Diacerhein treatment reduces the severity of osteoarthritis in the canine cruciate-deficiency model of osteoarthritis.
(17/1761)
OBJECTIVE: To determine if diacerhein protects against the early stages of joint damage in a canine model of osteoarthritis (OA). METHODS: OA was induced in 20 adult mongrel dogs by transection of the anterior cruciate ligament of the left knee. Beginning the day after surgery, dogs in the active treatment group were dosed twice a day with capsules of diacerhein, providing a total daily dose of 40 mg/kg, for 32 weeks. Dogs in the control group received placebo capsules on the same schedule. Pathology in the unstable knee was assessed arthroscopically 16 weeks after surgery and by direct observation when the dogs were killed 32 weeks after surgery. The severity of gross joint pathology was recorded, and samples of the medial femoral condyle cartilage and the synovial tissue adjacent to the central portion of the medial meniscus were collected for histologic evaluation. Water content and uronic acid concentration of the articular cartilage from the femoral condyle were determined, and collagenolytic activity in extracts of cartilage pooled from the medial and lateral tibial plateaus was assayed against 14C-labeled collagen fibers. RESULTS: Diacerhein treatment slowed the progression of OA, as measured by grading of gross changes in the unstable knee at arthroscopy 16 weeks after cruciate ligament transection (P = 0.04) and at the time the animals were killed, 32 weeks after surgery (P = 0.05). However, 32 weeks after ACL transection, the mean proteoglycan concentration and water content of the OA cartilage and the level of collagenolytic activity in extracts of the cartilage were not significantly different in the diacerhein treatment group than in the placebo treatment group. CONCLUSION: Diacerhein treatment significantly reduced the severity of morphologic changes of OA compared with placebo. These findings support the view that diacerhein may be a disease-modifying drug for OA. (+info)
Messenger RNA expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human hepatocellular carcinoma.
(18/1761)
BACKGROUND: The prognosis of patients with hepatocellular carcinoma is relatively poor because of the high rate of intrahepatic recurrences. We have previously demonstrated an association between enhanced secretion of active matrix metalloproteinases (MMPs; gelatinase A and matrilysin) and early recurrence in hepatocellular carcinoma. The aim of this study was to examine further the relationship between messenger RNA levels of metalloproteinases and their inhibitors and behavior of this carcinoma. METHODS: Messenger RNA expression of gelatinase A, gelatinase B, matrilysin and tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 were analyzed in 30 patients with hepatocellular carcinoma by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The results were contrasted with the clinicopathological data of the patients. RESULTS: Enhanced mRNA expression of gelatinase A, gelatinase B and matrilysin in tumor was observed in 20, 22 and 19 of 30 patients, respectively. Enhanced mRNA expression of gelatinase A or gelatinase B and of matrilysin showed trends toward presence of capsular invasion (P = 0.078) and intrahepatic metastasis (P = 0.064), respectively. Concomitant overexpression of gelatinase A and matrilysin was associated with portal invasion, intrahepatic metastasis and recurrence within the first postoperative year (P < 0.05). A modest increase of mRNA expression of TIMP-1 and TIMP-2 in tumor was observed in half of the patients, but did not correlate with any clinicopathological features. CONCLUSION: Our results suggest that semiquantitative RT-PCR analysis of MMPs may be helpful in disease management of patients with hepatocellular carcinoma. (+info)
Expression of matrix metalloproteinases during murine chorioallantoic placenta maturation.
(19/1761)
A large body of experimental evidence supports the participation of two groups of extracellular proteases, matrix metalloproteinases (MMPs), and plasminogen activators/plasmin, in tissue remodeling in physiological and pathological invasion. In the late mouse placenta, several tissue remodeling and cell invasion processes take place. Spongiotrophoblast migration into maternal decidua, as well as decidual extracellular matrix remodeling require the coordinated action of extracellular proteolytic enzymes. Via Northern and in situ hybridization, we have analyzed the spatio-temporal expression patterns of members of the MMP family (stromelysin-3, gelatinases A and B), as well as their inhibitors TIMP-1, -2 and -3 in late murine placenta (days 10.5 to 18.5 of gestation). Gelatinase activity in placental extracts was assessed by substrate zymography. Gelatinase A and stromelysin-3 were found to be prominently expressed in decidual tissue; shortly after midpregnancy, the decidual expression patterns of gelatinase A and stromelysin-3 became overlapping with each other, as well as with the expression domain of TIMP-2. On the other hand, gelatinase B transcripts were expressed only by trophoblast giant cells at day 10.5, and were downregulated at later stages. TIMP-1 and TIMP-3 transcripts were detected in decidual periphery at day 10.5, while later the expression was restricted to the endometrial stroma and spongiotrophoblasts, respectively. The areas of stromelysin-3 expression were the same (giant trophoblasts) or adjacent (decidua) to those where urokinase (uPA) transcripts were detected, suggesting a possible cooperation between these proteinases in placental remodeling. We generated mice doubly deficient for stromelysin-3 and uPA, and report here that these mice are viable and fertile. Furthermore, these animals do not manifest obvious placental abnormalities, thereby suggesting the existence of compensatory/redundant mechanisms involving other proteolytic enzymes. Our findings document the participation of MMPs and their inhibitors in the process of late murine placenta maturation, and warrant the characterization of other members of the MMP family, like membrane type-MMPs, in this process. (+info)
Paradoxical preservation of a lipopolysaccharide response in C3H/HeJ macrophages: induction of matrix metalloproteinase-9.
(20/1761)
C3H/HeJ mice carry a mutant allele (Lpsd) of a recently identified gene whose normal allele (Lpsn) confers responsiveness to bacterial LPS in C3H/HeN and most other mouse strains. Recently we reported differential display analysis of matched macrophage-derived cell lines from C3H/HeJ and C3H/HeN mice under LPS-free conditions. Of the approximately 12,000 transcripts evaluated, 4 were differentially expressed. One transcript represented secretory leukocyte protease inhibitor. In this study, we report another differentially expressed transcript, mouse matrix metalloprotease-9 (MMP-9). Like secretory leukocyte protease inhibitor, MMP-9 was expressed constitutively in the Lpsd macrophage cell line and not in the Lpsn cell line. Similarly, two additional macrophage cell lines that respond readily to LPS by producing nitric oxide and TNF expressed no MMP-9 under LPS-free conditions. However, in all four cell lines, LPS induced MMP-9 or augmented its expression. In primary macrophages, concentrations of LPS in the ng/ml range augmented the expression of MMP-9 mRNA. Paradoxically, macrophages from Lpsd mice expressed more MMP-9 transcripts than macrophages from Lpsn mice. In contrast, the induction of TNF in response to LPS was much more pronounced in Lpsn macrophages. The present findings with MMP-9 suggest that homozygosity at Lpsd does not so much prevent a response to LPS as dysregulate it, resulting in the suppression of some LPS signaling pathways and the preservation of others. (+info)
N-glycan structures of matrix metalloproteinase-1 derived from human fibroblasts and from HT-1080 fibrosarcoma cells.
(21/1761)
Matrix metalloproteinase-1 (MMP-1) is a collagenolytic metalloproteinase capable of cleaving native triple-helical forms of several collagen subtypes, as well as a number of non-collagenous substrates. The role of MMP-1 in various diseases affecting the connective tissue is well characterized. MMP-1 is secreted as both glycosylated and unglycosylated species, and the two forms have been shown to be identical with respect to substrate specificity, specific activity and inhibitory profile. No function for the glycan moiety of the enzyme has been ascribed to date. In the present study, we report on the detailed characterization of MMP-1-derived oligosaccharides. Using strategies based on sequential exoglycosidase digestion combined with matrix-assisted laser desorption ionization-time of flight MS and electrospray tandem MS, we have characterized the N-glycan structures of MMP-1, derived from human dermal fibroblasts and from the HT-1080 fibrosarcoma cell line. MMP-1 derived from fibroblasts was found to carry mainly alpha 2,3-sialylated complex-type diantennary glycans. On the other hand, HT-1080 cells produce MMP-1 that has a heterogeneous glycosylation pattern, comprising diantennary glycans carrying Lewis X, LacdiNAc, sialylated LacdiNAc and GalNAc beta 1,4 (Fuc alpha 1,3)GlcNAc (LacdiNAc analogue of Lewis X) as terminal elements. We also show that, of the two potential glycosylation sites in the MMP-1 sequence, only Asn120 is used. (+info)
Differential inhibition of collagenase and interleukin-1alpha gene expression in cultured corneal fibroblasts by TGF-beta, dexamethasone, and retinoic acid.
(22/1761)
PURPOSE: Expression of the genes for collagenase and interleukin-1alpha (IL-1alpha) are induced as stromal cells become activated to the repair fibroblast phenotype after injury to the cornea. This investigation examines the mechanisms whereby expression of these genes is inhibited by transforming growth factor-beta (TGF-beta), dexamethasone (DEX), or retinoic acid (RET A). METHODS: A model of freshly isolated cultures of corneal stromal cells and early passage cultures of corneal fibroblasts was used in these studies. This model reproduces the events of stromal cell activation in the corneal wound. RESULTS: In early passage cultures of corneal fibroblasts, expression of collagenase is under obligatory control by autocrine IL-1alpha. IL-1alpha controls its own expression through an autocrine feedback loop that is dependent on transcription factor NF-kappaB. TGF-beta, DEX, and RET A were each effective inhibitors of collagenase gene expression in these cells. Furthermore, these agents have the capacity to inhibit expression of IL-1alpha and this was correlated with their ability to affect DNA-binding activity of NF-kappaB. However, TGF-beta, DEX, and RET A were also effective inhibitors of the low level of collagenase expressed by freshly isolated corneal stromal cells that cannot express IL-1alpha. CONCLUSIONS: In cells with an active IL-1alpha autocrine loop there are at least two distinct signaling pathways by which collagenase gene expression can be modulated. The results of this study demonstrate that TGF-beta, DEX, and RET A differentially inhibit collagenase and IL-1alpha gene expression. This information will be useful in the design of therapeutic modalities for fibrotic disease in the cornea and other parts of the eye. (+info)
Residue 2 of TIMP-1 is a major determinant of affinity and specificity for matrix metalloproteinases but effects of substitutions do not correlate with those of the corresponding P1' residue of substrate.
(23/1761)
The unregulated activities of matrix metalloproteinases (MMPs) are implicated in disease processes including arthritis and tumor cell invasion and metastasis. MMP activities are controlled by four homologous endogenous protein inhibitors, tissue inhibitors of metalloproteinases (TIMPs), yet different TIMPs show little specificity for individual MMPs. The large interaction interface in the TIMP-1.MMP-3 complex includes a contiguous region of TIMP-1 around the disulfide bond between Cys1 and Cys70 that inserts into the active site of MMP-3. The effects of fifteen different substitutions for threonine 2 of this region reveal that this residue makes a large contribution to the stability of complexes with MMPs and has a dominant influence on the specificity for different MMPs. The size, charge, and hydrophobicity of residue 2 are key factors in the specificity of TIMP. Threonine 2 of TIMP-1 interacts with the S1' specificity pocket of MMP-3, which is a key to substrate specificity, but the structural requirements in TIMP-1 residue 2 for MMP binding differ greatly from those for the corresponding residue of a peptide substrate. These results demonstrate that TIMP variants with substitutions for Thr2 represent suitable starting points for generating more targeted TIMPs for investigation and for intervention in MMP-related diseases. (+info)
In response to cutaneous injury, expression of collagenase-1 is induced in keratinocytes via alpha2beta1 contact with native type I collagen, and enzyme activity is essential for cell migration over this substratum. However, the cellular mechanism(s) mediating integrin signaling remain poorly understood. We demonstrate here that treatment of keratinocytes cultured on type I collagen with epidermal growth factor receptor (EGFR) blocking antibodies or a specific receptor antagonist inhibited cell migration across type I collagen and the matrix-directed stimulation of collagenase-1 production. Additionally, stimulation of collagenase-1 expression by hepatocyte growth factor, transforming growth factor-beta1, and interferon-gamma was blocked by EGFR inhibitors, suggesting a required EGFR autocrine signaling step for enzyme expression. Collagenase-1 mRNA was not detectable in keratinocytes isolated immediately from normal skin, but increased progressively following 2 h of contact with collagen. In contrast, EGFR mRNA was expressed at high steady-state levels in keratinocytes isolated immediately from intact skin but was absent following 2 h cell contact with collagen, suggesting down-regulation following receptor activation. Indeed, tyrosine phosphorylation of the EGFR was evident as early as 10 min following cell contact with collagen. Treatment of keratinocytes cultured on collagen with EGFR antagonist or heparin-binding (HB)-EGF neutralizing antibodies dramatically inhibited the sustained expression (6-24 h) of collagenase-1 mRNA, whereas initial induction by collagen alone (2 h) was unaffected. Finally, expression of collagenase-1 in ex vivo wounded skin and re-epithelialization of partial thickness porcine burn wounds was blocked following treatment with EGFR inhibitors. These results demonstrate that keratinocyte contact with type I collagen is sufficient to induce collagenase-1 expression, whereas sustained enzyme production requires autocrine EGFR activation by HB-EGF as an obligatory intermediate step, thereby maintaining collagenase-1-dependent migration during the re-epithelialization of epidermal wounds. (+info)