bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of acetylation in Timp‐1 regulation
Authors: Young David A. , Edwards Dylan R. , Clark Ian M. 
Published: September 2009

Introduction  Aberrant matrix turnover, mediated by a family of proteases known as matrix metalloproteinases (MMPs), is involved in a number of pathologies including rheumatoid arthritis and osteoarthritis, tumour invasion and metastasis, and liver fibrosis. The active forms of all of the MMPs are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As inhibition represents a major level of control of MMP activity, a detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important in developing therapies for these diseases. Histone acetyl transferases (HATs) play a crucial role in gene regulation via acetylation of both histones (to loosen nucleosomal structure and to recruit accessory factors) and transcription factors. Here, we describe the effects of a histone deacetylase inhibitor (Trichostatin A – TSA) on the TGF‐β1 and Phorbol ester (PMA) induction of Timp‐1.Materials and methods  RNA isolation, TaqMan® real‐time RT‐PCR, cell culture, transient transfections and reporter gene assays along with electrophoretic mobility shift assays (EMSA) were performed essentially as described (Young et al. 2002).Results  In murine C3H10T1/2 cells both TGF‐β1 and PMA induce Timp‐1 expression in a protein synthesis‐dependent manner as measured by Northern blotting and TaqMan® real‐time RT‐PCR. TSA superinduces PMA‐induced Timp‐1 expression but represses TGF‐β‐induced Timp‐1 expression. A TSA dose–response experiment further demonstrates that TGF‐β and PMA stimulate Timp‐1 gene expression via different mechanisms. The effects of TGF‐β, PMA and TSA on Timp‐1 expression can be reiterated in transient transfection studies with Timp‐1 promoter containing reporter constructs. Deletion of the proximal AP‐1 motif from the promoter demonstrates that this sequence is critical for TGF‐β1‐induced reporter expression, while PMA appears to act via other sequences/mechanisms.Discussion  Repression of deacetylation by TSA differentially affects the TGF‐β or PMA induction of Timp‐1. These results will help elucidate the different molecular mechanisms by which these factors regulate the expression of Timp‐1 and other genes. Furthermore, the effects of acetylation on TGF‐β induction of Timp‐1 may act as a paradigm for other cytokine‐induced genes.This work was funded and supported by the Arthritis Research Campaign (ARC) and the Dunhill Medical Trust.
Discovery of potent and selective inhibitors of procollagen C‐proteinase for the treatment of fibrotic disorders
Authors: Butt R.P. , Huggins J.P. , Greiling D. , Hopkins B. , Gaboardi S. , Winslow D. , Ronald M. , Lewis S. , Ward S. , Levett E. , Owen J. , Burslem F. , Collis M. , Bailey S. , Fish P.V. , Whitlock G. , Billotte S. , James K. , Mcelroy A. , Blagg J. 
Published: September 2009

Introduction  Fibrosis is a component of many tissue pathologies leading to loss of normal tissue function, primarily due to excessive collagen deposition. Collagen is deposited following cleavage of the C‐ and N‐ terminal peptides from the pro‐collagen molecule. The cleavage of the globular C‐peptide by PCP reduces solubility of the fibrillar collagen molecule, resulting in deposition of insoluble collagen. Increased insoluble collagen deposition is a feature of all organ fibroses, with inhibition of this process, a key potential anti‐fibrotic mechanism. The aim of this work was to discover potent and selective PCP inhibitors as experimental, topically applied, anti‐fibrotic drugs for clinical evaluation.Materials and methods  PCP was cloned from human osteosarcoma cells and enzymatic activity demonstrated using a PCP‐specific peptide cleavage assay. Activities were confirmed by measuring cleavage of [3H]C‐peptide from type‐I pro‐collagen. A cell‐based fibroplasias model was employed to demonstrate compound efficacy using collagen deposition, liberated C‐peptide and histological endpoints. The activities of PCP inhibitors in fibroblast and epithelial in vitro cell proliferation and migration assays, and selectivity vs. a panel of MMPs were also determined.Discussion  In summary, we have identified and characterized potent and selective inhibitors of PCP for progression to clinical studies for investigation as a treatment paradigm for fibrotic disease.  Results

Assay
UK‐383,367
UK‐421,045

Inhibition of peptide cleavage (Ki, nM)
33.3 ± 1.92
8.7 ± 2.9

Inhibition of pro‐collagen cleavage (IC50, nM)
27.5 ± 3.2
3.4 ± 1.0

Maximal inhibition of collagen deposition in fibroplasia model
75.5% @10 µm
76.0% @10 µm

Maximal inhibition of C‐peptide liberation
(IC50 approximately 2 µm)
(IC50 approximately 1 µm)

n.d.
72.2% @10 µm

(IC50 approximately 1 µm)

Effect on fibroblast proliferation/migration
No effect
No effect

Selectivity vs. MMPs (fold vs. PCP Ki in peptide substrate assay)
MMP‐1,‐2,‐9,13,‐14 >2273
>10,000

MMP‐3 1684
9,000

TACE >500
>5,000

All values mean ± SEM; n ≥ 3 replicate experiments. Compounds: UK‐383,367 and UK‐421,045. MMP, matrix metalloproteinase; PCP, procollagen C‐proteinase.
Novel strategies for enhancing tissue integration in cartilage repair
Authors: Davies L.C. , Caterson B. , Duance V.C. 
Published: September 2009

Introduction  Articular cartilage is unable to initiate a spontaneous repair response when injured due to its avascular and aneural properties. Within adult cartilage, chondrocytes are entrapped within an extensive extracellular matrix and are unable to migrate to sights of injury to regulate tissue repair. Injury to this tissue therefore inevitably leads to degeneration of the cartilage and the development of degenerative diseases such as osteoarthritis.The surgical technique of autologous chondrocyte transplantation (ACT) was developed for the treatment of full‐thickness cartilage defects (Brittberg et al. 1994). Implantation of chondrocytes into the defect site repairs the injury site with a mixture of fibrocartilaginous and hyaline‐like tissue that poorly integrates with the existing cartilage and frequently degenerates with time. In this current study, we have developed an in vitro model to investigate methods for enhancing this integration and the development of a more biomechanically stable repair tissue.Materials and methods  Bovine articular cartilage explants from the metacarpalphalangeal joint were experimentally injured using a stainless steel trephine and cultured for a period of 28 days. Autologous chondrocytes in an agarose suspension were injected into the interface region at the injury site. Media was collected and analysed for proteoglycan and collagen content using the DMMB and hydroxyproline assays, respectively. Matrix metalloproteinase (MMP) expression was also analysed using zymography and an adapted collagen fibril assay.Results  Morphological analyses indicate attempts at repair and integration within both control and experimental treatment groups, although the presence of autologous chondrocytes appeared to amplify this repair response. Although not statistically significant, considerable differences in proteoglycan release between injured explants and the intact control group were seen. Collagen release into the media was only seen at day 28 within experimental cultures. An up‐regulation of MMP‐2 and MMP‐9 was seen within the experimental cultures compared to the controls. Preliminary data also suggest up‐regulation of collagenases in the experimental group when compared to controls.Discussion  As seen with clinical ACT treatment, the presence of autologous chondrocytes appears to enhance repair and integration attempts; however, morphologically, this repair tissue appears to be fibrocartilaginous. Further analysis will establish whether the repair tissue is true hyaline cartilage and monitor the synthesis and turnover of macromolecules within the established culture system.
Expression profiling of metalloproteinases and inhibitors in cartilage
Authors: Kevorkian Lara , Young David A. , Darrah Clare , Donell Simon T. , Shepstone Lee , Porter Sarah , Brockbank Sarah , Edwards Dylan R. , Parker Andrew E. , Clark Ian M. 
Published: September 2009

Objective  To profile the expression of all known members of the matrix metalloproteinase (MMP), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and tissue inhibitor of metalloproteinases (TIMPs) gene families in normal cartilage and that from patients with osteoarthritis (OA).Methods  Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur. Total RNA was purified and expression of genes assayed using quantitative real‐time PCR.Results  Several members of the above gene families were regulated in OA. Genes increasing in expression in OA were: at P < 0.001, MMP‐13, MMP‐28, ADAMTS‐16; at P < 0.01, MMP‐9, MMP‐16, ADAMTS‐2, ADAMTS‐14 and at P < 0.05, MMP‐2, TIMP‐3, ADAMTS‐12. Genes decreasing in expression in OA were: at P < 0.001, MMP‐1, MMP‐3, ADAMTS‐1; at P < 0.01, MMP‐10, TIMP‐1, ADAMTS‐9 and at P < 0.05, TIMP‐4, ADAMTS‐5, ADAMTS‐15. Correlation analysis revealed that groups of genes across the gene families are co‐expressed in cartilage.Conclusion  This is the first comprehensive expression profile of all known MMP, ADAMTS and TIMP genes in cartilage. Patterns of expression provide a foundation on which to understand mechanisms of gene regulation in OA and potentially for refining the specificity of anti‐proteolytic therapies.
TGF‐β1 induces epithelial–mesenchymal transition but not myofibroblast transdifferentiation in primary cultures of human epithelial renal tubular cells
Authors: Anglani Franca , Forino Monica , Murer Luisa , Vella Manuela Dalla , Prete Dorella Del , Ceol Monica , Gambaro Giovanni , D'Angelo Angela 
Published: September 2009

Introduction  Although the origin of renal interstitial myofibroblasts is still a matter of debate (Powell et al. 1999), emerging evidences suggest that they may derive from tubular epithelial cells. Since the well‐known mesenchymal origin of human tubular epithelial cells (HUTECs), our working hypothesis is that in renal fibrogenesis, fibrogenetic cytokines could reactivate the mesenchymal program [epithelial–mesenchymal transition (EMT)], switched‐off during renal nephrogenesis, leading to differentiation into MF. TGF‐β1 is a key modulator of the myofibroblast phenotype in fibroblasts during the process of wound healing and in mesangial cells in culture. TGF‐β1 is also able to induce EMT in a variety of cell types. However, most of these studies including the recent one on SV40‐transformed HKC‐8 human tubular cells (Yang & Liu 2001) have been carried out in immortalized cells.Materials and methods  Primary HUTEC cultures were established from histologically normal human renal cortexes, obtained from surgical biopsies performed following informed consent in paediatric patients undergoing surgery because of extrinsic pyelouretheral obstruction. HUTECs were cultured for 4 and 6 days on plastic or type‐I collagen‐coated plates with or without 1, 5, 10 and 50 ng/ml TGF‐β1. Time‐course experiments (24 h to 6 days) with 1 ng/ml TGF‐β1 were also performed on primary human dermal fibroblasts, used as control mesenchymal cells, and HUTECs. Control conditions were represented by cells maintained for 4 and 6 days in 1% serum without TGF‐β1. The EMT process was monitored by morphology and immunophenotyping for α‐SMA, cytokeratin 8–18, E‐cadherin, vimentin and Ki67. Quantitative comparative RT/PCR evaluated the expression of collagen‐III and ‐V, fibronectin, tenascin, MMP‐2, CTGF, E‐cadherin and cadherin 11 genes. TGF‐β1 regulation of α‐SMA was investigated at both transcriptional and translational (Western blot) levels.Results and discussion  TGF‐β1‐driven EMT was documented morphologically, biochemically and molecularly. HUTEC morphology changes following TGF‐β1 exposure were already evident at 24 h and with as little as 1 ng/ml of TGF‐β1; HUTECs acquired a spindle shape with front‐end/back‐end polarity, like fibroblasts. The transition was characterized by drastic up‐regulation of all the mesenchymal markers studied, including CTGF, and down‐regulation of cytokeratin and E‐cadherin expression. These phenomena were dose‐dependent and favuored by growth on collagen‐I. TGF‐β1 treatment did not induce MF conversion, because it induces neither de novo expression of α‐SMA gene nor the myofibroblast phenotype. We demonstrate that the TGF‐β1‐driven EMT is characterized by the re‐appearance of developmental gene networks which, in incomplete or uncoordinate fashion, could contribute to the pathogenesis of renal fibrosis.
Decorin affects endothelial cells by interacting with IGF‐I and its receptor
Authors: Schönherr Elke 
Published: September 2009

Introduction  Endothelial cells (ECs) undergoing angiogenesis start to synthesize decorin, a member of the family of small proteoglycans with leucine‐rich repeats. Previously, we could show that decorin synthesis in ECs leads to capillary formation and survival of ECs in an angiogenesis model (Schönherr et al. 1999). In this model, decorin enhanced the phosphorylation of protein kinase B (Akt) and subsequently induced the cyclin‐dependent kinase inhibitor p21 (Schönherr et al. 2001).Materials and methods  Cell culture: ECs of the permanent cell line EA.hy 926 were cultured on collagen type‐I‐coated dishes in Waymouth MAB 87/3 medium containing 1% heat‐inactivated fetal calf serum and antibiotics. Decorin purified from fibroblast cultures and/or the respective inhibitors were added.Binding studies:  Decorin and IGF‐I were labelled with 125I‐iodine. (1) Decorin was run on an SDS‐PAGE and transferred to nitrocellulose. Blots were probed with 125I‐IGF‐I and exposed to X‐ray films. (2) A Sepharose CL‐4B gel filtration column was equilibrated with or without decorin containing buffer and 125I‐IGF‐I (500.000 cpm) was applied. The elution profiles were monitored. (3) IGF‐receptor was immunoprecipitated from EA.hy 926 cells with the antibody (sc‐713) against the β‐chain of the receptor. The immune complex was incubated with labelled or unlabelled decorin or IGF‐I (as indicated). Immune complexes without receptor were used as controls.Results  To analyse how decorin activates Akt, inhibitors of different receptor tyrosine kinases were tested. The EGF‐receptor inhibitor tyrphostin AG1478 (10 µm) had no effect on decorin‐induced Akt posphorylation, but preincubation with tyrphostin AG1024 (10 µm), an inhibitor of the insulin and the IGF‐I receptor tyrosine kinases, inhibited Akt phophorylation and p21 expression. Combined addition of IGF‐I and decorin to ECs in culture had an additive effect on Akt phosphorylation. Binding experiments with 125I‐IGF‐I to decorin immobilized on nitrocellulose revealed that both the core protein and the proteoglycan bind 125I‐IGF‐I. Binding of 125I‐IGF‐I to decorin in solution showed the dose‐dependent formation of a high molecular weight complex that eluted in the included volume of the column. Immunoprecipitated IGF‐I receptor bound 125I‐‐IGF‐I as well as 125I‐decorin. In addition, 125I‐IGF‐I bound to the receptor could be displaced by unlabelled decorin.Discussion  These results suggest that decorin can bind to IGF‐I and to its receptor and that this interaction enhances Akt phosphorylation in ECs. The effect of decorin on the IGF‐I receptor could be mediated by direct interaction. However, a further possibility is that decorin couples trace amounts of IGF‐I from the medium or the collagen on the dishes to a larger complex, which can more effectively activate the IGF‐I receptor. These possibilities as well as the role of IGF binding proteins will need further investigation.
The isoprostane 8‐iso‐PGF2α suppresses monocyte adhesion to microvascular endothelial cells: a role in limiting inflammation and fibrosis?
Authors: Kumar Anila , Kingdon Edward , Norman Jill 
Published: September 2009

Introduction  Isoprostanes, produced in vivo by nonenzymatic free radical‐induced lipid peroxidation, are recognized markers of oxidative stress with elevated serum and urine levels of 8‐iso‐PGF2α (iP) reported in a variety of fibrotic diseases including end‐stage renal disease, systemic sclerosis and atherosclerosis. It has been suggested that iP may also have pathogenic functions. Many fibrotic diseases are characterized by early perivascular inflammatory infiltrates, and inflammation has been shown to be a critical component of the fibrotic process. Adhesion of monocytes to the capillary endothelium is an initiating event in inflammation and, in line with a proposed pathological role for iP, we hypothesized that iP might stimulate monocyte adhesion and thus promote fibrosis.Methods  Human monocytes (U937 or THP‐1) were added to confluent MECs and the number of adhered monocytes measured colorimetrically or by cell counting.Results  In dose–response assays (10−12−10−5 M), iP > 10−7 M inhibited monocyte adhesion (by 76%P < 0.01) to quiescent or proliferating human and rat renal MECs. In contrast, iP stimulated U937 adhesion to HUVEC (as reported by Leitinger et al. FASEB J 15,1254, 2001) demonstrating diverse effects of iP on different ECs. iP had no effect on viability (trypan blue exclusion), proliferation (3H‐thymidine incorporation) or apoptosis (Annexin V) of U937 or MECs. Inhibition of adhesion was dependent on preincubation (minimum 1–2 h) of iP; simultaneous addition of monocytes and iP to HMEC or treatment of U937 had no effect; suggesting that inhibition is due to iP‐induced changes in HMEC. These changes appear to be independent of de novo protein synthesis. Furthermore, iP, added either before or after TNF‐α, blocked TNF‐α‐induced monocyte adhesion. The inhibitory effect of iP was mimicked by a thromboxane receptor (TxR) agonist (U46619, 10 µm) and blocked by a receptor antagonist (SQ29548, 10 µm) indicating a TxR‐mediated process. Experiments using signal transduction pathway inhibitors (SB203580, curcumin and PD98059) implicate p38 and JNK, but not ERK, in iP‐induced suppression of monocyte adhesion. In addition to a direct effect, conditioned medium (CM) transfer experiments suggest that iP induces a secondary mediator which also suppresses monocyte adhesion but via an alternative mechanism which is TxR‐independent and dependent on new protein synthesis. This pathway also involves activation of p38 but is only partially dependent on JNK.Conclusion  The data show that iP can suppress the attachment of monocytes to MECs via two independent pathways indicating an unexpected, potentially protective effect of iP in the microvasculature. Characterization of the mechanisms and mediators involved in this process may provide novel targets for intervention in inflammation and subsequent fibrosis.
Investigating the PKR‐signalling pathway in the articular joint
Authors: Gilbert S.J. , Duance V.C. , Mason D.J. 
Published: September 2009

Introduction  Our previous studies have shown that the protein kinase, PKR and its activator, PACT are involved in TNF‐α signalling in articular cartilage (Gilbert et al. 2002). The sphingolipid ceramide is known to play an important role in signal transduction of TNF‐α and is a potent apoptotic agent. The PKR pathway is also known to be activated by ceramide (Ruvolo et al. 2001). Recent studies have shown that ceramide stimulates proteoglycan degradation and mRNA expression of MMP‐1, MMP‐3 and MMP‐13 in rabbit cartilage suggesting a role for this second messenger in cartilage degradation (Sabatini et al. 2000). In the current study, we investigated the role of ceramide, TNF‐α and PKR in bovine articular cartilage degradation. In addition, we treated human primary synoviocytes with TNF and investigated whether PKR is activated and if NFkB, a transcription factor downstream of PKR, is translocated to the nucleus.Materials and methods  Bovine articular cartilage explants were stimulated with C2‐ceramide (50 µm) or TNF‐α (100 ng/ml) for 24 h. To inhibit the activation of PKR, 2‐aminopurine (10 mm) was added to duplicate cultures 1 h prior to the addition of treatments. Media was collected and MMPs analysed by gelatin zymography. Proteoglycan release was measured by the DMMB assay and cell viability determined by the Cytotox® assay. Human primary synoviocytes were stimulated with TNF‐α (10 ng/ml) for 30 min, and phosphorylated PKR and the Rel A p65 subunit of NFκB were detected by immunofluorescence.Results  C2‐ceramide treatment resulted in a significant release of both pro and active MMP‐2. Explants incubated with the PKR inhibitor, 2‐aminopurine, prior to TNF‐α or C2‐ceramide treatment resulted in a marked reduction in both MMP‐2 and MMP‐9 activation. A significant increase in proteoglycan release was observed following treatment with TNF‐α and C2‐ceramide, which was significantly inhibited by 2‐aminopurine. A significant loss of cell viability was observed when explants were treated with C2‐ceramide, which was also found to be regulated by a PKR‐dependant pathway. Studies using antisense oligonucleotides to PACT and PKR are currently being undertaken to confirm these results.An increase in phosphorylation of PKR and NFkB translocation was observed in synoviocytes treated with TNF.Discussion  Our data has shown that PKR potentially is an important mediator of degradative and death pathways in chondrocytes. Collectively, these results suggest a novel role for the PKR pathway in the turnover of articular cartilage and support our hypothesis that PKR and PACT are implicated in the cartilage loss that occurs in arthritic disease.