Notch gain of function inhibits chondrocyte differentiation via Rbpj-dependent suppression of Sox9

Abstract

Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj-dependent and Rbpj-independent Notch signaling mechanisms might be involved. Cartilage specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by “butterfly” vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj deficient Notch GOF mice compared to Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/NICD transcription complex to Rbpj-binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis.

A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis

“…We and others have previously shown that sustained NICD activation in committed growth plate chondrocytes (17, 48) and articular chondrocytes (49) stimulated the expression of the catabolic factor Mmp13 , while permanent NOTCH inhibition within growth plate and primary articular chondrocyte cultures reduced Mmp13 expression (17, 48). Several lines of evidence have demonstrated that sustained NOTCH signaling in mesenchymal progenitors and growth plate chondrocytes suppresses Sox9, Col2a1 , and Acan (17, 48, 50, 51). Data presented by Mead and colleagues (18) and our lab (Kohn et al, unpublished data) have indicated that loss of NOTCH signaling leads to inappropriate expression of Sox9 in hypertrophic chondrocytes.…”

Repression of Sox9 by Jag1 Is Continuously Required to Suppress the Default Chondrogenic Fate of Vascular Smooth Muscle Cells

“…Specifically we found that between E13 and E14.5 Notch permits the establishment of the vSMC phenotype by repressing other fates through the regulation of Sox9, Pax1 and Scx transcript level but also regulation of Sox9 activity on its target genes Col2a1 and Sox6. In fact in chondrocytes, RBP-J/NICD complex binds and represses Sox9 transcription (Chen et al, 2013). Furthermore, the Notch target genes Hey1 and Hes1 bind and repress the Col2a1 promoter through competition with Sox9 (Grogan et al, 2008).…”

Mechanisms in Endocrinology: Notch signaling in skeletal health and disease

“…The role played by Notch in mature chondrocytes is less clear (22,23,24,25) . Notch signaling regulates osteoblast and osteoclast differentiation and function, and as a consequence controls bone remodeling.…”