Search

Backround Obesity has been recognized as a state of subclinical inflammation resulting in a loss of insulin receptors and decreased insulin sensitivity. We here studied in vivo the role of circulating matrix metaloproteinase-8 (MMP-8) among young healthy twin adults. Also in vitro analysis of the cleavage of human insulin receptor (INSR) by MMP-8 was investigated as well its inhibition by doxycycline and other MMP-8 inhibitor, Ilomastat/GM6001, which are broadspectrum MMP-inhibitors. Materials and methods We analyzed serum MMP-8 levels by a time-resolved immunofluorometric assay in obese (n=34), overweight (n=76) and normal weight (n=130) twin individuals. The effect of MMP-8 on INSR and the effects of synthetic MMP-8 inhibitors, doxycycline and Ilomastat/GM6001, were studied by SDS-PAGE. Results We found that in obese individuals relative to normal weight individuals the serum MMP-8 levels and MMP-8/TIMP-1 ratio were significantly increased (P=0.0031 and P=0.031, respectively). Among normal weight and obese individuals also smoking significantly increases serum MMP-8 and MMP-8/TIMP-1 ratio. In vitro we found that INSR was degraded by MMP-8 and this was inhibited by doxycycline and Ilomastat/GM6001. Conclusions Obesity associated with elevated circulating MMP-8 found among young adults may contribute to progression of insulin resistance by cleaving INSR. This INSR cleavage by MMP-8 can be inhibited by synthetic MMP-8 inhibitors such as doxycycline. In addition to obesity, also smoking independently explained increased MMP-8 levels. Our results suggest that MMP-8 is an essential mediator in systemic subclinical inflammatory response in obesity, and a potential drug target. This article is protected by copyright. All rights reserved.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Matrix metalloproteinases (MMPs), and especially membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), play a role in cancer progression and can have a prognostic value. Various synthetic broad-spectrum MMP inhibitors have been developed but have had little success in cancer patient treatment owing to side effects. Until recently, selective targeting of specific MMPs has not been possible due to lack of specific inhibitors. Here we have developed a selective MT1-MMP peptide-inhibitor GACFSIAHECGA, which did not affect the activities of many other MMPs including MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -15, -17 or -20. In a fluorescent peptide cleavage assay it displayed an IC(50) value of 150 microM. The peptide effectively inhibited the migration and invasion of cancer cell lines in vitro. Furthermore, in vivo the peptide reduced the growth of tongue carcinoma xenografts and prolonged the survival of mice. Overall these results suggest that selective MT1-MMP inhibitors may have utility as anticancer agents.

Proteolytic processing of defensins is a critical mode of posttranslational regulation of peptide activity. Because mouse alpha-defensin precursors are cleaved and activated by matrix metalloproteinase-7 (MMP-7), we determined if additional defensin molecules, namely human neutrophil defensin pro-HNP-1 and beta-defensins, are targets for MMP-7. We found that MMP-7 cleaves within the pro-domain of the HNP-1 precursor, a reaction that does not generate the mature peptide but produces a 59-amino acid intermediate. This intermediate, which retains the carboxyl-terminal end of the pro-domain, had antimicrobial activity, indicating that the residues important for masking defensin activity reside in the amino terminus of this domain. Mature HNP-1 was resistant to processing by MMP-7 unless the peptide was reduced and alkylated, demonstrating that only the pro-domain of alpha-defensins is normally accessible for cleavage by this enzyme. From the 47-residue HBD-1 precursor, MMP-7 catalyzed removal of 6 amino acids from the amino terminus. Neither a 39-residue intermediate form of HBD-1 nor the mature 36-residue form of HBD-1 was cleaved by MMP-7. In addition, both pro-HBD-2, with its shorter amino-terminal extension, and pro-HBD-3 were resistant to MMP-7. However, human and mouse beta-defensin precursors that lack disulfide bonding contain a cryptic MMP-7-sensitive site within the mature peptide moiety. These findings support and extend accumulating evidence that the native three-dimensional structure of both alpha- and beta-defensins protects the mature peptides against proteolytic processing by MMP-7. We also conclude that sites for MMP-7 cleavage are more common at the amino termini of alpha-defensin rather than beta-defensin precursors, and that catalysis at these sites in alpha-defensin pro-domains results in acquisition of defensin activity.

Squamous cell carcinoma (SCC) of the tongue is the most common cancer in the oral cavity and has a high mortality rate. A total of 90 mobile tongue SCC samples were analysed for Bryne's malignancy scores, microvascular density, and thickness of the SCC sections. In addition, the staining pattern of cyclooxygenase-2, alphavbeta6 integrin, the laminin-5 gamma2-chain, and matrix metalloproteinases (MMPs) -2, -7, -8, -9, -20, and -28 were analysed. The expression of MMP-8 (collagenase-2) was positively associated with improved survival of the patients and the tendency was particularly prominent in females. No sufficient evidence for a correlation with the clinical outcome was found for any other immunohistological marker. To test the protective role of MMP-8 in tongue carcinogenesis, MMP-8 knockout mice were used. MMP-8 deficient female mice developed tongue SCCs at a significantly higher incidence than wild-type mice exposed to carcinogen 4-Nitroquinoline-N-oxide. Consistently, oestrogen-induced MMP-8 expression in cultured HSC-3 tongue carcinoma cells, and MMP-8 cleaved oestrogen receptor (ER) alpha and beta. According to these data, we propose that, contrary to the role of most proteases produced by human carcinomas, MMP-8 has a protective, probably oestrogen-related role in the growth of mobile tongue SCCs.

Objective Aberrant matrix metalloproteinase (MMP) and human β-defensin (HBD) functions have been found in inflammatory diseases. The objectives of this study were to investigate the immunolocalisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2 in chronic and aggressive periodontitis and in peri-implantitis. The expression of MMP-25 by cultured human plasmacytoma cells and macrophages, and the effects of MMP-26 and Porphyromonas gingivalis trypsin-like proteinase on HBD-1 and -2 were also studied. Design Immunohistochemistry, immunofluorescent analysis, reverse transcriptase polymerase chain reaction and immunoblotting were used to assess localisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2. HBD degradation by MMP-26 and P. gingivalis proteinase was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results MMP-25 was present in plasma cells and polymorphonuclear leucocytes, and MMP-26 was present in oral and sulcular basement membrane zones. HBD-1 was distributed perivasculary in gingival connective tissue and in oral and sulcular epithelium, and HBD-2 was found to a lesser extent in the perivascular space. Low MMP-25, MMP-26, HBD-1 and HBD-2 mRNA expression was found. Immunoblot revealed 29–57-kDa MMP-25 in myeloma cell lysates, but not in macrophages, and partly activated MMP-25 and -26 in diseased gingival crevicular fluid and peri-implant sulcular fluid. P. gingivalis trypsin-like proteinase degraded HBD-1 and -2. Conclusions Both MMP-25 and -26 were expressed more strongly in extensively inflamed gingiva compared with healthy gingiva. The expression of HBD-1 was stronger than that of HBD-2 in periodontitis and peri-implantitis. De-novo expression of MMP-25 and -26 is associated with periodontal and peri-implant inflammation. Furthermore, P. gingivalis trypsin-like proteinase, but not MMP-26, can degrade HBD-1 and -2, which could lead to a weakened innate immune response.

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity.

Our study aimed to examine the molecular forms and gingival crevicular fluid (GCF) levels of laminin-5 gamma2-chain in patients with different periodontal disease, and compare the effects of P.gingivalis trypsin-like proteinase on intact laminin-5 gamma2-chain species.Eighteen patients with generalized aggressive periodontitis (G-AgP), 29 patients with chronic periodontitis (CP), 20 with gingivitis and 20 periodontally healthy subjects were included. Probing depth, clinical attachment loss, presence of bleeding on probing and plaque were recorded. Molecular forms and GCF laminin-5 gamma2-chain levels and the effects of P. gingivalis trypsin-like proteinase on intact laminin-5 gamma2-chain were analysed by computer-quantitated Western immunoblotting.Laminin-5 gamma2-chain 40 and 70 kDa fragments could be detected in all groups, in varying levels. The CP group had elevated GCF laminin-5 gamma2-chain fragment levels compared with the gingivitis and healthy groups (p0.008). The G-AgP group had GCF laminin-5 gamma2-chain fragment levels similar to the gingivitis and healthy groups (p0.008). GCF laminin-5 gamma2-chain fragments differed clearly from the multiple lower molecular size fragments of P.gingivalis trypsin-laminin-5 gamma2-chain proteinases.Increased GCF laminin-5 gamma2-chain fragments in periodontitis sites with deep periodontal pocket suggest that these cleaved 40 and 70 kDa fragments could reflect the extent of the inflammatory reaction in CP.

Matrix metalloproteinases (MMP-2 and MMP-9, or gelatinases) are involved in tongue SCC invasion, metastasis and angiogenesis. We have recently shown that a novel and selective hydrophobic cyclic CTTHWGFTLC (CTT1) peptide is inhibitor for MMP-2 and MMP-9 (Koivunen et al., Nat Biotechnol 1999; 17:768–74). In this study, we demonstrate that both the new hydrophilic derivate GRENYHGCTTHWGFTLC (CTT2) peptide and the CTT1 peptide inhibited specifically the human tongue squamous cell carcinoma (HSC-3) cell-derived gelatinolytic activity and in vitro invasion and migration of these cells (p ≤ 0.049). In situ zymography revealed that both peptides also inhibited clearly almost all of the gelatinolytic activity present in the human tongue SCC tissue sections, indicating that MMP-2 and MMP-9 are the major gelatinases detected in the tongue carcinomas. However, CTT2 did not inhibit the type I collagen degradation by human collagenases (MMP-1, MMP-8 and MMP-13). Furthermore, CTT2 reduced the blood vessel density (p ≤ 0.043) and clearly improved the survival of the mice bearing human tongue carcinoma xenografts (p ≤ 0.012). Overall, we suggest that CTT1 and CTT2 peptides being selective gelatinase inhibitors with significant anti-tumor properties could be useful to diminish the invasion and angiogenesis of human tongue carcinomas characterized by enhanced gelatinolytic activity in tumors. © 2005 Wiley-Liss, Inc.

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.

Matrix metalloproteinase (MMP)-2 and membrane type 1-MMP can process the laminin-5 (Ln-5) γ2-chain, revealing a cryptic site inducing epithelial cell migration. We investigated whether other MMPs process the Ln-5 γ2-chain and related their ability to induce epithelial cell migration. The N-terminal sequences of the MMP-3, -12, -13, and -20 processed 80 kDa Ln-5 γ2x-chains were identical whereas the N-terminus of the 80 kDa MMP-8 Ln-5 γ2x-chain was not. MMP-3, -13, -14, and -20 induced MCF-7 cell migration over Ln-5 while MMP-8 was a poor inducer of MCF-7 cell migration. In conclusion, several MMPs can process the Ln-5 γ2-chain and induce epithelial cell migration.

Laminin-5 (LN-5) is an important epithelial cell-derived structural and adhesive component in hemidesmosomes and basement membranes (BM). In peri-implant tissue, gingival BM underlies the junctional epithelium (JE) and reflects the peri-implant health. Matrix metalloproteinase-8 (MMP-8 or collagenase-2) is one of the key mediators of periodontal tissue destruction. Western immunoblotting with image analysis was used to quantitate the molecular forms of LN-5 gamma2-chain and MMP-8 in peri-implant sulcular fluid (PISF) from healthy and diseased implants. These observations were related to the recorded gingival (GI) and bone resorption (BR) indices of the studied sites. Altogether, 72 PISF samples from osseointegrated dental implants were examined. Significantly elevated levels of fragmented LN-5 gamma2-chain species (45 and 70 kDa) and MMP-8 immunoreactivities were observed in diseased PISF in relation to healthy PISF. The elevated levels of both LN-5 gamma2-chain 45 and 70 kDa fragments and MMP-8 in diseased PISF from peri-mucositis (BR = 0) and peri-implantitis (BR >/= 1) lesions strongly correlated with elevated GI. Low levels - almost comparable to those seen in healthy control PISF - were seen in PISF from peri-implantitis lesions (BR >/= 1) with no GI. Activation of 75 kDa neutrophil (PMN)-type proMMP-8 to 10 kDa lower-molecular-size active forms was especially detected in PISF from peri-implantitis with elevated GI. These cross-sectional findings indicate that elevated MMP-8 and LN-5 gamma2-chain fragment levels in PISF can reflect the active phase of the inflammatory peri-implant disease. Longitudinal studies are required to assess their use, either alone or in combination as molecular biochemical PISF markers, to predict the risk of progression of peri-implantitis, as well as to monitor the impact of treatment of the disease.

Dextran sulfate sodium-induced inflammatory bowel disease in mice resembles human ulcerative colitis. In inflammatory bowel diseases matrix metalloproteinases contribute to tissue degradation. Laminin-5 is an anchoring filament protein in the basement membrane area that can be cleaved by matrix metalloproteinases. We investigated the expression of matrix metalloproteinases-2 and -8 and laminin-5 γ2-chain in dextran sulfate sodium-induced mice by immunohistochemistry and in situ hybridization. Matrix metalloproteinase-8 expression was evidenced in the colon surface epithelial cells and the protein was more abundant in dextran sulfate sodium-induced mice colon. Matrix metallproteinase-2 and laminin-5 γ2-chain colocalized in the colon surface epithelial cells and in the basement membrane zone as demonstrated by double immunostaining. In dextran sulfate sodium-induced colon, matrix metalloproteinase-2 immunoreactivity was detected in epithelial cells in the lower parts of the crypt and surrounding the degraded crypts. Matrix metalloproteinase-2 and -8 could participate in the local epithelial inflammatory processes and tissue destruction. The presence of laminin-5 γ2-chain indicates alternative anchoring mechanisms in the colon, a compartment devoid of hemidesmosomes.

MMP-8 (collagenase-2) is the most effective collagenase to initiate type I collagen degradation. Since initiation of lysis of the surrounding collagen matrix is an essential prerequisite for carcinoma cells to spread, this study investigated the expression of MMP-8 in squamous cell carcinoma (SCC) of the head and neck in vivo and in vitro. Most of the recently established head and neck carcinoma cell lines (22/25), corresponding tumour (5/7) and dermal (2/2) fibroblasts, commercial tongue carcinoma (HSC-3 and SCC-25), and transformed keratinocyte cell lines of the tongue (IHGK) and skin (HaCaT) expressed MMP-8 mRNA analysed by the PCR method. Western blotting revealed a latent 50 kD band in concentrated culture media of carcinoma cells and corresponding tumour and dermal fibroblasts. The expression of immunoreactive MMP-8 protein was reduced 30% by transforming growth factor beta-1 (TGF-beta1) at 1 ng/ml concentration and 60% at 10 ng/ml concentration, but up-regulated 2- and 2.5-fold after 10 nM and 100 nM phorbol 12-myristate 13 acetate (PMA), respectively. Immunohistological staining localized MMP-8 protein in a few malignant invading tumour cell islands, certain fibroblasts, polymorphonuclear neutrophils (PMNs), and plasma cells. In situ hybridization revealed a faint sporadic signal in carcinoma cells of all eight tissue sections analysed. It is concluded that tissue from head and neck carcinomas can express MMP-8 both in vivo and in vitro. Since the amount of MMP-8 in carcinoma and stromal cells is rather low, MMP-8 may have a potential role, with other collagenases, in the proteolysis of connective tissue associated with the spreading of invasive carcinoma.

Background Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME). Methods TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1. Results Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1. Conclusions Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.

Matrix metalloproteinase-8 (MMP-8)-deficient mice (Mmp8-/-) exhibit delayed dermal wound healing, but also partly contradicting results have been reported. Using the Mmp8-/- mice we investigated the role of MMP-8 in acute wound healing of the mobile tongue, and analyzed the function of tongue fibroblasts in vitro. Interestingly, in the early phase the tongue wounds of Mmp8-/- mice healed faster than those of wild type (wt) mice resulting in significant difference in wound widths (P=0.001, 6–24 h). The Mmp8-/- wounds showed no change in myeloperoxidase positive myeloid cell count, but the level of transforming growth factor (TGF)-β1 was significantly increased (P=0.007) compared to the wt tongues. Fibroblasts cultured from wt tongues expressed MMP-8 and TGF-β1. However, higher TGF-β1 levels were detected in Mmp8-/- fibroblasts, and MMP-8 treatment decreased phosphorylated Smad-2 levels and α-smooth muscle actin expression in these fibroblasts suggesting reduced TGF-β1 signaling. Consistently, a degradation of recombinant TGF-β1 by MMP-8 decreased its ability to activate the signaling cascade in fibroblasts. Moreover, collagen gels with Mmp8-/- fibroblasts reduced more in size. We conclude that MMP-8 regulates tongue wound contraction rate and TGF-β1 levels. In vitro analyses suggest that MMP-8 may also play a role in regulating TGF-β1 signaling of stromal fibroblasts.

In situ zymography provides a tool to localize proteolytic activity in tissues in vivo. However, it has been difficult to discriminate between the proteases responsible for the detected activity. We used a selective tissue-permeable gelatinase inhibitor, the CTTHWGFTLC-peptide (CTT) in inflamed human gingiva. The CTT-peptide was evidenced to home, target to, and selectively inhibit the areas of gelatinolytic activity in inflamed human gingiva expressing MMP-2 and -9. Gelatinolytic activity, MMP-9 immunoreactivity, and mRNA expression as well as CD-45-positive inflammatory cells colocalized well in the inflamed human gingival connective tissue. Gelatinolytic activity corresponding to MMP-2 colocalized with laminin-5 gamma2-chain immunoreactivity and was detected in the close vicinity of the sulcular basement membrane region. Furthermore, the CTT-peptide inhibited beta-caseinolysis by human MMP-2 and MMP-9 as well as laminin-5 gamma2-chain degradation by MMP-2 in vitro. Thus, the CTT-peptide may prove to be a useful tool (i) to discriminate between gelatinolytic proteases detected by in situ zymography and (ii) to preventMMP-2-dependent induction of epithelial cell migration and gelatinase-dependent tissue destruction in inflammatory and malignant diseases.

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone-destructive lesions. This study examined the ability of immunoglobulin-producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP-8 and -13) in vivo and in vitro. Phorbol-12-myristate-13-acetate, interleukin-6, and tumour necrosis factor-alpha and heparin with the tumour promoter or cytokines potently enhanced (up to nine-fold) MMP-8 and -13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP-8 and -13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP-8 and MMP-13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP-13 was more frequently expressed than MMP-8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP-13 has a particularly important role in benign and malignant bone-destructive lesions.

Periodontal inflammation is characterized by irreversible degradation of periodontal ligament collagen fibers leading to loss of tooth attachment. Cultured gingival keratinocytes and fibroblasts express, in vitro, various matrix metalloproteinases (MMPs) which can degrade fibrillar collagens. We hypothesized that several MMPs are also synthesized in vivo by sulcular epithelium, and analyzed the collagenolytic MMPs (MMP-2, -8, -13, and -14) and matrilysin (MMP-7) in gingival tissue specimens and gingival crevicular fluid from adult and localized juvenile periodontitis patients by in situ hybridization, immunohistochemistry, and Western immunoblotting. MMP-2, -7, -8, and -13 were expressed in gingival sulcular epithelium. MMP-7 and -13 were also located in fibroblasts and macrophages, and MMP-8 in neutrophils. MMP-8- and -13-positive cells/mm2 were higher in periodontitis gingiva when compared with healthy control tissue (p < 0.01). In periodontal diseases, gingival sulcular epithelium expresses several, rather than a single, collagenolytic MMPs, and this proteolytic cascade is evidently responsible for the tissue destruction characteristic of adult and juvenile periodontitis.

Matrix metalloproteinase-8 (MMP-8) participates in skin wound healing and inflammation. We hypothesized that MMP-8 plays a role in wound healing after tooth extraction and in periapical inflammation. Bone formation, collagen metabolism, and inflammation in tooth extraction socket and in periapical lesions were analyzed in wild-type mice and in MMP-8-deficient (MMP-8(-/-)) mice. New trabecular bone area in the extraction sockets and in periapical lesions were similar in both groups. In extraction sockets significantly more type III procollagen was synthesized, and the neutrophil and MMP-9 levels were lower in MMP-8(-/-) mice. The amount of Fas ligand, identified as a substrate for MMP-8, was lower in alveolar mucosa but higher in alveolar bone of MMP-8(-/-) mice. These results indicate that MMP-8 can modulate inflammation and collagen metabolism of alveolar bone and mucosa.

Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 inPorphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8−/−) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses.P. gingivalisinfection induced accelerated site-specific alveolar bone loss in bothMMP8−/−and WT mice relative to uninfected mice. The most extensive bone degradation took place in theP. gingivalis-infectedMMP8−/−group. Surprisingly, MMP-8 significantly attenuated (P< 0.05)P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss inP. gingivalis-infectedMMP8−/−and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, theMMP8−/−mice underwent a shift toward a smaller HDL/VLDL particle sizes.P. gingivalisinfection increased the HDL/VLDL particle size in theMMP8−/−mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response toP. gingivaliswere significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially inMMP8−/−mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.

Wound healing involves highly controlled events including reepithelialization, neoangiogenesis, and reformation of the stromal compartment. Matrix metalloproteinases (MMPs) are a family of neutral zinc-dependent endopeptidases known to be essential for the wound-healing process. MMP-8 (collagenase-2) is a neutrophil-derived highly effective type I collagenase, recently indicated to be important for acute wound healing. MMP-26 is a more recent and less well-studied member of the MMP family. Our aim was to study the expression of MMP-8 and MMP-26 in human cutaneous wound repair and chronic wounds using histological methods and cell culture. MMP-8 expression was associated with epithelial cells, neutrophils, and other inflammatory cells in chronic human wounds. MMP-26 was prominently expressed in the extracellular compartment of most chronic wounds in close vicinity to the basement membrane area. MMP-26 was also expressed in acute day 1 wounds with declining expression thereafter. In vitro wound experiments showed that both MMP-8 and MMP-26 were expressed by migrating human mucosal keratinocytes. Inhibiting MMP-26 resulted in aberrant keratinocyte migration and proliferation. We conclude that MMP-8 and MMP-26 are differentially expressed in acute and chronic wounds.

Dextran sulfate sodium-induced inflammatory bowel disease in mice resembles human ulcerative colitis. In inflammatory bowel diseases matrix metalloproteinases contribute to tissue degradation. Laminin-5 is an anchoring filament protein in the basement membrane area that can be cleaved by matrix metalloproteinases. We investigated the expression of matrix metalloproteinases-2 and -8 and laminin-5 gamma2-chain in dextran sulfate sodium-induced mice by immunohistochemistry and in situ hybridization. Matrix metalloproteinase-8 expression was evidenced in the colon surface epithelial cells and the protein was more abundant in dextran sulfate sodium-induced mice colon. Matrix metallproteinase-2 and laminin-5 gamma2-chain colocalized in the colon surface epithelial cells and in the basement membrane zone as demonstrated by double immunostaining. In dextran sulfate sodium-induced colon, matrix metalloproteinase-2 immunoreactivity was detected in epithelial cells in the lower parts of the crypt and surrounding the degraded crypts. Matrix metalloproteinase-2 and -8 could participate in the local epithelial inflammatory processes and tissue destruction. The presence of laminin-5 gamma2-chain indicates alternative anchoring mechanisms in the colon, a compartment devoid of hemidesmosomes.

The aim of this study was to investigate the involvement of the MT1-MMP/MMP-2 cascade in induced sputum (IS) and bronchoalveolar lavage fluid (BALF) from bronchial asthma (BA) and bronchiectasis (BE) patients and healthy controls. The molecular forms and cellular origins of MT1-MMP and MMP-2 were determined by Western immunoblotting, immunohistochemistry and in situ hybridization. Elevated levels of soluble activated and autocatalyzed MT1-MMP species as well as activated forms of MMP-2 in IS and BALF samples from BA and BE patients were evidenced. The activation degrees of soluble MT1-MMP and MMP-2 were significantly correlated in BA and BE IS and BALF. Only low levels of both these MMPs were observed in healthy control IS and BALF. The co-expression of MMP-2 with MT1-MMP was evidenced by double immunostaining in bronchial epithelial cells, submucosal glandular cells, smooth muscle cells and monocyte/macrophages. The MT1-MMP/MMP-2 cascade is present and active in human inflammatory lung disease fluid and tissue samples. This cascade seemingly reflects the active destructive phases of these chronic lung diseases.

Background: Odontogenic keratocyst (KC) differs from other epithelial odontogenic cysts in regard to increased epithelial proliferation and a strong tendency to recur. Laminin-5 (Ln-5) is an epithelial anchoring filament component, which after modulation by certain matrix metalloproteinases (MMPs), like MMP-2 and MMP-13, induces epithelial cell migration. Methods: Using in situ hybridization and immunohistochemistry, we studied the Ln-5 gamma-2 chain expression related to the expression of MMP-2, -8, and -13 in different odontogenic cysts, including radicular cysts (RC; n = 11), follicular cysts (FC; n = 11), and odontogenic keratocysts (KC; n = 16). Results: Ln-5 mRNA was present in all cysts examined, while less than half of KCs and RCs (33 and 40%, respectively) demonstrated MMP-2 mRNA. MMP-13 mRNA was present in all KC samples. Ln-5 protein was located as a continuous ribbon in BM zone of all KCs, and MMP-2 and MMP-13 immunoreactivities colocated significantly with Ln-5 in that area. MMP-8 was expressed by stromal macrophages and epithelial goblet cells, but never located in BM zone. Conclusions: Our results indicate that the colocalization of Ln-5 with MMP-2 or MMP-13, but not with MMP-8, in BM zone of KCs, may be related to special characteristics of KC.

Matrix metalloproteinases (MMPs) contribute to extracellular matrix and basement membrane degradation in asthma. The present study analyzed molecular forms and degree of activation and expression of MMP-8 in bronchoalveolar lavage fluid (BALF), BALF cells, and bronchial tissue specimens from 14 steroid-naive asthma patients, 13 uncontrolled severe asthma patients, 13 controlled asthma patients, and 14 healthy subjects by Western immunoblotting, immunohistochemistry, and in situ hybridization. Immunohistochemistry and in situ hybridization revealed a prominent MMP-8 immunoreactivity in submucosal inflammatory, glandular, and shed, but not in intact bronchial epithelial cells of asthma patients. In BALF cytospins, both MMP-8 protein and mRNA expression were observed in epithelial cells, macrophages, and polymorphonuclear leukocytes (PMNs). MMP-8 was present in BALFs asthma patients in complex, pro- and active PMN-type, and pro- and active non-PMN–type forms. BALF MMP-8 was significantly converted to active form only in BALFs from steroid-naive and uncontrolled severe asthma patients, but not in BALFs from well-controlled asthma patients or healthy controls. A significant inverse correlation between BALF MMP-8 levels and FEV1 (r = −0.283, p = 0.04), and BALF activated MMP-8 forms and FEV1 (r = −0.427, p = 0.001) was detected. Overall, these data suggest that MMP-8 and its activation has an important role in the airway destruction, healing, remodeling, and treatment response in asthma.

Estrogen deficiency is associated with impaired cutaneous wound healing. Remodeling of the extracellular matrix in wound healing involves the action of matrix metalloproteinases on basement membrane zone components, especially laminin-5. We studied the effects of estrogen and a potent matrix metalloproteinase inhibitor, chemically modified non-antimicrobial tetracycline, CMT-8, on wound healing in ovariectomized rats. At the tissue level, laminin-5 gamma2-chain expression was decreased and the migration-inductive 80 kDa form of laminin-5 gamma2-chain was absent in ovariectomized rats when compared with sham and CMT-8- or estrogen-treated ovariectomized animals as detected by Western blotting. The highest levels of gelatinolytic activity (matrix metalloproteinase-2 and -9) were found in sham animals. Levels were reduced in ovariectomized rats and were lowest after treating ovariectomized rats with CMT-8 or estrogen as analyzed by functional activity assay and zymography. The total amount of membrane type 1-matrix metalloproteinase was unchanged in all groups. We conclude that CMT-8 and estrogen can promote wound healing in ovariectomized rats, not only by normalizing wound bed total collagen content and structure, but also by recovering the expression and processing of key molecules in wound healing, i.e., laminin-5 gamma2-chain. This study shows, for the first time, the role of estrogen and CMT-8 in laminin-5 gamma2-chain modulation in vivo.

An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation. One quarter of two cows was inoculated with endotoxin (Escherichia coli 055:B5). Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h. The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting. Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk. Five clinical E. coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement. Milk MMP levels increased 2h after the endotoxin challenge. Both MMP-2 and MMP-9 were involved in this early proteolytic event. These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC. Later, 6-12h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate. In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk. Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood-milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis. In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis.

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)-8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP-8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP-8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP-8 species were detected: 70-80 kD MMP-8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40-60 kD MMP-8 from non-PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP-8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP-8 complexes, evidently representing MMP-8 trapped by endogenous MMP inhibitors and/or MMP-8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP-8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP-8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP-8 in the inflamed lung. MMP-8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP-8 in the destruction of lung and bronchial tissues.

Gelatinolytic activity was analysed to study whether elevated activity previously found at the tracheal level of the respiratory tract of horses with chronic obstructive pulmonary disease (COPD) could also be found at the lower part of the respiratory tract. Furthermore, presence and significance of the gelatinolytic matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in respiratory secretions of healthy and COPD horses were determined. Elevated gelatinolytic matrix metalloproteinases were detected in bronchoalveolar and tracheobronchial secretions from COPD horses. The main pathologically elevated MMP was characterised to be MMP-9. Significantly increased MMP-9 activities as measured by gelatin zymography and Western blotting were found in all the respiratory samples from COPD horses compared to healthy horses. Elevation of active MMP-9 paralleled with increased gelatinase-associated lipocalin levels. Bronchoalveolar lavage fluid (BALF) epithelial cells, macrophages, neutrophils and lymphocytes expressed MMP-9 immunoreactivity demonstrated by immunocytochemistry and MMP-9 mRNA was expressed by bronchial epithelial cells of lung tissue section shown by in situ hybridisation. MMP-2 seems not to play a major role in chronic lung inflammation. No clear differences in MMP-2 or MMP-14 (a potent MMP-2 activator) levels were found when comparing the samples from COPD or healthy horses. These results suggests that MMP-9 could serve as a potential diagnostic marker for the active ongoing tissue remodelling in the acute phase of equine COPD. Increased gelatinolytic activity could be found at both tested respiratory tract levels. Therefore, tracheal epithelial lining fluid (TELF) samples can usefully serve as diagnostic material for detection of increased levels of the main gelatinolytic MMP, MMP-9, representing the entire diseased lung.

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.

Background: The tumor microenvironment (TME) promotes cancer growth, invasion and spread. Our human tissue (uterus leiomyoma) derived three-dimensional organotypic model (Nurmenniemi et al. Am J Pathol, 2009), approximates the natural TME and was developed for invasion studies. Here, the aim was to further characterize and modulate our model in order to better understanding the role of TME in cancer invasion. Methods: Intact or extensively rinsed (14 days) myoma discs were used for analyzing the invasion depth of mobile tongue carcinoma (HSC-3) cells and the degradation of type III collagen (IIICTP). Both broad spectrum (GM6001 and E64) and more specific protease inhibitors (peptide G for MT1-MMP and CTT2 for gelatinases) were added into the culture media. Invasion depth of HSC-3 cells, co-cultured either with (a) phorbol 12-myristate 13-acetate (PMA)-differentiated leukemic THP-1 macrophages (PMAM); (b) with PMA, lipo-poly-saccharine (LPS) and interferon gamma (IFN-γ) differentiated pro-inflammatory macrophages (M1); or with (c) PMA, interleukin-4 (IL-4) and IL-13 differentiated pro-tumorigenic macrophages (M2); were analyzed. In a set of experiments, HSC-3 cells were co-cultured with normal gingival fibroblasts (GF), or with human bone marrow derived mesenchymal stem cells (MSC). In addition, soluble myoma tissue rinsing samples or myoma tissue species were analyzed with gel electrophoresis, immunohistology and Western blot analyses. Results: We found that HSC-3 cells invaded twice as deep, but degraded six times less type III collagen when cultured on top of intact compared to rinsed myoma discs. Peptide G, GM6001 and E64 significantly inhibited (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 55. doi:1538-7445.AM2012-55