PPARs配体对巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子表达的影响

目的：观察PPAR α、γ配体对巨噬细胞、泡沫细胞细胞外基质金属蛋白酶诱导因子(EMMPRIN)表达的影响。方法：体外诱导THP-1单核细胞转化为巨噬细胞、泡沫细胞，分别加入PPAR α配体氯贝特（clofibrate）、PPARγ配体吡格列酮(pioglitazone)共同培养，应用Real-time RT-PCR和Western blotting测定巨噬细胞、泡沫细胞中EMMPRIN基因和蛋白表达，ELISA测定细胞培养上清液MMP-9浓度，Zymgraphy法测定MMP-9活性。结果：氯贝特和吡格列酮均能显著抑制巨噬细胞和泡沫细胞EMMPRIN的表达，此抑制作用与PPAR α、γ配体抑制MMP-9分泌及活性的趋势一致。结论：PPAR α、γ配体均可抑制巨噬细胞、泡沫细胞EMMPRIN的表达，下调EMMPRIN可能是PPARs配体抑制粥样斑块局部MMPs产生的机制之一。     

MMP-2、MMP-9及EMMPRIN在子宫内膜异位症中的表达及临床意义

目的研究基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、细胞外基质金属蛋白酶诱导因子（EMMPRIN）在子宫内膜异位症（EMs）的表达和意义。方法应用免疫组化二步法检测EMs患者异位内膜42例、在位内膜42例及正常内膜20例中的MMP-2、MMP-9、EMMPRIN的表达情况,并对它们的EMMPRIN、MMP-2、MMP-9蛋白表达水平进行相关性分析。结果 MMP-2、MMP-9、EMMPRIN在异位内膜组中阳性表达率分别为95.24%、92.86%和90.48%,显著高于在位内膜组、正常内膜组（P〈0.05）;而在位内膜组和正常内膜组差异无统计学意义（P〉0.05）。异位内膜组中,EMMPRIN分别和MMP-2,MMP-9呈正相关性（P〈0.01）。结论 MMP-2、MMP-9、EMMPRIN共同参与了子宫内膜异位症的发生发展;EMMPRIN可能通过促进MMP-2和MMP-9的合成与分泌发挥其作用。     

基因沉默PRDX1 表达对人结直肠癌SW480 细胞侵袭迁移的影响

目的:探讨RNA 干扰过氧化还原酶1(Peroxiredoxin 1,PRDX1)表达对人结直肠癌SW480 细胞侵袭转移能力的影响。方法:筛选RNA 干扰PRDX1 的慢病毒质粒,与阴性对照慢病毒质粒分组转染结直肠癌SW480 细胞,转染后的SW480 细胞可分为PRDX1 基因沉默组(si-PRDX1)和阴性对照组(Vector)。实时荧光定量PCR(qRT-PCR)和免疫印迹法(Western blot)分别检测两组细胞中PRDX1 mRNA 和蛋白表达;采用Transwell 侵袭和迁移实验检测基因沉默PRDX1 表达对结直肠癌细胞侵袭及迁移能力的影响;通过Western blot 检测两组细胞中基质金属蛋白酶(MMP)家族部分蛋白表达水平。结果:基因沉默PRDX1 表达可有效抑制结直肠癌SW480 细胞中PRDX1 mRNA 和蛋白水平的表达,与阴性对照组相比(Vector),差异均具有统计学意义(P<0.01),说明基因沉默PRDX1 的SW480 细胞系构建成功;Transwell 侵袭和迁移实验显示si-PRDX1组细胞的侵袭及迁移能力较对照组均明显降低(P<0.01);Western blot 结果显示,与Vector 组相比,si-PRDX1 组细胞中组织基质金属蛋白酶抑制剂2(TIMP-2)的表达明显增加,而MMP-2 及MMP-9 的表达显著下降,且差异均具有统计学意义(P<0.05)。结论:基因沉默人结直肠癌SW480 细胞的PRDX1 表达可有效抑制细胞的侵袭、迁移及转移能力,其机制可能会通过调控TIMP-2、MMP-2 及MMP-9 的表达介导。     

SM22α对结直肠癌细胞侵袭和迁移能力的影响

目的:探究平滑肌22α蛋白(SM22α)对结直肠癌细胞侵袭和迁移能力的影响,并探讨其分子机制。方法:通过慢病毒转染人结直肠癌细胞HCTL16构建SM22α过表达细胞,应用细胞划痕实验检测细胞迁移能力的变化;Transwell小室检测细胞侵袭和迁移能力的变化;使用RT-qPCR检测细胞SM22αmRNA表达的改变;通过Western blot法检测细胞外信号调节激酶(ERK)、p-ERK、基质金属蛋白酶9(MMP-9)和SM22α蛋白水平。结果:成功构建HCT116过表达SM22α细胞;SM22α过表达细胞的侵袭和迁移能力减弱(P0.05);SM22α过表达抑制p-ERK和MMP-9的蛋白水平(P0.05)。结论:SM22α通过抑制ERK/MMP-9信号通路调节结直肠癌细胞侵袭和迁移能力。     

低氧抑制猪肺动脉平滑肌细胞MMP-2和MMP-9的表达

目的探讨低氧对猪肺动脉平滑肌细胞（PASMC）分泌基质金属蛋白酶（MMPs）的影响。方法采用酶谱法测定PASMC培养基中MMP-2和MMP-9的酶活性，免疫印迹法检测培养基中MMP-2和MMP-9的蛋白表达，免疫组化法测定细胞原位MMP-2和MMP-9的蛋白表达，RT-PCR法检测mRNA的表达。结果低氧后PASMC分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；MMP-9酶活性、细胞外蛋白表达量下降，而细胞内蛋白表达无变化。结论低氧可抑制PASMC分泌MMP-2和MMP-9的酶活性，其机制可能是低氧影响PASMC中MMP-2基因的转录、影响MMP-9蛋白表达后的分泌与活化，导致MMP-2和MMP-9酶活性的改变。     

量子点免疫荧光技术检测EMMPRIN、MMP-2和 P53蛋白在人肺癌组织中的表达意义

目的: 本研究利用新型免疫荧光标记试剂-量子点(QDs)结合组织芯片技术检测细胞外基质金属蛋白酶诱导因子(EMMPRIN/CD147)、基质金属蛋白酶2(MMP-2)和P53 蛋白在人肺癌组织中的表达,并探讨EMMPRIN与肺癌恶性生物学行为的关系。方法: 利用QDs免疫荧光组织化学(QDs-IHC)技术分别检测人肺癌组织芯片中EMMPRIN、MMP-2和P53蛋白的表达,并利用QDs免疫荧光双标法同时检测了EMMPRIN与P53蛋白的共表达。结果: EMMPRIN、MMP-2和P53蛋白表达在肺癌组织的阳性率分别为:70.00%、77.14%和72.86%,与非癌变肺组织相比,差异均显著(P<0.05);而且与肺癌的TNM分期、淋巴结转移均显著相关(P<0.05),但与其它的临床病理特征均明显无关(P>0.05)。EMMPRIN和MMP-2、P53蛋白表达之间均呈显著正相关(P<0.01)。结论: EMMPRIN、MMP-2和P53蛋白均与肺癌的发生有关,EMMPRIN可能与MMP-2、P53协同促进肺癌的恶性进展。     

长链非编码RNA母系表达基因3对结直肠癌细胞侵袭和迁移能力的影响

目的:检测长链非编码RNA母系表达基因3(maternally expressed gene 3,MEG3)在结直肠癌细胞中的表达,并观察过表达MEG3对结直肠癌细胞侵袭和迁移能力的影响。方法:检测人正常结肠细胞NCM460及结直肠癌细胞SW48、Lo Vo中MEG3的水平,在SW48细胞和Lo Vo细胞中转染MEG3过表达质粒,利用Transwell小室及划痕实验观察过表达MEG3对细胞侵袭和迁移能力的影响,通过Western blotting检测基质金属蛋白酶(matrix metalloproteinase,MMP)家族相关蛋白的变化。结果:结直肠癌细胞SW48和Lo Vo中MEG3的水平明显低于人正常结肠细胞NCM460;在SW48和Lo Vo细胞中过表达MEG3后能够明显抑制细胞的侵袭和迁移能力;Transwell侵袭实验和迁移实验显示MEG3表达组SW48细胞的穿膜数及Lo Vo细胞的穿膜数与对照组比较明显减少。划痕实验中过表达MEG3后细胞间距较对照组明显增大,提示细胞运动能力减弱。同时过表达MEG3可明显降低细胞中MMP-2及MMP-9的表达,增高金属蛋白酶组织抑制物2(tissue inhibitor of metalloproteinase-2,TIMP-2)的表达。结论:结直肠癌细胞中MEG3水平较正常结直肠细胞明显降低;在结直肠癌中过表达MEG3可抑制细胞的侵袭、迁移运动能力,并且可能通过影响TIMP-2、MMP-2及MMP-9的表达发挥上述功能。     

启动子区DNA去甲基化增强子宫内膜异位症患者子宫内膜基质细胞MMP-9的表达

目的研究子宫内膜异位症中基质金属蛋白酶9(MMP-9)基因表达的DNA甲基化调控机制。方法采用甲基化特异性PCR检测在位与异位子宫内膜组织中MMP-9 mRNA的表达,采用免疫组织化学染色检测在位与异位子宫内膜组织中MMP-9蛋白的表达。原代培养子宫内膜异位基质细胞,细胞经5-杂氮-2'-脱氧胞苷(5-Aza-dC)处理后,分析MMP-9基因的表达及其启动子甲基化状态。结果异位子宫内膜组织中MMP-9 mRNA和蛋白的表达高于在位子宫内膜组织。异位子宫内膜组织中MMP-9基因启动子区DNA甲基化水平低于在位子宫内膜组织。5-Aza-dC处理异位子宫内膜细胞后,MMP-9的表达水平升高,MMP-9基因启动子区出现明显的去甲基化。结论 MMP-9基因启动子区DNA去甲基化增强子宫内膜异位症患者异位内膜基质细胞中MMP-9的表达。     

红景天苷对骨髓抑制贫血小鼠骨髓基质金属蛋白酶的影响

本研究用免疫组织化学方法和酶谱电泳法观察了红景天苷对骨髓抑制贫血小鼠骨髓基质金属蛋白酶-2和-9（MMP-2、MMP-9）的影响，并探讨其在造血调控中的可能作用。免疫组化结果显示，各组小鼠骨髓细胞中均检测到MMP-2和MMP-9表达。与对照组相比，模型组及高、中、低剂量红景天苷组骨髓细胞中MMP-2、MMP-9蛋白表达明显增强。在造模后第4d，中剂量红景天苷组中MMP-2和MMP-9表达最强。在8d，分别是低剂量和中剂量红景天苷组中MMP-2和MMP-9表达最强。酶谱电泳结果显示，在对照组骨髓中可检测到66kD（MMP-2的酶原形式，proMMP-2）、62kD（MMP-2的活性形式，MMP-2）、86kD（MMP-9活性形式，MMP-9）和94kD（MMP-9的酶原形式，proMMP-9）4条明胶酶的活性带，其中MMP-9的活性最强。造模后骨髓造血微环境中明胶酶的活性明显降低，不同剂量的红景天苷均能明显促进proMMP-9和MMP-9的活性增强，使proMMP-2的活性减弱。结果表明，红景天苷可能通过促进骨髓细胞中基质金属蛋白酶表达增强、骨髓造血微环境中基质金属蛋白酶活性升高，进而导致ECM中或基质细胞膜上细胞因子的释放、骨髓微血管损伤的修复以及HSCs增殖、迁移和分化能力的增加来促进骨髓造血功能的恢复。     

弗林蛋白酶抑制剂对乳腺癌细胞MCF-7迁移的影响

 目的：探讨乳腺癌转移的机制,为深入研究乳腺癌发生、发展机制提供理论基础。方法：不同浓度的弗林蛋白酶(furin)抑制剂处理人乳腺癌细胞MCF-7 48 h。细胞划痕实验（wound healing assay）和细胞趋化实验（Transwell assay）检测MCF-7细胞迁移和侵袭能力。Western blotting 检测细胞迁移相关蛋白膜型基质金属蛋白酶1（MT1-MMP）、血管内皮生长因子（VEGF）-C和VEGF-D水平。酶联免疫吸附法（ELISA）检测细胞培养液中基质金属蛋白酶(MMP)2和9水平。结果：与对照组相比,200 nmol/L的furin抑制剂α1-PDX即对细胞迁移及侵袭起显著抑制作用（均P<0.05）;细胞迁移相关的MT1-MMP、VEGF-C和VEGF-D表达水平均显著降低（P<0.05）;MCF-7细胞上清液中MMP2 和 MMP9的表达均显著降低（P<0.05）。结论：Furin抑制剂通过下调乳腺癌细胞MCF-7的MMPs及VEGFs表达抑制其迁移。     

Suppression of oxLDL‐Induced MMP‐9 and EMMPRIN Expression by Berberine via Inhibition of NF‐κB Activation in Human THP‐1 Macrophages

Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF‐κB activation regulates many key inflammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP‐9 and EMMPRIN expression, the role of NF‐κB activation in oxLDL‐stimulated macrophages, and the possible mechanism in which NF‐κB activation is involved were investigated. Berberine inhibited the expression of MMP‐9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IκB‐α and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF‐κB activation was inhibited by berberine in oxLDL‐stimulated macrophages. Overall, berberine suppressed the expression of MMP‐9 and EMMPRIN by at least reducing partly the activity of NF‐κB in oxLDL‐induced macrophages. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

EMMPRIN modulates epithelial barrier function through a MMP-mediated occludin cleavage: implications in dry eye disease

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure.     

Soluble EMMPRIN (extra-cellular matrix metalloproteinase inducer) stimulates the migration of HEp-2 human laryngeal carcinoma cells, accompanied by increased MMP-2 production in fibroblasts

The basement membrane functions as a barrier against the invasion of cancer cells. It is therefore important to investigate the mechanism of basement membrane degradation by matrix metalloproteinases (MMPs). Previously, cancer cells were long considered to be the major source of MMPs; however, current evidence indicates that most MMPs in cancer tissue are produced by stromal rather than cancer cells. A glycoprotein highly expressed on the cancer-cell membrane, EMMPRIN (extra-cellular matrix metalloproteinase inducer), exhibits the potential role of the MMP inductor in stromal cells. Depending on the cell type, EMMPRIN can stimulate the production of MMP-1, MMP-2, and MMP-3. We here report that soluble full-length EMMPRIN is liberated from HEp-2 human laryngeal epidermoid carcinoma cells, probably via microvesicle shedding. Soluble EMMPRIN stimulates human fibroblasts to produce MMP-2, after which the augmented migration of HEp-2 cells occurs, as observed in an invasion chamber assay with separately cultured fibroblasts. An anti-EMMPRIN function-blocking antibody reduced MMP-2 activity in the conditioned medium and inhibited the migration of HEp-2; obviously, EMMPRIN activity contributes to cancer-cell migration. We postulate that soluble EMMPRIN probably triggers the promotion of cancer invasion in vivo.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

GM-CSF activates RhoA, integrin and MMP expression in human monocytic cells

Monocyte migration is one of the key events occurring in the early stage of atherosclerosis. This process includes monocytic adhesion to and penetration through the arterial intima. In such an environment, many factors stimulate the monocytes to enhance integrin activation and extracellular matrix degradation. To investigate the coordinative operation of these two events in relation to monocyte migration, we paid particular attention to the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on monocytes in terms of RhoA activation and matrix metalloproteinase (MMP) expression. RhoA and integrin clustering were activated by GM-CSF, monocyte chemoattractant protein-1 (MCP-1) and platelet-derived growth factor-BB (PDGF-BB) in human monocytic cell lines. Furthermore, enhancement of migration was observed with stimulation by MCP-1 and PDGF-BB. Granulocyte-macrophage colony-stimulating factor did not enhance the migration, even though it activated RhoA and integrin. However, GM-CSF is known to stimulate monocytes to express MCP-1, suggesting the presence of an indirect mechanism for GM-CSF-mediated migratory activity. In contrast, only GM-CSF enhanced the expression of MMP-1 and MMP-9. These results provide evidence that GM-CSF has multiple functions enhancing monocytic migration via RhoA and integrin activation, and via MMP expression.     

Modulation of matrix metalloproteinase-9 secretion from tumor-associated macrophage-like cells by proteolytically processed laminin-332 (laminin-5)

Macrophages infiltrating tumor tissues (tumor-associated macrophages, TAM) affect the malignant behaviors of tumor cells. We previously reported that monocytes were differentiated into TAM-like cells secreting matrix metalloproteinase (MMP)-9 by co-culture with tumor cells, and that cell adhesion to extracellular matrix (ECM) proteins played a critical role in the differentiation. In this study, we found that the monocyte differentiation was promoted by laminin-332 (laminin-5), a major epithelial ECM component. We also demonstrated that the proteolytic processing of the γ2 chain of laminin-332 was essential for its activity but that the N-terminal short arm of the γ2 chain inhibited MMP-9 secretion. These results indicate that the activity of laminin-332 for monocyte differentiation is dynamically regulated by the proteolytic processing of the γ2 chain.     

EMMPRIN-mediated MMP regulation in tumor and endothelial cells

Tumor invasion and metastasis are multistep processes which require extracellular matrix remodeling by proteolytic enzymes such as matrix metalloproteinases (MMPs). The production of these enzymes is stimulated by many soluble or cell-bound factors. Among these factors, extracellular matrix metalloproteinase inducer (EMMPRIN) is known to increase in vitro stromal cell production of MMP-1, MMP-2 and MMP-3. In this study, we demonstrated that EMMPRIN-transfected MDA-MB-436 tumor cells displayed a more invasive capacity than vector-transfected cells in a modified Boyden chamber invasion assay. Using gelatin zymography and protein analyses, we showed that EMMPRIN-transfected cancer cells produced significantly more latent and active MMP-2 and MMP-3 than vector-transfected cancer cells. We found that EMMPRIN did not regulate MMP-1, MMP-9, membrane type-1 MMP (MT1-MMP) expression and had also no effect on the production of the specific tissue inhibitors of MMPs (TIMPs), TIMP-1 and TIMP-2. We also demonstrated that tumor-derived EMMPRIN stimulated MMP-1, -2, and -3 without modification of MMP-9, MT1-MMP, TIMP-1 and TIMP-2 production in human umbilical vein endothelial cells (HUVEC). These data provide support for the role of EMMPRIN in tumor invasion, metastasis, and neoangiogenesis by stimulating extracellular matrix remodeling around tumor cell clusters, stroma, and blood vessels. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential expression of extracellular matrix metalloproteinase inducer (CD147) in normal and ulcerated corneas: role in epithelio-stromal interactions and matrix metalloproteinase induction

Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally identified on the tumor cell surface as an inducer of matrix metalloproteinase (MMP) production in neighboring fibroblasts. Here we demonstrate a role for EMMPRIN in MMP induction during corneal wound healing. MMP and EMMPRIN expression was analyzed in normal and ulcerated human corneas, as well as in corneal epithelial and stromal cells in culture using confocal microscopy, zymography, immunoblots, and real-time polymerase chain reaction. In normal cornea EMMPRIN was predominantly expressed in the epithelium but was markedly induced in the anterior stroma of ulcerated corneas. This coincided with MMP-2 induction that co-localized with EMMPRIN at the epithelio-stromal boundary. The role of epithelial-stromal interaction in MMP induction was investigated in an in vitro co-culture system and demonstrated an induction and co-localization of EMMPRIN and MMP-2 in the fibroblasts at the interface with epithelial cells. Direct contact of fibroblasts with EMMPRIN-containing purified epithelial cell membranes also induced MMP-1, MMP-2, and EMMPRIN and this was inhibited by a blocking anti-EMMPRIN antibody, suggesting that EMMPRIN was primarily responsible for this induction. These findings, and the up-regulation of EMMPRIN by epidermal growth factor and transforming growth factor-beta, demonstrate a role for EMMPRIN in wound healing and suggest that sustained local up-regulation of EMMPRIN and MMPs in chronic situations in which healing is delayed may lead to excessive matrix degradation and corneal melts.