Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Adrenomedullin production is increased in colorectal adenocarcinomas; its relation to matrix metalloproteinase-9

Adrenomedullin (AM) is highly expressed in various cancer cell lines, suggesting a possible association with cancer growth. In the present study, we examined the expression and/or concentration of AM, its related peptide, adrenomedullin2/intermedin (AM2/IMD) and their receptors in human colorectal cancer and the surrounding normal tissue. In addition, we assessed the correlation between the expression of AM and AM2/IMD with that of vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9. Using a specific immunoradiometric assay, we found that AM concentrations were 2-11-fold higher in colorectal cancer tissues than in the surrounding normal tissues. Moreover, real-time quantitative RT-PCR showed that the expression levels of preproAM (+548%), preproAM2/IMD (+2674%), calcitonin receptor-like receptor (CLR) (+518%), receptor activity modifying protein (RAMP)2 (+281%), RAMP3 (+178%), VEGF-A (+277%) and MMP-9 (+864%) mRNAs were significantly higher in cancer tissues than in the surrounding normal tissues, and there was a positive correlation between the gene expressions of MMP-9 and preproAM (r = 0.352; p = 0.005), but not with preproAM2/IMD (r = 0.041, p = 0.406). Both AM and AM2/IMD immunoreactivity were detected mainly within cancer cells, whereas MMP-9 immunoreactivity was mostly seen in the surrounding stroma. These findings suggest that AM produced in colorectal tumors acts in concert with MMP-9 in the stroma to contribute to the pathogenesis of colorectal cancer.     

Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy

Diabetic retinopathy represents the most common causes of vision loss in patients affected by diabetes mellitus. The cause of vision loss in diabetic retinopathy is complex and remains incompletely understood. One of the earliest changes in the development of retinopathy is the accelerated apoptosis of retinal microvascular cells and the formation of acellular capillaries by unknown mechanism. Results of a recent research suggest an important role of matrix metalloproteinases (MMPs) in the development of diabetic retinopathy. MMPs are a large family of proteinases that remodel extracellular matrix components, and under pathological condition, its induction is considered as a negative regulator of cell survival; and in diabetes, latent MMPs are activated in the retina and its capillary cells, and activation of MMP-2 and -9 induces apoptosis of retinal capillary cells. This review will focus on the MMP-2 and MMP-9 in the diabetic retina with special reference to oxidative stress, mitochondria dysfunction, inflammation and angiogenesis, as well as summarizing the current information linking these proteins to pathogenesis of diabetic retinopathy.     

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.     

Inhibition of matrix metalloproteinase-9 by interferons and TGF-beta1 through distinct signalings accounts for reduced monocyte invasiveness

Cytokines may provide signals for regulating human monocyte matrix metalloproteinase-9 (MMP-9) activity. In this study, we investigated the roles of interferons (IFN) type I/II and transforming growth factor-beta1 (TGF-beta1) in MMP-9-mediated invasiveness. MMP-9 antibody and inhibitor, IFNs and TGF-beta1 inhibited monocyte transmigration through Matrigel. IFNs and TGF-beta1 downregulated MMP-9 mRNA, protein and activity levels. The inhibitory action of IFNs was associated with the STAT1/IRF-1 pathway since the JAK inhibitor AG490 blocked STAT1 phosphorylation, IRF-1 synthesis and counteracted the blockade of MMP-9 release. TGF-beta1-mediated MMP-9 inhibition appeared STAT1/IRF-1-independent but reversed by the protein tyrosine kinase inhibitor tyrphostin 25. Our data point out the importance of IFNs and TGF-beta1 in the control of monocyte MMP-9-mediated extravasation.     

Inhibition of matrix metalloproteinase-9 activity by doxycycline ameliorates RANK ligand-induced osteoclast differentiation in vitro and in vivo

Tetracycline antibiotics, including doxycycli\e (DOX), have been used to treat bone resorptive diseases, partially because of their activity to suppress osteoclastogenesis induced by receptor activator of nuclear factor kappa B ligand (RANKL). However, their precise inhibitory mechanism remains unclear. Therefore, the present study examined the effect of Dox on osteoclastogenesis signaling induced by RANKL, both in vitro and in vivo. Although Dox inhibited RANKL-induced osteoclastogenesis and down-modulated the mRNA expression of functional osteoclast markers, including tartrate-resistant acid phosphatase (TRAP) and cathepsin K, Dox neither affected RANKL-induced MAPKs phosphorylation nor NFATc1 gene expression in RAW264.7 murine monocytic cells. Gelatin zymography and Western blot analyses showed that Dox down-regulated the enzyme activity of RANKL-induced MMP-9, but without affecting its protein expression. Furthermore, MMP-9 enzyme inhibitor also attenuated both RANKL-induced osteoclastogenesis and up-regulation of TRAP and cathepsin K mRNA expression, indicating that MMP-9 enzyme action is engaged in the promotion of RANKL-induced osteoclastogenesis. Finally, Dox treatment abrogated RANKL-induced osteoclastogenesis and TRAP activity in mouse calvaria along with the suppression of MMP9 enzyme activity, again without affecting the expression of MMP9 protein. These findings suggested that Dox inhibits RANKL-induced osteoclastogenesis by its inhibitory effect on MMP-9 enzyme activity independent of the MAPK-NFATc1 signaling cascade.     

cAMP elevating agents suppress secretory phospholipase A(2)-induced matrix metalloproteinase-2 activation

Phospholipase A2 proteins are major regulators of the arachidonic acid cascade and are involved in various cellular responses. Previously, we reported that group IB PLA2 proteins stimulate MMP-2 activation and subsequent cell migration. Here, we describe a novel mechanism whereby sPLA2-induced proMMP-2 activation is regulated by intracellular cAMP in HT1080 cells, although sPLA2 itself had no effect on the regulation of cAMP levels. Exogenous dibutyryl cAMP (a cAMP analogue) strongly inhibited proMMP-2 activation, and cAMP elevating agents, namely, cholera toxin (a Gs activator) and forskolin (an adenylyl cyclase activator), abrogated basal and sPLA2-induced proMMP-2 activation. We also found that the down-regulation of TIMP-2 expression and extracellular signal-regulated kinase (ERK)1/2 activation by sPLA2 were blocked by increasing the intracellular cAMP level. Taken together, our data indicate that sPLA2-induced proMMP-2 activation is influenced by intracellular cAMP levels via the modulations of TIMP-2 expression and ERK1/2 activation.     

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.     

Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.