Preventing obesity, insulin resistance and type 2 diabetes by targeting MT1-MMP.

Obesity is one of the predominant risk factors for type 2 diabetes. Despite all the modern advances in medicine, an effective drug treatment for obesity without overt side effects has not yet been found. The discovery of growth and differentiation factor 15 (GDF15), an appetite-regulating hormone, created hopes for the treatment of obesity. However, an insufficient understanding of the physiological regulation of GDF15 has been a major obstacle to mitigating GDF15-centric treatment of obesity. Our recent studies revealed how a series of proteolytic events predominantly mediated by membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP14), a key cell-surface metalloproteinase involved in extracellular remodeling, contribute to the pathogenesis of metabolic disorders, including obesity and diabetes. The MT1-MMP-mediated cleavage of the GDNF family receptor-α-like (GFRAL), a key neuronal receptor of GDF15, controls the satiety center in the hindbrain, thereby regulating non-homeostatic appetite and bodyweight changes. Furthermore, increased activation of MT1-MMP does not only lead to increased risk of obesity, but also causes age-associated insulin resistance by cleaving Insulin Receptor in major metabolic tissues. Importantly, inhibition of MT1-MMP effectively protects against obesity and diabetes, revealing the therapeutic potential of targeting MT1-MMP for the management of metabolic disorders.

Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding.

Overexpression of the transmembrane matrix metalloproteinase MT1-MMP/MMP14 promotes cancer cell invasion. Here we show that MT1-MMP-positive cancer cells turn MT1-MMP-negative cells invasive by transferring a soluble catalytic ectodomain of MT1-MMP. Surprisingly, this effect depends on the presence of TKS4 and TKS5 in the donor cell, adaptor proteins previously implicated in invadopodia formation. In endosomes of the donor cell, TKS4/5 promote ADAM-mediated cleavage of MT1-MMP by bridging the two proteases, and cleavage is stimulated by the low intraluminal pH of endosomes. The bridging depends on the PX domains of TKS4/5, which coincidently interact with the cytosolic tail of MT1-MMP and endosomal phosphatidylinositol 3-phosphate. MT1-MMP recruits TKS4/5 into multivesicular endosomes for their subsequent co-secretion in extracellular vesicles, together with the enzymatically active ectodomain. The shed ectodomain converts non-invasive recipient cells into an invasive phenotype. Thus, TKS4/5 promote intercellular transfer of cancer cell invasiveness by facilitating ADAM-mediated shedding of MT1-MMP in acidic endosomes.

Curcumin Inhibits Bladder Cancer by Inhibiting Invasion via AKT/MMP14 Pathway.

BACKGROUND: Bladder cancer is a malignant tumor of the urinary and reproductive tract that seriously threatens human health. It is urgent to develop new drugs for bladder cancer. This study aims to explore whether curcumin could inhibit bladder cancer and the potential mechanism. METHODS: Firstly, network pharmacology was applied to explore the potential target of curcumin in bladder cancer. Among the potential target of curcumin on bladder cancer, the role of matrix metalloproteinase-14 (MMP14) was further explored by bioinformatic analysis and the expression of MMP14 was confirmed by immunohistochemistry staining. The effect of curcumin on bladder cancer was then studied using the cell counting kit-8 (CCK-8) assay, clone formation assay, apoptosis assay, and Transwell assay. Finally, AKT, MMP14, E-cadherin and N-cadherin were analyzed by Western blot assay to confirm whether curcumin could inhibit bladder cancer by inhibiting invasion via AKT/MMP14 pathway. RESULTS: In the present study, we found that the target of curcumin for bladder cancer includes signal transducer and activator of transcription 3 (STAT3), AKT, cyclin A2 (CCNA2), epidermal growth factor receptor (EGFR), E1A binding protein p300 (EP300) and MMP14. MMP14 was highly expressed in bladder cancer than in normal tissues and was associated with a worse prognosis (p < 0.05). Curcumin could inhibit the proliferation and migration of bladder cancer cells (p < 0.05), while promoting cell apoptosis by inhibiting the AKT/MMP14 pathway (p < 0.05). CONCLUSION: Curcumin could inhibit bladder cancer by inhibiting invasion through the AKT/MMP14 pathway.

MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis.

Fibrotic hypersensitivity pneumonitis (FHP) is a fatal interstitial pulmonary disease with limited treatment options. Lung macrophages are a heterogeneous cell population that exhibit distinct subsets with divergent functions, playing pivotal roles in the progression of pulmonary fibrosis. However, the specific macrophage subpopulations and underlying mechanisms involved in the disease remain largely unexplored. In this study, a decision tree model showed that matrix metalloproteinase-14 (MMP14) had higher scores for important features in the up-regulated genes in macrophages from mice exposed to the Saccharopolyspora rectivirgula antigen (SR-Ag). Using single-cell RNA sequencing (scRNA-seq) analysis of hypersensitivity pneumonitis (HP) mice profiles, we identified MMP14high macrophage subcluster with a predominant M2 phenotype that exhibited higher activity in promoting fibroblast-to myofibroblast transition (FMT). We demonstrated that suppressing toll-like receptor 2 (TLR2) and nuclear factor kappa-B (NF-κB) could attenuate MMP14 expression and exosome secretion in macrophages stimulation with SR-Ag. The exosomes derived from MMP14-overexpressing macrophages were found to be more effective in regulating the transition of fibroblasts through exosomal MMP14. Importantly, it was observed that the transfer of MMP14-overexpressing macrophages into mice promoted lung inflammation and fibrosis induced by SR-Ag. NSC-405020 binding to the hemopexin domain (PEX) of MMP-14 ameliorated lung inflammation and fibrosis induced by SR-Ag in mice. Thus, MMP14-overexpressing macrophages may be an important mechanism contributing to the exacerbation of allergic reactions. Our results indicated that MMP14 in macrophages has the potential to be a therapeutic target for HP.

Simvastatin induces degradation of the extracellular matrix in human leiomyomata: novel in vitro, in vivo, and patient level evidence of matrix metalloproteinase involvement.

OBJECTIVES: To assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. DESIGN: Laboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. SETTING: Academic research center. PATIENT(S): Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. INTERVENTION(S): Simvastatin treatment. MAIN OUTCOME MEASURE(S): Serum concentrations, xenograft volumes, and protein expression. RESULTS: Mice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls; 12 treated with activated simvastatin at 10 mg/kg body weight; and 15 at 20 mg/kg body weight. Simvastatin was detected in the serum of mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase was increased in vitro and in vivo. Matrix metalloproteinase 2 and low-density lipoprotein receptor-related protein 1 were increased in vitro. CONCLUSIONS: Simvastatin exhibited antitumoral activity with ECM degradation and decreased leiomyoma tumor volume in vivo. Activation of the matrix metalloproteinase 2, membrane type 1 matrix metalloproteinase, and low-density lipoprotein receptor-related protein 1 pathway may explain these findings.

[Inhibition of GMA-induced ubiquitination process in 16HBE malignantly transformed cells on protein CD44 and MMP14].

OBJECTIVE: To observe the effect of the ubiquitination process on the expression of CD44 antigen(CD44) and matrix metalloproteinase-14(MMP14) in human bronchial epithelial(16HBE) malignantly transformed cells induced by glycidyl methacrylate(GMA). METHODS: Successfully resuscitated 16HBE cells were cultured using a final concentration of 8 µg/mL GMA as the treatment group and 1 µg/mL dimethyl sulfoxide as the solvent control group, each time stained for 72 h, and then stained again after an interval of 24 h. After repeating the staining three times, the cells were cultured in passages respectively. The 40th generation(P40) GMA-treated group and the same-generation solvent control group were subjected to soft agar colony formation assay and concanavalin A(ConA) agglutination test to confirm that the 40th generation of GMA-induced malignant transformed 16HBE cells possessed malignant transformed cell characteristics.5, 10, 20, 40, 60 µmol/L anacardic acid were used to inhibit the ubiquitination process of GMA-induced malignant transformed 16HBE cells. The protein expression of CD44 and MMP14 were detected by western blotting, while the transcript levels of CD44, MMP14, and TFAP2A were assessed by real-time fluorescence quantitative PCR(qPCR). RESULTS: (1) In the soft agar colony formation assay, the number of clones formed by the cells in the solvent control group was 22, and the number of clones created by the malignantly transformed cells in the GMA-treated group was 208. In the ConA agglutination test, the cells in the solvent control group were uniformly dispersed in ConA solution, and no obvious agglutination occurred for 30 min, whereas the cells in the GMA-treated group were agglutinated in the 5th min, and the agglutinated cells were larger and more rapidly agglutinated. The agglomerates were more significant and faster, and the sensitivity of agglutination was increased. (2) After differential inhibition of GMA-induced ubiquitination in malignantly transformed 16HBE cells, the expression levels of CD44 and MMP14 were reduced in GMA-induced malignantly transformed 16HBE cells compared with the control group(P<0.05). The transcript levels of MMP14 and CD44 decreased with increasing inhibitor concentration(P<0.05), and the transcript levels of the upstream transcription factor TFAP2A were also simultaneously reduced(P<0.05). CONCLUSION: Inhibition of the cellular ubiquitination process mediates the down-regulation of protein expression and transcriptional expression of CD44 and MMP14 in GMA-induced malignantly transformed 16HBE cells.

Mmp14 is required for matrisome homeostasis and circadian rhythm in fibroblasts.

The circadian clock in tendon regulates the daily rhythmic synthesis of collagen-I and the appearance and disappearance of small-diameter collagen fibrils in the extracellular matrix. How the fibrils are assembled and removed is not fully understood. Here, we first showed that the collagenase, membrane type I-matrix metalloproteinase (MT1-MMP, encoded by Mmp14), is regulated by the circadian clock in postnatal mouse tendon. Next, we generated tamoxifen-induced Col1a2-Cre-ERT2::Mmp14 KO mice (Mmp14 conditional knockout (CKO)). The CKO mice developed hind limb dorsiflexion and thickened tendons, which accumulated narrow-diameter collagen fibrils causing ultrastructural disorganization. Mass spectrometry of control tendons identified 1195 proteins of which 212 showed time-dependent abundance. In Mmp14 CKO mice 19 proteins had reversed temporal abundance and 176 proteins lost time dependency. Among these, the collagen crosslinking enzymes lysyl oxidase-like 1 (LOXL1) and lysyl hydroxylase 1 (LH1; encoded by Plod2) were elevated and had lost time-dependent regulation. High-pressure chromatography confirmed elevated levels of hydroxylysine aldehyde (pyridinoline) crosslinking of collagen in CKO tendons. As a result, collagen-I was refractory to extraction. We also showed that CRISPR-Cas9 deletion of Mmp14 from cultured fibroblasts resulted in loss of circadian clock rhythmicity of period 2 (PER2), and recombinant MT1-MMP was highly effective at cleaving soluble collagen-I but less effective at cleaving collagen pre-assembled into fibrils. In conclusion, our study shows that circadian clock-regulated Mmp14 controls the rhythmic synthesis of small diameter collagen fibrils, regulates collagen crosslinking, and its absence disrupts the circadian clock and matrisome in tendon fibroblasts.

CLDN1 silencing suppresses the proliferation and migration of airway smooth muscle cells by modulating MMP14.

Airway remodeling is an important pathologic factor in the progression of asthma. Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) are important pathologic mechanisms in severe asthma. In the current study, claudin-1 (CLDN1) was identified as an asthma-related gene and was upregulated in ASMCs stimulated with platelet-derived growth factor BB (PDGF-BB). Cell counting kit-8 and EdU assays were used to evaluate cell proliferation, and transwell assay was carried out to analyze cell migration and invasion. The levels of inflammatory factors were detected using enzyme-linked immunosorbent assay. The results showed that CLDN1 knockdown inhibited the proliferation, migration, invasion, and inflammation of ASMCs treated with PDGF-BB, whereas overexpression of CLDN1 exhibited the opposite effects. Protein-protein interaction assay and co-immunoprecipitation revealed that CLDN1 directly interacted with matrix metalloproteinase 14 (MMP14). CLDN1 positively regulated MMP14 expression in asthma, and MMP14 overexpression reversed cell proliferation, migration, invasion, and inflammation induced by silenced CLDN1. Taken together, CLDN1 promotes PDGF-BB-induced cell proliferation, migration, invasion, and inflammatory responses of ASMCs by upregulating MMP14 expression, suggesting a potential role for CLDN1 in airway remodeling in asthma.

ETV4 facilitates angiogenesis in hepatocellular carcinoma by upregulating MMP14 expression.

Abnormal vascularization plays a crucial role in cell proliferation, tumor invasion and metastasis of hepatocellular carcinoma (HCC). It has been reported that ETV4 functions as an oncogenic gene in driving the carcinogenesis and progression, and promoting invasion and metastasis of HCC. However, the function of ETV4 on angiogenesis in HCC remains unclear. In the current study, immunohistochemistry showed that knockdown of ETV4 reduced angiogenesis in HCC xenograft tumor tissues. In vitro, tube formation assay verified that ETV4 expression promoted angiogenesis through simulating the angiogenic environment in HCC cells. Transcriptome sequencing indicated that MMP14 was one of the differentially expressed genes enriched in angiogenesis process. Subsequently, it was confirmed that MMP14 was regulated by ETV4 at the transcription level in HCC cells, clinical tissue samples and online databases. Further, we demonstrated that MMP14 induced angiogenesis in ETV4-mediated HCC microenvironment. Collectively, this research further reveals the biological mechanism of ETV4 in promoting the migration and invasion of HCC, and provides novel mechanistic insights and strategic guidance for anti-angiogenic therapy in HCC.

Assessment of MMP14, CAV2, CLU and SPARCL1 expression profiles in endometriosis.

Endometriotic cells exhibit a notable degree of invasiveness and some characteristics of tissue remodeling underlying lesion formation. In this regard, do matrix metalloproteinases 14 (MMP14) and other related genes such as SPARC-like protein 1 (SPARCL1), caveolin 2 (CAV2), and clusterin (CLU) exert any significant influence in the processes of endometriosis development and pathophysiology is not apparent. We aim to assess whether these genes could serve as potential diagnostic biomarkers in endometriosis. Microarray-based gene expression analysis was performed on total RNA extracted from endometriotic tissue samples treated with and without gonadotropin-releasing hormone agonist (GnRHa). The GnRHa untreated patients were considered the control group. The validation of genes was performed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis showed significant downregulation in the expression of MMP14 (p = 0.024), CAV2 (p = 0.017), and upregulation of CLU (p = 0.005) in endometriosis patients treated with GnRHa. SPARCL1 did not show any significant (p = 0.30) change in the expression compared to the control group. These data have the potential to contribute to the comprehension of the molecular pathways implicated in the remodeling of the extracellular matrix, which is a vital step for the physiology of the endometrium. Based on the result, it is concluded that changes in the expression of MMP14, CAV2, and CLU post-treatment imply their role in the pathophysiology of endometriosis and may serve as a potential diagnostic biomarker of endometriosis in response to GnRHa treatment in patients with ovarian endometrioma.

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs.

The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis.

Expression of MMP-14 and its role in bone destruction in middle ear cholesteatoma: A prospective observational study.

Cholesteatoma is a noncancerous cystic lesion caused by an abnormal growth of keratinizing squamous epithelium which is invasive and capable of destroying structures. A prospective study on the expression of membrane type1-matrix metalloproteinases (MMP-14) and its related influencing factors in middle ear cholesteatoma was conducted to fully understand the pathogenesis of cholesteatoma in the molecular level. We examined the expression of MMP-14 by immunohistochemical staining 39 middle ear cholesteatoma specimens and 10 external auditory meatus epithelial cell specimens. The cholesteatoma specimens were divided into 4 groups according to the degree of destruction of the ossicles during surgery. The associated factors affecting MMP-14 expression were analyzed using statistical methods; The positive expression of MMP-14 in the epithelium of the external auditory canal was significantly different between middle ear cholesteatoma and normal patients (P < .05); Gender, age, and the degree of hearing loss had no statistically significant effect on MMP-14 expression (P > .05); The expression of MMP-14 was positively correlated with the severity of bone destruction (R = 0.535, P < .05); MMP-14 plays an important role in the pathological development of the epithelium of cholesteatoma; MMP-14 expression in middle ear cholesteatoma tissue was not strongly correlated with the level of hearing loss, age or gender, but was positively correlated with the degree of middle ear bone destruction.

Reflux conditions induce E-cadherin cleavage and EMT via APE1 redox function in oesophageal adenocarcinoma.

OBJECTIVE: Chronic gastro-oesophageal reflux disease, where acidic bile salts (ABS) reflux into the oesophagus, is the leading risk factor for oesophageal adenocarcinoma (EAC). We investigated the role of ABS in promoting epithelial-mesenchymal transition (EMT) in EAC. DESIGN: RNA sequencing data and public databases were analysed for the EMT pathway enrichment and patients' relapse-free survival. Cell models, pL2-IL1ß transgenic mice, deidentified EAC patients' derived xenografts (PDXs) and tissues were used to investigate EMT in EAC. RESULTS: Analysis of public databases and RNA-sequencing data demonstrated significant enrichment and activation of EMT signalling in EAC. ABS induced multiple characteristics of the EMT process, such as downregulation of E-cadherin, upregulation of vimentin and activation of ß-catenin signalling and EMT-transcription factors. These were associated with morphological changes and enhancement of cell migration and invasion capabilities. Mechanistically, ABS induced E-cadherin cleavage via an MMP14-dependent proteolytic cascade. Apurinic/apyrimidinic endonuclease (APE1), also known as redox factor 1, is an essential multifunctional protein. APE1 silencing, or its redox-specific inhibitor (E3330), downregulated MMP14 and abrogated the ABS-induced EMT. APE1 and MMP14 coexpression levels were inversely correlated with E-cadherin expression in human EAC tissues and the squamocolumnar junctions of the L2-IL1ß transgenic mouse model of EAC. EAC patients with APE1high and EMThigh signatures had worse relapse-free survival than those with low levels. In addition, treatment of PDXs with E3330 restrained EMT characteristics and suppressed tumour invasion. CONCLUSION: Reflux conditions promote EMT via APE1 redox-dependent E-cadherin cleavage. APE1-redox function inhibitors can have a therapeutic role in EAC.

Vimentin-mediated myosin 10 aggregation at tips of cell extensions drives MT1-MMP-dependent collagen degradation in colorectal cancer.

Colorectal cancer (CRC) is a high prevalence adenocarcinoma with progressive increases in metastasis-related mortality, but the mechanisms governing the extracellular matrix (ECM) degradation important for metastasis in CRC are not well-defined. We investigated a functional relationship between vimentin (Vim) and myosin 10 (Myo10), and whether this relationship is associated with cancer progression. We tested the hypothesis that Vim regulates the aggregation of Myo10 at the tips of cell extensions, which increases membrane-type 1 matrix metalloproteinase (MT1-MMP)-associated local collagen proteolysis and ECM degradation. Analysis of CRC samples revealed colocalization of Vim with Myo10 and MT1-MMP in cell extensions adjacent to sites of collagen degradation, suggesting an association with local cell invasion. We analyzed cultured CRC cells and fibroblasts and found that Vim accelerates aggregation of Myo10 at cell tips, which increases the cell extension rate. Vim stabilizes the interaction of Myo10 with MT1-MMP, which in turn increases collagenolysis. Vim depletion reduced the aggregation of Myo10 at the cell extension tips and MT1-MMP-dependent collagenolysis. We propose that Vim interacts with Myo10, which in turn associates with MT1-MMP to facilitate the transport of these molecules to the termini of cell extensions and there enhance cancer invasion of soft connective tissues.

Simultaneous quantification of exosomal MMP14 expression and proteolysis activity on a spherical dual-probe-based fluorescent nanosensor.

Among various exosomal proteins, matrix metalloproteinases (MMPs) are a family of membrane associated endopeptidases and have been considered as potential biomarkers in liquid biopsy owing to their multiple roles in pathological processes. However, the potential of MMP14 expression (MMP14-E) and MMP14 proteolytic activity (MMP14-A) in clinical diagnosis is still not clear due to the lack of sensitive and simultaneous detection techniques. Herein, we propose a fluorescent nanosensor for the simultaneous detection of MMP14-E and MMP14-A using a spherical aptamer/peptide dual-probe strategy. The aptamer and peptide probes were sequentially immobilized on Fe3O4 magnetic nanoparticles coated with gold nanoparticles (m-AuNPs) using a disulfide linker. MMP14 can be specifically recognized by the aptamer, while the proteolytic-active MMP14 can cleave the peptide probe. While achieving simultaneous detection, the proposed sensor provides better analytical performances than traditional MMP14 sensors owing to the m-AuNP-based spherical dual-probe strategy. This sensor has been successfully applied for the detection of exosomal MMP14 from cell culture media and real serum samples. Levels of both MMP14-E and MMP14-A increase in serum from cancer patients, indicating their potential applications as biomarkers in liquid biopsy for disease diagnosis and real-time surveillance.

Expression analysis of MMP14: Key enzyme action in modulating visceral adipose tissue plasticity in patients with obesity.

Compromised adipose tissue plasticity is a hallmark finding of obesity orchestrated by the intricate interplay between various extracellular matrix components. Collagen6 (COL6) is well characterized in obese visceral adipose tissue (VAT), not much is known about MMP14 which is hypothesized to be the key player in matrix reorganization. Subjects with obesity (BMI ≥40; n = 50) aged 18-60 years undergoing bariatric surgery and their age-matched controls (BMI < 25; n = 30) were included. MMP14, Col6A3 and Tissue inhibitor of metalloproteinase 2 (TIMP2) mRNA expression was assessed in VAT and their serum levels along with endotrophin were estimated in both groups preoperatively and post-operatively in the obese group. The results were analysed statistically and correlated with anthropometric and glycaemic parameters, namely fasting glucose and insulin, HbA1c, HOMA-IR, HOMA-ß and QUICKI. Circulating levels as well as mRNA expression profiling revealed significant differences between the individuals with and without obesity (p < .05), more so in individuals with diabetes and obesity (p < .05). Follow-up serum analysis revealed significantly raised MMP14 (p < .001), with decreased Col6A3, endotrophin and TIMP2 levels (p < .01, p < .001 and p < .01, respectively). A rise in serum MMP14 protein, simultaneous with post-surgical weight loss and decreased serum levels of associated extracellular matrix (ECM) remodellers, suggests its crucial role in modulating obesity-associated ECM fibrosis and pliability of VAT.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate.

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Soluble Sema4D cleaved from osteoclast precursors by TACE suppresses osteoblastogenesis.

Bone remodelling is mediated by orchestrated communication between osteoclasts and osteoblasts which, in part, is regulated by coupling and anti-coupling factors. Amongst formally known anti-coupling factors, Semaphorin 4D (Sema4D), produced by osteoclasts, plays a key role in downmodulating osteoblastogenesis. Sema4D is produced in both membrane-bound and soluble forms; however, the mechanism responsible for producing sSema4D from osteoclasts is unknown. Sema4D, TACE and MT1-MMP are all expressed on the surface of RANKL-primed osteoclast precursors. However, only Sema4D and TACE were colocalized, not Sema4D and MT1-MMP. When TACE and MT1-MMP were either chemically inhibited or suppressed by siRNA, TACE was found to be more engaged in shedding Sema4D. Anti-TACE-mAb inhibited sSema4D release from osteoclast precursors by ~90%. Supernatant collected from osteoclast precursors (OC-sup) suppressed osteoblastogenesis from MC3T3-E1 cells, as measured by alkaline phosphatase activity, but OC-sup harvested from the osteoclast precursors treated with anti-TACE-mAb restored osteoblastogenesis activity in a manner that compensates for diminished sSema4D. Finally, systemic administration of anti-TACE-mAb downregulated the generation of sSema4D in the mouse model of critical-sized bone defect, whereas local injection of recombinant sSema4D to anti-TACE-mAb-treated defect upregulated local osteoblastogenesis. Therefore, a novel pathway is proposed whereby TACE-mediated shedding of Sema4D expressed on the osteoclast precursors generates functionally active sSema4D to suppress osteoblastogenesis.

The selective regulation of immune responses by matrix metalloproteinase MMP14 in Ostrinia furnacalis.

Matrix metalloproteinases (MMPs) are crucial for tissue remodeling and immune responses in insects, yet it remains unclear how MMPs affect the various immune processes against pathogenic infections and whether the responses vary among insects. In this study, we used the lepidopteran pest Ostrinia furnacalis larvae to address these questions by examining the changes of immune-related gene expression and antimicrobial activity after the knockdown of MMP14 and bacterial infections. We identified MMP14 in O. furnacalis using the rapid amplification of complementary DNA ends (RACE), and found that it was conserved and belonged to the MMP1 subfamily. Our functional investigations revealed that MMP14 is an infection-responsive gene, and its knockdown reduces phenoloxidase (PO) activity and Cecropin expression, while the expressions of Lysozyme, Attacin, Gloverin, and Moricin are enhanced after MMP14 knockdown. Further PO and lysozyme activity determinations showed consistent results with gene expression of these immune-related genes. Finally, the knockdown of MMP14 decreased larvae survival to bacterial infections. Taken together, our data indicate that MMP14 selectively regulates the immune responses, and is required to defend against bacterial infections in O. furnacalis larvae. Conserved MMPs may serve as a potential target for pest control using a combination of double-stranded RNA and bacterial infection.

Evidence that enolase-phosphatase 1 exacerbates early cerebral ischemia injury and blood-brain barrier breakdown by enhancing extracellular matrix destruction and inhibiting the interaction between ADI1 and MT1-MMP.

Enolase-phosphatase 1 (ENOPH1) is a newly identified enzyme associated with stress responses and cell proliferation. Our previous study found that ENOPH1 mediates cerebral microvascular endothelial cell apoptosis under cerebral ischemia conditions. In this study, we systematically provide mechanistic insights into the regulation of ENOPH1 in blood-brain barrier (BBB) dysfuction induced by early ischemia. ENOPH1 knockout mice (ENOPH1 KO) and wild type (WT) mice were exposed to transient middle cerebral artery occlusion (tMCAO) for 90 min followed by 3 h of reperfusion in vivo, and brain microvascular endothelial cell lines (bEnd.3 cells) were exposed to oxygen-glucose deprivation (OGD) in vitro. BEnd.3 cells were transfected with ENOPH1 shRNA to knockdown ENOPH1 expression. Brain ischemic damage and nerve function was assessed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological scores. BBB permeability and tight junction (TJ) protein and adherens junction (AJ) proteins expression were analyzed by FITC-dextran staining, western blotting and coimmunofluorescence. The MMP-2/9 activity was analyzed by gelatin zymography. Differential protein expression was assessed by quantitative proteomics. The interaction between ADI1 and MT1-MMP was measured by coimmunoprecipitation assay and coimmunofluorescence. Knockout of ENOPH1 ameliorated cerebral ischemic injury, decreased BBB permeability, inhibited the activity of MMP-2/9, upregulated the expression of TJ/AJ proteins and reversed extracellular matrix destruction after ischemia in vivo. Mechanistic studies have shown that ENOPH1 silencing enhanced the interaction between ADI1 and MT1-MMP by promoting the nuclear translocation of ADI1 to inhibit MT1-MMP in bEnd.3 cells after OGD and decreasing the expression of Tnc and Fn1 to inhibit ECM degradation. Our results reveal that ENOPH1 increases the activity of MMP-2/9, then promotes TJ protein and extracellular matrix degradation, and eventually destroys the stability of the BBB. Therefore, ENOPH1 is a new therapeutic target for ischemic stroke.