Notch signalling in chondrocytes. The involvement of notch signalling and HES genes for articular cartilage development, chondrogenic progenitor cells, and osteoartrithis

Abstract

Notch signalling and its downstream mediators, the helix-loop-helix (HLH) family of transcription factors, are characterized by a broad spectrum of functions, from cell proliferation and differentiation to apoptosis. The aim of this thesis was to investigate if the Notch signalling pathway controls chondrogenesis. Further, we wanted to investigate the role of Notch signalling in osteoarthritis (OA) and Notch as a marker for chondroprogenitor cells. We found that chondrocytes during chondrogenesis in vitro differentiate towards a hyaline cartilage-like phenotype in a process mimicking foetal cartilage development. Since this process during development starts with condensation of mesenchymal stem cells (MSCs), it is of interest to study whether adult MSCs also possesses the ability to differentiate towards the chondrogenic lineage. MSCs and articular chondrocytes differentiated towards different cartilage phenotypes, hyaline and hypertrophic respectively. Several HLH genes, including HES1, HES5, ID1, and Dec1, were differentially expressed between chondrocytes and MSCs as well as during differentiation, thus being potential regulators of chondrogenesis. We then focused our attention on one of these genes, the at the time unknown human HES5 gene and showed that the expression of HES5 was regulated by Notch signalling. Transcription of several Notch receptors, their ligands, and HES5, were significantly reduced during chondrogenesis. Transfection of HES5 to ATDC5 cells demonstrated that HES5 especially regulates genes controlling the early stages of chondrogenesis. Transgenic mice lacking both the HES1 and the HES5 genes displayed no apparent defects in their formation of cartilage and long bones, which probably is explained by functional redundancy between the genes belonging to the family of HES genes. Although Notch1 is considered a marker for progenitor cells in several tissues as well as in cartilage from young donors, we found no evidence for Notch1 as a marker for chondroprogenitors in adult cartilage. Since chondrocytes in patients with OA are in another phenotypical state than chondrocytes from a healthy joint, we then investigated whether these cells had an aberrant Notch signalling. An abundant expression of HES5, Notch1, and Jagged 1 were detected in OA cartilage. Microarray analysis revealed that activated Notch signalling induces and represses genes that could potentially contribute to the OA phenotype. TNF-á or IL-1â significantly repressed transcription of several Notch markers but did not affect the cellular localization of Notch intracellular domain 1 (NICD1). The increased production of cytokines characteristic for OA thus does not explain the abundant expression of Notch1, Jagged1, or HES5 detected in OA. Cytokines repressed HES5 transcription via induction of NFkB rather than the canonical NICD pathway. The results presented in this thesis thus show that Notch signalling influences several different chondrogenic processes.