
2023 Impact Factor
1Department of Molecular Medicine, Tissue Injury Defense Research Center, Ewha Womans University Medical School, Seoul 158-710
2Department of Molecular Medicine, Kyongbuk National University, Daegu 700-842, Republic of Korea
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate cell-matrix composition and are also involved in processing various bioactive molecules such as cell-surface receptors, chemokines, and cytokines. Our group recently reported that MMP-3, -8, and -9 are upregulated during microglial activation and play a role as proinflammatory mediators (
Matrix metalloproteinases (MMPs), also called matrixins, are Zn2+-dependent endopeptidases that degrade extracellular matrix proteins. MMPs are involved in normal brain development, plasticity, angiogenesis, and repair following brain injury (Agrawal
Tumor necrosis factor-α (TNF-α), a pleiotropic pro-inflammatory cytokine, mediates inflammation, cell activation, and cell migration (Aggarwal
Recently, we demonstrated that MMP-3, -8, and -9 mediate inflammatory reactions through cleavage and activation of protease-activated receptor-1 in α-synuclein-stimulated microglia (Lee
LPS (
The immortalized murine BV2 microglial cells (Bocchini
BV2 cells (1×105 cells per well in a 48-well plate) were pretreated with various concentrations (0?100 μM) of NNGH, M8I, or M9I for 1 h and stimulated with LPS (100 ng/ml) for 3 h. The supernatants of the cultured microglia were then collected, and the TNF-α concentration was measured by ELISA according to the procedure recommended by the supplier (BD Biosciences, San Jose, CA, USA). To measure the amount of TNF-α in cell lysates, BV2 cells were lysed in PBS by 10 passes through a 26-gauge needle. Cells were then centrifuged for 10 min at 14,000×g, and supernatant was taken for determination of intracellular TNF-α level by ELISA.
TACE activity was assayed using the SensoLyteTM 520 TACE activity assay kit (AnaSpec, Fremont, CA, USA). Recombinant protein (rhTACE or rhMMP, 250 ng) with TAPI-0 or MMP inhibitor (0.1, 0.5, or 1 μM) were incubated with TACE substrate. TACE activity was then determined by continuous detection of peptide cleavage in wells for 30?60 min using a fluorescence plate reader. TACE activity was expressed as the change in fluorescence intensity at excitation of 490 nm/emission of 520 nm.
A liquid chromatography-mass spectrometry (LC-MS)-based pro-TNF-α cleavage assay was performed to identify interactions between pro-TNF-α and MMP-3 or MMP-9 using residues 71?82 (Ac-S71PLAQAVRSSSR82-NH2) (Peptron, Daejeon, South Korea) (Minond
Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. The data are presented as means ± S.E.M., and statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test.
To examine the effects of MMP inhibitors on TNF-α secretion, BV2 cells were pretreated with one of the three kinds of MMP inhibitor for 1 h and stimulated with LPS (100 ng/ml). After a 3-h incubation with LPS, the supernatants were removed from cultured cells, and the TNF-α concentration was measured. The percent release of TNF-α was determined by dividing the amount of TNF-α in the supernatant by the total amount of TNF-α (cell-associated+secreted TNF-α). As shown in Fig. 2A, LPS led to secretion of approximately 88% of TNF-α into the cell culture media, whereas pretreatment with MMP inhibitors suppressed LPS-induced TNF-α secretion in a dose-dependent manner. Among the three types of inhibitors, the inhibitory effect of M8I was most prominent, followed by NNGH and M9I. Intriguingly, the cell-associated TNF-α levels were not significantly altered by the MMP inhibitors. The results suggest that MMP inhibitors are mainly involved in the secretion of the active form of TNF-α.
TNF-α is produced as a proform (26 kDa) and secreted in an active form (17 kDa) after cleavage of its prodomain by TACE (Bahia and Silakari, 2010). To further dissect the mechanism of the effects of MMP inhibitors on TNF-α secretion, we examined whether three types of MMP inhibitors inhibit TACE activity using recombinant human TACE protein (rhTACE). Consistent with the TNF-α secretion data, the MMP inhibitors inhibited TACE activity of rhTACE in an efficacy order of M8I>NNGH>M9I. Of note, M8I and NNGH suppressed the TACE activity significantly more than TAPI-0, a general TACE inhibitor. The IC50 of each inhibitor of TACE activity of rhTACE are shown in Fig. 3B. We previously reported that MMP-8 itself has TACE activity (Lee
We previously reported that MMP-8 has TACE activity by demonstrating the cleavage of the active site of proTNF-α (A76/V77, A74/Q75) (Lee
In the present study, we compared the efficacy of chemical inhibitors of MMPs in the regulation of TNF-α in the context of microglial activation. We observed that MMP inhibitors inhibited TNF-α secretion and TACE activity in an efficacy order of M8I>NNGH>M9I (Fig. 2, 3). Interestingly, we found that MMP-3, -8, and -9 themselves have TACE enzymatic activity by cleaving the prodomain of TNF-α (Fig. 3). A subsequent TNF-α cleavage assay identified cleavage sites of the prodomain of TNF-α by each MMP (Fig. 4). Although MMP-3, -8, and -9 commonly cleaved a conventional cleavage site in the N-terminal propeptide of TNF-α, one or two additional cleavage sites were identified depending on the MMP (Fig. 4E).
Because MMPs are involved in various acute and chronic diseases such as arthritis, multiple sclerosis, atherosclerosis, stroke, and cancer, many pharmaceutical companies are actively developing compounds that can be used to block their action. The major synthetic inhibitors of MMPs are based on a hydroxamate structure (Hu
We previously reported that administration of M8I significantly inhibits microglial activation and expression/secretion of TNF-α in the brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice (Lee