Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte

The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-α, increased the number of cells exhibiting an elongated morphology. TNF-α potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-α and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-α and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-α or by EGF, alone or in combination. EGF alone did not activate NF-κB or alter NF-κB activation by TNF-α. Pharmacologic studies indicated that the effects of TNF-α and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-α attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-α or basal activity. TNF-α and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals

Egr-1 inhibits the expression of extracellular matrix genes in chondrocytes by TNFα-induced MEK/ERK signalling

Introduction TNFα is increased in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. TNFα activates mitogen-activated kinase kinase (MEK)/extracellular regulated kinase (ERK) in chondrocytes; however, the overall functional relevance of MEK/ERK to TNFα-regulated gene expression in chondrocytes is unknown. Methods Chondrocytes were treated with TNFα with or without the MEK1/2 inhibitor U0126 for 24 hours. Microarray analysis and real-time PCR analyses were used to identify genes regulated by TNFα in a MEK1/2-dependent fashion. Promoter/ reporter, immunoblot, and electrophoretic mobility shift assays were used to identify transcription factors whose activity in response to TNFα was MEK1/2 dependent. Decoy oligodeoxynucleotides bearing consensus transcription factor binding sites were introduced into chondrocytes to determine the functionality of our results. Results Approximately 20% of the genes regulated by TNFα in chondrocytes were sensitive to U0126. Transcript regulation of the cartilage-selective matrix genes Col2a1, Agc1 and Hapln1, and of the matrix metalloproteinase genes Mmp-12 and Mmp-9, were U0126 sensitive – whereas regulation of the inflammatory gene macrophage Csf-1 was U0126 insensitive. TNFα-induced regulation of Sox9 and NFKB activity was also U0126 insensitive. Conversely, TNFα-increased early growth response 1 (Egr-1) DNA binding was U0126 sensitive. Transfection of chondrocytes with cognate Egr-1 oligodeoxynucleotides attenuated the ability of TNFα to suppress Col2a1, Agc1 or Hapln1 mRNA expression. Conclusions Our results suggest that MEK/ERK and Egr1 are required for TNFα-regulated catabolic and anabolic genes of the cartilage extracellular matrix, and hence may represent potential targets for drug intervention in osteoarthritis or rheumatoid arthritis

Regulation of Sox9 activity by crosstalk with nuclear factor-κB and retinoic acid receptors

Introduction: Sox9 and p300 cooperate to induce expression of cartilage-specific matrix proteins, including type II collagen, aggrecan and link protein. Tumour necrosis factor (TNF)-alpha, found in arthritic joints, activates nuclear factor-kappaB (NF-kappaB), whereas retinoic acid receptors (RARs) are activated by retinoid agonists, including all-trans retinoic acid (atRA). Like Sox9, the activity of NF-kappaB and RARs depends upon their association with p300. Separately, both TNF-alpha and atRA suppress cartilage matrix gene expression. We investigated how TNF-alpha and atRA alter the expression of cartilage matrix genes. Methods: Primary cultures of rat chondrocytes were treated with TNF-alpha and/or atRA for 24 hours. Levels of transcripts encoding cartilage matrix proteins were determined by Northern blot analyses and quantitative real-time PCR. Nuclear protein levels, DNA binding and functional activity of transcription factors were assessed by immunoblotting, electrophoretic mobility shift assays and reporter assays, respectively. Results: Together, TNF-alpha and atRA diminished transcript levels of cartilage matrix proteins and Sox9 activity more than each factor alone. However, neither agent altered nuclear levels of Sox9, and TNF-alpha did not affect protein binding to the Col2a1 48-base-pair minimal enhancer sequence. The effect of TNF-alpha, but not that of atRA, on Sox9 activity was dependent on NF-kappaB activation. Furthermore, atRA reduced NF-kappaB activity and DNA binding. To address the role of p300, we over-expressed constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 to increase p300 acetylase activity. caMEKK1 enhanced basal NF-kappaB activity and atRA-induced RAR activity. Over-expression of caMEKK1 also enhanced basal Sox9 activity and suppressed the inhibitory effects of TNF-alpha and atRA on Sox9 function. In addition, over-expression of p300 restored Sox9 activity suppressed by TNF-alpha and atRA to normal levels. Conclusion: NF-kappaB and RARs converge to reduce Sox9 activity and cartilage matrix gene expression, probably by limiting the availability of p300. This process may be critical for the loss of cartilage matrix synthesis in inflammatory joint diseases. Therefore, agents that increase p300 levels or activity in chondrocytes may be useful therapeutically

The induction of CCN2 by TGFβ1 involves Ets-1

CCN2 is encoded by an immediate-early gene induced in mesenchymal cells during the formation of blood vessels, bone and connective tissue. It plays key roles in cell adhesion and migration, as well as matrix remodeling. CCN2 is overexpressed in fibrosis, arthritis and cancer; thus, an understanding of how to control CCN2 expression is likely to have importance in developing therapies to combat these pathologies. Previously, we found that the promoter sequence GAGGAATG is important for Ccn2 gene regulation in NIH 3T3 fibroblasts. In this report, we show that this sequence mediates activation of the CCN2 promoter by the ETS family of transcription factors. Endogenous Ets-1 binds this element of the CCN2 promoter, and dominant negative Ets-1 and specific Ets-1 small interfering RNA block induction of CCN2 expression by TGFβ. In the absence of added TGFβ1, Ets-1, but not the related fli-1, synergizes with Smad 3 to activate the CCN2 promoter. Whereas the ability of transfected Ets-1 to activate the CCN2 promoter is dependent on protein kinase C (PKC), Ets-1 in the presence of co-transfected Smad3 does not require PKC, suggesting that the presence of Smad3 bypasses the requirement of Ets-1 for PKC to activate target promoter activity. Our results are consistent with the notion that Smad3 and Ets-1 cooperate in the induction of the CCN2 promoter by TGFβ1. Antagonizing Ets-1 might be of benefit in attenuating CCN2 expression in fibrosis, arthritis and cancer, and may be useful in modulating the outcome of these disorders

Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis

Preclinical osteoarthritis (OA) models are often employed in studies investigating disease-modifying OA drugs (DMOADs). In this study we present a comprehensive, longitudinal evaluation of OA pathogenesis in a rat model of OA, including histologic and biochemical analyses of articular cartilage degradation and assessment of subchondral bone sclerosis. Male Sprague-Dawley rats underwent joint destabilization surgery by anterior cruciate ligament transection and partial medial meniscectomy. The contralateral joint was evaluated as a secondary treatment, and sham surgery was performed in a separate group of animals (controls). Furthermore, the effects of walking on a rotating cylinder (to force mobilization of the joint) on OA pathogenesis were assessed. Destabilization-induced OA was investigated at several time points up to 20 weeks after surgery using Osteoarthritis Research Society International histopathology scores, in vivo micro-computed tomography (CT) volumetric bone mineral density analysis, and biochemical analysis of type II collagen breakdown using the CTX II biomarker. Expression of hypertrophic chondrocyte markers was also assessed in articular cartilage. Cartilage degradation, subchondral changes, and subchondral bone loss were observed as early as 2 weeks after surgery, with considerable correlation to that seen in human OA. We found excellent correlation between histologic changes and micro-CT analysis of underlying bone, which reflected properties of human OA, and identified additional molecular changes that enhance our understanding of OA pathogenesis. Interestingly, forced mobilization exercise accelerated OA progression. Minor OA activity was also observed in the contralateral joint, including proteoglycan loss. Finally, we observed increased chondrocyte hypertrophy during pathogenesis. We conclude that forced mobilization accelerates OA damage in the destabilized joint. This surgical model of OA with forced mobilization is suitable for longitudinal preclinical studies, and it is well adapted for investigation of both early and late stages of OA. The time course of OA progression can be modulated through the use of forced mobilization

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

The seeds of divergence: the economy of French North America, 1688 to 1760

Generally, Canada has been ignored in the literature on the colonial origins of divergence with most of the attention going to the United States. Late nineteenth century estimates of income per capita show that Canada was relatively poorer than the United States and that within Canada, the French and Catholic population of Quebec was considerably poorer. Was this gap long standing? Some evidence has been advanced for earlier periods, but it is quite limited and not well-suited for comparison with other societies. This thesis aims to contribute both to Canadian economic history and to comparative work on inequality across nations during the early modern period. With the use of novel prices and wages from Quebec—which was then the largest settlement in Canada and under French rule—a price index, a series of real wages and a measurement of Gross Domestic Product (GDP) are constructed. They are used to shed light both on the course of economic development until the French were defeated by the British in 1760 and on standards of living in that colony relative to the mother country, France, as well as the American colonies. The work is divided into three components. The first component relates to the construction of a price index. The absence of such an index has been a thorn in the side of Canadian historians as it has limited the ability of historians to obtain real values of wages, output and living standards. This index shows that prices did not follow any trend and remained at a stable level. However, there were episodes of wide swings—mostly due to wars and the monetary experiment of playing card money. The creation of this index lays the foundation of the next component. The second component constructs a standardized real wage series in the form of welfare ratios (a consumption basket divided by nominal wage rate multiplied by length of work year) to compare Canada with France, England and Colonial America. Two measures are derived. The first relies on a “bare bones” definition of consumption with a large share of land-intensive goods. This measure indicates that Canada was poorer than England and Colonial America and not appreciably richer than France. However, this measure overestimates the relative position of Canada to the Old World because of the strong presence of land-intensive goods. A second measure is created using a “respectable” definition of consumption in which the basket includes a larger share of manufactured goods and capital-intensive goods. This second basket better reflects differences in living standards since the abundance of land in Canada (and Colonial America) made it easy to achieve bare subsistence, but the scarcity of capital and skilled labor made the consumption of luxuries and manufactured goods (clothing, lighting, imported goods) highly expensive. With this measure, the advantage of New France over France evaporates and turns slightly negative. In comparison with Britain and Colonial America, the gap widens appreciably. This element is the most important for future research. By showing a reversal because of a shift to a different type of basket, it shows that Old World and New World comparisons are very sensitive to how we measure the cost of living. Furthermore, there are no sustained improvements in living standards over the period regardless of the measure used. Gaps in living standards observed later in the nineteenth century existed as far back as the seventeenth century. In a wider American perspective that includes the Spanish colonies, Canada fares better. The third component computes a new series for Gross Domestic Product (GDP). This is to avoid problems associated with using real wages in the form of welfare ratios which assume a constant labor supply. This assumption is hard to defend in the case of Colonial Canada as there were many signs of increasing industriousness during the eighteenth and nineteenth centuries. The GDP series suggest no long-run trend in living standards (from 1688 to circa 1765). The long peace era of 1713 to 1740 was marked by modest economic growth which offset a steady decline that had started in 1688, but by 1760 (as a result of constant warfare) living standards had sunk below their 1688 levels. These developments are accompanied by observations that suggest that other indicators of living standard declined. The flat-lining of incomes is accompanied by substantial increases in the amount of time worked, rising mortality and rising infant mortality. In addition, comparisons of incomes with the American colonies confirm the results obtained with wages— Canada was considerably poorer. At the end, a long conclusion is provides an exploratory discussion of why Canada would have diverged early on. In structural terms, it is argued that the French colony was plagued by the problem of a small population which prohibited the existence of scale effects. In combination with the fact that it was dispersed throughout the territory, the small population of New France limited the scope for specialization and economies of scale. However, this problem was in part created, and in part aggravated, by institutional factors like seigneurial tenure. The colonial origins of French America’s divergence from the rest of North America are thus partly institutional

Regulatory influences of microenvironmental factors on osseous cell proliferation and differentiation in vitro

The cellular activity in bone and cartilage tissue is controlled by substances present in the osseous environment. Using representative cell lines, modulation of osteoblastic and chondrocytic phenotypic markers such as hormone-responsiveness, enzymatic activities, and matrix production by osteotropic factors was examined in vitro. In UMR 106 cells, a rat osteoblast-derived osteosarcoma cell line, a differential distribution of EGF and PTH receptors over proliferating and quiescent cells, respectively, was observed. EGF treatment resulted in expansion of a less differentiated population characterized by decreased PTH- and CT-stimulated adenylate cyclase and binding capacity, and increased CGRP-responsiveness. Using immunocytolysis, a mixed osseous cell population (OBCK6) was derived from fetal rat calvarial cells. Subsequent dilution cloning yielded two cell lines, the CFK1 line with osteoblastic characteristics and the CFK2 line with chondrocytic properties. Although only the OBCK6 cells deposited a mineralized matrix in vitro, organic matrix components were produced by the other two lines. EGF stimulated proliferation in both CFK1 and CFK2 cells. Retinoic acid decreased cell proliferation, PTH-responsiveness, and alkaline phosphatase activity, caused cell shape change and population reorganization, and increased EGF receptors in CFK1 cells. Induction of cartilage specific matrix components (type II collagen, proteoglycan core protein, link protein, and thrombospondin) was observed in CFK2 cells maintained in culture without passaging and could be regulated by EGF, PTH, dexamethasone, and retinoic acid. Thus, peptidergic and steroidal factors have been employed to modulate functional activity and cell cycle kinetics, and have resulted in the characterization of discrete stages of differentiation of osteoblastic and chondrocytic populations in vitro