Notch Signaling and the Skeleton

Abstract

Notch 1 to 4 receptors are important determinants of cell fate and function, and Notch signaling plays an important role in skeletal development and bone remodeling. After direct interactions with ligands of the Jagged and Delta-like families, a series of cleavages release the Notch intracellular domain (NICD), which translocates to the nucleus where it induces transcription of Notch target genes. Classic gene targets of Notch are hairy and enhancer of split (Hes) and Hes-related with YRPW motif (Hey). In cells of the osteoblastic lineage, Notch activation inhibits cell differentiation and causes cancellous bone osteopenia because of impaired bone formation. In osteocytes, Notch1 has distinct effects that result in an inhibition of bone resorption secondary to an induction of osteoprotegerin and suppression of sclerostin with a consequent enhancement of Wnt signaling. Notch1 inhibits, whereas Notch2 enhances, osteoclastogenesis and bone resorption. Congenital disorders of loss- and gain-of-Notch function present with severe clinical manifestations, often affecting the skeleton. Enhanced Notch signaling is associated with osteosarcoma, and Notch can influence the invasive potential of carcinoma of the breast and prostate. Notch signaling can be controlled by the use of inhibitors of Notch activation, small peptides that interfere with the formation of a transcriptional complex, or antibodies to the extracellular domain of specific Notch receptors or to Notch ligands. In conclusion, Notch plays a critical role in skeletal development and homeostasis, and serious skeletal disorders can be attributed to alterations in Notch signaling.

Figure 1.

Domains of the four Notch receptors. The upper panel shows the domain and motif organization of a generic human/murine Notch receptor before cleavage at the S1 site by furin-like convertases in the Golgi compartment. The extracellular domain contains a leader peptide (LP) and multiple EGF-like tandem repeats followed by Lin12-Notch repeats (LNR) and the HD. The transmembrane domain (TMD) is located between the extracellular and intracellular domains. The NICD contains a RAM, a nuclear localization sequence (NLS), ANK repeats, and tandem NLS, which are followed by a PEST domain. The lower panel shows the domains and motifs of heterodimeric individual receptors; the NRR is formed by the LNR and HD following cleavage at the S1 site. Notch1 and Notch2 have 36 EGF-like repeats; in green are those required for binding of Notch1 and Notch2 to cognate DSL ligands. Notch1 and Notch2 have a similar NICD, and Notch3 has 34 EGF-like repeats and a shorter NICD than Notch1 and Notch2. Notch4 has 29 EGF-like repeats and an NICD that is shorter than that of other receptors and lacks the tandem NLS located between the ANK repeats and the PEST domain.

Figure 2.

Activation of Notch receptors. DSL ligands expressed by a signal-sending cell (top) engage Notch receptors on a signal-receiving cell (bottom). The DSL ligand bound to the extracellular domain of Notch is internalized by the ligand-expressing cell, a process termed trans-endocytosis, exposing the S2 cleavage site of Notch to the activity of the metalloprotease disintegrin and metalloprotease domain (ADAM) 10. This proteolytic event creates a peptide consisting of the transmembrane and intracellular regions of Notch and exposes the intramembranous S3 and S4 sites, which are recognized by the γ-secretase complex. Cleavage at the S3 and S4 sites by γ-secretase activity releases the Notch intracellular domain, which translocates to the nucleus to regulate transcription.

Figure 3.

Transcriptional events regulated by Notch receptors. A, In the absence of the NICD, Rbpjκ associates with transcriptional repressors and histone deacetylase complexes (HDAC). These interactions occur at the promoter region of Notch transcriptional targets, such as members of the hairy enhancer of split (Hes) and HES-related with YRPW motif (Hey) gene families. B, After nuclear translocation, NICD associates with Rbpjκ and Maml to form a ternary NICD/Rbpjκ/Maml complex. These events lead to the recruitment of transcriptional activators, displacement of the transcriptional repressors, and subsequent expression of Hes and Hey. C, Maml associates with CDK8, which phosphorylates (P) the PEST domain of the NICD. D, Maml recruits the E3 ubiquitin-ligase F-box and WD repeat domain-containing (Fbxw) 7, which ubiquitinates (U) the NICD and leads to its proteasomal degradation. As a result, the active transcriptional complex is disassembled, allowing reinstitution of transcriptional suppression after association of Rbpjκ with the transcriptional repressors and the HDAC (A).

Figure 4.

Clinical features of AOS. A, Brachydactyly of the left foot and missing toes on right foot. B, Bald area on the scalp. C, Brachydactyly of toes. D, Brachydactyly of fingers. E, Aplasia cutis congenita. G, Short distal phalanges and symphalangism of index finger. [Reproduced from J. A. Meester et al: Heterozygous loss-of-function mutations in DLL4 cause Adams-Oliver syndrome. Am J Hum Genet. 2015;97(3):475–482 (171), with permission. © American Society of Human Genetics.]

Figure 5.

Alagille syndrome butterfly vertebrae. In A and B, arrows point to butterfly vertebrae identified in two patients with Alagille syndrome. Note more severe clefting of vertebrae in patient on the left. [Reproduced from I. D. Krantz et al: Alagile syndrome. J Med Genet. 1997;34:152–157 (316), with permission. © BMJ Publishing Group Ltd.].

Figure 6.

Spondylocostal dysostosis due to homozygous mutations in DLL3. A, All vertebrae show abnormal segmentation, and the ribs show irregular points of fusion along their length. However, there is an overall symmetry to the thoracic cage. (Image courtesy of Yanick Crow.) B, Because of the similarity to smooth, eroded pebbles on a beach, the authors of the original publication suggested calling the radiological appearance the “pebble beach” sign. [Reproduced from P. D. Turnpenny, et al: Abnormal vertebral segmentation and the notch signaling pathway in man. Dev Dyn. 2007;236(6):1456–1474 (194), with permission. © John Wiley & Sons, Inc.]

Figure 7.

Phenotypic characteristics of syndromic recessive brachydactyly caused by a frameshift mutation of CHSY1. C, X-ray radiograph of male proband's right hand when he was 8 years of age, showing partial duplications of proximal phalanges in digits 1, 2, and 3 (inset: hand photograph). D, X-ray radiograph and pictures of male proband's right foot showing severe skeletal anomalies. The big toe exhibits short and duplicated metatarsals and proximal phalanges. The second and fourth proximal phalanges are duplicate as well. [Reproduced from J. Tian et al: Loss of CHSY1, a secreted FRINGE enzyme, causes syndromic brachydactyly in humans via increased NOTCH signaling. Am J Hum Genet. 2010;87(6):768–778 (207), with permission. © American Society of Human Genetics.]

Figure 8.

Identification of NOTCH2 mutations in individuals with Hajdu-Cheney syndrome. A, Facial dysmorphy with micrognathia, thick eyebrows, long philtrum, hypertelorism, and low-set and posteriorly rotated ears. Written consent to publish photographs of this individual was obtained by the authors of the original publication. Phalanges radiograph showing acro-osteolysis of all distal phalanges. Skull radiograph showing characteristic findings of HCS, including platybasia, thickened occipital bone, open sutures, and wormian bones. [Reproduced from B. Isidor et al: Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis. Nat Genet. 2011;43(4):306–308 (221). © Nature Publishing Group.]

Figure 9.

Cranial and central nervous system features of LMS. A, Thickened cranial vault, mild dilatation of the subarachnoideal spaces, sellar arachnocele, and verticalization of the tentorium cerebellum at magnetic resonance imaging. B, Cranial vault sclerosis. C–H, Magnetic resonance images showing thickening of the neurocranium and increased distance between cortex and meninges (C) and an empty sella filled with cerebrospinal fluid (D); lateral meningocele (arrows) at the thoracic (E and F); and at lumbar metameres (G and H). [Reproduced from M. Castori et al: Late diagnosis of lateral meningocele syndrome in a 55-year-old woman with symptoms of joint instability and chronic musculoskeletal pain. Am J Med Genet A. 2014;164A(2):528–534 (317), with permission. © Wiley-Liss, Inc.]

Figure 10.

Notch signaling and regulation of bone remodeling. DSL ligands induce cleavage of Notch receptors and the generation of the NICD. Activation of Notch1 in osteoblast precursors suppresses osteoblastogenesis by inhibiting osterix (Osx), runt-related transcription factor (Runx)2 and cytosolic β-catenin, thereby impairing bone formation. The Notch2 NICD in osteoclast precursors associates with Nf-κB) and induces Nf of activated T cells (Nfatc)1 transcription and osteoclastogenesis. Impaired bone formation and increased bone resorption lead to a decrease in bone mass (left). Activation of Notch1 in osteoblastic cells induces osteoprotegerin (Opg), an inhibitor of Rankl, leading to suppressed cancellous bone resorption. Activation of Notch1 in osteocytes suppresses sclerostin and dickkopf (Dkk)1, enhances Wnt signaling, and increases cortical bone formation. Decreased cancellous bone resorption and enhanced cortical bone formation lead to an in increase in bone mass (right).