Priya S. Chockalingam

Protein Kinase Cζ Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-κB by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes

Protein kinase Cζ (PKCζ) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCζ is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCζ may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCζ, implicating PKCζ activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCζ activity in NF-κB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-α caused an increase in NF-κB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCζ-sparing inhibitor calphostin C. A cell-permeable PKCζ pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-κB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCζ protein effectively prevented cytokine-mediated NF-κB activation in primary chondrocytes. These data implicate PKCζ as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor

OBJECTIVE To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. METHOD An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. RESULTS Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. CONCLUSIONS Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage

BackgroundTenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.MethodsTN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.ResultsTN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.ConclusionsTN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.

Tenascin-C levels in synovial fluid are elevated after injury to the human and canine joint and correlate with markers of inflammation and matrix degradation

OBJECTIVE We have previously shown the capacity of tenascin-C (TN-C) to induce inflammatory mediators and matrix degradation in vitro in human articular cartilage. The objective of the present study was to follow TN-C release into knee synovial fluid after acute joint injury or in joint disease, and to correlate TN-C levels with markers of cartilage matrix degradation and inflammation. METHOD Human knee synovial fluid samples (n = 164) were from a cross-sectional convenience cohort. Diagnostic groups were knee healthy reference, knee anterior cruciate ligament rupture, with or without concomitant meniscus lesions, isolated knee meniscus injury, acute inflammatory arthritis (AIA) and knee osteoarthritis (OA). TN-C was measured in synovial fluid samples using an enzyme-linked immunosorbent assay (ELISA) and results correlated to other cartilage markers. TN-C release was also monitored in joints of dogs that underwent knee instability surgery. RESULTS Statistically significantly higher levels of TN-C compared to reference subjects were observed in the joint fluid of all human disease groups and in the dogs that underwent knee instability surgery. Statistically significant correlations were observed between the TN-C levels in the synovial fluid of the human patients and the levels of aggrecanase-dependent Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments and matrix metalloproteinases 1 and 3. CONCLUSIONS We find highly elevated levels of TN-C in human knee joints after injury, AIA or OA that correlated with markers of cartilage degradation and inflammation. TN-C in synovial fluid may serve dual roles as a marker of joint damage and a stimulant of further joint degradation.

Immortalized mouse articular cartilage cell lines retain chondrocyte phenotype and respond to both anabolic factor BMP‐2 and pro‐inflammatory factor IL‐1

Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM‐Sv/HP and MM‐Sv/HP‐2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q‐PCR) were performed on cells in monolayer culture and cells embedded in a three‐dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP‐2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro‐inflammatory IL‐1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP‐13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP‐2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL‐1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase‐cleaved aggrecan fragments. In summary, MM‐Sv/HP and MM‐Sv/HP‐2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology. J. Cell. Physiol. 215: 68–76, 2008.

Soluble Fn14 Is Detected and Elevated in Mouse and Human Kidney Disease.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tissue injury to mediate local tissue responses including inflammation and tissue remodeling. We found that in various models of kidney disease, Fn14 expression (mRNA and protein) is upregulated in the kidney. These models include: lupus nephritis mouse models (Nephrotoxic serum Transfer Nephritis and MRL.Faslpr/lpr), acute kidney injury models (Ischemia reperfusion injury and Folic acid injury), and a ZSF-1 diabetic nephropathy rat model. Fn14 expression levels correlate with disease severity as measured by disease histology. We have also shown for the first time the detection of soluble Fn14 (sFn14) in the urine and serum of mice. Importantly, we found the sFn14 levels are markedly increased in the diseased mice and are correlated with disease biomarkers including proteinuria and MCP-1. We have also detected sFn14 in human plasma and urine. Moreover, sFn14 levels, in urine are significantly increased in DN patients and correlated with proteinuria and MCP-1 levels. Thus our data not only confirm the up-regulation of Fn14/TWEAK pathway in kidney diseases, but also suggest a novel mechanism for its regulation by the generation of sFn14. The correlation of sFn14 levels and disease severity suggest that sFn14 may serve as a potential biomarker for both acute and chronic kidney diseases.

Continued exploration of biphenylsulfonamide scaffold as a platform for aggrecanase-1 inhibition.

Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 μg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.

Immortalized cell lines from mouse xiphisternum preserve chondrocyte phenotype

Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP‐2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL‐1 results in the increased expression of catabolic effectors including MMP‐13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP‐2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL‐1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase‐cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3‐dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA. J. Cell. Physiol. 209: 551–559, 2006.