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1.  Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis 
Annals of the Rheumatic Diseases  1999;58(11):691-697.
OBJECTIVE—To define the pattern of mRNA expression of all human matrix metalloproteinases (MMPs) described to date in rheumatoid arthritis (RA) and traumatic synovial membrane, in order to differentiate between a physiological tissue remodelling pattern and that associated with inflammatory tissue destruction.
METHODS—Analysis of SwissProt protein and EMBL/GenBank nucleotide sequence banks, protein sequence alignment, reverse transcriptase-polymerase chain reaction and nucleotide sequencing were used.
RESULTS—MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-11 (stromelysin-3) and MMP-19 were constitutively expressed. MMP-1 (fibroblast type collagenase), MMP-9 (gelatinase B) and MMP-14 (MT1-MMP) were expressed in all RA, but only in 55-80% of trauma samples. MMP-13 (collagenase-3) and MMP-15 (MT2-MMP) were expressed exclusively in RA (80-90% of the samples). MMP-20 (enamelysin) was absent and MMP-8 (collagenase-2) was rarely found in RA or trauma. All other MMPs (-7, -10, -12, -16, -17) had an intermediate pattern of expression.
CONCLUSIONS—Some MMPs without interstitial collagenase activity seem to have a constitutive pattern of expression and probably participate in physiological synovial tissue remodelling. Some MMPs are exclusively associated to RA synovitis, for example, MMP-13, which preferentially degrades type II collagen and aggrecan, and MMP-15, which activates proMMP-2 and proMMP-13 and is involved in tumour necrosis factor α processing. This clear cut rheumatoid/inflammatory MMP profile, more complex than has been previously appreciated, may facilitate inflammatory tissue destruction in RA.


PMCID: PMC1752794  PMID: 10531073
2.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
Introduction:
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
Aims:
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Methods:
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
Results:
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Discussion:
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
3.  TNF-α stimulates activation of pro-MMP2 in human skin through NF-κB mediated induction of MT1-MMP 
Journal of cell science  2001;114(Pt 1):131-139.
SUMMARY
Tumor necrosis factor-alpha (TNF-α) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-α are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with proinflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-α stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing.
To investigate this observation at the cellular and molecular levels, we examined TNF-α mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-α substantially promoted activation of pro-MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-α individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro-MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-α significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-α and collagen activate the NF-κB pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-κB is essential to induce MT1-MMP expression in fibroblasts after TNF-α exposure. First, SN50, a peptide inhibitor for NF-κB nuclear translocation, simultaneously blocked the TNF-α and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-α induced IκB to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-κB activation. Lastly, a consensus binding site for p65 NF-κB (TGGAGCTTCC) was found in the 5′-flanking region of human mt1-mmp gene.
Based on these results and previous reports, we propose a model to explain TNF-α activation of MMP-2 in human skin. Activation of NF-κB signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-α may affect matrix remodeling during wound healing and other physiological and pathological processes.
PMCID: PMC2435089  PMID: 11112697
TNF-alpha; MMP-2; MT1-MMP; NF-kappa B; Collagen; Type IV collagenase; Gelatinase A; Inflammatory cytokine; Wound healing; Skin
4.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
5.  Induction of multiple matrix metalloproteinases in human dermal and synovial fibroblasts by Staphylococcus aureus: implications in the pathogenesis of septic arthritis and other soft tissue infections 
Infections of body tissue by Staphylococcus aureus are quickly followed by degradation of connective tissue. Patients with rheumatoid arthritis are more prone to S. aureus-mediated septic arthritis. Various types of collagen form the major structural matrix of different connective tissues of the body. These different collagens are degraded by specific matrix metalloproteinases (MMPs) produced by fibroblasts, other connective tissue cells, and inflammatory cells that are induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF). To determine the host's contribution in the joint destruction of S. aureus-mediated septic arthritis, we analyzed the MMP expression profile in human dermal and synovial fibroblasts upon exposure to culture supernatant and whole cell lysates of S. aureus. Human dermal and synovial fibroblasts treated with cell lysate and filtered culture supernatants had significantly enhanced expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, and MMP-11 compared with the untreated controls (p < 0.05). In the S. aureus culture supernatant, the MMP induction activity was identified to be within the molecular-weight range of 30 to >50 kDa. The MMP expression profile was similar in fibroblasts exposed to a combination of IL-1/TNF. mRNA levels of several genes of the mitogen-activated protein kinase (MAPK) signal transduction pathway were significantly elevated in fibroblasts treated with S. aureus cell lysate and culture supernatant. Also, tyrosine phosphorylation was significantly higher in fibroblasts treated with S. aureus components. Tyrosine phosphorylation and MAPK gene expression patterns were similar in fibroblasts treated with a combination of IL-1/TNF and S. aureus. Mutants lacking staphylococcal accessory regulator (Sar) and accessory gene regulator (Agr), which cause significantly less severe septic arthritis in murine models, were able to induce expression of several MMP mRNA comparable with that of their isogenic parent strain but induced notably higher levels of tissue inhibitors of metalloproteinases (TIMPs). To our knowledge, this is the first report of induction of multiple MMP/TIMP expression from human dermal and synovial fibroblasts upon S. aureus treatment. We propose that host-derived MMPs contribute to the progressive joint destruction observed in S. aureus-mediated septic arthritis.
doi:10.1186/ar2086
PMCID: PMC1794521  PMID: 17129374
6.  Implication of interleukin 18 in production of matrix metalloproteinases in articular chondrocytes in arthritis: direct effect on chondrocytes may not be pivotal 
Annals of the Rheumatic Diseases  2005;64(5):735-742.
Objective: To clarify the effect of interleukin (IL) 18 on cartilage degeneration by studying the profile of IL18 receptor (IL18R) on chondrocytes and the direct effect of IL18 on production of matrix metalloproteinases (MMPs), aggrecanases, and tissue inhibitors of metalloproteinases (TIMPs) in articular chondrocytes.
Methods: Monolayer cultured human articular chondrocytes were isolated from non-arthritic subjects and patients with rheumatoid arthritis or osteoarthritis. Gene expression of IL18, IL18Rα, IL18Rß, MMPs, and aggrecanases was detected by RT-PCR. Protein levels of IL18Rα were analysed by flow cytometry. Protein levels of IL18, MMPs, and TIMPs were measured by ELISA. Aggrecanase-2 mRNA expression was quantitatively analysed by real time RT-PCR. Protein levels of signalling molecules were assayed by western blotting.
Results: IL18 mRNA was constitutively expressed in chondrocytes, and was enhanced by IL1ß stimulation. Flow cytometric analysis showed that IL1ß, tumour necrosis factor α, and IL18 up regulated IL18Rα expression levels. The level of IL18Rß mRNA was much lower than that of IL18Rα, and was slightly up regulated by IL1ß. In chondrocytes responding to IL18, IL18 (1–100 ng/ml) slightly increased the production of MMP-1, MMP-3, and MMP-13, which was blocked by NF-κB inhibitor and p38 mitogen activated protein kinase inhibitor. IL18 up regulated mRNA expression of aggrecanase-2, but not aggrecanase-1. IL18 also slightly stimulated TIMP-1 production?through extracellular signal regulated kinase activation.
Conclusion: IL18 induces production of MMPs from chondrocytes in inflammatory arthritis. Although the direct effect of IL18 on chondrocytes may not be pivotal for the induction of cartilage degeneration, IL18 seems to play some part in the degradation of articular cartilage in arthritis.
doi:10.1136/ard.2004.026088
PMCID: PMC1755478  PMID: 15834055
7.  Suppressive effect of secretory phospholipase A2 inhibitory peptide on interleukin-1β-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts, and its antiarthritic activity in hTNFtg mice 
Arthritis Research & Therapy  2009;11(5):R138.
Introduction
Secretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.
Methods
Gene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.
Results
PIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.
Conclusions
Our results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
doi:10.1186/ar2810
PMCID: PMC2787297  PMID: 19765281
8.  Attachment to laminin‐111 facilitates transforming growth factor β‐induced expression of matrix metalloproteinase‐3 in synovial fibroblasts 
Annals of the Rheumatic Diseases  2006;66(4):446-451.
Background
In the synovial membrane of patients with rheumatoid arthritis (RA), a strong expression of laminins and matrix degrading proteases was reported.
Aim
To investigate the regulation of matrix metalloproteinases (MMPs) in synovial fibroblasts (SFs) of patients with osteoarthritis (OA) and RA by attachment to laminin‐1 (LM‐111) and in the presence or absence of costimulatory signals provided by transforming growth factor β (TGFβ).
Methods
SFs were seeded in laminin‐coated flasks and activated by addition of TGFβ. The expression of genes was investigated by quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR), immunocytochemistry and ELISA, and intracellular signalling pathways by immunoblotting, and by poisoning p38MAPK by SB203580, MEK‐ERK by PD98059 and SMAD2 by A‐83‐01.
Results
Attachment of SF to LM‐111 did not activate the expression of MMPs, but addition of TGFβ induced a fivefold higher expression of MMP‐3. Incubation of SF on LM‐111 in the presence of TGFβ induced a significant 12‐fold higher expression of MMP‐3 mRNA, and secretion of MMP‐3 was elevated 20‐fold above controls. Functional blocking of LM‐111–integrin interaction reduced the laminin‐activated MMP‐3 expression significantly. Stimulation of SF by LM‐111 and TGFβ activated the p38MAPK, ERK and SMAD2 pathways, and inhibition of these pathways by using SB203580, PD98059 or A‐83‐01 confirmed the involvement of these pathways in the regulation of MMP‐3.
Conclusion
Attachment of SF to LM‐111 by itself has only minor effects on the expression of MMP‐1 or MMP‐3, but it facilitates the TGFβ‐induced expression of MMP‐3 significantly. This mode of MMP‐3 induction may therefore contribute to inflammatory joint destruction in RA independent of the proinflammatory cytokines interleukin (IL)1β or tumour necrosis factor (TNF)α.
doi:10.1136/ard.2006.060228
PMCID: PMC1856036  PMID: 17124250
9.  Inhibitory Effects of Insulin-like Growth Factor-1 and Osteogenic Protein-1 on Fibronectin Fragment- and Interleukin-1β-stimulated Matrix Metalloproteinase-13 Expression in Human Chondrocytes* 
The Journal of biological chemistry  2003;278(28):25386-25394.
Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1β or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1β in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment- or IL-1β-stimulated transcription of the MMP-13 gene in a dose-dependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragment-stimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to down-regulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-κB and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis.
doi:10.1074/jbc.M302048200
PMCID: PMC2895259  PMID: 12734180
10.  Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover 
Gut  2003;52(2):275-282.
Background: Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies suggest that activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrogenesis via increased synthesis of ECM proteins. However, the role of these cells in ECM protein degradation has not been fully elucidated.
Aims: To determine: (i) whether PSCs secrete matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and, if so (ii) whether MMP and TIMP secretion by PSCs is altered in response to known PSC activating factors such as tumour necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), ethanol, and acetaldehyde.
Methods: Cultured rat PSCs (n=3–5 separate cell preparations) were incubated at 37°C for 24 hours with serum free culture medium containing TNF-α (5–25 U/ml), TGF-β1 (0.5–1 ng/ml), IL-6 (0.001–10 ng/ml), ethanol (10–50 mM), or acetaldehyde (150–200 μM), or no additions (controls). Medium from control cells was examined for the presence of MMPs by zymography using a 10% polyacrylamide-0.1% gelatin gel. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine gene expression of MMP9 and the tissue inhibitors of metalloproteinases TIMP1 and TIMP2. Western blotting was used to identify a specific MMP, MMP2 (a gelatinase that digests basement membrane collagen and the dominant MMP observed on zymography) and a specific TIMP, TIMP2. Reverse zymography was used to examine functional TIMPs in PSC secretions. The effect of TNF-α, TGF-β1, and IL-6 on MMP2 secretion was assessed by densitometry of western blots. The effect of ethanol and acetaldehyde on MMP2 and TIMP2 secretion was also assessed by this method.
Results: Zymography revealed that PSCs secrete a number of MMPs including proteinases with molecular weights consistent with MMP2, MMP9, and MMP13. RT-PCR demonstrated the presence of mRNA for metalloproteinase inhibitors TIMP1 and TIMP2 in PSCs while reverse zymography revealed the presence of functional TIMP2 in PSC secretions. MMP2 secretion by PSCs was significantly increased by TGF-β1 and IL-6, but was not affected by TNF-α. Ethanol and acetaldehyde induced secretion of both MMP2 and TIMP2 by PSCs.
Conclusions: Pancreatic stellate cells have the capacity to synthesise a number of matrix metalloproteinases, including MMP2, MMP9, and MMP13 and their inhibitors TIMP1 and TIMP2. MMP2 secretion by PSCs is significantly increased on exposure to the proinflammatory cytokines TGF-β1 and IL-6. Both ethanol and its metabolite acetaldehyde increase MMP2 as well as TIMP2 secretion by PSCs.
Implication: The role of pancreatic stellate cells in extracellular matrix formation and fibrogenesis may be related to their capacity to regulate the degradation as well as the synthesis of extracellular matrix proteins.
PMCID: PMC1774949  PMID: 12524413
11.  Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis 
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis.
In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCδ inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCδ, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.
doi:10.1186/ar2021
PMCID: PMC1779381  PMID: 16872482
12.  In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts 
BMC Cancer  2006;6:18.
Background
Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult.
Methods
We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state.
Results
In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma.
Conclusion
We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.
doi:10.1186/1471-2407-6-18
PMCID: PMC1397851  PMID: 16430785
13.  Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration 
Introduction
The longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.
Methods
Forty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.
Results
Quantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.
Conclusions
This rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.
doi:10.1186/ar3764
PMCID: PMC3446417  PMID: 22394620
14.  Matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the K/BxN serum-transfer arthritis model 
Arthritis Research & Therapy  2010;12(6):R224.
Introduction
Rheumatoid arthritis is an autoimmune disease in which joint inflammation leads to progressive cartilage and bone erosion. Matrix metalloproteinases (MMPs) implicated in homeostasis of the extracellular matrix play a central role in cartilage degradation. However, the role of specific MMPs in arthritis pathogenesis is largely unknown. The aim of the present study was to investigate the role of Mmp-8 (collagenase-2) in an arthritis model.
Methods
Arthritis was induced in Mmp8-deficient and wildtype mice by K/BxN serum transfer. Arthritis severity was measured by a clinical index and ankle sections were scored for synovial inflammation, cartilage damage and bone erosion. cDNA microarray analysis, real-time PCR and western blot were performed to identify differential changes in gene expression between mice lacking Mmp8 and controls.
Results
Mmp8 deficiency increased the severity of arthritis, although the incidence of disease was similar in control and deficient mice. Increased clinical score was associated with exacerbated synovial inflammation and bone erosion. We also found that the absence of Mmp8 led to increased expression of IL-1β, pentraxin-3 (PTX3) and prokineticin receptor 2 (PROKR2) in arthritic mice joints.
Conclusions
Lack of Mmp-8 is accompanied by exacerbated synovial inflammation and bone erosion in the K/BxN serum-transfer arthritis model, indicating that this Mmp has a protective role in arthritis.
doi:10.1186/ar3211
PMCID: PMC3046537  PMID: 21190566
15.  Inhibition of interleukin 1‐induced matrix metalloproteinase 13 expression in human chondrocytes by interferon γ 
Annals of the Rheumatic Diseases  2006;66(6):782-789.
Background
Despite well‐documented immunomodulation by interferon γ (IFNγ), its role and mechanism of regulation of matrix metalloproteinase 13 (MMP13) gene expression in human chondrocytes is unknown.
Objective
To investigate the ability and mechanism of IFNγ to suppress interleukin 1 (IL1)‐induced MMP13 expression in articular chondrocytes.
Methods
Human chondrocytes were treated with IFNγ or IL1β alone or in combination. MMP13 mRNA was analysed by semiquantitative reverse transcriptase‐PCR. MMP13 protein, phospho‐signal transducer and activator of transcription 1 (STAT1) and p44/42 mitogen‐activated protein kinase levels were measured by western blotting. MMP13 promoter luciferase, cytomegalovirus cyclic AMP response element‐binding protein (CBP)/p300 plasmids and STAT1 small interfering RNA (siRNA) were transfected by the calcium phosphate method. IFNγ receptor was also neutralised. Activator protein (AP) 1 activity was monitored by the TransAM transcription factor kit. STAT1‐CBP/p300 interaction was studied by immunoprecipitation.
Results
IFNγ potently suppressed IL1‐induced expression of MMP13 and promoter activity. Blockade with neutralising IFNγ R1 antibody revealed that MMP13 inhibition by IFNγ is mediated by the IFN receptor. IFNγ‐stimulated activation of STAT1 was directly correlated with MMP13 suppression. Knockdown of the STAT1 gene by specific siRNA or its inhibition with fludarabine partially restored the IL1β induction of MMP13 expression and promoter activity. IFNγ did not alter AP1 binding ability but promoted physical interaction of STAT1 and CBP/p300 coactivator. p300 overexpression reversed IFNγ inhibition of endogenous MMP13 mRNA expression and exogenous MMP13 promoter activity.
Conclusion
IFNγ, through its receptor, activates STAT1, which binds with CBP/p300 coactivator, sequesters it from the cell system, and thus inhibits transcriptional induction of the MMP13 gene in chondrocytes. IFNγ and its signalling pathways could be targeted therapeutically for diminishing IL1‐induced cartilage degradation by MMP13 in patients with arthritis.
doi:10.1136/ard.2006.060269
PMCID: PMC1954643  PMID: 17179173
16.  N-acetyl-Ser-Asp-Lys-Pro inhibits interleukin-1β-mediated matrix metalloproteinase activation in cardiac fibroblasts 
Myocardial matrix turnover involves a dynamic balance between collagen synthesis and degradation, which is regulated by matrix metalloproteinases (MMPs). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) is a small peptide that inhibits cardiac inflammation and fibrosis. However, its role in MMP regulation is not known. Thus, we hypothesized that Ac-SDKP promotes MMP activation in cardiac fibroblasts and decreases collagen deposition via this mechanism. To that end, we tested the effects of Ac-SDKP on interleukin-1β (IL-1β; 5 ng/ml)-stimulated adult rat cardiac fibroblasts. We measured total collagenase activity, MMP-2, MMP-9, and MMP-13 expressions, and activity along with their inhibitors, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. In order to examine the effects of Ac-SDKP on the signaling pathway that controls MMP transcription, we also measured nuclear factor-κB (NFκB) and p42/44 mitogen-activated protein kinase (MAPK) activation. Ac-SDKP did not alter collagenase or gelatinase activity in cardiac fibroblasts under basal conditions, but blunted the IL-1β-induced increase in total collagenase activity. Similarly, Ac-SDKP normalized the IL-1β-mediated increase in MMP-2 and MMP-9 activities and MMP-13 expression. Inhibition of MMPs by Ac-SDKP was associated with increased TIMP-1 and TIMP-2 expressions. Collagen production was not affected by Ac-SDKP, IL-1β, or a combination of both agents. Ac-SDKP blocked IL-1β-induced p42/44 phosphorylation and NFκB activation in cardiac fibroblasts. We concluded that the Ac-SDKP-inhibited collagenase expression and activation was associated with increased expression of TIMP-1 and TIMP-2. These pharmacological effects of Ac-SDKP may be linked to the inhibition of MAPK and NFκB pathway.
doi:10.1007/s00424-013-1262-8
PMCID: PMC4240013  PMID: 23652767
Myocardial matrix turnover; Synthesis and degradation; Matrix metalloproteinases; N-acetyl-Ser-Asp-Lys-Pro; Cardiac fibroblasts; NFκB; MAPK
17.  Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis 
Annals of the Rheumatic Diseases  2000;59(6):455-461.
OBJECTIVE—Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA.
METHODS—Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [3H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated.
RESULTS—Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF.
CONCLUSIONS—Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.


doi:10.1136/ard.59.6.455
PMCID: PMC1753174  PMID: 10834863
18.  Tumor necrosis factor-α-accelerated degradation of type I collagen in human skin is associated with elevated matrix metalloproteinase (MMP)-1 and MMP-3 ex vivo 
Tumor necrosis factor (TNF)-α induces matrix metalloproteinases (MMPs) that may disrupt skin integrity. We have investigated the effects and mechanisms of exogenous TNF-α on collagen degradation by incubating human skin explants in defined serum-free media with or without TNF-α (10 ng/ml) in the absence or presence of the nonselective MMP inhibitor GM6001 for 8 days. The basal culture conditions promoted type I collagen catabolism that was accelerated by TNF-α (p < 0.005) and accomplished by MMPs (p < 0.005). Levels of the collagenases MMP-8 and MMP-13 were insignificant and neither MMP-2 nor MMP-14 were associated with increased collagen degradation. TNF-α increased secretion of MMP-1 (p < 0.01) but had no impact on MMP-1 quantities in the tissue. Immunohistochemical analysis confirmed similar tissue MMP-1 expression with or without TNF-α with epidermis being the major source of MMP-1. Increased tissue-derived collagenolytic activity with TNF-α exposure was blocked by neutralizing MMP-1 monoclonal antibody and was not due to down-regulation of tissue inhibitor of metalloproteinase-1. TNF-α increased production (p < 0.01), tissue levels (p < 0.005) and catalytic activity of the endogenous MMP-1 activator MMP-3. Type I collagen degradation correlated with MMP-3 tissue levels (rs = 0.68, p < 0.05) and was attenuated with selective MMP-3 inhibitor. Type I collagen formation was down-regulated in cultured compared with native skin explants but was not reduced further by TNF-α. TNF-α had no significant effect on epidermal apoptosis. Our data indicate that TNF-α augments collagenolytic activity of MMP-1, possibly through up-regulation of MMP-3 leading to gradual loss of type I collagen in human skin.
doi:10.1016/j.ejcb.2014.10.001
PMCID: PMC4300401  PMID: 25457675
Aging; Cytokine; Extracellular matrix proteins; Protease inhibitors; UK370106; C-terminal telopeptide of type I collagen; Type I C-terminal collagen propeptide
19.  Matrix Metalloproteinase and G Protein Coupled Receptors: Co-conspirators in the pathogenesis of autoimmune disease and cancer 
Journal of autoimmunity  2009;33(3-4):214-221.
Similarities in the pathologies of autoimmune diseases and cancer have been noted for at least 30 years. Inflammatory cytokines and growth factors mediate cell proliferation, and proteinases, especially the collagenase, Matrix Metalloproteinase-1 (MMP-1), contribute to disease progression by remodeling the extracellular matrix and modulating the microenvironment. This review focuses on two cancers (melanoma and breast) and on the autoimmune disorder, rheumatoid arthritis (RA), and discusses the activated stromal cells found in these diseases. MMP-1 was originally thought to function only to degrade interstitial collagens, but recent studies have revealed novel roles for MMP-1 involving the G protein-coupled receptors: the chemokine receptor, CXCR-4, and Protease Activated Receptor-1 (PAR-1). Cooperativity between MMP-1 and CXCR4/SDF-1 signaling influences the behavior of activated fibroblasts in both RA and cancer. Further, MMP-1 is a vital part of an autocrine/paracrine MMP-1/PAR-1 signal transduction axis, a function that amplifies its potential to remodel the matrix and to modify cell behavior. Finally, new therapeutic agents directed at MMP-1 and G protein-coupled receptors are emerging. Even though these agents are more specific in their targets than past therapies, these targets are often shared between RA and cancer, underscoring fundamental similarities between autoimmune disorders and some cancers.
doi:10.1016/j.jaut.2009.09.011
PMCID: PMC2783549  PMID: 19800199
rheumatoid arthritis; collagenase; CXCR4; PAR-1; synovial fibroblasts; carcinoma associated fibroblasts; endothelial cells
20.  Borrelia burgdorferi-Induced Expression of Matrix Metalloproteinases from Human Chondrocytes Requires Mitogen-Activated Protein Kinase and Janus Kinase/Signal Transducer and Activator of Transcription Signaling Pathways  
Infection and Immunity  2004;72(5):2864-2871.
Elevations in matrix metalloproteinase 1 (MMP-1) and MMP-3 have been found in patients with Lyme arthritis and in in vitro models of Lyme arthritis using cartilage explants and chondrocytes. The pathways by which B. burgdorferi, the causative agent of Lyme disease, induces the production of MMP-1 and MMP-3 have not been elucidated. We examined the role of the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in MMP induction by B. burgdorferi. Infection with B. burgdorferi results in rapid phosphorylation of p38 and JNK within 15 to 30 min. Inhibition of JNK and p38 MAPK significantly reduced B. burgdorferi-induced MMP-1 and MMP-3 expression. Inhibition of ERK1/2 completely inhibited the expression of MMP-3 in human chondrocytes following B. burgdorferi infection but had little effect on the expression of MMP-1. B. burgdorferi infection also induced phosphorylation and nuclear translocation of STAT-3 and STAT-6 in primary human chondrocytes. Expression of MMP-1 and MMP-3 was significantly inhibited by inhibition of JAK3 activity. Induction of MMP-1 and -3 following MAPK and JAK/STAT activation was cycloheximide sensitive, suggesting synthesis of intermediary proteins is required. Inhibition of tumor necrosis factor alpha (TNF-α) significantly reduced MMP-1 but not MMP-3 expression from B. burgdorferi-infected cells; inhibition of interleukin-1β (IL-1β) had no effect. Treatment of B. burgdorferi-infected cells with JAK and MAPK inhibitors significantly inhibited TNF-α induction, consistent with at least a partial role for TNF-α in B. burgdorferi-induced MMP-1 expression in chondrocytes.
doi:10.1128/IAI.72.5.2864-2871.2004
PMCID: PMC387916  PMID: 15102798
21.  p38 Mitogen-Activated Protein Kinase-Dependent Activation of Protein Phosphatases 1 and 2A Inhibits MEK1 and MEK2 Activity and Collagenase 1 (MMP-1) Gene Expression 
Molecular and Cellular Biology  2001;21(7):2373-2383.
Degradation of collagenous extracellular matrix by collagenase 1 (also known as matrix metalloproteinase 1 [MMP-1]) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, chronic ulcers, and tumor invasion and metastasis. Here, we have investigated the role of distinct mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-1 gene expression. The activation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (designated ERK1,2) pathway by oncogenic Ras, constitutively active Raf-1, or phorbol ester resulted in potent stimulation of MMP-1 promoter activity and mRNA expression. In contrast, activation of stress-activated c-Jun N-terminal kinase and p38 pathways by expression of constitutively active mutants of Rac, transforming growth factor β-activated kinase 1 (TAK1), MAPK kinase 3 (MKK3), or MKK6 or by treatment with arsenite or anisomycin did not alone markedly enhance MMP-1 promoter activity. Constitutively active MKK6 augmented Raf-1-mediated activation of the MMP-1 promoter, whereas active mutants of TAK1 and MKK3b potently inhibited the stimulatory effect of Raf-1. Activation of p38 MAPK by arsenite also potently abrogated stimulation of MMP-1 gene expression by constitutively active Ras and Raf-1 and by phorbol ester. Specific activation of p38α by adenovirus-delivered constitutively active MKK3b resulted in potent inhibition of the activity of ERK1,2 and its upstream activator MEK1,2. Furthermore, arsenite prevented phorbol ester-induced phosphorylation of ERK1,2 kinase-MEK1,2, and this effect was dependent on p38-mediated activation of protein phosphatase 1 (PP1) and PP2A. These results provide evidence that activation of signaling cascade MKK3-MKK3b→p38α blocks the ERK1,2 pathway at the level of MEK1,2 via PP1-PP2A and inhibits the activation of MMP-1 gene expression.
doi:10.1128/MCB.21.7.2373-2383.2001
PMCID: PMC86870  PMID: 11259586
22.  Modulatory effect of interleukin-1α on expression of structural matrix proteins, MMPs and TIMPs in human cardiac myofibroblasts: Role of p38 MAP kinase 
Matrix Biology  2010;29(7-6):613-620.
The proinflammatory cytokine interleukin-1 (IL-1) elicits catabolic effects on the myocardial extracellular matrix (ECM) early after myocardial infarction but there is little understanding of its direct effects on cardiac myofibroblasts (CMF), or the role of p38 mitogen-activated protein kinase (MAPK). We used a focused RT-PCR microarray to investigate the effects of IL-1α on expression of 41 ECM genes in CMF cultured from different patients, and explored regulation by p38 MAPK.
IL-1α (10 ng/ml, 6 h) had minimal effect on mRNA expression of structural ECM proteins, including collagens, laminins, fibronectin and vitronectin. However, it induced marked increases in expression of specific ECM proteases, including matrix metalloproteinases MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (gelatinase-B) and MMP-10 (stromelysin-2). Conversely, IL-1α reduced mRNA and protein expression of ADAMTS1, a metalloproteinase that suppresses neovascularization. IL-1α increased expression of TIMP-1 slightly, but not TIMP-2. Data for MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and ADAMTS1 were confirmed by quantitative real-time RT-PCR. Tumor necrosis factor-alpha (TNFα), another important myocardial proinflammatory cytokine, did not alter expression of these metalloproteinases. IL-1α strongly activated the p38 MAPK pathway in human CMF. Pharmacological inhibitors of p38-α/β (SB203580) or p38-α/β/γ/δ (BIRB-0796) reduced MMP-3 and ADAMTS1 mRNA expression, but neither inhibitor affected MMP-9 levels. MMP-1 and MMP-10 expression were inhibited by BIRB-0796 but not SB203580, suggesting roles for p38-γ/δ.
In summary, IL-1α induces a distinct pattern of ECM protein and protease expression in human CMF, in part regulated by distinct p38 MAPK subtypes, affirming the key role of IL-1α and CMF in post-infarction cardiac remodeling.
doi:10.1016/j.matbio.2010.06.007
PMCID: PMC3004031  PMID: 20619343
ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin motifs; CMF, cardiac myofibroblast; DMSO, dimethylsulfoxide; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSP27, heat shock protein-27; IL-1, intereukin-1; MAPK, mitogen-activated protein kinase; MI, myocardial infarction; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor; Interleukin-1; Matrix metalloproteinase; Cardiac fibroblast; Extracellular matrix; p38 MAP kinase; Microarray
23.  Basic Fibroblast Growth Factor Stimulates Matrix Metalloproteinase-13 via the Molecular Cross-talk between the Mitogen-activated Protein Kinases and Protein Kinase Cδ Pathways in Human Adult Articular Chondrocytes* 
The Journal of biological chemistry  2007;282(15):11110-11121.
Excessive release of basic fibroblast growth factor (bFGF) during loading and/or injury of the cartilage matrix may contribute to the onset or progression of osteoarthritis. This pathological role may be related to the ability of bFGF to decrease proteoglycan synthesis and to antagonize the activity of anabolic growth factors in cartilage such as insulin-like growth factor-1 and bone morphogenetic protein 7 (BMP7 or OP-1). Matrix metalloproteinase-13 (MMP-13), a catabolic cartilage-degrading enzyme, is dramatically up-regulated by inflammatory cytokines or by fibronectin fragments in articular chondrocytes. In this study, we investigated MMP-13 production by bFGF using human articular chondrocytes. Endogenous concentration of bFGF in synovial fluids collected from arthritis patients and asymptomatic subjects showed a good linear correlation with the endogenous levels of MMP-13. bFGF stimulation of MMP-13 was mediated at the transcriptional level and, at least in part, by stimulation of interleukin-1 production. Also, our findings suggest that bFGF stimulation of MMP-13 required the activation of multiple MAPKs (ERK, p38, and JNK) by bFGF, and more importantly, bFGF activation of protein kinase C (PKC) δ played a key role in the MMP-13 stimulation. Indeed, PKCδ is the only isoform associated with MMP-13 stimulation among the PKC isoforms tested. PKCδ controls the bFGF response by regulating multiple MAPK pathways. Our results suggest that PKCδ activation is a principal rate-limiting event in the bFGF-dependent stimulation of MMP-13 in human adult articular chondrocytes. We propose that deregulation of cross-talk between MAPK and PKCδ signaling may contribute to the etiology of osteoarthritis in human patients.
doi:10.1074/jbc.M609040200
PMCID: PMC2895271  PMID: 17311929
24.  Matrix Metalloproteinase 9 Expression Is Induced by Epstein-Barr Virus Latent Membrane Protein 1 C-Terminal Activation Regions 1 and 2 
Journal of Virology  1999;73(7):5548-5555.
Nasopharyngeal carcinoma (NPC), which is closely associated with the Epstein-Barr virus (EBV), is a highly metastatic malignant tumor. An important activity in tumor invasion and metastasis is that of the 92-kDa type IV collagenase or gelatinase, matrix metalloproteinase 9 (MMP-9), which mediates the degradation of the basement membrane and extracellular matrix. The expression of MMP-9 has been shown to be enhanced by the EBV oncoprotein, latent membrane protein 1 (LMP-1). LMP-1, which is expressed in NPC, has two essential signaling domains within the carboxy terminus, termed C-terminal activation regions 1 (CTAR-1) and CTAR-2. This study reveals that either signaling domain can activate the MMP-9 promoter and induce MMP-9 activity; however, LMP-1 deletion mutants lacking either CTAR-1 or CTAR-2 had a decreased ability to induce MMP-9 expression. The deletion of both activation regions completely abolished the induction of MMP-9 activity, while the cotransfection of both the CTAR-1 and CTAR-2 deletion mutants restored MMP-9 activity to levels produced by wild-type LMP-1. The NF-κB and activator protein 1 (AP-1) binding sites in the MMP-9 promoter were essential for the activation of MMP-9 gene expression by both CTAR-1 and CTAR-2. The induction of MMP-9 expression by LMP-1 and both CTAR-1 and CTAR-2 mutants was blocked by the overexpression of IκB. The tumor necrosis factor receptor-associated factor (TRAF) pathway also contributed to the activation of the MMP-9 promoter as shown by the use of TRAF-2 and TRAF-3 dominant-negative constructs. These data indicate that the activation of both the NF-κB and AP-1 pathways by LMP-1, CTAR-1, and CTAR-2 is necessary for the activation of MMP-9 expression. In NPC, LMP-1 may contribute to invasiveness and metastasis through the induction of MMP-9 transcription and enzymatic activity.
PMCID: PMC112612  PMID: 10364303
25.  Detection of Functional Matrix Metalloproteinases by Zymography 
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.
doi:10.3791/2445
PMCID: PMC3159606  PMID: 21085107

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