Notch signaling controls chondrocyte hypertrophy via indirect regulation of Sox9

Abstract

RBPjk-dependent Notch signaling regulates both the onset of chondrocyte hypertrophy and the progression to terminal chondrocyte maturation during endochondral ossification. It has been suggested that Notch signaling can regulate Sox9 transcription, although how this occurs at the molecular level in chondrocytes and whether this transcriptional regulation mediates Notch control of chondrocyte hypertrophy and cartilage development is unknown or controversial. Here we have provided conclusive genetic evidence linking RBPjk-dependent Notch signaling to the regulation of Sox9 expression and chondrocyte hypertrophy by examining tissue-specific Rbpjk mutant (Prx1Cre;Rbpjk ^f/f ), Rbpjk mutant/Sox9 haploinsufficient (Prx1Cre;Rbpjk ^f/f ;Sox9 ^f/+ ), and control embryos for alterations in SOX9 expression and chondrocyte hypertrophy during cartilage development. These studies demonstrate that Notch signaling regulates the onset of chondrocyte maturation in a SOX9-dependent manner, while Notch-mediated regulation of terminal chondrocyte maturation likely functions independently of SOX9. Furthermore, our in vitro molecular analyses of the Sox9 promoter and Notch-mediated regulation of Sox9 gene expression in chondrogenic cells identified the ability of Notch to induce Sox9 expression directly in the acute setting, but suppresses Sox9 transcription with prolonged Notch signaling that requires protein synthesis of secondary effectors.

Cartilage and bone formation: Knowledge of molecular mechanisms taken up a Notch

A study of cartilage and bone maturation has taken us a step closer to understanding the molecular mechanisms involved and how they malfunction in disease. Research led by Matthew Hilton from Duke University School of Medicine, US, investigated how a signaling pathway called Notch regulates the protein SOX9, which is itself a key regulator of cartilage and bone development. Some evidence suggests that SOX9 regulation is mediated by a protein called RBPjk, which alters gene expression in response to Notch signaling. By manipulating expression of SOX9 and RBPjk in mice and cultured cells, the researchers confirmed that Notch signaling regulates SOX9, but showed that RBPjk is not directly involved. Instead, proteins that are made in response to Notch signaling seem to be required. Further studies that identify these proteins will provide a greater understanding of cartilage and bone development.

References

[1] Kronenberg HM. Developmental regulation of the growth plate. Nature, 2003, 423: 332-336

[2] Akiyama H, Lefebvre V. Unraveling the transcriptional regulatory machinery in chondrogenesis. J Bone Miner Metab, 2011, 29: 390-395

[3] Foster JW, Dominguez-Steglich MA, Guioli S et al Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature, 1994, 372: 525-530

[4] Wagner T, Wirth J, Meyer J et al Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell, 1994, 79: 1111-1120

[5] Bi W, Huang W, Whitworth DJ et al Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 2001, 98: 6698-6703

[6] Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev, 2002, 16: 2813-2828

[7] Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B. Sox9 is required for cartilage formation. Nat Genet, 1999, 22: 85-89

[8] Lefebvre V, Li P, de Crombrugghe B. A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J, 1998, 17: 5718-5733

[9] Hattori T, Muller C, Gebhard S et al SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development, 2010, 137: 901-911

[10] Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C. Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell, 2005, 8: 727-738

[11] Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun, 2005, 328: 651-657

[12] Yoon BS, Lyons KM. Multiple functions of BMPs in chondrogenesis. J Cell Biochem, 2004, 93: 93-103

[13] Engin F, Lee B. NOTCHing the bone: insights into multi-functionality. Bone, 2010, 46: 274-280

[14] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell, 2009, 137: 216-233

[15] Bray SJ. Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol, 2006, 7: 678-689

[16] Chiba S. Notch signaling in stem cell systems. Stem Cells, 2006, 24: 2437-2447

[17] Dong Y, Jesse AM, Kohn A et al RBPjkappa-dependent Notch signaling regulates mesenchymal progenitor cell proliferation and differentiation during skeletal development. Development, 2010, 137: 1461-1471

[18] Mead TJ, Yutzey KE. Notch pathway regulation of chondrocyte differentiation and proliferation during appendicular and axial skeleton development. Proc Natl Acad Sci U S A, 2009, 106: 14420-14425

[19] Hilton MJ, Tu X, Wu X et al Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med, 2008, 14: 306-314

[20] Kohn A, Dong Y, Mirando AJ et al Cartilage-specific RBPjkappa-dependent and -independent Notch signals regulate cartilage and bone development. Development, 2012, 139: 1198-1212

[21] Han H, Tanigaki K, Yamamoto N et al Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int Immunol, 2002, 14: 637-645

[22] Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. Genesis, 2002, 33: 77-80

[23] Hilton MJ, Tu X, Cook J, Hu H, Long F. Ihh controls cartilage development by antagonizing Gli3, but requires additional effectors to regulate osteoblast and vascular development. Development, 2005, 132: 4339-4351

[24] Hilton MJ, Tu X, Long F. Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage. Dev Biol, 2007, 308: 93-105

[25] Hu H, Hilton MJ, Tu X, Yu K, Ornitz DM, Long F. Sequential roles of Hedgehog and Wnt signaling in osteoblast development. Development, 2005, 132: 49-60

[26] McLeod MJ. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology, 1980, 22: 299-301

[27] Zhao Q, Eberspaecher H, Lefebvre V, De Crombrugghe B. Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev Dyn, 1997, 209: 377-386

[28] Chen S, Tao J, Bae Y et al Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9. J Bone Miner Res, 2013, 28: 649-659

[29] Tun T, Hamaguchi Y, Matsunami N, Furukawa T, Honjo T, Kawaichi M. Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic Acids Res, 1994, 22: 965-971

[30] Fischer A, Gessler M. Delta-Notch--and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res, 2007, 35: 4583-4596

[31] Bray S, Bernard F. Notch targets and their regulation. Curr Top Dev Biol, 2010, 92: 253-275

[32] Akiyama H, Kim JE, Nakashima K et al Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci U S A, 2005, 102: 14665-14670

[33] Lefebvre V, de Crombrugghe B. Toward understanding SOX9 function in chondrocyte differentiation. Matrix Biol, 1998, 16: 529-540

[34] Dao DY, Jonason JH, Zhang Y et al Cartilage-specific beta-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development. J Bone Miner Res, 2012, 27: 1680-1694

[35] Akiyama H, Lyons JP, Mori-Akiyama Y et al Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev, 2004, 18: 1072-1087

[36] Chen S, Tao J, Bae Y et al Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9. J Bone Miner Res, 2013, 28: 649-659

[37] Haller R, Schwanbeck R, Martini S et al Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation. Cell Death Differ, 2012, 19: 461-469

[38] Muto A, Iida A, Satoh S, Watanabe S. The group E Sox genes Sox8 and Sox9 are regulated by Notch signaling and are required for Muller glial cell development in mouse retina. Exp Eye Res, 2009, 89: 549-558

[39] Seymour PA, Shih HP, Patel NA et al A Sox9/Fgf feed-forward loop maintains pancreatic organ identity. Development, 2012, 139: 3363-3372