Nanoemulsions as novel nanocarrieres for drug delivery across the skin: In-vitro, in-vivo evaluation of miconazole nanoemulsions for treatment of Candidiasis albicans.

In the current research, attempt is made to fabricate a nanoemulsion (NE) containing an antifungal agent. The prepared formulation has been expected to enhance skin penetration. It is also studied for in vitro drug release and toxicity assessment. Spontaneous titration method was used for preparation of NE. Prepared NE were characterized for their charge, size, morphology, rheological behaviour, drug release profile, skin permeability. The drug permeation and skin irritation were investigated. The in vitro antifungal activity was inspected using the well agar diffusion method. Miconazole NE showed good penetration in the skin as compared to marketed products. SEM showed semispherical shapes of the droplets. Zeta potential and zeta sizer showed that size was in nano ranges having positive charge.

Cartilage regeneration by selected chondrogenic clonal mesenchymal stem cells in the collagenase-induced monkey osteoarthritis model.

Osteoarthritis (OA) is the most common form of arthritis, in which cartilage is irreversibly degraded, causing severe pain and disability. Current therapeutic strategies cannot repair damaged cartilage. We evaluated the repair potential of selected chondrogenic clonal MSCs (sC-MSCs) by delivering them into the injured cartilage site in a collagenase-induced OA model in Cynomolgus monkeys. In vitro characterization showed that the isolated monkey sC-MSCs and polyclonal MSCs (P-MSCs) expressed mesenchymal stem cell markers and could differentiate into chondrocytes. The articular cartilage lesions in animals were treated with normal saline (NS), autologous P-MSCs and sC-MSCs, respectively, by direct delivery. The clinical parameters, radiographic images, histological and immunohistochemical examinations at weeks 8, 16 and 24 post-treatment demonstrated that the abrasions of articular cartilage were significantly improved and repaired by MSC-based treatment, particularly in the sC-MSC-treated group, which displayed consistently higher histological scores than those of other groups. In summary, treatment with sC-MSCs can effectively improve the healing of cartilage lesions in the Cynomolgus monkey collagenase-induced OA model. Due to the genetic proximity of monkey and human, the therapeutic strategy presented in this study will have broad applications in clinical practice.

Reconstruction of cartilage with clonal mesenchymal stem cell-acellular dermal matrix in cartilage defect model in nonhuman primates.

OBJECTIVE: Articular cartilage defects are commonly associated with trauma, inflammation and osteoarthritis. Mesenchymal stem cell (MSC)-based therapy is a promising novel approach for repairing articular cartilage. Direct intra-articular injection of uncommitted MSCs does not regenerate high-quality cartilage. This study explored utilization of a new three-dimensional, selected chondrogenic clonal MSC-loaded monkey acellular dermal matrix (MSC-ADM) scaffold to repair damaged cartilage in an experimental model of knee joint cartilage defect in Cynomolgus monkeys. METHODS: MSCs were characterized for cell size, cell yield, phenotypes, proliferation and chondrogenic differentiation capacity. Chondrogenic differentiation assays were performed at different MSC passages by sulfated glycosaminoglycans (sGAG), collagen, and fluorescence activated cell sorter (FACS) analysis. Selected chondrogenic clonal MSCs were seeded onto ADM scaffold with the sandwich model and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy. Cartilage defects were treated with normal saline, clonal MSCs and clonal MSC-ADM grafts, respectively. The clinical parameters, and histological and immunohistochemical examinations were evaluated at weeks 8, 16, 24 post-treatment, respectively. RESULTS: Polyclonal and clonal MSCs could differentiate into the chondrogenic lineage after stimulation with suitable chondrogenic factors. They expressed mesenchymal markers and were negative for hematopoietic markers. Articular cartilage defects were considerably improved and repaired by selected chondrogenic clonal MSC-based treatment, particularly, in MSC-ADM-treated group. The histological scores in MSC-ADM-treated group were consistently higher than those of other groups. CONCLUSION: Our results suggest that selected chondrogenic clonal MSC-loaded ADM grafts could improve the cartilage lesions in Cynomolgus monkey model, which may be applicable for repairing similar human cartilage defects.

Role of Sp1 transcription factor in Interleukin-1-induced ADAMTS-4 (aggrecanase-1) gene expression in human articular chondrocytes.

Proinflammatory cytokines such as interleukin-1 beta (IL-1ß) stimulate cartilage extracellular matrix aggrecan degradation by aggrecanases or ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) during the pathogenesis of arthritis. Human aggrecanase-1 (ADAMTS-4) gene promoter contains at least one specificity protein-1 (Sp1)-transcription factor-binding site. We investigated the previously unknown role of Sp1 in the regulation of ADAMTS-4 gene expression in human articular chondrocytes. Mithramycin and WP631, the specific inhibitors of guanine cytosine (GC)-rich Sp1 DNA binding, partially suppressed IL-1-induced ADAMTS-4 expression and activity. Genetic inhibition of Sp1 by antisense oligonucleotide or by small interfering RNA (siRNA)-mediated Sp1 knockdown partially inhibited ADAMTS-4 induction by IL-1. Sense oligonucleotide and negative control siRNA had no effect. In contrast, cytomegalovirus promoter-driven Sp1 overexpression further enhanced IL-1-induced ADAMTS-4 expression and activity. Constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was not affected by any of the agents. These results provide pharmacological and genetic evidence for the importance of Sp1 in ADAMTS-4 gene regulation by IL-1. Thus, Sp1 could be potentially targeted to reduce arthritis-associated cartilage aggrecan loss.

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.

Molecular targets of natural health products in arthritis.

Patients with rheumatoid arthritis (RA) and osteoarthritis (OA) consume 'natural health products' (NHPs) whose therapeutic efficacy, toxicity and mechanisms of action are poorly understood. In a previous issue of Arthritis Research and Therapy, Haqqi and colleagues characterized IL-1-activated mitogen-activated protein kinase kinase 3 (MKK3) and p38-mitogen-activated protein kinase (MAPK) isoforms in human OA chondrocytes. The cartilageprotective mechanisms of pomegranate extract involve diminishing MKK3-activated p38α, JNK, NF-κB and Runx2 pathways, which regulate inflammatory proteins and cartilage-destroying proteases. Epigallocatechin- 3-gallate, resveratrol, curcumin and other NHP active ingredients suppress multiple inflammatory and catabolic molecular mediators of arthritis. Non-toxicity, reduced severity and incidence of arthritis in animal models warrant testing NHP active ingredients for preventing human OA and RA.

Involvement of H-Ras and reactive oxygen species in proinflammatory cytokine-induced matrix metalloproteinase-13 expression in human articular chondrocytes.

Proinflammatory cytokines such as interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) enhance degradation of cartilage-specific, type II collagen by matrix metalloproteinase-13 (MMP-13). We investigated the previously unknown role of H-Ras and reactive oxygen species (ROS) in the cytokine induction of MMP-13 gene expression in human articular chondrocytes by using pharmacological inhibitors, RNA interference (RNAi) and antioxidants. Manumycin A, an inhibitor of H-Ras farnesylation by farnesyltransferase, suppressed IL-1ß- and TNF-α-induced MMP-13 mRNA and protein expression. Small interfering RNA (siRNA)-mediated H-Ras silencing down-regulated MMP-13 mRNA and protein induction by IL-1ß and TNF-α. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) inhibitor, diphenyleneiodonium (DPI) suppressed cytokine-induced MMP-13 expression and superoxide production. Apocynin, another NOX inhibitor, also diminished MMP-13 induction. Deoxyglucose an antimetabolite of glucose metabolism reduced MMP-13 increase. Role of NOX-mediated ROS production was reaffirmed by the observation that the antioxidants, trolox, nordihydroguaiaretic acid (NDGA), quercetin and resveratrol downregulated cytokine-induced MMP-13 mRNA and protein expression. These results provide strong pharmacological and genetic evidence for the implication of H-Ras and NADPH oxidase-generated superoxide production in MMP-13 gene regulation by IL-1ß and TNF-α. These proteins could be potentially targeted for therapeutic inhibition of MMP-13-driven cartilage erosion by using H-Ras and NOX inhibitors and antioxidants.

Enhanced expression of tissue inhibitor of metalloproteinases-4 gene in human osteoarthritic synovial membranes and its differential regulation by cytokines in chondrocytes.

OBJECTIVE: Tissue inhibitors of metalloproteinases (TIMPs) are multi-functional proteins with matrix metalloproteinases-inhibiting activities. We studied expression of anti-inflammatory, TIMP-4 gene in human joint tissues and its regulation by arthritis-associated cytokines. RESULTS: TIMP-4 RNA expression originating from synovial fibroblasts was significantly (2.4 fold; p<0.001) elevated in 8 osteoarthritic (OA) versus 7 non-arthritic synovial membranes. Non-arthritic and OA femoral head and knee chondrocytes displayed substantial but variably constitutive expression of the TIMP-4 mRNA. In articular chondrocytes, transforming growth factor beta (TGF-ß1) and oncostatin M (OSM) upregulated TIMP-4 RNA and protein expression while interleukin-1 (IL-1ß) and tumor necrosis factor alpha (TNF-α) did not, suggesting differential regulation by arthritis-associated cytokines. Interleukin 17 (IL-17) mildly induced TIMP-4 mRNA. TGF-ß1 induction of TIMP-4 expression was partly inhibited by ERK pathway and Sp1 transcription factor inhibitors. CONCLUSION: Enhanced TIMP-4 gene expression in OA synovial membranes and cartilage may be due to induction by TGF-ß1, OSM and IL-17, suggesting its pathophysiological role in tissue remodeling in human joints. TGF-ß1 induction of TIMP-4 expression is mediated partly by ERK pathway and Sp1 transcription factor.

Adaptor proteins and Ras synergistically regulate IL-1-induced ADAMTS-4 expression in human chondrocytes.

Aggrecanases (a disintegrin [corrected] and metalloproteinase with thrombospondin motif, ADAMTSs) are principal proteases involved in cartilage extracellular matrix aggrecan degradation. The role and relative contribution of MyD88, IRAK1, and TRAF6 adaptor proteins in IL-1beta regulation of aggrecanase-1 (ADAMTS-4) is unknown. By small interfering RNAs-mediated knockdown, we show that IL-1beta-induced up-regulation of ADAMTS-4 in chondrocytes requires MyD88, IRAK1, and TRAF6 adaptor proteins. However, partial inhibition of ADAMTS-4 induction by their knockdown suggested the involvement of additional signaling proteins. Because IL-1beta is also known to induce reactive oxygen species (ROS) through Ras-mediated activation of NADPH oxidase, we investigated the implication of Ras in ADAMTS-4 regulation. Ras knockdown, or inhibition of ROS by antioxidants along with the ablation of MyD88, IRAK1, or TRAF6 more potently down-regulated IL-1beta-induced ADAMTS-4. In addition, IL-1beta-induced phosphorylation of downstream effectors, IkappaB kinase alphabeta, IkappaBalpha, and activation of transcription factor NF-kappaB was significantly reduced in the MyD88-, IRAK1-, TRAF6-, or Ras-deficient cells. The combined knockdown of Ras and individual adaptor proteins strongly blocked the activation of IKKalphabeta, IkappaBalpha, and NF-kappaB. These findings suggest that Ras, ROS along with MyD88, IRAK1, or TRAF6 synergistically mediate ADAMTS-4 regulation by IL1-beta. Thus, complete ablation of ADAMTS-4 induction could be achieved by combined inhibition of Ras and individual adaptor proteins, which may be of therapeutic value in arthritis.

High-efficiency transfection of nucleic acids by the modified calcium phosphate precipitation method in chondrocytes.

Extracellular matrix (ECM)-rich cartilage-derived chondrocytes are difficult to transfect with DNA/RNA. We modified the classical calcium phosphate transfection method by detaching adherent chondrocytes with trypsin and resuspending in CaPo4-nucleic acid precipitate followed by readherence for 24h. Due to the absence of ECM, chondrocytes could be transfected with 80% efficiency. Potent gene silencing with several antisense oligonucleotides and small interfering RNAs and strong promoter-luciferase activity could be achieved. This approach is applicable to any adherent or suspended cells and has utility in gene knockdown, ectopic overexpression, promoter regulation studies, and gene delivery in tissue engineering and gene therapy applications.

Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.

Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.

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.

MyD88, IRAK1 and TRAF6 knockdown in human chondrocytes inhibits interleukin-1-induced matrix metalloproteinase-13 gene expression and promoter activity by impairing MAP kinase activation.

Interleukin-1 (IL-1) is the major prototypic proinflammatory cytokine that stimulates degradation of cartilage in arthritis by inducing prominent collagen II-degrading matrix metalloproteinase-13 (MMP-13). Nothing is known about the involvement of adaptor proteins, MyD88, IRAK1 and TRAF6 in MMP-13 regulation. Here we investigated for the first time the role of these proteins in IL-1-regulated MMP-13 expression in chondrocytes. MyD88 homodimerization inhibitory peptide diminished the expression of MMP-13 gene, promoter activity, phosphorylation of mitogen-activated protein kinases (MAPKs), c-Jun and activating protein 1 (AP-1) activity. Knockdown of MyD88, IRAK1 and TRAF6 by RNA interference (RNAi) drastically down-regulated the expression of IL-1-induced MMP-13 mRNA and protein levels and MMP-13 promoter-driven luciferase activity. Non-specific control siRNA had no effect. Mechanisms of MMP-13 inhibition involved reduced phosphorylation of ERK, p38, JNK and c-Jun as well as AP-1 transcription factor binding activity. The genetic evidence presented here demonstrates that MyD88, IRAK1 and TRAF6 proteins are crucial early mediators for the IL-1-induced MMP-13 regulation through MAPK pathways and AP-1 activity. These proteins could constitute important therapeutic targets for arthritis-associated cartilage loss by MMP-13.

Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-beta in human chondrocytes.

Transforming growth factor beta (TGF-beta1) induces cartilage extracellular matrix synthesis and tissue inhibitor of metalloproteinases-3 (TIMP-3), an important natural inhibitor of matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme, which are implicated in cartilage degradation and joint inflammation. This study tested the hypothesis that Akt/protein kinase B signaling pathway could mediate TGF-beta1 induction of TIMP-3 in human articular chondrocytes. TGF-beta activated phosphorylation of Akt in a delayed and sustained fashion that correlated with TIMP-3 mRNA induction. Phosphatidylinositol kinase (PI3K) inhibitors, Wortmannin and LY294002 and Akt inhibitor (NL-71-101) significantly inhibited TGF-beta-induced Akt phosphorylation, TIMP-3 expression, TIMP-3 promoter (-940 to +376)-driven luciferase activity and Sp1 transcription factor binding. PI3K p85, Akt and Sp1 small interfering RNA (siRNA)-driven knockdown of the respective gene products significantly suppressed TGF-beta-induced TIMP-3 gene expression. TGF-beta-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-beta induces TIMP-3 gene expression in human chondrocytes partly through PI3K/Akt pathway and Sp1 transcription factor and by translational mechanisms via mammalian target of rapamycin (mTOR) signaling. TGF-beta induction of pro-survival Akt cascade and TIMP-3 may be related to strengthening of cartilage extracellular matrix, increased chondrocyte viability and maintenance of joint tissue integrity.

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.

Mithramycin downregulates proinflammatory cytokine-induced matrix metalloproteinase gene expression in articular chondrocytes.

Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-alpha) are the main proinflammatory cytokines implicated in cartilage breakdown by matrix metalloproteinase (MMPs) in arthritic joints. We studied the impact of an anti-neoplastic antibiotic, mithramycin, on the induction of MMPs in chondrocytes. MMP-3 and MMP-13 gene expression induced by IL-1beta, TNF-alpha and IL-17 was downregulated by mithramycin in human chondrosarcoma SW1353 cells and in primary human and bovine femoral head chondrocytes. Constitutive and IL-1-stimulated MMP-13 levels in bovine and human cartilage explants were also suppressed. Mithramycin did not significantly affect the phosphorylation of the mitogen-activated protein kinases, extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase. Despite effective inhibition of MMP expression by mithramycin and its potential to reduce cartilage degeneration, the agent might work through multiple unidentified mechanisms.

Triptolide suppresses proinflammatory cytokine-induced matrix metalloproteinase and aggrecanase-1 gene expression in chondrocytes.

A hallmark of rheumatoid- and osteoarthritis (OA) is proinflammatory cytokine-induced degeneration of cartilage collagen and aggrecan by matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS). Effects of the Chinese herb, Tripterygium wilfordii Hook F (TWHF), on cartilage and its anti-arthritic mechanisms are poorly understood. This study investigated the impact of a purified derivative of TWHF, PG490 (triptolide), on cytokine-stimulated expression of the major cartilage damaging proteases, MMP-3, MMP-13, and ADAMTS4. PG490 inhibited cytokine-induced MMP-3, MMP-13 gene expression in primary human OA chondrocytes, bovine chondrocytes, SW1353 cells, and human synovial fibroblasts. Triptolide was effective at low doses and blocked the induction of MMP-13 by IL-1 in human and bovine cartilage explants. TWHF extract and PG490 also suppressed IL-1-, IL-17-, and TNF-alpha-induced expression of ADAMTS-4 in bovine chondrocytes. Thus, PG490 could protect cartilage from MMP- and aggrecanase-driven breakdown. The immunosuppressive, cartilage protective, and anti-inflammatory properties could make PG490 potentially a new therapeutic agent for arthritis.

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.

Transforming growth factor Beta1 induction of tissue inhibitor of metalloproteinases 3 in articular chondrocytes is mediated by reactive oxygen species.

Transforming growth factor beta1 (TGF-beta1) stimulates cartilage extracellular matrix synthesis but, in excess, evokes synovial inflammation, hyperplasia, and osteophyte formation in arthritic joints. TGF-beta1 induces tissue inhibitor of metalloproteinases 3 (TIMP-3), an inhibitor of cartilage-damaging matrix metalloproteianases and aggrecanases. We investigated the role of reactive oxygen species (ROS) in TIMP-3 induction by TGF-beta1. In primary human and bovine chondrocytes, ROS scavenger and antioxidant N-acetylcysteine (NAC) inhibited TGF-beta1-induced TIMP-3 mRNA and protein increases. Ebselen and ascorbate also reduced this induction. TGF-beta1 time-dependently induced ROS production that was suppressed by NAC. Hydrogen peroxide, a ROS, induced TIMP-3 RNA. The TIMP-3 increase induced by TGF-beta1 was partly Smad2-dependent. TGF-beta1-stimulated Smad2 phosphorylation was inhibited by NAC. Reduced glutathione and L-cysteine also blocked Smad2 and TIMP-3 induction by TGF-beta1, whereas a nonthiol, N-acetylalanine, did not. Smad2 was not activated by H2O2. Smad2 phosphorylation was independent, and TIMP-3 expression was dependent, on new protein synthesis. TGF-beta-stimulated ERK and JNK phosphorylation was also inhibited by NAC. However, inhibitory actions of NAC were not mediated by ERK activation. Thus, ROS mediate TGF-beta1-induced TIMP-3 gene expression. Blocking TGF-beta1-induced gene expression by modulating cellular redox status with thiols can be potentially beneficial for treating arthritic and other disorders caused by excessive TGF-beta1.