 Woods A, Wang G, Beier F. Regulation of chondrocyte differentiation by the actin cytoskeleton and adhesive interactions. J Cell Physiol. 2007; 213: 1–8.
- 52 Uribe R, Jay D. A review of actin binding proteins: new perspectives. Mol Biol Rep. 2009; 36: 121–5.
- 53 Loy CJ, Sim KS, Yong EL. Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions. Proc Natl Acad Sci USA. 2003; 100: 4562–7.
- 54 Gerber HP, Vu TH, Ryan AM, et al . VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med. 1999; 5: 623–8.
- 55 Shen Z, Gantcheva S, Mansson B, et al . Chondradherin expression changes in skeletal development. Biochem J. 1998; 330: 549–57.
- 56 Dreier R, Opolka A, Grifka J, et al . Collagen IX-deficiency seriously compromises growth cartilage development in mice. Matrix Biol. 2008; 27: 319–29.
- 57 Hasky-Negev M, Simsa S, Tong A, et al . Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate. J Anim Sci. 2008; 86: 1306–15.
- 58 Steinmayr M, Andre E, Conquet F, et al . Staggerer phenotype in retinoid-related orphan receptor alpha-deficient mice. Proc Natl Acad Sci USA. 1998; 95: 3960–5.
- 59 Raspe E, Duez H, Gervois P, et al . Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORalpha. J Biol Chem. 2001; 276: 2865–71.
- 60 Meyer T, Kneissel M, Mariani J, et al . In vitro and in vivo evidence for orphan nuclear receptor RORalpha function in bone metabolism. Proc Natl Acad Sci USA. 2000; 97: 9197–202.
- 61 Kopmels B, Mariani J, Delhaye-Bouchaud N, et al . Evidence for a hyperexcitability state of staggerer mutant mice macrophages. J Neurochem. 1992; 58: 192–9.
- 62 Wang G, Beier F. Rac1/Cdc42 and RhoA GTPases antagonistically regulate chondrocyte proliferation, hypertrophy, and apoptosis. J Bone Miner Res. 2005; 20: 1022–31.
- 63 Seabra MC. Membrane association and targeting of prenylated Ras-like GTPases. Cell Signal. 1998; 10: 167–72.
- 64 Sidman RL, Lane PW, Dickie MM. Staggerer, a new mutation in the mouse affecting the cerebellum. Science. 1962; 137: 610–2.
- 65 Wu S, De Luca F. Role of cholesterol in the regulation of growth plate chondrogenesis and longitudinal bone growth. J Biol Chem. 2004; 279: 4642–7.
Citing Literature
