Manipulating co-regulators of RUNX2 and SOX9 to enhance the chondrogenic potential of chondrogenic progenitor cells in osteoarthritis

Osteoarthritis (OA) is a degenerative disease of the joint leading to the degradation of the articular cartilage. Current therapies cannot halt or reverse the disease, leading to a severe impact on life quality accompanied by immense health care and society cost. One major characteristic of OA is the replacement of articular cartilage by a fibrocartilaginous repair tissue. Chondrogenic progenitor cells (CPCs) reside in the late-stage OA fibrocartilage and express the fibrotic marker collagen type 1 (COL1). However, these cells can undergo chondrogenic differentiation in vitro and produce the articular cartilage marker collagen type 2 (COL2). The aim of this project is to analyze how the CPCs’ chondrogenic potential can be increased to lay the foundation for future therapeutic interventions in OA. The chondrogenic transcription factor SRY (sex determining region Y)-box 9 (SOX9) and the osteogenic transcription factor runt-related transcription factor 2 (RUNX2) are key regulators in CPCs and control their chondrogenic differentiation. Chapter I of this thesis analyzes the effect of the extra-cellular matrix (ECM) protein biglycan (BGN) and of the transforming growth factor β (TGF-β) and epidermal growth factor (EGF) signaling pathways on SOX9 and RUNX2 expression. Histological staining confirmed the presence of these molecules in late-stage OA fibrocartilage specimens. Analysis on the RNA and protein level demonstrated that BGN modulates TGF and EGF signaling events and vice versa. TGF and EGF stimulation of CPCs resulted in elevated osteochondrogenic marker expression, including SOX9 and RUNX2. Chapter II deals with a direct manipulation of the osteochondrogenic regulators SOX9 and RUNX2. Despite its osteogenic role in chondrocyte hypertrophy that precedes bone formation, RUNX2 was confirmed to have a pro-chondrogenic effect. Next, the early endosomal marker ras-related protein Rab-5C (RAB5C) was identified as potential interaction partner of SOX9 in CPCs. The deletion of RAB5C using CRISPR/Cas9 increased the chondrogenic potential of CPCs and did not impair their ability to migrate. Finally, our findings suggest that altered bone morphogenetic protein, notch and interleukin signaling mediate this pro-chondrogenic effect. In summary, this thesis discusses novel strategies to manipulate the chondrogenic potential of CPCs. First, the modulation of chondrogenic signaling events in CPCs by BGN highlights the role of the ECM on the chondrogenic potential. Secondly, RAB5C was identified as potential co-regulator of SOX9 in CPCs and resembles a candidate for future research on therapeutic interventions in OA.2021-11-2