Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2019.
Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation that affects all tissues within the synovial joint. OA is associated with an enormous public health burden, as it is the most common cause of disability in the United States. Two critical risk factors for development of OA are advanced age and prior traumatic joint injury. Age-associated chronic, low-grade inflammation is now recognized as a likely contributor to disease, as are both the acute and chronic phases of inflammation experienced by the joint following traumatic injury. We have characterized the age-associated spontaneous development of OA in a C57BL/6J mouse model and both male and female mice develop a joint phenotype consistent with early-stage OA; this includes proteoglycan staining loss within the articular cartilage extracellular matrix (ECM), synovial hyperplasia and immune cell infiltration, and an increase in the overall size and mineralized area of the meniscus. Using a transcriptomics approach, we see that aged articular cartilage has increased expression of factors that act as positive regulators of IKKβ/NF-κB signaling relative to younger articular cartilage, and that several factors involved in the DNA damage response are also upregulated. Similarly, upregulation of inflammatory factors regulated by NF-κB signaling, such as TNF-α and CCL20, is seen in various tissues within the knee joint following administration of a meniscal-ligamentous injury (MLI). Based on this collective set of data, we make the central hypothesis that inflammation driven by activation of canonical NF-κB signaling functions as an initiating event in the pathogenesis of OA, and that activation of the pathway in a cartilage-specific manner using a genetic approach in young, uninjured joints could accelerate onset and progression of OA. To test this hypothesis, we generated postnatal chondrocyte-specific IKKβ gain-of-function (GOF) mice. Remarkably, IKKβ GOF mice develop a phenotype strikingly similar to the age-associated joint pathology seen in wild C57BL/6J mice, but it develops at a largely accelerated rate, suggesting that IKKβ GOF enhances spontaneous OA development. These IKKβ GOF chondrocytes are capable of upregulating markers of cellular senescence and negative regulators of apoptosis, and can produce a senescence-associated secretory phenotype (SASP) in vitro. Using this model, we have identified CCL20 as a highly upregulated downstream target of IKKβ in chondrocytes that may play a critical role in recruitment and activation of immune cells to the knee joint during the process of aging and following joint traumatic injury. Strikingly, initial experiments performing meniscal-ligamentous injury on knockout animals for the receptor for CCL20, CCR6, reveal that CCR6 knockout mice are protected from joint degeneration following traumatic knee injury.
In experiments parallel to these, we examined the ability of inflammation induced by traumatic injury to regulate the process of chondrocyte hypertrophy during OA pathogenesis. Chondrocyte hypertrophy, characterized by expression of factors catabolic to the cartilage matrix, is essential for endochondral bone formation, but is only observed within the articular chondrocytes during OA development. RUNX2 is a transcription factor essential for induction of chondrocyte hypertrophy. Thus, we hypothesized that RUNX2 overexpression in adult articular chondrocytes would promote the onset and/or progression of OA. We generated postnatal chondrocyte-specific RUNX2 overexpression (OE) mice to test this hypothesis, but found that, surprisingly, overexpression of RUNX2 alone was not sufficient to promote the onset of OA. We then subjected the RUNX2 OE mice to either sham injury or a meniscal-ligamentous injury (MLI). RUNX2 OE mice showed more severe induction of OA following meniscal-ligamentous injury (MLI), as evidenced by histology and histomorphometric analysis. MMP13, a direct downstream target of RUNX2, was significantly upregulated in the RUNX2 OE MLI group, as was chondrocyte apoptosis as measured by TUNEL staining. This suggests that the environment within the injured joint was permissive to elevated induction of chondrocyte hypertrophy and can accelerate the pace of injury-induced OA progression.
Taken together, the findings presented here identify a critical role for inflammation, and more specifically, canonical NF-κB signaling, in the onset and progression of OA. Further, we have identified CCL20, a chemokine downstream of IKKβ that is highly upregulated in IKKβ GOF chondrocytes as well as in a variety of joint tissues following traumatic joint injury, as a molecule of interest for future studies that may further elucidate the molecular mechanisms underlying the effects of IKKβ signaling during OA pathogenesis. Finally, we have shown that the injured joint environment may work in concert with predisposing genetic factors to promote the onset and accelerate the progression of OA following traumatic joint injury.
