Muhammad Zafarullah

Molecular mechanisms of N-acetylcysteine actions.

Abstract. Oxidative stress generated by an imbalance between reactive oxygen species (ROS) and antioxidants contributes to the pathogenesis of arthritis, cancer, cardiovascular, liver and respiratory diseases. Proinflammatory cytokines and growth factors stimulate ROS production as signaling mediators. Antioxidants such as N-acetylcysteine (NAC) have been used as tools for investigating the role of ROS in numerous biological and pathological processes. NAC inhibits activation of c-Jun N-terminal kinase, p38 MAP kinase and redox-sensitive activating protein-1 and nuclear factor kappa B transcription factor activities regulating expression of numerous genes. NAC can also prevent apoptosis and promote cell survival by activating extracellular signal-regulated kinase pathway, a concept useful for treating certain degenerative diseases. NAC directly modifies the activity of several proteins by its reducing activity. Despite its nonspecificity, ability to modify DNA and multiple molecular modes of action, NAC has therapeutic value for reducing endothelial dysfunction, inflammation, fibrosis, invasion, cartilage erosion, acetaminophen detoxification and transplant prolongation.

Inhibition of interleukin-1-stimulated MAP kinases, activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) transcription factors down-regulates matrix metalloproteinase gene expression in articular chondrocytes

Interleukin-1 (IL-1), the main cytokine instigator of cartilage degeneration in arthritis, induces matrix metalloproteinase-3 (MMP-3) and MMP-13 RNA and protein in chondrocytes. The molecular mechanisms of this induction were investigated with specific inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and activating protein (AP-1) and nuclear factor kappa B (NF-kappa B) transcription factors. IL-1 rapidly induced the activation of extracellular-signal regulated kinase (ERK), protein 38 (p38) and c-Jun N-terminal kinase (JNK) MAPKs in the first-passage human femoral head OA chondrocytes. The ERK-MAPK pathway inhibitor, PD98059, attained 46-53% (MMP-3) and 59-66% (MMP-13) inhibition of RNA induction in human OA and 47-52% (MMP-3) and 69-73% (MMP-13) inhibition in bovine chondrocytes. U0126 conferred 37-77% (MMP-3) and 43-73% (MMP-13) suppression in human and 77-100% (MMP-3) and 96-100% (MMP-13) in bovine chondrocytes. P38 and JNK inhibitor, SB203580 caused 35-37% reduction of MMP-3 and MMP-13 RNA in human and 36-46% (MMP-3) and 60-88% (MMP-13) in bovine chondrocytes. Inhibitor of JNK, AP-1 and NF-kappa B, curcumin, achieved 48-99% suppression of MMP-3 and 45-97% of MMP-13 in human and 8-100% (MMP-3) and 32-100% (MMP-13) in bovine chondrocytes. NF-kappaB inhibitor, pyrrolidine dithiocarbamate yielded 83-84% reduction of MMP-3 and 38-55% for MMP-13 in human chondrocytes. In bovine chondrocytes, the induction decreased by 54-64% for MMP-3 and 74-93% for MMP-13 RNA. These results suggest the involvement of MAPKs, AP-1 and NF-kappa B transcription factors in the IL-1 induction of MMPs in chondrocytes. Inhibition of IL-1 signal transduction by these agents could be useful for reducing cartilage resorption by MMPs in arthritis.

Induction of matrix metalloproteinase-13 gene expression by TNF-α is mediated by MAP kinases, AP-1, and NF-κB transcription factors in articular chondrocytes

Abstract Tumor necrosis factor alpha (TNF-α), a major proinflammatory cytokine, induces arthritic joint inflammation and resorption of cartilage by matrix metalloproteinase-13 (MMP-13). RNA for MMP-13 is increased in human arthritic femoral cartilage. Mechanisms of this induction were investigated by pretreating primary human osteoarthritic (OA) femoral head chondrocytes or chondrosarcoma cells with the potential inhibitors of TNF-α signal transduction and downstream target transcription factors followed by stimulation with TNF-α and analysis of MMP-13 RNA/protein. TNF-α rapidly activated phosphorylation of extracellular signal-regulated kinases (ERKs), p38, and c-jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in human chondrocytes. Inhibitors of ERK (U0126, PD98059, and ERK1/2 antisense phosphorothioate oligonucleotide), JNK (SB203580, SP600125, and curcumin), and p38 (SB203580 and SB202190) pathways down-regulated the TNF-stimulated expression of MMP-13. Inhibitors of the transcription factors AP-1 (nordihydroguaiaretic acid, NDGA) and NF-κB (curcumin, proteasome inhibitors, and Bay-11-7085) suppressed TNF-α-induced MMP-13 expression in primary chondrocytes and SW1353 cells. These results suggest that induction of the MMP-13 gene by TNF-α is mediated by ERK, p38, and JNK MAP kinases as well as AP-1 and NF-κB transcription factors. Blockade of TNF-α signaling and its target transcription factors by the approaches tested here may be beneficial for reducing cartilage breakdown by MMP-13 in arthritis.

TGF-β-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‐β1) 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‐β‐induced TIMP‐3 gene in chondrocytes and chondrosarcoma cells. TGF‐β time‐dependently induced a sustained phosphorylation of ERK‐MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF‐β‐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‐β‐inducible TIMP‐3 expression. Indeed, mithramycin blocked TGF‐β‐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‐β‐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‐β in chondrocytes.

Expression of the tissue inhibitor of metalloproteinases (TIMP) gene family in normal and osteoarthritic joints

Abstract The pathophysiological and biological significance of tissue inhibitor of the metalloproteinases-3 (TIMP-3) gene compared to other TIMPs was investigated in osteoarthritic (OA) human and normal bovine joint tissues. Human OA synovial fibroblasts in culture constitutively expressed TIMP mRNAs. TIMP-3, TIMP-1 and gelatinase A mRNAs were elevated in most human OA synovia over controls, while TIMP-2 expression was similar. TIMP-3 and TIMP-1 mRNAs present in bovine cartilage were inducible by serum factors. Transforming growth factor beta (TGF-β1) induced TIMP-3 RNA and protein in human OA and normal bovine chondrocytes. TIMP mRNAs were low (TIMP-1) or undetectable in human fetal chondrocytes but were expressed at all other ages. Thus, the two main joint tissues, synovial membranes and cartilage, express TIMP genes. Due to their matrix protecting activities, the presence of multiple TIMPs may be beneficial for normal joints, while increased TIMP-3 and TIMP-1 expression in arthritic joints may be associated with pathological remodeling.

Up‐regulation of tissue inhibitor of metalloproteinases‐3 gene expression by TGF‐β in articular chondrocytes is mediated by serine/threonine and tyrosine kinases

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn‐over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF‐β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP‐3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF‐β‐mediated induction of TIMP‐3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12‐myristate 13‐acetate (PMA), a PKC activator, increased TIMP‐3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP‐3 gene to TGF‐β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF‐β induction of TIMP‐3. H7 and genistein also suppressed TGF‐β‐induced TIMP‐3 protein expression. These results suggest that TGF‐β signaling for TIMP‐3 gene induction involves H7‐sensitive serine/threonine kinase as well as herbimycin A‐ and genistein‐sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998.

Tissue inhibitor of metalloproteinases-4 (TIMP-4) gene expression is increased in human osteoarthritic femoral head cartilage

Tissue inhibitor of metalloproteinases‐4 (TIMP‐4), the newest member of the TIMP family, blocks the activities of several matrix metalloproteinases (MMPs) implicated in the arthritic cartilage erosion. By utilizing semi‐quantitative RT‐PCR, immunoblotting, and immunohistochemistry, we investigated whether the TIMP‐4 gene is expressed in human non‐arthritic and osteoarthritic (OA) cartilage. Directly analyzed femoral head cartilage showed TIMP‐4 RNA expression in 2 of 9 non‐arthritic and 12 of 14 OA patients. Femoral head cartilage from 6 of 9 OA patients had elevated TIMP‐4 protein compared to the low‐level expression in 3 of 8 non‐arthritic controls. In most patients, there was correlation between TIMP‐4 RNA and protein expression. TIMP‐4 protein was also detected immunohistochemically in the upper zone of OA cartilage. The widespread TIMP‐4 RNA and protein expression and augmentation in femoral OA cartilage suggests its important role in joint tissue remodeling and pathogenesis of OA. Increased TIMP levels in arthritic cartilage may not be a sufficiently effective defense against cartilage resorption by excessive multiple MMPs and aggrecanases. J. Cell. Biochem. 85: 295–303, 2002.

Expression of c-fos, c-jun, jun-B, metallothionein and metalloproteinase genes in human chondrocyte

Normal and osteoarthritic (OA) human articular cartilage chondrocytes, released enzymatically in the presence of 0.5% fetal calf serum, display constitutive expression of early response activating protein (AP‐1) genes; c‐fos, c‐jun and jun‐B. Among the late AP‐1 responsive genes, total matallothionein (MT) and stromelysin mRNAs were expressed at high levels in both normal and OA chondrocytes, while collagenase and hMT‐IIA mRNA levels were elevated only in OA individuals. Despite the common AP‐1 sequences present in their promoter regions, the three late genes were differentially expressed.

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

Interleukin-1 (IL-1), IL-17 and tumor necrosis factor alpha (TNF-α) 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-1β, TNF-α 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.

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.