A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-dependent manner

Reelin is a glycoprotein essential for brain development and functions. Reelin is subject to specific proteolysis at two distinct (N-t and C-t) sites, and these cleavages significantly diminish Reelin activity. The decrease of Reelin activity is detrimental for brain function, but the protease that catalyzes specific cleavage of Reelin remains elusive. Here we found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-specific manner. Among ADAMTS-4 isoforms, p50 cleaves the N-t site only, while p75 cleaves both sites. This is the first report identifying a protease that can specifically cleave Reelin.     

AHR,a novel acute hypoxia‐response sequence,drives reporter gene expression under hypoxia in vitro and in vivo

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an early immediate gene. We have previously reported that ADAMTS1 was strongly induced by hypoxia. In this study, we investigated whether ADAMTS1 promoter‐driven reporter signal is detectable by acute hypoxia. We constructed the GFP (green fluorescent protein) expression vector [AHR (acute hypoxia‐response sequence)‐GFP] under the control of ADAMTS1 promoter and compared it with the constitutive GFP‐expressing vector under the control of CMV (cytomegalovirus promoter‐GFP). We transduced AHR‐GFP and examined whether GFP signals can be detected under the acute hypoxia. When the human umbilical vein [HUVEC (human umbilical vein endothelial cells)] was transduced under normoxia, there were few GFP signals, while CMV‐GFP showed considerable GFP signals. When HUVEC was stimulated with hypoxia, GFP signals from AHR‐GFP gene were induced under hypoxic conditions. Notably, the GFP signals peaked at 3 h under hypoxia. In ischaemic hind limb model, transduced AHR‐GFP showed hypoxic induction of GFP signals. In summary, we have demonstrated that the AHR system induced the reporter gene expression by acute hypoxia, and its induction is transient. This is the first report showing the unique acute hypoxia‐activated gene expression system.     

HDAC inhibition upregulates the expression of angiostatic ADAMTS1

HDAC inhibitors are promising anticancer agents that induce cell cycle arrest and apoptosis. However, the role of HDACs in cancer progression, such as angiogenesis and metastasis, remains largely unexplored. Among various HDAC inhibitors, we demonstrate that TSA and SAHA upregulated the expression of angiostatic ADAMTS1 in A549 cells. HDAC6 inhibitor tubacin, and knockdown of HDAC6, also lead to ADAMTS1 upregulation. By reporter, DAPA, and ChIP assays, the proximal GC boxes were demonstrated to be essential for ADAMTS1 induction. Decreased binding of SP1 and HDAC6 to the ADAMTS1 promoter after TSA treatment was also seen. These data suggest the involvement of HDAC6 and SP1 in the HDACi-induced expression of angiostatic ADAMTS1.     

含凝血酶敏感素1型基序的解聚素样金属蛋白酶在冠心病中作用的研究进展

含凝血酶敏感素1型基序的解聚素样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs 1,ADAMTS-1)可降解细胞外基质中的多能蛋白聚糖,促进平滑肌细胞的迁移,参与动脉粥样硬化的发生,并能进一步活化粥样斑块,促进斑块破裂;还可抑制血管的新生、参与胶原代谢加速心室重构、心肌纤维化等病理过程的进展,与各种类型的冠心痛如稳定型心绞痛(stable angina,SA)、急性冠脉综合征(acute coronary syndrome,ACS)、缺血性心肌病(ischemic cardiomyopathy,ICM)均密切相关.研究表明,ADAMTS-1有助于冠脉狭窄程度评估、斑块稳定性判断、ACS的辅助诊断及治疗策略的选择,有望用于冠心病的危险分层和预后评估,并可能成为动脉粥样硬化和各种类型冠心病预防和治疗的新靶点.本文将就ADAMTS-1在冠心病中作用的研究进展进行综述.     

The N-terminal fragment of Reelin is generated after endocytosis and released through the pathway regulated by Rab11

Reelin is a large secreted glycoprotein essential for brain formation, but its trafficking and function at the molecular level remain incompletely understood. After binding to its receptor, Reelin is internalized by endocytosis. Here we show that internalized Reelin is subject to specific proteolysis within the cell and its N-terminal fragment is re-secreted. This re-secretion is inhibited by bafilomycin A₁ or by expression of a mutant of Rab11, a regulator of the recycling pathway. As the N-terminal fragment does not bind to Reelin receptor but has homology to F-spondin, its recycling may be involved in the regulation of extracellular matrix.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.     

Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes

Here, we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1β, TNFα, IL-6 and IL-8. Furthermore, we show the participation of ADAMTS4 and ADAMTS5 in the in vitro degradation of matrilin-3. We provide evidence for a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1β. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage.     

Structural determinants of the ADAM inhibition by TIMP-3: crystal structure of the TACE-N-TIMP-3 complex

TIMP-3 (tissue inhibitor of metalloproteinases 3) is unique among the TIMP inhibitors, in that it effectively inhibits the TNF-α converting enzyme (TACE). In order to understand this selective capability of inhibition, we crystallized the complex formed by the catalytic domain of recombinant human TACE and the N-terminal domain of TIMP-3 (N-TIMP-3), and determined its molecular structure with X-ray data to 2.3 Å resolution. The structure reveals that TIMP-3 exhibits a fold similar to those of TIMP-1 and TIMP-2, and interacts through its functional binding edge, which consists of the N-terminal segment and other loops, with the active-site cleft of TACE in a manner similar to that of matrix metalloproteinases (MMPs). Therefore, the mechanism of TIMP-3 binding toward TACE is not fundamentally different from that previously elucidated for the MMPs. The Phe34 phenyl side chain situated at the tip of the relatively short sA-sB loop of TIMP-3 extends into a unique hydrophobic groove of the TACE surface, and two Leu residues in the adjacent sC-connector and sE-sF loops are tightly packed in the interface allowing favourable interactions, in agreement with predictions obtained by systematic mutations by Gillian Murphy's group. The combination of favourable functional epitopes together with a considerable flexibility renders TIMP-3 an efficient TACE inhibitor. This structure might provide means to design more efficient TIMP inhibitors of TACE.     

Matrix metalloproteinases in inflammation

Background

Matrix metalloproteinases (MMPs) are a family of ubiquitously expressed zinc-dependent endopeptidases with broad substrate specificity and strictly regulated tissue specific expression. They are expressed in physiological situations and pathological conditions involving inflammation. MMPs regulate several functions related to inflammation including bioavailability and activity of inflammatory cytokines and chemokines. There is also evidence that MMPs regulate inflammation in tumor microenvironment, which plays an important role in cancer progression.

Scope of review

Here, we discuss the current view on the role of MMPs in the regulation of inflammation.

Major conclusions

MMPs modulate inflammation by regulating bioavailability and activity of cytokines, chemokines, and growth factors, as well as integrity of physical tissue barriers. MMPs are also involved in immune evasion of tumor cells and in regulation of inflammation in tumor microenvironment.

General significance

There is increasing evidence for non-matrix substrates of MMPs that are related to regulation of inflammatory processes. New methods have been employed for identification of the substrates of MMPs in inflammatory processes in vivo. Detailed information on the substrates of MMPs may offer more specific and effective ways of inhibiting MMP function by blocking the cleavage site in substrate or by inhibition of the bioactivity of the substrate. It is expected, that more precise information on the MMP–substrate interaction may offer novel strategies for therapeutic intervention in inflammatory diseases and cancer without blocking beneficial actions of MMPs. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Proteases involved in cartilage matrix degradation in osteoarthritis

Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely thought to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Colon tumour secretopeptidome: Insights into endogenous proteolytic cleavage events in the colon tumour microenvironment

The secretopeptidome comprises endogenous peptides derived from proteins secreted into the tumour microenvironment through classical and non-classical secretion. This study characterised the low-M_r (< 3 kDa) component of the human colon tumour (LIM1215, LIM1863) secretopeptidome, as a first step towards gaining insights into extracellular proteolytic cleavage events in the tumour microenvironment. Based on two biological replicates, this secretopeptidome isolation strategy utilised differential centrifugal ultrafiltration in combination with analytical RP-HPLC and nanoLC-MS/MS. Secreted peptides were identified using a combination of Mascot and post-processing analyses including MSPro re-scoring, extended feature sets and Percolator, resulting in 474 protein identifications from 1228 peptides (≤ 1% q-value, ≤ 5% PEP) — a 36% increase in peptide identifications when compared with conventional Mascot (homology ionscore thresholding). In both colon tumour models, 122 identified peptides were derived from 41 cell surface protein ectodomains, 23 peptides (12 proteins) from regulated intramembrane proteolysis (RIP), and 12 peptides (9 proteins) generated from intracellular domain proteolysis. Further analyses using the protease/substrate database MEROPS, (http://merops.sanger.ac.uk/), revealed 335 (71%) proteins classified as originating from classical/non-classical secretion, or the cell membrane. Of these, peptides were identified from 42 substrates in MEROPS with defined protease cleavage sites, while peptides generated from a further 205 substrates were fragmented by hitherto unknown proteases. A salient finding was the identification of peptides from 88 classical/non-classical secreted substrates in MEROPS, implicated in tumour progression and angiogenesis (FGFBP1, PLXDC2), cell–cell recognition and signalling (DDR1, GPA33), and tumour invasiveness and metastasis (MACC1, SMAGP); the nature of the proteases responsible for these proteolytic events is unknown. To confirm reproducibility of peptide fragment abundance in this study, we report the identification of a specific cleaved peptide fragment in the secretopeptidome from the colon-specific GPA33 antigen in 4/14 human CRC models. This improved secretopeptidome isolation and characterisation strategy has extended our understanding of endogenous peptides generated through proteolysis of classical/non-classical secreted proteins, extracellular proteolytic processing of cell surface membrane proteins, and peptides generated through RIP. The novel peptide cleavage site information in this study provides a useful first step in detailing proteolytic cleavage associated with tumourigenesis and the extracellular environment. This article is part of a Special Issue entitled: An Updated Secretome.     

Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells

Amyloid precursor protein (APP) is a type I transmembrane protein, and one of its processed forms, β-amyloid, is considered to play a central role in the development of Alzheimer’s disease. We previously showed that APP is a primary androgen-responsive gene in prostate cancer and that its increased expression is correlated with poor prognosis for patients with prostate cancer. APP has also been implicated in several human malignancies. Nevertheless, the mechanism underlying the pro-proliferative effects of APP on cancers is still not well-understood. In the present study, we explored a pathophysiological role for APP in prostate cancer cells using siRNA targeting APP (siAPP). The proliferation and migration of LNCaP and DU145 prostate cancer cells were significantly suppressed by siAPP. Differentially expressed genes in siAPP-treated cells compared to control siRNA-treated cells were identified by microarray analysis. Notably, several metalloproteinase genes, such as ADAM10 and ADAM17, and epithelial–mesenchymal transition (EMT)-related genes, such as VIM, and SNAI2, were downregulated in siAPP-treated cells as compared to control cells. The expression of these genes was upregulated in LNCaP cells stably expressing APP when compared with control cells. APP-overexpressing LNCaP cells exhibited enhanced migration in comparison to control cells. These results suggest that APP may contribute to the proliferation and migration of prostate cancer cells by modulating the expression of metalloproteinase and EMT-related genes.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu^373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser^341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu^2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.     

Metallothionein-III increases ADAM10 activity in association with furin,PC7, and PKCα during non-amyloidogenic processing

A-disintegrin and metalloproteinase 10 (ADAM10) is involved in the generation of amyloid-β (Aβ) during amyloid precursor protein (APP) processing, and has a protective effect against Aβ neurotoxicity. We explored how metallothionein-III (MT-III) is regulated in the non-amyloidogenic pathway to generate soluble APPα (sAPPα). MT-??? increased sAPPα levels and reduced Aβ peptide levels, but did not affect ADAM10 expression. However, MT-III increased the activity of ADAM10. MT-???-induced sAPPα secretion, and Aβ peptide formation was blocked by specific inhibitors of furin, proprotein convertase7 (PC7), and PKCα. These results demonstrate that MT-??? increases the amount of active ADAM10 in association with furin, PC7 and PKCα.     

The N-terminal cleavage of cellular prion protein in the human brain

Human brain cellular prion protein (PrP(c)) is cleaved within its highly conserved domain at amino acid 110/111/112. This cleavage generates a highly stable C-terminal fragment (C1). We examined the relative abundance of holo- and truncated PrP(c) in human cerebral cortex and we found important inter-individual variations in the proportion of C1. Neither age nor postmortem interval explain the large variability observed in C1 amount. Interestingly, our results show that high levels of C1 are associated with the presence of the active ADAM 10 suggesting this zinc metalloprotease as a candidate for the cleavage of PrP(c) in the human brain.     

The function of neurofascin155 in oligodendrocytes is regulated by metalloprotease-mediated cleavage and ectodomain shedding

Formation of the paranodal axo-glial junction requires the oligodendrocyte-specific 155-kDa isoform of neurofascin (NF155). Here, we report the presence of two peptides in cultured oligodendrocytes, which are recognized by distinct NF155-specific antibodies and correspond to a membrane anchor of 30 kDa and a 125 kDa peptide, which is shed from the cells, indicating that it consists of the NF155 ectodomain. Transfection of OLN-93 cells with NF155 verified that both peptides originate from NF155 cleavage, and we present evidence that metalloproteases mediate NF155 processing. Interestingly, metalloprotease activity is required for NF155 transport into oligodendrocyte processes supporting the functional significance of NF155 cleavage. To further characterize NF155 cleavage and function, we transfected MDCK cells with NF155. Although ectodomain shedding was observed in polarized and non-polarized MDCK cells, surface localization of NF155 was restricted to the lateral membrane of polarized cells consistent with a role in cell-cell adhesion. Aggregation assays performed with OLN-93 cells confirmed that NF155 accelerates cell-cell adhesion in a metalloprotease-dependent manner. The physiological relevance of NF155 processing is corroborated by the presence of NF155 cleavage products in heavy myelin, suggesting a role of NF155 ectodomain shedding for the generation and/or stabilization of the nodal/paranodal architecture.     

Lost in disruption: Role of proteases in glioma invasion and progression

A characteristic feature of malignant glial tumors (gliomas) is their tendency to diffusely infiltrate the nervous system preventing their complete surgical resection. Proteases play a decisive role in this malignant process, either by degradation of brain extracellular matrix (ECM) components, adhesion molecules, or by regulating the activity of growth and chemotactic factors. Secreted matrix metalloproteinases (MMPs) and ADAMTS proteases (ADAMs with thrombospondin motifs) cleave different ECM components like the proteoglycans (lecticans) aggrecan, versican, neurocan and brevican with selective preferences; they are further regulated by endogenous inhibitors and activating metallo- and serine proteases. Cell surface proteases of the ADAM family (A Disintegrin And Metalloproteinase), but also serine proteases regulate the activity of growth factors and chemokines that act as autocrine / paracrine stimulators within gliomas. Thus, proteases play a decisive role for the spread and growth of gliomas and are prominent targets for their therapy.     

Reciprocal effects between microRNA-140-5p and ADAM10 suppress migration and invasion of human tongue cancer cells

ADAM10, overexpressed in tongue squamous cell carcinoma (TSCC), has been well documented for its role in tumor progression and metastasis. In the present study, we evaluated the inhibition effect of microRNAs (miRNAs) on the TSCC and identified that miR-140-5p could directly targets ADAM10 and inhibits the invasion and migration of TSCC cells. LAMC1, HDAC7 and PAX6, clustered into migration-related genes, were validated to be direct targets of miR-140-5p, while IGF1R and PSEN1 were not responsible to the regulation. Most intriguingly, ERBB4 was upregulated by miR-140-5p even though the interaction between ERBB4 3′UTR and miR-140-5p existed simultaneously. Meanwhile, ADAM10 is involved in the “positive” regulation of ERBB4 and negative regulation of PAX6 by miR-140-5p. Taken together, our results suggest that miR-140-5p play a role in TSCC cell migration and invasion, and two brand new relationships between miRNA and its targets emerged: (1) ADAM10 is not just a direct target of miR-140-5p, the repressed ADAM10 also helps to enhance the effect of miR-140-5p to other target genes: ERBB4 and PAX6; (2) ERBB4 is “positively” regulated by miR-140-5p.