LRP1 Controls TNF Release via the TIMP-3/ADAM17 Axis in Endotoxin-Activated Macrophages.

The metalloproteinase ADAM17 plays a pivotal role in initiating inflammation by releasing TNF from its precursor. Prolonged TNF release causes many chronic inflammatory diseases, indicating that tight regulation of ADAM17 activity is essential for resolution of inflammation. In this study, we report that the endogenous ADAM17 inhibitor TIMP-3 inhibits ADAM17 activity only when it is bound to the cell surface and that cell surface levels of TIMP-3 in endotoxin-activated human macrophages are dynamically controlled by the endocytic receptor LRP1. Pharmacological blockade of LRP1 inhibited endocytic clearance of TIMP-3, leading to an increase in cell surface levels of the inhibitor that blocked TNF release. Following LPS stimulation, TIMP-3 levels on the surface of macrophages increased 4-fold within 4 h and continued to accumulate at 6 h, before a return to baseline levels at 8 h. This dynamic regulation of cell surface TIMP-3 levels was independent of changes in TIMP-3 mRNA levels, but correlated with shedding of LRP1. These results shed light on the basic mechanisms that maintain a regulated inflammatory response and ensure its timely resolution.

TIMP-3 suppression induces choroidal neovascularization by moderating the polarization of macrophages in age-related macular degeneration.

PURPOSE: To investigate the role of tissue inhibitor of metalloproteinases-3 (TIMP-3) as a key moderator of macrophage polarization in choroidal neovascularization (CNV) lesions of model mice and in bone marrow-derived macrophage (BMDM). METHOD: We used siR-TIMP-3 to transfect BMDM and gave an intravitreal injection of siR-TIMP-3 to laser-induced CNV mice model, real time-PCR and western blot were applied for detecting the expressions of TIMP-3 and macrophages' biomarker. Besides, CNV lesions in different treatment groups of animal model were examined by the optical coherence tomography angiography (OCTA). RESULTS: Our experimental data showed that lack of TIMP-3 stimulated M2 polarization proved by real time-PCR and western blot in BMDMs and CNV mice model. Moreover, intravitreal injection of siR-TIMP-3 accelerated CNV formation using OCTA, which indicated that TIMP-3 suppression is related to pro-angiogenesis of M2 macrophage. CONCLUSION: We showed that the absence of TIMP-3 leads to a more pro-angiogenic microenvironment, playing a key role in CNV formation by positively modulating M2 polarization. The role of TIMP-3 in the regulating inflammation and novel therapeutic target of nAMD needs to be further studied.

MicroRNA-206 regulates the secretion of inflammatory cytokines and MMP9 expression by targeting TIMP3 in Mycobacterium tuberculosis-infected THP-1 human macrophages.

Tuberculosis (TB) is a serious disease that is characterized by Mycobacterium tuberculosis (M.tb)-triggered immune system impairment and lung tissue damage shows limited treatment options. MicroRNAs (miRNAs) are regulators of gene expression that play critical roles in many human diseases, and can be up- or downregulated by M.tb infection in macrophage. Recently, tissue inhibitor of matrix metalloproteinase (TIMP) 3 has been found to play roles in regulating macrophage inflammation. Here, we found that TIMP3 expression was regulated by miR-206 in M.tb-infected THP-1 human macrophages. In THP-1 cells infected with M.tb, the miR-206 level was significantly upregulated and the expression of TIMP3 was markedly decreased when the secretion of inflammatory cytokines was increased. Inhibition of miR-206 markedly suppressed inflammatory cytokine secretion and upregulated the expression of TIMP3. In contrast, the upregulation of miR-206 promoted the matrix metalloproteinase (MMP) 9 levels and inhibited TIMP3 levels. Using a dual-luciferase reporter assay, a direct interaction between miR-206 and the 3'-untranslated region (UTR) of TIMP3 was confirmed. SiTIMP3, the small interfering RNA (siRNA) specific for TIMP3, significantly attenuated the suppressive effects of miR-206-inhibitor on inflammatory cytokine secretion and MMP9 expression. Our data suggest that miR-206 may function as an inflammatory regulator and drive the expression of MMP9 in M.tb-infected THP-1 cells by targeting TIMP3, indicating that miR-206 is a potential therapeutic target for patients with TB.

Effect of Xianziyizhen Recipe Capsule on PGI2-PPARδ Signaling Pathway in Embryo Implantation Dysfunction Mice.

PROBLEM: We investigated the effect of Xianziyizhen recipe capsule (XRC), a kidney-tonifying herb, on the PGI2-PPARδ signaling pathway at the maternal-fetal interface in embryo implantation dysfunction (EID) mice. METHOD OF STUDY: Intragastric administration of Progynova (estradiol) or XRC was performed in EID mouse model, following experimental induction of kidney deficiency by co-treatment with chemotherapy drug hydroxyurea and antiprogesterone mifepristone. The PPARδ and IL-11 mRNA expression in endometrium were detected by real-time relative reverse transcription-polymerase chain reaction (RT-PCR). Further, the protein expression of COX-2, PGI2, MMP-9, and TIMP-3 was detected in endometrial glandular epithelium and in stromal cells by immunohistochemical (IHC) assay. RESULTS: The results showed that hydroxyurea and mifepristone-induced EID were associated with significantly lower PPARδ and IL-11 mRNA levels in endometrium and reduced COX-2, PGI2, MMP-9, and TIMP-3 levels in endometrial glandular epithelium, compared with normal controls. However, XRC and Progynova treatment reversed these effects, leading to significant increases in PPARδ and IL-11 mRNA expression, and COX-2, PGI2, MMP-9 and TIMP-3 protein levels, when compared with the levels observed in EID mice. CONCLUSION: These results strongly suggested that XRC is beneficial in EID treatment and that XRC may mediate its effects through regulation of the PGI2-PPARδ signaling pathway.

The endogenous protease inhibitor TIMP-1 mediates protection and recovery from cutaneous photodamage.

UVB exposure is well known to induce skin photodamage and photoaging that correlates with qualitative and quantitative deterioration of the dermal extracellular matrix (ECM) because of the upregulation of matrix metalloproteinases (MMPs). Although inhibitory effects of tissue inhibitor of metalloproteinases (TIMPs) on most MMPs have been reported, the protective role of TIMP-1 against photodamage is poorly understood. To address this, TIMP-1 function was augmented or abolished in a human skin xenograft photodamage model after the confirmation of significantly diminished TIMP-1 expression both in photoaged and intrinsically aged skins. During a chronic UVB exposure regimen, pre-treatment with a lentiviral vector overexpressing TIMP-1 or concomitant administration of an anti-TIMP-1-neutralizing antibody (NAB) led to photoprotection or more severe photodamage, respectively. Overexpression of TIMP-1 resulted in significant inhibition of UVB-induced ECM degradation, as well as suppression of decreased skin elasticity and roughness, whereas the NAB-mediated inhibition of TIMP-1 had opposite effects. Furthermore, UVB-induced production of the pro-inflammatory cytokine, tumor necrosis factor α, was inhibited by TIMP-1 treatment of human keratinocytes. Taken together, these data shed light on the important role of TIMP-1 in protection and recovery from cutaneous photodamage because of its suppression of ECM degradation and inflammation.

Stromal TIMP3 regulates liver lymphocyte populations and provides protection against Th1 T cell-driven autoimmune hepatitis.

Lymphocyte infiltration into epithelial tissues and proinflammatory cytokine release are key steps in autoimmune disease. Although cell-autonomous roles of lymphocytes are well studied in autoimmunity, much less is understood about the stromal factors that dictate immune cell function. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls systemic cytokine bioavailability and signaling by inhibiting the ectodomain shedding of cytokines and their receptors. The role of TIMP3 in cytokine biology is emerging; however, its contribution to cellular immunology remains unknown. In this study, we show that TIMP3 produced by the hepatic stroma regulates the basal lymphocyte populations in the liver and prevents autoimmune hepatitis. TIMP3 deficiency in mice led to spontaneous accumulation and activation of hepatic CD4(+), CD8(+), and NKT cells. Treatment with Con A in a model of polyclonal T lymphocyte activation resulted in a greatly enhanced Th1 cytokine response and acute liver failure, which mechanistically depended on TNF signaling. Bone marrow chimeras demonstrated that TIMP3 derived from the stromal rather than hematopoietic compartment provided protection against autoimmunity. Finally, we identified hepatocytes as the major source of Timp3 in a resting liver, whereas significant Timp3 gene transcription was induced by hepatic stellate cells in the inflamed liver. These results uncover metalloproteinase inhibitors as critical stromal factors in regulating cellular immunity during autoimmune hepatitis.

TACE inhibition amplifies TNF-alpha-mediated colonic epithelial barrier disruption.

Inflammatory bowel diseases (IBD) are characterized by tumor necrosis factor alpha (TNF-alpha)-mediated epithelial barrier disruption. TNF-alpha production and the bioavailability of its receptors on the cell surface are regulated by TACE (TNF-alpha converting enzyme), a pleiotropic metalloprotease also known as ADAM17, and its specific inhibitor TIMP3. We therefore examined ADAM17 and TIMP3 expression in human intestinal epithelial cells (IEC) using immunohistochemistry on tissue microarrays and real-time PCR on preparations of IEC isolated from human normal and IBD colon. The effects of TACE inhibition by TIMP3 or a pharmacological inhibitor were assessed in inflammatory conditions on a TIMP3-deficient colonic cell line HT29-Cl.16E. Both TACE and TIMP3 were found to be constitutively expressed by intestinal epithelial cells in the normal and inflammatory human intestinal barrier. In the TIMP3-deficient cell line, the addition of recombinant human TIMP3 or of Tapi-2, a pharmacological ADAM17 inhibitor, i) sensitized the cells to TNF-alpha-mediated hyperpermeability, ii) down-regulated tight junction-associated protein expression and iii) inhibited TNFRI shedding. In conclusion, our data showed that TACE and TIMP3 were co-expressed in the human intestinal barrier and that TACE inhibition, either physiologically or pharmacologically, amplified TNF-alpha-mediated hyperpermeability. TIMP3 could thus play a major role in inflammatory conditions by creating an autocrine effect leading to amplified epithelial barrier hyperpermeability.

Interleukin-10 regulates TNF-alpha-converting enzyme (TACE/ADAM-17) involving a TIMP-3 dependent and independent mechanism.

IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.

Tissue inhibitor of metalloproteinase 3 regulates TNF-dependent systemic inflammation.

Host response to infectious agents must be rapid and powerful. One mechanism is the release of presynthesized membrane-bound TNF. TNF shedding is mediated by TNF-alpha converting enzyme, which is selectively inhibited by the tissue inhibitor of metalloproteinase 3 (TIMP3). We show that loss of TIMP3 impacts innate immunity by dysregulating cleavage of TNF and its receptors. Cultured timp3-/- macrophages release more TNF in response to LPS than wild-type macrophages. In timp3-/- mice, LPS causes serum levels of TNF and its receptors to rise more rapidly and remain higher compared with wild-type mice. The altered kinetics of ligand and receptor shedding enhances TNF signaling in timp3-/- mice, indicated by elevated serum IL-6. Physiologically, timp3-/- mice are more susceptible to LPS-induced mortality. Ablation of the TNF receptor gene p55 (Tnfrsf1a) or treatment with a synthetic metalloproteinase inhibitor rescues timp3-/- mice. Thus, TIMP3 is essential for normal innate immune function.

A microarray study to characterize the molecular mechanism of TIMP-3-mediated tumor rejection.

Glial cell invasion is a multistep cellular process that involves a complex system of tightly regulated proteases (matrix metalloproteinases; MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases; TIMPs) to mediate the degradation of the basement membrane and extracellular matrix. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a matrix-bound inhibitor of MMPs. In the present study, we have overexpressed the TIMP3 gene in human glioma cells with a herpes simplex virus type 1 amplicon-based vector. Oligonucleotide DNA arrays were employed to identify genes that were differentially modulated by the overexpression of TIMP-3. Consistent with the function of TIMP-3, genes associated with angiogenesis, growth factors, cytokines, death receptors, and substrates of the various MMPs were found to be up-regulated. Furthermore, caspases are important in the signaling pathway of cellular apoptosis, and the overexpression of TIMP-3 in glioma cells is tightly associated with the activation of caspases, including caspase-1, at both the mRNA level (P=0.0371) and the protein level. Moreover, the activation of an apoptotic pathway via the overexpression of TIMP-3 induced apoptosis of transduced human glioma cells in vitro and the growth inhibition of human glioma tumor xenografts in immunodeficient mice.

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering.

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.

Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation.

BACKGROUND AND AIM: Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and ulceration in inflammatory bowel disease and coeliac disease. Studies to date have concluded that stromelysin 1 is functionally involved in mucosal degradation. However, there are many other MMPs whose function in the gut is currently unknown. This work had two aims: firstly, to use gene array technology to measure changes in MMP and tissue inhibitor of metalloproteinase (TIMP) expression in a model of T cell mediated injury in the gut, and secondly, to correlate data from gene arrays with that generated by in situ hybridisation. METHODS: T cells in explants of human fetal gut were activated with pokeweed mitogen or anti-CD3 plus interleukin 12. Gene array analysis and in situ hybridisation were performed to investigate changes in MMP gene expression. RESULTS: Both gene array analysis and in situ hybridisation indicated marked upregulation of stromelysin 2 and macrophage metalloelastase expression in the explants associated with mucosal destruction. The arrays also confirmed our previous observation that interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), and gelatinase B (MMP-9) are upregulated but there was no change in MMP-2, -7, -8, -9, -11, -13, -14-17, or -19. Following T cell activation, transcripts for TIMPs were reduced. CONCLUSIONS: These results show that there is differential upregulation of MMPs during T cell responses in the gut and suggest that further studies on the role of stromelysin 2 and macrophage metalloelastase may show that they have a functional role. In addition, the increase in MMPs and reduction in TIMPs suggest that the protease/antiprotease balance in the mucosa may determine the extent of mucosal degradation.