Adenoviral overexpression of Smad-7 and Smad-6 differentially regulates TGF-β- mediated chondrocyte proliferation and proteoglycan synthesis A Scharstuhl,

Slides:

Advertisements

Similar presentations

IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,

Advertisements

Supplemental Figure 2 A B vehicle

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

Chondro-protective effects of low intensity pulsed ultrasound

BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium- derived progenitor cells cultured in three-dimensional alginate hydrogel

CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes T. Fujisawa, Ph.D., D.D.S.,

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is increased in osteoarthritis and regulates chondrocyte catabolic and anabolic activities

Muscle cell-derived factors inhibit inflammatory stimuli-induced damage in hMSC- derived chondrocytes R.S. Rainbow, H. Kwon, A.T. Foote, R.C. Preda, D.L.

Regulation of mesenchymal stem cell chondrogenesis by glucose through protein kinase C/transforming growth factor signaling T.-L. Tsai, P.A. Manner,

Induction of chondrogenic phenotype in synovium-derived progenitor cells by intermittent hydrostatic pressure K. Sakao, M.D., K.A. Takahashi, M.D., Ph.D.,

Insulin-like growth factor-1 boosts the developing process of condylar hyperplasia by stimulating chondrocytes proliferation Y. Chen, J. Ke, X. Long,

Upregulated ank expression in osteoarthritis can promote both chondrocyte MMP-13 expression and calcification via chondrocyte extracellular PPi excess

Glutamate signaling in chondrocytes and the potential involvement of NMDA receptors in cell proliferation and inflammatory gene expression T. Piepoli,

The cartilage chondrolytic mechanism of fibronectin fragments involves MAP kinases: comparison of three fragments and native fibronectin L. Ding, Ph.D.,

M. -H. Moon, J. -K. Jeong, Y. -J. Lee, J. -W. Seol, C. J. Jackson, S

The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFβ1 A. van Caam, E. Blaney Davidson, A.

Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus T. Nishida,

AG-041R, a novel indoline-2-one derivative, stimulates chondrogenesis in a bipotent chondroprogenitor cell line CL-1 Hidetomo Kitamura, D.V.M., Ph.D.,

Endoglin differentially regulates TGF-β-induced Smad2/3 and Smad1/5 signalling and its expression correlates with extracellular matrix production and.

Interleukin 6 mediates selected effects of Notch in chondrocytes

Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes G. Zhu, Y. Tang, X. Liang,

TGFβ inhibition during expansion phase increases the chondrogenic re-differentiation capacity of human articular chondrocytes R. Narcisi, L. Signorile,

Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1β signaling in osteoarthritis fibroblast-like.

MicroRNA-558 regulates the expression of cyclooxygenase-2 and IL-1β-induced catabolic effects in human articular chondrocytes S.J. Park, E.J. Cheon,

Fibroblast growth factor-2 induced chondrocyte cluster formation in experimentally wounded articular cartilage is blocked by soluble Jagged-1 I.M. Khan,

GRIP1 enhances estrogen receptor α-dependent extracellular matrix gene expression in chondrogenic cells M. Kato, H. Takaishi, M. Yoda, T. Tohmonda, J.

P.M. van der Kraan, E.N. Blaney Davidson, A. Blom, W.B. van den Berg

Volume 69, Issue 8, Pages (April 2006)

Hyaluronan promotes the chondrocyte response to BMP-7

Volume 16, Issue 4, Pages (October 2009)

Differential regulation of cytokine-induced MMP-1 and MMP-13 expression by p38 kinase inhibitors in human chondrosarcoma cells: potential role of Runx2.

Agonists of peroxisome proliferators-activated receptors (PPAR) α, β/δ or γ reduce transforming growth factor (TGF)-β-induced proteoglycans' production.

Volume 116, Issue 5, Pages (May 1999)

S. Varghese, Ph. D. , P. Theprungsirikul, B. S. , S. Sahani, B. S. , N

CaMKII inhibition in human primary and pluripotent stem cell-derived chondrocytes modulates effects of TGFβ and BMP through SMAD signaling B. Saitta,

Bone Morphogenetic Proteins and articular cartilage

Glucosamine promotes chondrogenic phenotype in both chondrocytes and mesenchymal stem cells and inhibits MMP-13 expression and matrix degradation A.

Unloading results in rapid loss of TGFβ signaling in articular cartilage: role of loading- induced TGFβ signaling in maintenance of articular chondrocyte.

R. Sommaggio, M. Pérez-Cruz, J.L. Brokaw, R. Máñez, C. Costa

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte.

Differential metabolic effects of glucosamine and N-acetylglucosamine in human articular chondrocytes A.R. Shikhman, D.C. Brinson, J. Valbracht, M.K.

Involvement of Gas7 along the ERK1/2 MAP kinase and SOX9 pathway in chondrogenesis of human marrow-derived mesenchymal stem cells Y. Chang, M.D., S.W.N.

S.A. Nazli, R.F. Loeser, S. Chubinskaya, J.S. Willey, R.R. Yammani

Expression of proteinase-activated receptors (PAR)-2 in articular chondrocytes is modulated by IL-1β, TNF-α and TGF-β Y. Xiang, M.D., K. Masuko-Hongo,

The effect of hyaluronan on CD44-mediated survival of normal and hydroxyl radical- damaged chondrocytes P. Brun, Ph.D., S. Panfilo, B.S., D. Daga Gordini,

Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method Dr. K. Masuda, M.D., B.E.

Role of p38 MAPK in Transforming Growth Factor β Stimulation of Collagen Production by Scleroderma and Healthy Dermal Fibroblasts Madoka Sato, Daniel.

A predominantly articular cartilage-associated gene, SCRG1, is induced by glucocorticoid and stimulates chondrogenesis in vitro Kensuke Ochi, M.D., Ph.D.,

P.M. van der Kraan, E.N. Blaney Davidson, A. Blom, W.B. van den Berg

TGF-β is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5- Smad2/3 pathway. Potential role in OA related pain? E.N. Blaney.

Volume 12, Issue 4, Pages (October 2003)

Glutamate signaling in chondrocytes and the potential involvement of NMDA receptors in cell proliferation and inflammatory gene expression T. Piepoli,

Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin Y. Luan, Ph.D., M.D., L. Kong, Ph.D.,

Halofuginone, an Inhibitor of Type-I Collagen Synthesis and Skin Sclerosis, Blocks Transforming-Growth-Factor-β-Mediated Smad3 Activation in Fibroblasts

Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds.

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

JNK Regulates Autocrine Expression of TGF-β1

R. Parekh, M.K. Lorenzo, S.Y. Shin, A. Pozzi, A.L. Clark

Volume 116, Issue 6, Pages (June 1999)

Bone Morphogenetic Proteins and articular cartilage

Gene expression analysis of OA synovium reveals elevation of TGF-ß responsive genes in OA-related fibrosis D.F. Remst, A.B. Blom, E.N. Blaney Davidson,

Chondroitin sulfate modulation of matrix and inflammatory gene expression in IL-1β- stimulated chondrocytes – study in hypoxic alginate bead cultures

Influence of pro-inflammatory (IL-1α, IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-4) cytokines on chondrocyte function A.J Schuerwegh, M.D., Ph.D.,

N. Takahashi, Ph. D. , K. Rieneck, M. D. , P. M. van der Kraan, Ph. D

IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,

Nitric oxide decreases IGF-1 receptor function in vitro; glutathione depletion enhances this effect in vivo R.K. Studer, Ph.D. Osteoarthritis and Cartilage

Adenovirus-mediated gene transfer of glutamine: fructose-6-phosphate amidotransferase antagonizes the effects of interleukin-1β on rat chondrocytes1 1.

R.D. Chavez, G. Coricor, J. Perez, H.-S. Seo, R. Serra

Volume 41, Issue 2, Pages (August 2004)

Chondro-protective effects of low intensity pulsed ultrasound

Presentation transcript:

Adenoviral overexpression of Smad-7 and Smad-6 differentially regulates TGF-β- mediated chondrocyte proliferation and proteoglycan synthesis A Scharstuhl, R Diepens, J Lensen, E Vitters, H van Beuningen, P van der Kraan, W van den Berg Osteoarthritis and Cartilage Volume 11, Issue 11, Pages 773-782 (November 2003) DOI: 10.1016/S1063-4584(03)00165-1

Fig. 1 Smad-6 and Smad-7 effects on TGF-β-induced PAI-1 promoter driven luciferase activity in MLEC. MLEC were transfected with Ad-DL70-3, Ad-Smad-6 or Ad-Smad-7 at MOI 1, 10 and 100 in serum-free DMEM for 2h. Next, medium was replaced with DMEM/10% FCS. After another 5h, 1.56ng/ml TGF-β was added. After 16h incubation luciferase activity was measured via the Bright-Glo luciferase assay reagent. The effect of adenoviral transfection was calculated from TGF-β standards added to mock transfected cells. Standards and samples were tested in quadruplicate and results were averaged and analyzed via ANOVA. Osteoarthritis and Cartilage 2003 11, 773-782DOI: (10.1016/S1063-4584(03)00165-1)

Fig. 2 Immunocytochemical localization of Smad-6 and Smad-7 after adenoviral overexpression. H1 chondrocytes were cultured in Lab-Tek II chamber slides at a concentration of 2×105cells/well. The next day, cells were transfected with MOI 10 with Ad-Luc, Ad-Smad-6 or Ad-Smad-7 or were mock transfected. The next day, I-Smad overexpression was studied using anti-Smad-6 or anti-Smad-7 Ab followed by incubation with the appropriate biotin-labeled secondary Ab. Ad-Smad-6 (A and C) and Ad-Smad-7 (B and D) transfected chondrocytes incubated with anti-Smad-6 or anti-Smad-7 Ab, respectively. Note, the intense staining of cytoplasm and perinuclear staining of the nucleus. Staining is absent from cytoplasmic vesicles and certain regions of the nucleus. (E) Ad-Smad-6 transfected chondrocytes incubated with anti-Smad-7 Ab. Little Smad-7 staining is observed. (F) Ad-Smad-7 transfected chondrocytes incubated with anti-Smad-6 Ab. Few positive cells are stained. Ad-Luc transfected chondrocytes incubated with anti-Smad-6 (G) or anti-Smad-7 Ab (H). Only faint staining is visible. Osteoarthritis and Cartilage 2003 11, 773-782DOI: (10.1016/S1063-4584(03)00165-1)

Fig. 3 Smad-6 and Smad-7 block TGF-β-induced chondrocyte proliferation. H1 chondrocytes were transfected at MOI 10 with Ad-Luc, Ad-Smad-6 or Ad-Smad-7 or were mock transfected in DMEM/F12 without serum. After 2h, medium was changed to DMEM/F12+10% FCS. After another 5h, 10ng/ml TGF-β was added and cells were incubated for 48h. Chondrocyte proliferation was studied via measurement of DNA content via staining with the fluorescent dye Picogreen. The DNA content was taken as an indirect measure for cell number. Fluorescence was measured using the Fluorstar Galaxy. Gain was adjusted to the highest concentration in the standard curve. Results were analyzed via ANOVA. ∗Significantly more proliferation than in non-stimulated (−TGF-β) group. Osteoarthritis and Cartilage 2003 11, 773-782DOI: (10.1016/S1063-4584(03)00165-1)

Fig. 4 Smad-7 inhibits TGF-β-induced PG synthesis in chondrocytes. H1 chondrocytes were transfected at MOI 10 with Ad-Luc, Ad-Smad-6 or Ad-Smad-7 or were mock transfected in DMEM/F12 without serum. After 2h, medium was changed to DMEM/F12+10% FCS. After another 5h, 10ng/ml TGF-β was added and cells were incubated for 48h, pulse-labeled with 35SO42−(20μCi/well), followed by incubation with papain-carrier solution. Subsequently, glycosaminoglycans were precipitated and incorporated 35SO42−was counted using a liquid scintillation counter. From the paired results from 35SO42−incorporation and proliferation the 35SO42−incorporation per cell was calculated. Triplicates were averaged and differences were analyzed via ANOVA. Osteoarthritis and Cartilage 2003 11, 773-782DOI: (10.1016/S1063-4584(03)00165-1)