Interleukin-1 induction of aggrecanase gene expression in human articular chondrocytes is mediated by mitogen-activated protein kinases.

BACKGROUND/AIMS: We investigated the unknown molecular mechanisms of Interleukin-1 (IL-1ß)-induced cartilage aggrecan degeneration by aggrecanase (ADAMTS-A Disintegrin And Metalloproteinase with ThromboSpondin motifs) in human articular chondrocytes, a model mimicking human arthritis. METHODS: Chondrocytes were pretreated with various pharmacological inhibitors and then stimulated with IL-1ß for 24 h. ADAMTS-4 expression or activity was studied by RT-PCR or ELISA and other proteins measured by Western blotting. RESULTS: MAP kinase kinase-specific inhibitor, U0126 inhibited IL-1-induced phosphorylation of ERK1/2 and down-regulated ADAMTS-4 expression and activity. Protein 38 inhibitor, SB203580 down-regulated the phosphorylation of p38 and its target, activating transcription factor-2 (ATF-2), ADAMTS-4 mRNA and activity. C-Jun N-terminal kinase (JNK) inhibitor, SP600125 diminished IL-1-stimulated JNK phosphorylation, ADAMTS-4 mRNA expression and enzyme activity. A c-fos/lipoxygenase pathway inhibitor and antioxidant, nordihydroguaiaretic acid (NDGA) significantly suppressed ADAMTS-4 mRNA induction and activity. Activating protein (AP-1) and nuclear factor kappa B (NF-ĸB) transcription factor inhibitors, curcumin and pyrrolidine dithiocarbamate (PDTC) partially inhibited ADAMTS-4 induction and activity. CONCLUSION: These results suggest partial involvement of ERK-, p38-and JNK-MAPKs as well as AP-1, ATF-2 and NF-ĸB transcription factors in IL-1-induced ADAMTS-4 in chondrocytes. Inhibition of these targets by the specific pharmacological agents could be useful for reducing aggrecanase-driven cartilage resorption in arthritis.

Interleukin-4 antagonizes oncostatin M and transforming growth factor beta-induced responses in articular chondrocytes.

Oncostatin M (OSM) stimulates cartilage degradation in rheumatoid arthritis (RA) by inducing matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS; a disintegrin and metalloproteinase with thrombospondin motif). Transforming growth factor beta (TGF-beta1) induces cartilage repair in joints but in excessive amounts, promotes inflammation. OSM and TGF-beta1 also induce tissue inhibitor of metalloproteinase-3 (TIMP-3), an important natural inhibitor of MMPs, aggrecanases, and tumor necrosis factor alpha converting enzyme (TACE), the principal proteases involved in arthritic inflammation and cartilage degradation. We studied cartilage protective mechanisms of the antiinflammatory cytokine, interleukin-4 (IL-4). IL-4 strongly (MMP-13 and TIMP-3) or minimally (ADAMTS-4) suppressed OSM-induced gene expression in chondrocytes. IL-4 did not affect OSM-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), protein 38 (p38), c-Jun N-terminal kinase (JNK) and Stat1. Lack of additional suppression with their inhibitors suggested that MMP-13, ADAMTS-4, and TIMP-3 inhibition was independent of these mediators. IL-4 also downregulated TGF-beta1-induced TIMP-3 gene expression, Smad2, and JNK phosphorylation. Additional suppression of TIMP-3 RNA by JNK inhibitor suggests JNK implication. The cartilage protective effects of IL-4 in animal models of arthritis may be due to its inhibition of MMPs and ADAMTS-4 expression. However, suppression of TIMP-3 suggests caution for using IL-4 as a cartilage protective therapy.

Signaling pathways implicated in oncostatin M-induced aggrecanase-1 and matrix metalloproteinase-13 expression in human articular chondrocytes.

Molecular mechanisms of oncostatin M (OSM)-stimulated cartilage extracellular matrix catabolism and signaling pathways were investigated in human arthritic chondrocytes. OSM, alone or with Interleukin-1 (IL-1beta), increased glycosaminoglycan release and induced ADAMTS-4 and MMP-13 protein expression in human cartilage explants. OSM dose- and time-dependently increased ADAMTS-4 mRNA and MMP-13 protein expression in human femoral head chondrocytes. Extracellular signal-regulated kinases (ERK1/2)-MAPK pathway inhibitor, U0126, down-regulated ADAMTS-4 and MMP-13 induction by OSM. Janus kinase 2 (JAK2) inhibitor, AG490, suppressed OSM-induced ADAMTS-4 mRNA expression but did not affect MMP-13 levels while JAK3 pharmacological inhibitor and siRNA transfection suppressed both. Parthenolide, a signal transducer and activator of transcription (STAT1 and STAT3) phosphorylation inhibitor, reduced OSM-induced ADAMTS-4 and MMP-13 gene expression and prevented STAT1/3 DNA binding activity. Additionally, OSM-enhanced ADAMTS-4 mRNA and MMP-13 expression was down-regulated by phosphatidylinositol 3-kinase (PI3K) and Akt/PKB inhibitors, LY294002 and NL-71-101. Furthermore, JAK3 inhibition time-dependently down-regulated Akt but not ERK1/2 phosphorylation suggesting that Akt is a downstream target of JAK3. These results suggest that OSM-stimulated ADAMTS-4 and MMP-13 expression is mediated by ERK1/2, JAK3/STAT1/3 and PI3K/Akt and by cross talk between these pathways. The inhibitors of these cascades could block OSM-evoked degeneration of cartilage by ADAMTS-4 and MMP-13.

PPAR-gamma inhibits ANG II-induced cell growth via SHIP2 and 4E-BP1.

The present study evaluated the effects of peroxisome proliferator-activated receptor (PPAR)-gamma activators on ANG II-induced signaling pathways and cell growth. Vascular smooth muscle cells (VSMC) derived from rat mesenteric arteries were treated with ANG II, with/without the AT1 receptor blocker valsartan or the AT2 receptor blocker PD-123319, after pretreatment for 24 h with the PPAR-gamma activators 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or rosiglitazone. Both 15d-PGJ2 and rosiglitazone decreased ANG II-induced DNA synthesis. Rosiglitazone treatment increased nuclear PPAR-gamma expression and activity in VSMC. However, rosiglitazone did not alter expression of PPAR-alpha/beta, ERK 1/2, Akt, or ANG II receptors. 15d-PGJ2 and rosiglitazone decreased ERK 1/2 and Akt peak activity, both of which were induced by ANG II via the AT1 receptor. Rosiglitazone inhibited ANG II-enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), as well as Src homology (SH) 2-containing inositol phosphatase 2 (SHIP2). PPAR-gamma activation reduced ANG II-induced growth associated with inhibition of ERK 1/2, Akt, 4E-BP1, and SHIP2. Modulation of these pathways by PPAR-gamma activators may contribute to regression of vascular remodeling in hypertension.

TGF-beta-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor.

Transforming growth factor (TGF-beta1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 transcription factor in TGF-beta-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-beta time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF-beta-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Sp1-binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF-beta-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-beta-stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (-940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-beta-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Sp1 transcription factor play a pivotal role in the induction of TIMP-3 by TGF-beta in chondrocytes.

SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension.

We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85alpha and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85alpha and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85alpha-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR.

Structure, endothelial function, cell growth, and inflammation in blood vessels of angiotensin II-infused rats: role of peroxisome proliferator-activated receptor-gamma.

BACKGROUND: Pioglitazone and rosiglitazone, thiazolidinedione peroxisome proliferator-activated receptor-gamma (PPARgamma) activators, reduce blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that pioglitazone or rosiglitazone would prevent BP elevation and vascular dysfunction in angiotensin (Ang) II-infused rats by direct vascular effects. METHODS AND RESULTS: Sprague-Dawley rats received Ang II (120 ng x kg(-1) x min(-1) SC) with or without pioglitazone (10 mg x kg(-1) x d(-1)) or rosiglitazone (5 mg x kg(-1) x d(-1)) for 7 days. Systolic BP, elevated in Ang II-infused rats (176+/-5 mm Hg) versus controls (109+/-2 mm Hg, P<0.01), was reduced by pioglitazone (134+/-2 mm Hg) or rosiglitazone (123+/-2 mm Hg). In mesenteric small arteries studied in a pressurized myograph, media/lumen ratio was increased (P<0.05) and acetylcholine-induced relaxation impaired in Ang II-infused rats (P<0.05); both were normalized by the thiazolidinediones. In Ang II-infused rats, vascular DNA synthesis (by 3H-thymidine incorporation); expression of cell cycle proteins cyclin D1 and cdk4, angiotensin II type 1 receptors, vascular cell adhesion molecule-1, and platelet and endothelial cell adhesion molecule; and nuclear factor-kappaB activity were increased. These changes were abrogated by pioglitazone or rosiglitazone. CONCLUSIONS: Thiazolidinedione PPAR-gamma activators attenuated the development of hypertension, corrected structural abnormalities, normalized cell growth, and improved endothelial dysfunction induced by Ang II and prevented upregulation of angiotensin II type 1 receptors, cell cycle proteins, and proinflammatory mediators. Thiazolidinediones may be useful in the prevention and/or treatment of hypertension, particularly when it is associated with insulin resistance or diabetes mellitus.

Effect of AT(1) receptor blockade on cardiac apoptosis in angiotensin II-induced hypertension.

Angiotensin II (ANG II) via AT(1) receptors induces apoptosis in cardiomyocytes in vitro. We tested the hypothesis that in vivo AT(1) receptor stimulation is accompanied by cardiac apoptosis and attempted to elucidate the molecular mechanisms involved in the death signaling pathway. Male Sprague-Dawley rats received ANG II (120 ng x kg(-1) x min(-1) sc) for 7 days with or without the AT(1) receptor antagonist losartan (10 mg x kg(-1) x day(-1) orally). Cardiac function was assessed by echocardiography. Apoptosis in the heart was detected and quantified by in situ TdT-mediated dUTP nick-end labeling (TUNEL) and radiolabeled DNA laddering. Expression of bax, bcl-2, caspase 3, and AT(1) and AT(2) receptors was examined by Western blot analysis. Activity of caspase 3 was also measured by a fluorometric immunosorbent enzyme assay. Tail cuff systolic blood pressure was elevated (P < 0.01, n = 6) in ANG II-infused rats (173 +/- 3 mmHg) versus controls (111 +/- 2 mmHg) and reduced by losartan (134 +/- 4 mmHg). Cardiac function was essentially unchanged in ANG II-infused rats. Increased internucleosomal DNA cleavage by TUNEL assay and radiolabeled DNA laddering showed results compatible with enhanced cardiomyocyte apoptosis in the hearts of ANG-II infused rats. The bax-to-bcl-2 ratio, expression of the active form of caspase 3 (17 kDa), and activity of caspase 3 in the hearts of the ANG II group increased more than twofold above controls. Protein expression of AT(1) and AT(2) receptors was significantly increased in ANG II-infused rats compared with control rats. Losartan-treated ANG II-infused rats exhibited normalized apoptosis, bax, caspase 3 activity, and AT(1) receptors. ANG II stimulation of AT(1) receptors in the heart in vivo is associated with an increased rate of apoptosis without major hemodynamic consequences. Bax and caspase 3 are involved in the apoptotic signaling pathway in this experimental paradigm.