The effect of Ro 28-2653, a synthetic matrix metalloproteinase (MMP) inhibitor, on adipose tissue development was studied in mice kept on a high fat diet (HFD). Five-week-old male wild-type (C57Bl/6J) mice were fed the HFD (42% kcal as fat, 20.1 kJ/g) and received daily p.o. instillations of inhibitor (30 mg/kg) or vehicle. After 15 weeks of the HFD, the body weight gain was lower in the inhibitor-treated group (7.4 +/- 0.88 g versus 10 +/- 1.4 g) whereas the weights of the isolated subcutaneous (SC) or gonadal (GON) fat deposits were 10-15% lower. The number of adipocytes in adipose tissues of the inhibitor-treated mice was somewhat higher (10-17%) but their diameter was smaller (about 10%). In situ zymography showed reduced gelatinolytic activity in SC (about 2.7-fold) and GON (1.4-fold) adipose tissue of inhibitor-treated mice, whereas their fibrillar collagen content was higher (1.5- and 4.7-fold, respectively). In both SC and GON adipose tissues of inhibitor-treated mice, MMP-2 (gelatinase A) and MMP-14 (membrane type-1 MMP) were 2- to 3-fold upregulated, whereas MMP-9 (gelatinase B) mRNA levels were not affected. Thus, in this in vivo model partial inhibition of gelatinolytic activity is associated with moderate effects on adipose tissue development and cellularity. Possibly, enhanced MMP expression to some extent counteracts the in vivo effect of the inhibitor in adipose tissue.

Preeclampsia is a devastating pregnancy-related syndrome characterized by the onset of hypertension, proteinuria and edema. Insufficient invasion of trophoblasts is well-known to be correlated with preeclampsia development. The present study was performed to investigate the functional role microRNA (miRNA)-204 in trophoblastic invasion in vitro. We here found that the invasive capabilities of BeWo and JEG3 trophoblast-like cells were suppressed by miR-204 mimics, whereas enhanced by its inhibitor. Matrix metalloproteinase-9 (MMP9) was first confirmed to play a role in regulating trophoblast invasion through loss- or gain-of-function experiment. Notably, we demonstrated MMP9 as a direct target of miR-204 in BeWo cells by using the dual-luciferase assay. Moreover, forced overexpression of MMP9 was noted to partly attenuate the inhibitory effects of miR-204 on BeWo cell invasion. Taken together, our study indicates that miR-204 may contribute to the development of preeclampsia by inhibiting trophoblastic invasion, and that MMP9 is involved in miR-204-mediated trophoblast cell invasion. Our study suggests miR-204 as a novel therapeutic target for preeclampsia.

Release and activation of matrix metalloproteinases (MMPs) significantly contribute to myocardial stunning injury immediately after ischemia and reperfusion, however, their role in preconditioning remains unknown. We therefore examined the effects of preconditioning and subsequent ischemia/reperfusion on MMP activity in isolated rat hearts. Hearts were subjected to a preconditioning protocol (three consecutive 5-min periods of global ischemia interspersed with 5 min of reperfusion) followed by 30 min ischemia and 5 min reperfusion. To measure MMP release, coronary effluent was collected: (a) during aerobic perfusion, (b) in reperfusion following each preconditioning ischemia, and (c) during the final reperfusion following test ischemia. MMP-2 activities could be detected by gelatin zymography in the ventricles and coronary effluent samples from the perfused hearts. The levels of MMP-2 activity in the effluent were markedly increased in effluent following test ischemia from control hearts without preconditioning. This was accompanied by a decrease in corresponding tissue MMP activities. Preconditioning significantly decreased the MMP-2 activity in the coronary effluent following test ischemia/reperfusion and preserved the MMP-2 protein content and activity in the myocardium. Our results demonstrate that classic preconditioning inhibits ischemia/reperfusion induced release and activation of MMP-2. These results suggest that preconditioning may exert part of its cardioprotective effects through the reduction of MMP-2 release.

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, β1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4β9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved β1-3 but not α4β9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted β1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored β1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the β1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

Matrix metalloproteinase-9 (MMP-9) is a secreted glycoprotein with a major role in shaping the extracellular matrix and a detailed understanding of the secretory mechanism could help identify methods to correct diseases resulting from dysregulation of secretion. MMP-9 appears to follow a canonical secretory pathway through a quality control cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Through a complementation assay, we determined that LMAN1, a well-studied lectin-carrier protein, interacts with a secretion-competent N-glycosylated MMP-9 in the ER while N-glycosylation-deficient secretion-compromised MMP-9 does not. In contrast, co-immunoprecipitation demonstrated protein interaction between LMAN1 and secretion-compromised N-glycosylation-deficient MMP-9. MMP-9 secretion was reduced in the LMAN1 knockout cell line compared to control cells confirming the functional role of LMAN1. These observations support the role of LMAN1 as a lectin-carrier protein mediating efficient MMP-9 secretion.

The cornea is the most sensitive tissue in the human body, with the dense nerve endings of the cornea being derived from the first division of the ophthalmic nerve. The existence of such organized nerve fibers reflects the role of neural regulation in corneal homeostasis, with the proper distribution and function of these nerve fibers thus being required for maintenance of a healthy cornea. We recently established an in vitro model, based on the coculture of human corneal epithelial cells and fibroblasts on opposite sides of a collagen vitrigel membrane. We have now examined the role of neural cells in corneal homeostasis with the use of a similar coculture system. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that the presence of neural cells (differentiated PC12 cells) increased the expression of matrix metalloproteinase-1 (MMP-1) in human corneal fibroblasts at both the mRNA and protein levels. The expression of MMP-2 and MMP-9 in corneal fibroblasts was not affected by PC12 cells. Furthermore, a multiplex assay showed that, among various cytokines assayed, only the release of interleukin-6 in cocultures of the two cell types was markedly greater than that in cultures of corneal fibroblasts alone. These results thus suggest that factors released from neural cells may play an important role in regulation of the function of corneal fibroblasts and thereby contribute to the maintenance of corneal structure and function.

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5' untranslated region (UTR) of 51 bp, a 3' UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.

The extracellular microenvironment plays a key role in regulation of cellular functions and growth control. We show here that membrane-type 1 matrix metalloproteinase (MT1-MMP) acts as a growth promoter in confluent culture. When MT1-MMP was silenced in HT1080 fibrosarcoma cells, cells created three dimensional (3D) fibronectin matrix in a confluent culture, and growth of cells embedded within it was retarded. Formation of 3D fibronectin matrix initiated by MT1-MMP silencing was impeded by knockdown of either FN or integrin β₁, which resulted in restoration of cell growth. When cells in 3D fibronectin matrix were treated with integrin β₁ inhibitory antibody, cells underwent S phase entry. These results suggest that MT1-MMP prevents growth suppression by 3D fibronectin matrix, which is mediated through integrin β₁.

Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.

Chondrocyte apoptosis is the most important element of development and progression of osteoarthritis (OA). Nitric oxide (NO) was used as the agent to induce chondrocyte apoptosis. Carboxymethylated chitosan (CMCS) has anti-apoptosis effect on many cell types in vitro. This study was designed to investigate the protective effect of CMCS on NO-induced chondrocyte apoptosis and the probable molecular mechanisms. The newborn Sprague-Dawley (SD) rats were used in this study for isolation of chondrocytes. The cell viability was determined by cell counting kit (CCK-8), cell apoptosis was detected by Annexin-V/PI double staining assay kit. The levels of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt), Bcl-2 and Bax were determined by Western blot analysis. The caspase-3 activity was determined by a quantitative colorimetric assay. Results showed that pretreatment with CMCS could inhibit the apoptosis induced by NO. CMCS could decrease the activity of NO and decrease the expression of Bcl-2, p-PI3K and p-Akt, increase the expression of Bax, cytochrome c and caspase-3. CMCS also could reverse the effect of NO that prompted matrix metalloproteinase-13 (MMP-13) and inhibited tissue inhibitor of metalloproteinase-1 (TIMP-1) activity. All the present results indicated that CMCS can protect NO induced chondrocytes apoptosis by activate PI3K/Akt signaling pathway.

Chronic obstructive pulmonary disease (COPD) is an inflammatory process characterized by airway mucus hypersecretion. Lipopolysaccharides (LPS) are known to stimulate the production of mucin 5AC (MUC5AC) via epidermal growth factor receptor (EGFR) in human airway cells. Noteworthy, we have previously demonstrated that EGFR/Rac1/reactive oxygen species (ROS)/matrix metalloproteinase 9 (MMP-9) is a key signaling cascade regulating MUC5AC production in airway cells challenged with LPS. Various reports have shown an inverse association between the intake of polyunsaturated fatty acids (PUFA) of the n-3 (omega-3) family or fish consumption and COPD. In the present study, we investigated the influence of docosahexaenoic acid (DHA), one of the most important omega-3 PUFA contained in fish oil, on the production of MUC5AC in LPS-challenged human airway cells NCI--H292. Our results indicate that DHA is capable of counteracting MUC5AC overproduction in LPS-stimulated cells by abrogating both EGFR phosphorylation and its downstream signaling pathway. This signaling pathway not only includes Rac1, ROS and MMP-9, but also NF-κB, since we have found that ROS require NF-κB activity to induce MMP-9 secretion and activation.

Exposure to particulate matter 2.5 (PM2.5) has been linked to ocular surface diseases, yet knowledge of the molecular mechanism impacted on retina pathogenesis is limited. Therefore, the purpose of this study was to explore the effects and involved factors of PM2.5 exposure in human retinal pigment epithelial APRE-19 cells. Our data revealed a decreased cell viability and an increased migratory ability in APRE-19 cells after PM2.5 stimulation. The MMP-2 and MMP-9 protein levels were markedly increased while the MMPs regulators TIMP-1 and TIMP-2 were significantly reduced in PM2.5-exposed APRE-19 cells. PM2.5 also increased pro-MMP-2 expression in the cell culture supernatants. Additionally, PM2.5 promoted the EMT markers through the activation of PI3K/AKT/mTOR pathway. Moreover, the ICAM-1 production was also remarkably increased by PM2.5 but reduced by PI3K/AKT inhibitor LY294002 in APRE-19 cells. Taken together, these results suggest that PM2.5 promotes EMT in a PI3K/AKT/mTOR-dependent manner in the retinal pigment epithelium.

Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air-liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24-48h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.

The extracellular matrix not only provides a structural scaffold for cells to inhabit but also forms a conduit by which mechanical information may be transmitted. Fibroblasts undergo a variety of changes when activated, including upregulating matrix metalloproteinase (MMP) activity and establishing a smooth muscle-like contractile apparatus. The relationship between MMP activity and matrix contraction has yet to be established. Here we report that inhibition of MMP activity correlates with a significant reduction in collagen gel contraction, however, force development does not change respective to MMP activity. These results suggest cellular controls of contractile forces are independent of MMP activity. Our results also raise the possibility that the material properties of the matrix dynamically change during remodeling.

Biochanin-A, a phytoestrogen derived from herbal plants, protected from the IL-1β-induced loss of proteoglycans through the suppression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, and ADAMTS-5 in primary rat chondrocytes and the knee articular cartilage. It also suppressed the expression of IL-1β-induced catabolic factors such as nitric oxide synthase 2, cyclooxygenase-2, prostaglandin E2, and inflammatory cytokines. Furthermore, biochanin-A suppressed the IL-1β-induced phosphorylation of NFκB, and inhibited its nuclear translocation in primary rat chondrocytes. These results indicate that biochanin-A antagonizes the IL-1β-induced catabolic effects through its anti-inflammatory activity that involves the modulation of NFκB signaling.

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.

Matrix metalloproteinase-9 (MMP-9) plays roles in cancer progression by degrading the extracellular matrix and basement membrane. Many growth factors including Transforming growth factor-beta1 (TGF-beta1) could induce MMP-9 expression. We demonstrated that TGF-beta1 induced MMP-9 mRNA and protein in human head and neck squamous cell carcinoma cell lines. Application of TGF-beta receptor type I inhibitor (SB505124) reduced the MMP-9 expression markedly. Whilst, inhibitor of Myosin light chain kinase (MLCK) could reduce the level of secreted MMP-9 in both the supernatants and cell lysate but not the level of MMP-9 mRNA. These suggested that MLCK might regulate MMP-9 expression post-transcriptionally. Application of SB505124 and siRNA Smad2/3 reduced the phosphorylation of myosin light chain (MLC) suggested that MLC is downstream to TbetaRI/Smad2/3 signaling pathway. In conclusion, these results describe a novel mechanism for the potentiation of TGF-beta1 signaling to induce MMP-9 expression via Smad and MLCK.

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.

Diabetic retinopathy (DR) is a key reason for legal blindness worldwide. Currently, it is urgently necessary to determine the etiology and pathological molecular mechanism of DR to search for resultful therapies. Dickkopf-1 (DKK1) is inhibitive for canonical Wnt signaling via negative feedback, and has been reported as a biomarker for DR. However, the related mechanisms are still unclear. In this work, our data showed that DKK1 was decreased in the vitreous tissues at an early stage of diabetes triggered by streptozotocin (STZ) injection in rats. We subsequently found that DKK1 intravitreal injection significantly ameliorated the physiological function of retina in STZ-challenged rats, accompanied by improved retinal structure. Surprisingly, our results indicated that DKK1 injection remarkably suppressed PANoptosis in retinal tissues of STZ-challenged rats with DR, as proved by ameliorated pyroptosis, apoptosis and necroptosis, which were mainly through the blockage of cleaved Gasdermin-D (GSDMD), Caspase-3 and receptor-interacting protein kinase-3 (RIPK3). Additionally, Wnt signaling including the expression of Wnt, β-catenin and LDL receptor-related protein 5/6 (LRP5/6) was also highly prohibited in retina of DKK1-injected rats with DR. Furthermore, retinal neovascularization and acellular vessel in DR rats were also considerably abolished after DKK1 injection, accompanied by reduced expression levels of retinal vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). More in vitro experiments showed that DKK1 treatment markedly repressed the proliferative and migratory ability of endothelial cells via inhibiting angiogenesis-related molecules. Together, all our results broaden the knowledge of the correlation between DKK1 and DR, and then provide a novel therapeutic strategy for the suppression of management of DR.