Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. 80% of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation, MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQQ209L transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901 or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a uveal melanoma xenograft model. Our data identifies PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations, and demonstrates combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.

Immortalization with SV40-TAg results in altered integrin expression by chondrocytes. Changes in the relative levels of alpha1, alpha2, and alpha3 subunits may significantly alter the manner in which chondrocytes interact with types II and VI collagen in the extracellular matrix.

Conclusion. ESE-1 is expressed in synovial tissues in RA and, to a variable extent, in OA, and is specifically induced in synovial fibroblasts, chondroDr. Libermann

Type II collagen, encoded by the COL2A1 gene, is the major collagen of the extracellular matrix of mature articular cartilage. Together with the other cartilage-specific type IX and XI collagens, the highly cross-linked fibrils of triple helical type II collagen molecules form a fibrillar network that confers tensile strength to articular surfaces (1). Chondrocytes comprise the single cellular component of adult hyaline cartilage and are responsive to a number of growth factors and cytokines that either stimulate or inhibit type II collagen synthesis (2). Interleukin-1 (IL-1)

Matrix metalloproteinase (MMP)-13 has a pivotal, rate-limiting function in cartilage remodeling and degradation due to its specificity for cleaving type II collagen. The proximal MMP13 promoter contains evolutionarily conserved E26 transformation-specific sequence binding sites that are closely flanked by AP-1 and Runx2 binding motifs, and interplay among these and other factors has been implicated in regulation by stress and inflammatory signals. Here we report that ELF3 directly controls MMP13 promoter activity by targeting an E26 transformation-specific sequence binding site at position ؊78 bp and by cooperating with AP-1. In addition, ELF3 binding to the proximal MMP13 promoter is enhanced by IL-1␤ stimulation in chondrocytes, and the IL-1␤-induced MMP13 expression is inhibited in primary human chondrocytes by siRNA-ELF3 knockdown and in chondrocytes from Elf3 ؊/؊ mice. Further, we found that MEK/ERK signaling enhances ELF3-driven MMP13 transactivation and is required for IL-1␤-induced ELF3 binding to the MMP13 promoter, as assessed by chromatin immunoprecipitation. Finally, we show that enhanced levels of ELF3 co-localize with MMP13 protein and activity in human osteoarthritic cartilage. These studies define a novel role for ELF3 as a procatabolic factor that may contribute to cartilage remodeling and degradation by regulating MMP13 gene transcription.The matrix metalloproteinases (MMPs) 3 are a family of enzymes that coordinately degrade components of the extracellular matrix in physiological/normal matrix remodeling processes (1) and in disease states wherein their aberrant and enhanced expression contributes to exacerbated matrix degradation (2, 3). Type II collagen is a major constituent of articular cartilage that contributes to its structural and functional properties by conferring tensile strength, and its degradation is the pivotal event that determines the irreversible progression of osteoarthritis (OA), in which articular cartilage is slowly and progressively destroyed (2). OA occurs in conjunction with changes in the synovium and subchondral bone that are associated with dysregulated chondrocyte physiology exemplified in part by the abnormal expression of catabolic and anabolic gene products (2). In this context, proinflammatory cytokines have been shown to trigger a diverse array of intracellular signaling pathways leading to the overexpression of a variety of matrix-degrading enzymes, including MMPs (2).Because collagen degradation is mediated almost exclusively by MMPs, those with higher collagenolytic activity (collagenases) are the rate-limiting, major players in irreversible cartilage destruction (4), and MMP13 (collagenase 3) plays a very prominent role here. MMP13 preferentially and more potently cleaves type II collagen compared with other collagenases (5-7). Moreover, MMP13 levels and activity are enhanced in OA cartilage, associated with degenerative changes and co-localizing with MMP13-specific type II collagen cleavage products, inflammatory cytokines, and their receptors (4,8). Furt...

The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1β (IL-1β). We reported previously that early activation of EGR-1 by IL-1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-κB-dependent inhibition by . Of the ETS factors tested, this response was specific to ESE-1, since ESE-3, which was also induced by IL-1β, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS-1 increased COL2A1 promoter activity and blocked the inhibition by IL-1β. These responses to ESE-1 and ETS-1 were confirmed using siRNA-ESE1 and siRNA-ETS1. In transient cotransfections, the inhibitory responses to ESE-1 and IL-1β colocalized in the -577/-132 bp promoter region, ESE-1 bound specifically to tandem ETS sites at 403/ 381 bp, and IL-1-induced binding of ESE-1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE-1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS-1, and other IL-1-induced factors, including EGR-1 at any given time. Intracellular ESE-1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE-1 in cartilage degeneration and suppression of repair.The chondrocyte is a specialized mesenchymal cell that synthesizes matrix proteins responsible for the tensile strength and resistance to mechanical loading of the articular cartilage (Poole, 2005). In adult articular cartilage, the chondrocytes maintain a low turnover rate of replacement of cartilage matrix proteins. Collagen turnover has been estimated to occur with a half-life of greater than 100 years, whereas the glycosaminoglycan constituents on the aggrecan core protein are more readily replaced and the half-life of aggrecan subfractions is in the range of 3-24 years. Nevertheless, chondrocytes in vivo respond to NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript structural changes in the surrounding cartilage matrix as occurs during the initial stages of osteoarthritis (OA) when increased cell proliferation and synthesis of matrix proteins, proteinases, and cytokines are observed. The early changes in synthetic activity are viewed as an attempt to regenerate the matrix with cartilage-specific components, including types II, IX, and XI collagens and aggrecan, and the presence of collagens not normally found in adult articular cartilage is evidence of phenotypic modulation (Sandell and Aigner, 2001). Genomic and proteomic analyses of global gene expression cartilage have confirmed the increased levels of type II collagen (COL2A1) mRNA and protein in OA cartilage (Bau et al., 2002;Hermansson et al., 2004), possibly ...

The chondrocyte is a specialized mesenchymal cell that synthesizes cartilage-specific matrix proteins, including type II collagen and the large aggregating proteoglycan, aggrecan, which are responsible for the tensile strength and resistance to mechanical loading of the articular cartilage (1). Although adult articular chondrocytes maintain a low turnover rate of replacement of cartilage matrix proteins, their limited capacity to repair

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