MTA2 silencing attenuates the metastatic potential of cervical cancer cells by inhibiting AP1-mediated MMP12 expression via the ASK1/MEK3/p38/YB1 axis.

Metastasis-associated protein 2 (MTA2) is a transcription factor that is highly associated with matrix metalloproteinase 12 (MMP12). Thus, we hypothesized that MTA2 may regulate MMP12 expression and is involved in cervical cancer metastasis. Results showed that MTA2 and MMP12 were highly expressed in cervical cancer cells, and MTA2 knockdown reduced MMP12 expression and inhibited the metastasis of cervical cancer cells in xenograft mice. MMP12 knockdown did not influence the viability of cervical cancer cells but clearly inhibited cell migration and invasion both in vitro and in vivo. MMP12 was highly expressed in cervical tumor tissues and correlated with the poor survival rate of patients with cervical cancer. Further investigations revealed that p38 mitogen-activated protein kinase (p38), mitogen-activated protein kinase kinase 3 (MEK3), and apoptosis signal-regulating kinase 1 (ASK1) were involved in MMP12 downregulation in response to MTA2 knockdown. Results also demonstrated that p38-mediated Y-box binding protein1 (YB1) phosphorylation disrupted the binding of AP1 (c-Fos/c-Jun) to the MMP12 promoter, thereby inhibiting MMP12 expression and the metastatic potential of cervical cancer cells. Collectively, targeting both MTA2 and MMP12 may be a promising strategy for the treatment of cervical cancer.

LPS aggravates lung inflammation induced by RSV by promoting the ERK-MMP-12 signaling pathway in mice.

BACKGROUND: RSV can lead to persistent airway inflammation and airway hyperresponsiveness (AHR), and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. Lipopolysaccharide (LPS) is also implicated in the onset and exacerbation of asthma. However, whether inhalation of LPS can boost airway inflammation induced by RSV is not clear. In this study, we utilized an LPS- and RSV-superinfected mouse model to explore underlying pathogenesis. METHODS: Mice were infected with RSV on day 0 and inoculated with LPS from day 35 to day 41, samples were collected on day 42. Inflammatory cells, lung histopathology and AHR were measured. Cytokines were detected by ELISA and ERK, JNK, p38 was determined by western blot. MMP408, PD98059, SP600125 and SB203580 were used to inhibit MMP-12, ERK, JNK and p38 respectively. RESULTS: LPS exposure superimposed on RSV-infected lungs could lead to more vigorous cellular influx, lung structures damage, augmented AHR and higher MMP-12 levels. Inhibition of MMP-12 or ERK signaling pathway in vivo both diminished LPS-driven airway inflammation and AHR. CONCLUSIONS: Exposure to LPS in RSV-infected mice is associated with enhanced increases in ERK-MMP-12 expression that translates into increased lung inflammation and AHR. These findings contribute novel information to the field investigating the onset of post-RSV bronchiolitis recurrent wheezing as a result of LPS exposure.

Six cases of perforating pilomatricoma: Anetodermic changes with expression of matrix metalloproteinases.

Perforating pilomatricoma (PP) is a rare clinical variant of pilomatricoma presenting as a crusted or ulcerated nodule. Previous reports have suggested that the tumor cells perforate the epidermis through a process of transepithelial elimination. Here, we report six cases of PP and examine the mechanism of transepithelial elimination in PP. Histologically, the dermis above or around the tumor nest exhibited edema, dilated vascular spaces, sparse collagen bundles and absence of elastic fibers, suggesting anetodermic changes in all cases. Immunohistochemistry demonstrated many CD68-positive macrophages around the tumor nests. Matrix metallopeptidase (MMP)-9 and MMP-12 were expressed in the inflammatory cells and tumor cells, and were also present in the epidermis and fibroblasts in all cases. We speculate that in PP anetodermic change caused by MMP and elastases including MMP-9 and MMP-12 may precede elimination of the tumor.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

Gingival Tissue Inflammation Promotes Increased Matrix Metalloproteinase-12 Production by CD200Rlow Monocyte-Derived Cells in Periodontitis.

Irreversible tissue recession in chronic inflammatory diseases is associated with dysregulated immune activation and production of tissue degradative enzymes. In this study, we identified elevated levels of matrix metalloproteinase (MMP)-12 in gingival tissue of patients with the chronic inflammatory disease periodontitis (PD). The source of MMP12 was cells of monocyte origin as determined by the expression of CD14, CD68, and CD64. These MMP12-producing cells showed reduced surface levels of the coinhibitory molecule CD200R. Similarly, establishing a multicellular three-dimensional model of human oral mucosa with induced inflammation promoted MMP12 production and reduced CD200R surface expression by monocyte-derived cells. MMP12 production by monocyte-derived cells was induced by CSF2 rather than the cyclooxygenase-2 pathway, and treatment of monocyte-derived cells with a CD200R ligand reduced CSF2-induced MMP12 production. Further, MMP12-mediated degradation of the extracellular matrix proteins tropoelastin and fibronectin in the tissue model coincided with a loss of Ki-67, a protein strictly associated with cell proliferation. Reduced amounts of tropoelastin were confirmed in gingival tissue from PD patients. Thus, this novel association of the CD200/CD200R pathway with MMP12 production by monocyte-derived cells may play a key role in PD progression and will be important to take into consideration in the development of future strategies to diagnose, treat, and prevent PD.

Effects of MMP12 on cell motility and inflammation during corneal epithelial repair.

Corneal epithelial defects are a common cause of ocular morbidity and can result in corneal scarring if they do not heal properly. Matrix metalloproteinases (MMPs) are extracellular matrix proteinases that regulate multiple aspects of corneal repair. We have previously shown that MMP12 has a protective effect on corneal fibrosis through its regulation of neutrophil and macrophage infiltration and angiogenesis in a chemical injury model involving full thickness damage to the cornea. However, the role of MMP12 in injuries limited to the corneal epithelium is relatively unknown. This study investigates the reparative effects of MMP12 following isolated corneal epithelial injury. Using a corneal epithelial debridement injury model performed on corneas of wild-type (WT) mice, we show that Mmp12 is expressed early following corneal epithelial injury with highest expression levels at 8 h after injury and lower expression levels at 4 and 8 days after injury. We investigated whether MMP12 has an effect on the rate of epithelial repair and cell migration using in vivo and in vitro scratch assays performed on WT and Mmp12-/- mice. We found that loss of MMP12 results in a slower scratch wound repair rate both in vivo and in vitro. We also found that corneas of Mmp12-/- mice have decreased neutrophil infiltration following injury. Loss of MMP12, however, does not affect cell proliferation in the center of the wounds. These data support a role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration.

Effect of TGF-beta1 on MMP/TIMP and TGF-beta1 receptors in great saphenous veins and its significance on chronic venous insufficiency.

Objectives Transforming growth factor-beta1 (TGF-ß1) may participate in local chronic inflammatory processes in varicose veins and in venous wall structure modifications through regulation of matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitor of metalloproteinase (TIMP)). The aim of this study was to analyze the effect of TGF-ß1 in the vein wall, namely on the gene expression of selected MMP, TIMP and TGF-ß1 receptors. Methods Healthy vein samples were harvested from eight subjects who underwent coronary bypass graft surgery with great saphenous vein. Each vein sample was divided into two segments, which were cultivated separately in vitro (one of the segments had TGF-ß1 added) and then submitted to gene expression analysis. Results In the TGF-ß1 supplemented group, there was a general increase in the mean gene expression. Specifically, expression of MMP9, MMP12, TIMP1 and TIMP2 were statistically significant. Conclusion The results of this study demonstrate that the gene expression of MMP9, MMP12, TIMP1 and TIMP2 was influenced by the addition of TGF-ß1. These results may be translated to chronic venous insufficiency framework and suggest involvement of TGF-ß1 in the vein wall pathology.

Type III Secretion System of Pseudomonas aeruginosa Affects Matrix Metalloproteinase 12 (MMP-12) and MMP-13 Expression via Nuclear Factor κB Signaling in Human Carcinoma Epithelial Cells and a Pneumonia Mouse Model.

The type III secretion system (T3SS) in Pseudomonas aeruginosa has been linked to severe disease and poor clinical outcomes in animal and human studies. We aimed to investigate whether the ExoS and ExoT effector proteins of P. aeruginosa affect the expression of matrix metalloproteinase 12 (MMP-12) and MMP-13 via nuclear factor κB (NF-κB) signaling pathways. To understand the T3SS, we used ΔExoS, ΔExoT, and ExsA::Ω mutants, as well as P. aeruginosa strain K (PAK)-stimulated NCI-H292 cells. We investigated the effects of ΔExoS, ΔExoT, and ExsA::Ω on the development of pneumonia in mouse models. We examined the effects of ΔExoS, ΔExoT, and ExsA::Ω on MMP-12 and MMP-13 production in NCI-H292 cells. ΔExoS and ΔExoT markedly decreased the neutrophil count in bronchoalveolar lavage fluid, with a reduction in proinflammatory mediators, MMP-12, and MMP-13. ΔExoS and ΔExoT reduced NF-κB phosphorylation, together with MMP-12 and MMP-13 expression in PAK-infected mouse models and NCI-H292 cells. To conclude, P. aeruginosa infection induced the expression of MMPs, and P. aeruginosa T3SS appeared to be a key player in MMP-12 and MMP-13 expression, which is further controlled by NF-κB signaling. These findings might be useful in devising a novel therapeutic approach to chronic pulmonary infections that involves decreasing the ExoS and ExoT levels.

Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse.

Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δ(D910A/D910A)), we show that fetuses carried by p110δ(D910A/D910A) females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δ(D910A/D910A) females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.

Matrix metalloproteinase 12 modulates high-fat-diet induced glomerular fibrogenesis and inflammation in a mouse model of obesity.

Obesity-induced kidney injury contributes to albuminuria, which is characterized by a progressive decline in renal function leading to glomerulosclerosis and renal fibrosis. Matrix metalloproteinases (MMPs) modulate inflammation and fibrosis by degrading a variety of extracellular matrix and regulating the activities of effector proteins. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm, atherosclerosis, and emphysema, but the underlying mechanisms remain unclear. The present study examined the function of MMP-12 in glomerular fibrogenesis and inflammation using apo E(-/-) or apo E(-/-)MMP-12(-/-) mice and maintained on a high-fat-diet (HFD) for 3, 6, or 9 months. MMP-12 deletion reduced glomerular matrix accumulation, and downregulated the expression of NADPH oxidase 4 and the subunit-p67(phox), indicating the inhibition of renal oxidative stress. In addition, the expression of the inflammation-associated molecule MCP-1 and macrophage marker-CD11b was decreased in glomeruli of apo E(-/-)MMP-12(-/-) mice fed HFD. MMP-12 produced by macrophages infiltrating into glomeruli contributed to the degradation of collagen type IV and fibronectin. Crescent formation due to renal oxidative stress in Bowman's space was a major factor in the development of fibrogenesis and inflammation. These results suggest that regulating MMP-12 activity could be a therapeutic strategy for the treatment of crescentic glomerulonephritis and fibrogenesis.

Matrix Metalloproteinase-12 Induces Blood-Brain Barrier Damage After Focal Cerebral Ischemia.

BACKGROUND AND PURPOSE: Matrix metalloproteinases (MMPs) have a central role in compromising the integrity of the blood-brain barrier (BBB). The role of MMP-12 in brain damage after ischemic stroke remains unknown. The main objective of the current study is to investigate the effect of MMP-12 suppression at an early time point before reperfusion on the BBB damage in rats. METHODS: Sprague-Dawley rats were subjected to middle cerebral artery occlusion and reperfusion. MMP-12 shRNA-expressing plasmids formulated as nanoparticles were administered at a dose of 1 mg/kg body weight. The involvement of MMP-12 on BBB damage was assessed by performing various techniques, including Evans blue dye extravasation, 2,3,5-triphenyltetrazolium chloride staining, immunoblot, gelatin zymography, and immunofluorescence analysis. RESULTS: MMP-12 is upregulated ≈31-, 47-, and 66-fold in rats subjected 1-, 2-, or 4-hour ischemia, respectively, followed by 1-day reperfusion. MMP-12 suppression protected the BBB integrity by inhibiting the degradation of tight-junction proteins. Either intravenous or intra-arterial delivery of MMP-12 shRNA-expressing plasmid significantly reduced the percent Evans blue dye extravasation and infarct size. Furthermore, MMP-12 suppression reduced the endogenous levels of other proteases, such as tissue-type plasminogen activator and MMP-9, which are also known to be the key players involved in BBB damage. CONCLUSIONS: These results demonstrate the adverse role of MMP-12 in acute brain damage that occurs after ischemic stroke and, thereby, suggesting that MMP-12 suppression could be a promising therapeutic target for cerebral ischemia.

Altered Expression Levels of MMP1, MMP9, MMP12, TIMP1, and IL-1ß as a Risk Factor for the Elevated IOP and Optic Nerve Head Damage in the Primary Open-Angle Glaucoma Patients.

The aim of presented work was to analyze the impact of particular polymorphic changes in the promoter regions of the -1607 1G/2G MMP1, -1562 C/T MMP9, -82 A/G MMP12, -511 C/T IL-1ß, and 372 T/C TIMP1 genes on their expression level in POAG patients. Blood and aqueous humor samples acquired from 50 patients with POAG and 50 control subjects were used for QPCR and protein levels analysis by ELISA. In vivo promoter activity assays were carried on HTM cells using dual luciferase assay. All studied subjects underwent ophthalmic examination, including BCVA, intraocular pressure, slit-lamp examination, gonioscopy, HRT, and OCT scans. Patients with POAG are characterized by an increased mRNA expression of MMP1, MMP9, MMP12, and IL-1ß genes as compared to the control group (P < 0.001). Aqueous humor acquired from patients with POAG displayed increased protein expression of MMP1, MMP9, MMP12, and IL-1ß compared to the control group (P < 0.001). Allele -1607 1G of MMP1 gene possesses only 42,91% of the -1607 2G allele transcriptional activity and allele -1562 C of MMP9 gene possesses only 21,86% of the -1562 T allele. Increased expression levels of metalloproteinases can be considered as a risk factor for the development of POAG.

Matrix metalloproteinase-12 expression is increased in cutaneous melanoma and associated with tumor aggressiveness.

Cutaneous melanoma is the most malignant form of skin cancer characterized by aggressive invasion. Matrix metalloproteinases play essential roles in tumor invasion due to their ECM degrading capacity. However, the clinical significance of matrix metalloproteinasis (MMP)-12 in human cutaneous melanoma has not been addressed yet. In the present study, we investigated MMP-12 expression level in 298 patients with cutaneous melanoma and 60 normal skin tissue specimens by immunohistochemistry assay. Appropriate statistical analysis was utilized to determine the association of MMP-12 with clinical features and prognosis of melanoma. Results showed that MMP-12 expression was increased in cutaneous melanoma compared with that in normal skin. It was also found that MMP-12 expression in melanoma was significantly associated with tumor invasion and metastasis. Univariate survival analysis indicated that patients with melanoma of high MMP-12 expression had unfavorable overall survival compared with those of low MMP-12 expression. Cox's proportional hazards analysis showed that MMP-12 expression was an independent prognostic marker of overall survival for patients with cutaneous melanoma. These results proved that MMP-12 expression was increased in cutaneous melanoma and associated with tumor progression. It also provided the first evidence that MMP-12 level could be an independent prognostic marker for patients with cutaneous melanoma.

Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential of ERA and warrant further investigation of their utility in chronic inflammatory airway diseases.

Early matrix metalloproteinase-12 inhibition worsens post-myocardial infarction cardiac dysfunction by delaying inflammation resolution.

RATIONALE: Matrix metalloproteinases (MMPs) regulate remodeling of the left ventricle (LV) post-myocardial infarction (MI). MMP-12 has potent macrophage-dependent remodeling properties in the atherosclerotic plaque; however, post-MI roles have not been examined. OBJECTIVE: The goal was to determine MMP-12 post-MI mechanisms. METHODS AND RESULTS: Male C57BL/6J mice (3-6 months old) were subjected to left coronary artery ligation. Saline or the RXP 470.1 MMP-12 inhibitor (MMP-12i; 0.5mg/kg/day) was delivered by osmotic mini-pump beginning 3h post-MI, and mice were sacrificed at day (d)1, 3, 5 or 7 post-MI and compared to d0 controls (mice without MI; n=6-12/group/time). MMP-12 expression increased early post-MI, and contrary to expected, neutrophils were a surprising early cellular source for MMP-12. MMP-12i reduced MMP-12 activity 33 ± 1% at d1 post-MI. Despite similar infarct areas and survival rates, MMP-12i led to greater LV dilation and worsened LV function. At d7 post-MI, MMP-12i prolonged pro-inflammatory cytokine upregulation (IL1r1, IL6ra, IL11, and Cxcr5) and decreased CD44 (both gene and protein levels). Hyaluronan (HA), a CD44 ligand, was elevated at d1 and d7 post-MI with MMP12i, as a result of decreased fragmentation. Because CD44-HA regulates neutrophil removal, apoptosis markers were evaluated. Caspase 3 increased, while cleaved caspase 3 levels decreased in MMP-12i group at d7 post-MI, indicating reduced neutrophil apoptosis. In isolated neutrophils, active MMP-12 directly stimulated CD44, caspase 3, and caspase 8 expression. CONCLUSION: Our results reveal a novel protective mechanism for MMP-12 in neutrophil biology. Post-MI, MMP-12i impaired CD44-HA interactions to suppress neutrophil apoptosis and prolong inflammation, which worsened LV function.

Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme.

The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of "targeted therapies" as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a "personalized therapy", without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis.

Sulfur compounds block MCP-1 production by Mycoplasma fermentans-infected macrophages through NF-κB inhibition.

BACKGROUND AND AIMS: Hydrogen sulfide (H2S), together with nitric oxide (NO) and carbon monoxide (CO), belongs to a family of endogenous signaling mediators termed "gasotransmitters". Recent studies suggest that H2S modulates many cellular processes and it has been recognized to play a central role in inflammation, in the cardiovascular and nervous systems. By infecting monocytes/macrophages with Mycoplasma fermentans (M.F.), a well-known pro-inflammatory agent, we evaluated the effects of H2S. METHODS: M.F.-infected cells were analyzed by ELISA and real time RT-PCR to detect the M.F. effects on MCP-1 and on MMP-12 expression. The role of two different H2S donors (NaHS and GYY4137) on MF-infected cells was determined by treating infected cells with H2S and then testing the culture supernatants for MCP-1 and on MMP-12 production by ELISA assay. In order to identify the pathway/s mediating H2S- anti-inflammatory activity, cells were also treated with specific pharmaceutical inhibitors. Cytoplasmic and nuclear accumulation of NF-κB heterodimers was analyzed. RESULTS: We show that H2S was able to reduce the production of pro-inflammatory cytokine MCP-1, that was induced in monocytes/macrophages during M.F. infection. Moreover, MCP-1 was induced by M.F. through Toll-like receptor (TLR)-mediated nuclear factor-κB (NF-κB) activation, as demonstrated by the fact that TLR inhibitors TIRAP and MyD88 and NF-κB inhibitor IKK were able to block the cytokine production. In contrast H2S treatment of M.F. infected macrophages reduced nuclear accumulation of NF-κB heterodimer p65/p52. CONCLUSIONS: Our data demonstrate that under the present conditions H2S is effective in reducing Mycoplasma-induced inflammation by targeting the NF-κB pathway. This supports further studies for possible clinical applications.

Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma.

BACKGROUND: Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC. METHODS: We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231. RESULTS: MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning -42 to -33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells. CONCLUSIONS: Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but additional validation studies are warranted.

Retinal MMP-12, MMP-13, TIMP-1, and TIMP-2 expression in murine experimental retinal detachment.

PURPOSE: Matrix metalloproteinases (MMPs) and their inhibitors play a role in the pathobiology of retinal detachment (RD) and proliferative vitreoretinopathy (PVR). Proliferative vitreoretinopathy is facilitated by chronic retinal detachment and involves excessive deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinase-2 and -13 are important modulators of the ECM which have not been evaluated in RD. The purpose of this study was to investigate the retinal expression of select MMPs, including MMP-12, MMP-13, and associated inhibitors in a murine model of retinal detachment. METHODS: Transient or chronic retinal detachments (RDs) were induced by subretinal injection of either saline (SA) or hyaluronic acid (HA) in C57BL/6 mice. To confirm that the HA-RD model has features consistent with PVR-like changes, glial activation and subretinal fibrosis were evaluated with immunofluorescence, dilated fundus examination, and spectral-domain optical coherence tomography (SD-OCT). Gene expression was quantified by qRT-PCR. Proteins were assayed by immunoblot and immunohistochemistry. RESULTS: Hyaluronic acid RD eyes developed gliosis and subretinal fibrosis on dilated exam, SD-OCT, and immunofluorescence analysis. Gene expression of Mmp-12 and Mmp-13, and Timp-1 was strongly upregulated at all time points in RD compared with controls. Timp-2, Mmp-2, and Mmp-9 expression was modest. Hyaluronic acid RDs exhibited more MMP and TIMP expression than SA-RDs. MMP-12, -13, and TIMP-1 proteins were elevated in RDs compared with controls. Immunohistochemistry revealed moderate to strong MMP-13 levels in subretinal space macrophages. CONCLUSIONS: Fibrosis can develop in the HA-RD model. There is an upregulation of select MMPs that may modulate the wound healing process following RD.

Human amniotic epithelial stem cells inhibit microglia activation through downregulation of tumor necrosis factor-α, interleukin-1ß and matrix metalloproteinase-12 in vitro and in a rat model of intracerebral hemorrhage.

BACKGROUND AIMS: The molecular mechanisms by which stem cell transplantation improves functional recovery after intracerebral hemorrhage (ICH) are not well understood. Accumulating evidence suggests that microglia cells are activated shortly after ICH and that this activation contributes to secondary ICH-induced brain injury. We studied the effect of human amniotic epithelial stem cells (HAESCs) on microglia activation. METHODS: To study the effect of HAESCs in vitro, we used thrombin to activate the microglia cells. Twenty-four hours after thrombin treatment, the levels of tumor necrosis factor-α and interleukin-1ß were measured by enzyme-linked immunosorbent assay. In vivo, the HAESCs were transplanted into the rat striatum 1 day after collagenase-induced ICH. The expression levels of matrix metalloproteinase (MMP)-12 and microglia infiltration in the peri-hematoma tissues were determined 7 days after ICH through the use of reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, respectively. RESULTS: Thrombin-activated microglia expression of tumor necrosis factor-α, interleukin-1ß and MMP-12 was significantly reduced through contact-dependent and paracrine mechanisms when the HAESCs were co-cultured with microglia cells. After transplantation of HAESCs in rat brains, the expression levels of MMP-12 and microglia infiltration in the peri-hematoma tissues were significantly reduced. CONCLUSIONS: Our observations suggest that microglia activation could be inhibited by HAESCs both in vitro and in vivo, which may be an important mechanism by which the transplantation of HAESCs reduces brain edema and ameliorates the neurologic deficits after ICH. Therefore, we hypothesize that methods for suppressing the activation of microglia and reducing the inflammatory response can be used for designing effective treatment strategies for ICH.