Browsing by Subject "Chondrocyte"

Selenium (Se) is an essential trace element and metalloid involved in several key metabolic activities: protection against oxidative damage, regulation of immune and thyroid function, and fertility. Several recent lines of evidence from epidemiology, genetic, and transgenic animal studies suggest that Se may play a protective role in Osteoarthritis (OA). However, the exact protective mechanism of Se is still unclear.

In this study, we hypothesized that Se exerts its chondroprotective benefit via an anti-oxidative and anti-inflammatory effect mediated by specific selenoproteins that neutralize cytokine-induced inflammatory responses in chondrocytes. We established an in vitro system for studying the effect of Se in the chondrosarcoma cell line SW-1353 and in human primary chondrocytes. Selenomethionine (SeMet) induced gene expression and enzyme activity of both antioxidative enzymes glutathione peroxidase (GPX) and thioredoxin reductase (TR) in SW-1353 cells. Our data suggest that Se may be protective against oxidative stress through regulation of the activity of these antioxidative enzymes.

As IL-1β is one of the primary pro-inflammatory cytokines contributing to the progression in OA, we next investigated the effect of Se on the gene expression induced by physiological doses of IL-1β. SeMet inhibited IL-1β induced catabolic gene expression of matrix metalloproteinase 1 (MMP1) and MMP13 as well as total MMP activity in chondrocytes. Similarly, SeMet inhibited chondrocyte gene expression of IL-1β induced nitric oxide synthase (iNOS) and cyclooxygenase (COX2) with corresponding reductions in nitric oxide (NO) and prostaglandin E2 (PGE2) production. In addition, SeMet pretreatment attenuated the IL-1β induced activation of p38 MAPK but not the ERK, JNK or NFkB pathways. Taken together, our results suggest that Se inhibits IL-1β induced expression of inflammatory and catabolic genes, partly through inhibition of IL-1β cell signaling.

Since Se may function through selenoproteins, we evaluated the role of three specific major selenoproteins, GPX1, TR1 and DIO2, in modifying the inflammatory response stimulated by IL-1β in chondrocytes by RNA interference. Based on RNA interference results, DIO2 and TR1 mediated the inhibitory effect of SeMet on IL-1β induced COX2 gene expression, while GPX1 did not show a significant inhibitory effect on Se. Depletion of DIO2 increased the IL-1β induced COX2 gene expression. This suggests that DIO2 may negatively modulate the IL-1β response. Our data also suggest that part of this inhibitory effect of DIO2 could be through regulation of IL-1β gene expression itself. These results highlight a potential new role of DIO2 in modulating the inflammatory response in chondrocytes

In summary, the result of this study suggests that Se may exert its chondroprotective effect through specific selenoproteins which neutralize oxidative stress and modify the inflammatory response in chondrocytes.

With growing numbers of increasingly younger patients suffering debilitating arthropathies, the need for simple models that recapitulate the complex interplay between distinct joint tissues, and grafts that emulate these joint structures in their biological properties and their strength have become more urgent. The objective of this study is to engineer constructs of multiple tissue types by controlling the morphogenetic factors that direct stem cell differentiation and tissue formation either exogenously or via transduction of expression vectors. Our hypothesis is that sequential changes in exogenous growth factor delivery and also scaffold-mediated inducible regulation of morphogenetic gene expression and signaling in 3D-constructs of murine iPSCs will lead to the formation of both bone and cartilage tissue types, both as separate tissues, and as osteochondral constructs. In the first study, osteochondral organoids were grown in a scaffold-free system from a single iPSC cell source, creating tissues containing a distinct core with the genetic and extracellular matrix profile of articular cartilage surrounded by a shell with the genetic and extracellular matrix profile of bone. In the second study, chondrogenic, osteogenic, and osteochondral tissue grafts were grown by scaffold-mediated lentiviral delivery of differentiation factors expressed both constitutively and in a temporally inducible manner. These constructs will provide an excellent platform to study diseases of the osteochondral junction, to screen pharmacologic therapies affecting both cartilage and bone tissue, and as a next step toward making an implantable osteochondral graft for the direct treatment of joint defects.

Enchondroma and chondrosarcoma are common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. Mutant IDH enzymes gain a neomorphic function of producing D-2-hydroxyglutarate (D-2HG) from ?-ketoglutarate. Expression of a mutant Idh1 gene is sufficient for enchondroma initiation but inhibiting mutant IDH enzymes did not cause a consistent change in the tumorigenic properties of chondrosarcomas. It is unclear how mutations of isocitrate dehydrogenases regulate cartilage tumors from initiation to cancer progression and maintenance. I hypothesize that mutations in IDH enzymes could regulate cartilage tumors through changes in gene expression and cellular metabolism. To address these questions, I examined the transcriptional regulation and metabolic regulations of mutant isocitrate dehydrogenases in chondrocytes and chondrosarcomas and identified cholesterol biosynthesis and glutamine metabolism as two key pathways dictating tumor behavior.

To understand the transcriptional regulation of IDH1 mutation in cartilage tumors, I performed RNA-sequencing analysis in chondrocytes from Col2a1Cre;Idh1LSL/+ mutant animals and their littermate wildtype controls and identified that cholesterol biosynthesis pathway was upregulated. Genetic inhibition of cholesterol biosynthesis in an enchondroma mouse model and pharmacological inhibition of cholesterol biosynthesis in human patient chondrosarcoma samples suppressed tumor development in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.

From a metabolic perspective, I found that chondrocytes and chondrosarcomas with mutations in IDH1/2 genes had enhanced glutamine utilization for downstream metabolism. Using genetic and pharmacological approaches, I demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1/2 mutations. Genetic ablation of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. Pharmacological inhibition of glutaminase led to decreased tumor weight of chondrosarcoma xenograft. During enchondroma development, glutamine-derived -ketoglutarate plays important roles in regulating chondrocyte differentiation and proliferation. In chondrosarcoma, glutamine-derived non-essential amino acids are important in preventing cell apoptosis.

In summary, findings in this dissertation described transcriptional and metabolic regulations by mutations in isocitrate dehydrogenases in cartilage tumors enchondroma and chondrosarcoma and provide novel insights for developing therapies for these diseases.