| Articular cartilage lines the ends of bones in articulating joints. Articular cartilage is composed of a specialized extracellular matrix (ECM) that includes type II collagen, aggrecan and link protein. Chondrocytes are responsible for the deposition and maintenance of the cartilage ECM. The expression of the ECM components is regulated by signalling pathways and transcription factors, including sry-type high mobility group box 9 (SOX9). SOX9 activity is required for the expression of type II collagen, aggrecan and link protein from the Col2a1, Agc1 and Hapln1 genes, respectively. In osteoarthritis (OA) and rheumatoid arthritis (RA), there is a bias in cartilage homeostasis towards catabolism. Extracellular factors mediating this catabolic shift include tumour necrosis factor alpha (TNF-alpha) and prostaglandin E2 (PGE2). In chondrocytes, TNF-alpha induces the activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signalling pathway. PGE2 signalling through EP2 and EP4 prostanoid receptors induces intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. However, specific mechanisms integrating the effects of TNF-alpha-induced NF-kappaB and MEK/ERK signalling or PGE 2/cAMP signalling on SOX9 activity and cartilage ECM gene expression are poorly characterized. In Chapter 2, I determined that the SOX9 transcriptional co-factor, p300, is limited in its activity and intracellular level when TNF-alpha activates NF-kappaB. In Chapter 3, I showed that TNF-alpha-activated MEK/ERK signalling is primarily responsible for regulating mRNA transcripts of genes whose products are localized to the extracellular space. These mRNAs include Col2a1, Agc1 and Hapln1. The transcription factor, early growth response protein 1 (EGR-1), was identified to be at least partially responsible for the reduction of these mRNAs. Finally, in Chapter 4, I found that PGE2/cAMP signalling reduces mRNA levels of cartilage-selective ECM genes and elevates chondrocyte metabolism. I determined that these effects likely involve inhibition of glycogen synthase kinase 3 (GSK3) and downstream beta-catenin-regulated transcription. In summary, I have elucidated key points of integration among multiple signalling pathways activated by factors upregulated in arthritis that inhibit normal chondrocyte function. Developing treatment strategies that target these multiple integration points (i.e., p300, MEK/ERK/EGR-1, cAMP/GSK3/beta-catenin) within TNF-alpha- and PGE2-induced signalling pathways may ultimately be useful for treatment of arthritic diseases.;Keywords: articular cartilage, chondrocyte, adenosine 3',5'-cyclic monophosphate, dedifferentiation, electrophoretic mobility shift assay, extracellular matrix, immunoblotting, metabolism, microarray, osteoarthritis, pharmacological inhibitors, prostaglandins, primary cell culture, quantitative real-time polymerase chain reaction, retinoic acid, rheumatoid arthritis, signal transduction, tumour necrosis factor alpha, transcription factors, transfection. |
