The LRP1-independent mechanism of PAI-1-inudced migration in CpG-ODN activated macrophages

CpG-oligodeoxynucleotides (CpG-ODNs) induces plasminogen activator inhibitor type-1 (PAI-1) expression in macrophages, leading to enhanced migration through vitronectin. However, the precise role of low-density lipoprotein receptor-related protein 1 (LRP1) in PAI-1 induced migration of macrophages in the inflammatory environment is not known. In this study, we elucidated a novel mechanism describing the altered role of LRP1 in macrophage migration depending on the activation state of the cells. Experimental evidence clearly shows that the blocking of LRP1 function inhibited the PAI-induced migration of resting RAW 264.7 cells through vitronectin but exerted a pro-migratory effect on CpG-ODN-activated cells. We also demonstrate that CpG-ODN downregulates the protein and mRNA levels of LRP1 both in vivo and in vitro, a function that depends on the NF-κB signaling pathway, resulting in reduced internalization of PAI-1. This work illustrates the distinct mechanism that PAI-1-induced migration of CpG-ODN-activated cells through vitronectin depends on the interaction of PAI-1 with vitronectin but not LRP1 unlike in the resting cells, where the migration is LRP1 dependent and vitronectin independent. In conclusion, our experimental results demonstrate the altered function of LRP1 in the migration of resting and activated macrophages in the context of microenvironmental extracellular matrix components.     

同型半胱氨酸对人脐静脉血管内皮细胞tPA 及PAI-1基因表达的影响

目的探讨同型半胱氨酸(Hcy)对纤溶系统的影响,观察Hcy在转录水平对人脐静脉血管内皮细胞(HUVEC)表达组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制剂1(PAI1)的影响。方法将体外培养的HUVEC分为生理浓度(10μmol/LHcy)组,病理浓度(50、200、500μmol/L)Hcy组及单纯培养基组(0μmol/LHcy),培养24h后,提取RNA,反转录聚合酶链反应分析(RTPCR)法分析各组tPA及PAI1基因表达水平。结果500μmol/LHcy组与10μmol/LHcy组相比,tPAmRNA基因表达明显下调(P<0.05),PAI1mRNA表达则明显上调(P<0.05)。而与单纯培养基组相比,10μmol/LHcy组tPAmRNA表达明显增高(P<0.05)。结论生理浓度Hcy可以增加纤溶系统活性,减少血栓性疾病的发生。高Hcy(病理浓度)则抑制纤溶系统活性,促进缺血性心脑血管疾病的发生。     

tPA, uPA, PAI-1和 PAI-2在人和恒河猴胎盘中的原位杂交和荧光免疫定位(英文)

用原位杂交和荧光免疫定位方法研究了组织型（ｔ）和尿激酶型（ｕ）纤溶酶原激活因子ｔＰＡ、ｕＰＡ和相应的抑制因子ＰＡＩ－１、ＰＡＩ－２在人和恒河猴胎盘中的定位和分布。结果表明：（１）激活因子ｔＰＡ、ｕＰＡ（Ｆｉｇ．１＆４）和抑制因子ＰＡＩ－１（Ｆｉｇ．２）、ＰＡＩ－２（Ｆｉｇ．３）一般都在不同程度上定位于两者胎盘的相同部位;（２）它们主要分布在绒毛干和蜕膜的血管壁、 ＲＯｈｒ’ｓ和Ｎｉｔａｂｕｃｈ’ｓ纹间的基盘外绒毛滋养层细胞、滋养壳、蜕膜细胞和腺体细胞。并且发现;ｔＰＡ和它的抑制因子ＰＡＩ－１更明显地定位于邻近母体组织离层界面的区域,而ｕＰＡ和抑制因子ＰＡＩ－１更集中在绒毛滋养层和外绒毛滋养细胞中;（３）激活因子和抑制因子的ｍＲＮＡ和蛋白的定位和分布基本上一致,但是、在绒毛核体滋养层细胞上未发现其ｍＲＮＡ表达,却有很强的免疫荧光的分布;（４）激活因子和抑制因子合成和分布部位与其作用底物,即纤蛋白类分子的产生部位一致;（５）未发现上述分子在人和恒河猴胎盘分布上的不同。上述实验结果说明,在妊娠的各个阶段 ＰＡ和它的抑制因子协同表达,局限在作用范围很小的特定产生底物的区域。它们的相互作用可能是维持正常妊娠所必需。在妊     

Mac-1 promotes FcgammaRIIA-dependent cell spreading and migration on immune complexes

The integrin Mac-1 plays a critical role in Fc receptor (FcR)-mediated antibody-dependent cellular cytotoxicity (ADCC). However, the mechanism by which Mac-1 facilitates the functions of FcgammaRIIA, a major FcR expressed on human leukocytes, is not fully understood. We report here that Mac-1 sustains cell adhesion, enhances cell spreading, and accelerates cell migration on preformed immune complexes (ICs) by directly interacting with FcgammaRIIA but not with the IC substrate. Coupling Mac-1 to FcgammaRIIA allows FcgammaRIIA to reside in the leading front of actin polymerization at the filopodial extension and thus could potentially enhance FcgammaRIIA-mediated cell spreading and migration. The direct interaction between Mac-1 and FcgammaRIIA is demonstrated by co-immunoprecipitation, by cell surface co-localization, and by solid-phase binding assays using recombinant alpha(M)I-domain and soluble FcgammaRIIA. Further mutational analysis identifies the E(253)-R(261) sequence within the alpha(M)I-domain as part of the FcgammaRIIA binding interface within Mac-1. Altogether, these results demonstrate that FcgammaRIIA recognizes Mac-1 via the alpha(M)I-domain but not the lectin domain, a distinct feature from other FcRs, and that Mac-1 binding confers FcgammaRIIA with the ability to prolong cell adhesion as well as to spread and migrate on the ICs, leading to effective cell killing by ADCC.     

The PAI-1 swing: microenvironment and cancer cell migration

Cancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process, we need to study each one of the levels involved, which allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymo-amaeboid transition. This matricellular protein should be considered as a potential 'launcher' of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level.     

CYLD coordinates with EB1 to regulate microtubule dynamics and cell migration

Cylindromatosis (CYLD), a deubiquitinase involved in inflammation and tumorigenesis via the modulation of cell signaling, has recently been identified as a critical regulator of microtubule dynamics. CYLD has also been shown to stimulate cell migration and thereby contribute to normal physiological processes. However, it remains elusive how the regulation of microtubule dynamic properties by CYLD is connected to its role in mediating cell migration. In this study, we performed yeast 2-hybrid screening with CYLD as bait and identified 7 CYLD-interacting proteins, including end-binding protein 1 (EB1). The CYLD–EB1 interaction was confirmed both in cells and in vitro, and these 2 proteins colocalized at the plus ends of microtubules. Interestingly, the association of CYLD with EB1 was significantly increased upon the stimulation of cell migration. CYLD coordinated with EB1 to orchestrate tail retraction, centrosome reorientation, and leading-edge microtubule stabilization in migratory cells. In addition, CYLD acted in concert with EB1 to regulate microtubule assembly in vitro, microtubule nucleation at the centrosome, and microtubule growth at the cell periphery. These data provide mechanistic insights into the actions of CYLD in the regulation of microtubule dynamics and cell migration. These findings also support the notion that coordinated actions of microtubule-binding proteins are critical for microtubule-mediated cellular events.     

Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.

To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.     

How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration

The interaction of the plasma protein vitronectin with plasminogen activator inhibitor-1 (PAI-1) is central to human health. Vitronectin binding extends the lifetime of active PAI-1, which controls hemostasis by inhibiting fibrinolysis and has also been implicated in angiogenesis. The PAI-1-vitronectin binding interaction also affects cell adhesion and motility. For these reasons, elevated PAI-1 activities are associated both with coronary thrombosis and with a poor prognosis in many cancers. Here we show the crystal structure at a resolution of 2.3 A of the complex of the somatomedin B domain of vitronectin with PAI-1. The structure of the complex explains how vitronectin binds to and stabilizes the active conformation of PAI-1. It also explains the tissue effects of PAI-1, as PAI-1 competes for and sterically blocks the interaction of vitronectin with cell surface receptors and integrins. Structural understanding of the essential biological roles of the interaction between PAI-1 and vitronectin opens the prospect of specifically designed blocking agents for the prevention of thrombosis and treatment of cancer.     

Regulation of IL-1 and TNF receptor expression and function by endogenous macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.     

PAI-1过表达促进食管鳞癌细胞的侵袭和迁移

食管癌是常见的恶性肿瘤之一。由SERPINE1基因编码的纤溶酶原激活物抑制因子1(plasminogen activator inhibitor-1,PAI-1)已被报道在多种类型癌症患者的肿瘤组织中存在高表达并参与癌症进展。为探讨PAI-1蛋白在食管鳞癌中的作用及其分子机制,本研究首先利用Westernblot实验和酶联免疫吸附实验(enzyme linked immunosorbent assay, ELISA)检测各食管鳞癌细胞系中PAI-1的表达和分泌水平,结果显示,PAI-1高表达的食管鳞癌细胞系分泌至细胞外的PAI-1水平相对较高。进一步选取PAI-1表达及分泌水平均较高的KYSE150和KYSE450细胞系作为研究模型,通过si RNA(小干扰RNA)瞬时转染和Transwell实验证实敲降SERPINE1可显著抑制食管鳞癌KYSE150和KYSE450细胞的侵袭和迁移。同时,构建了慢病毒介导的SERPINE1稳定敲降细胞株KYSE150和KYSE450,将SERPINE1稳定敲降的细胞培养基中外源加入PAI-1蛋白进行Transwell回复实验,结果表明PAI-1过表达可增强食管鳞癌细胞的侵袭和迁移能力。体内实验结果显示,降低PAI-1表达可显著抑制食管鳞癌细胞的成瘤和肺转移能力。分子水平检测表明PAI-1过表达可激活AKT和ERK信号通路,免疫共沉淀(co-immunoprecipitation,Co-IP)实验结果进一步显示PAI-1可能与膜受体LRP1(LDLreceptor related protein1)存在相互作用。上述研究结果表明,PAI-1可能通过与LRP1相互作用进而促进食管鳞癌细胞的侵袭和迁移。     

Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.     

Silencing of DLC1 upregulates PAI-1 expression and reduces migration in normal prostate cells

Deleted in liver cancer 1 (DLC1) is a GTPase-activating protein (GAP) domain containing tumor suppressor that localizes to focal adhesions. In cancer cells, loss of DLC1 is known to enhance cancer cell migration. However, the role of DLC1 in normal cell migration has not been well studied. Here, we show that silencing of DLC1 (shDLC1) in normal prostate epithelial cells reduces cell migration in both Transwell and wound-healing assays. This migration defect is mainly due to upregulation of plasminogen activator inhibitor 1 (PAI-1). Silencing of PAI-1 rescues the shDLC1-reduced migration phenotype. Reexpression of DLC1 suppresses PAI-1 and restores the migration defect as well. In contrast, DLC1-K714E (GAP inactive) mutant neither decreases the PAI-1 level nor rescues the shDLC1 migration defect. Interestingly, DLC1-Y442F (tensin-binding and focal adhesion-localizing defective) mutant is able to suppress PAI-1 expression but does not restore the migration defect. Furthermore, PAI-1 upregulation in shDLC1 cells is EGFR-MEK pathway dependent and is able to promote in vitro angiogenesis. Together, our results show that at least the following two new mechanisms are involved in DLC1-mediated normal cell migration: (i) DLC1 modulates the expression of PAI-1, which is a negative regulator for cell migration, in a GAP domain and EGFR-MEK-dependent manner and (ii) Independent of PAI-1, the interaction of DLC1 with tensin members positively regulates cell migration.     

Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.     

P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX₇ and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX₇ pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX₇ pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX₇ for neurovascular protection during SE.     

Beta-2-glycoprotein 1-dependent macrophage uptake of apoptotic cells. Binding to lipoprotein receptor-related protein receptor family members

The recognition and removal of apoptotic cells is critical to development, tissue homeostasis, and the resolution of inflammation. Many studies have shown that phagocytosis is regulated by signaling mechanisms that involve distinct ligand-receptor interactions that drive the engulfment of apoptotic cells. Studies from our laboratory have shown that the plasma protein beta-2-glycoprotein 1 (beta2GP1), a member of the short consensus repeat superfamily, binds phosphatidylserine-containing vesicles and apoptotic cells and promotes their bridging and subsequent engulfment by phagocytes. The phagocyte receptor for the protein/apoptotic cell complex, however, is unknown. Here we report that a member of the low density lipoprotein receptor-related protein family on phagocytes binds and facilitates engulfment of beta2GP1-phosphatidylserine and beta2GP1-apoptotic cell complexes. Using recombinant beta2GP1, we also show that beta2GP1-dependent uptake is mediated by bridging of the target cell to the phagocyte through the protein C- and N-terminal domains, respectively.     

The mouse leukocyte adhesion proteins Mac-1 and LFA-1: studies on mRNA translation and protein glycosylation with emphasis on Mac-1

Translation in vitro of mRNA and immunoprecipitation with specific rabbit antisera showed that the unglycosylated precursor polypeptides of the mouse Mac-1 and lymphocyte function associated antigen (LFA-1) alpha subunits are 130,000 Mr and 140,000 Mr, respectively. Furthermore, polysomes purified by using anti-Mac-1 IgG yielded a similar major product of translation in vitro of Mr = 130,000. The Mac-1 and LFA-1 alpha subunit translation products are immunologically noncross-reactive, showing that differences between these related proteins are not due to post-translational processing. Mac-1 and LFA-1 alpha subunits could only be in vitro translated from mRNA from cell lines the surfaces of which express the corresponding Mac-1 and LFA-1 alpha-beta complexes, showing tissue-specific expression is regulated at the mRNA level. The glycosylation of Mac-1 was examined by both translation in vitro in the presence of dog pancreas microsomes and by biosynthesis in vivo and treatment with tunicamycin, endoglycosidase H, and the deglycosylating agent trifluoromethane sulfonic acid. High mannose oligosaccharides are added to the Mac-1 alpha and beta polypeptide backbones of Mr = 130,000 and 72,000, respectively, to yield precursors of Mr = 164,000 and 91,000, respectively. The alpha and beta subunit precursors are then processed with partial conversion of high mannose to complex type carbohydrate to yield the mature subunits of Mr = 170,000 and 95,000, respectively.     

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.