ABSTRACT
Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited human platelet alloantigens (HPAs) present on the surface of fetal and neonatal platelets. There are currently no approved therapies for the prevention of FNAIT. We report herein the ability of 2 human HPA-1a-specific therapeutic candidates, one a polyclonal, and the other a monoclonal antibody, to prevent alloimmunization in a novel preclinical mouse model of FNAIT. Both antibody preparations effected the rapid and complete elimination of HPA-1a+ platelets from circulation and prevented the development of HPA-1a alloantibodies. HPA-1a- female mice treated prophylactically with anti-HPA-1a antibody prior to exposure to HPA-1a+ platelets gave birth to HPA-1a+/- pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish both the potential and threshold exposure targets for prophylactic treatment with HPA-1a-specific antibodies for the prevention of FNAIT in humans.
Subject(s)
Antigens, Human Platelet , Thrombocytopenia, Neonatal Alloimmune , Pregnancy , Humans , Female , Mice , Animals , Thrombocytopenia, Neonatal Alloimmune/prevention & control , Isoantibodies , Integrin beta3 , Prenatal Care , FetusABSTRACT
BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) ensues from parental incompatibility for platelet alloantigens with maternal sensitization. HPA-1a/1b incompatibility is the most common cause of FNAIT in Caucasians. Placental villitis and lower birthweight in FNAIT suggest anti-HPA-1a may have effects beyond inducing thrombocytopenia. OBJECTIVES: Does FNAIT secondary to anti-HPA-1a result in smaller newborns and, the corollary, does antenatal management of FNAIT increase birthweight? STUDY DESIGN: Birthweights of 270 FNAIT-affected newborns from a randomized clinical trial and a NAITbabies.org survey (135 paired siblings) were compared with those of published controls and treated to untreated FNAIT-affected siblings. Birthweights were converted to percentiles to account for gestational age, sex, and role of birth order in birth weight. Body weights of FNAIT-affected and -unaffected pups in a mouse FNAIT model were analyzed. RESULTS: Untreated siblings in both the clinical trial and NAITbabies.org cohorts were not small, compared with normal controls. However, treated siblings in both cohorts had significantly higher birthweight percentiles compared with their previous untreated affected sibling. After accounting for gestational age, sex, and birth order, increased birthweight percentile in treated compared with the untreated siblings remained significant in both cohorts. FNAIT-affected neonatal mice had lower bodyweights than FNAIT-unaffected pups. CONCLUSIONS: Untreated FNAIT-affected newborns were not small; however, treatment of FNAIT-affected pregnancies increased newborn birthweights despite corrections to account for other factors that might have influenced the results. High dose IVIG is believed to "block" FcRn and lower maternal anti-HPA-1a levels, and thus increase birthweights by reducing levels of maternal anti-HPA-1a and reducing placental villitis.
Subject(s)
Antigens, Human Platelet , Thrombocytopenia, Neonatal Alloimmune , Animals , Female , Humans , Infant, Newborn , Mice , Pregnancy , Birth Weight , Fetus , Gestational Age , Placenta , Thrombocytopenia, Neonatal Alloimmune/therapy , Male , Randomized Controlled Trials as TopicABSTRACT
There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbß3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood-brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.
Subject(s)
Anaphylaxis/immunology , Antigen-Antibody Complex/immunology , Blood Platelets/immunology , Serotonin/immunology , Shock, Septic/immunology , Adult , Anaphylaxis/blood , Anaphylaxis/genetics , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Platelet Activation , Platelet Count , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/immunology , Receptors, IgG/genetics , Receptors, IgG/immunology , Shock, Septic/blood , Shock, Septic/genetics , Young AdultABSTRACT
Platelet endothelial cell adhesion molecule 1 (PECAM-1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM-1 functions, we generated mice in which PECAM1, the gene encoding PECAM-1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3' loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo-pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT-flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 flox/flox offspring, which expressed PECAM-1 at normal levels and had no overt phenotype. PECAM1 flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY-box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 del/WT ), which were crossbred to generate Sox2Cre; PECAM1 del/del offspring. Sox2Cre; PECAM1 del/del mice recapitulated the phenotype of conventional global PECAM-1 knockout mice. PECAM1 flox/flox mice will be useful for studying distinct roles of PECAM-1 in tissue specific contexts and to gain insights into the roles that PECAM-1 plays in blood and vascular cell function.
Subject(s)
Gene Knockout Techniques/methods , Platelet Endothelial Cell Adhesion Molecule-1/genetics , Animals , Integrases/metabolism , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolismABSTRACT
Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin ß3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.
Subject(s)
Antigens, Human Platelet/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/immunology , Clustered Regularly Interspaced Short Palindromic Repeats , Isoantigens/genetics , Antigens, Human Platelet/immunology , Base Sequence , Cells, Cultured , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/immunology , Epitopes/genetics , Epitopes/immunology , Humans , Integrin beta3/genetics , Integrin beta3/immunology , Isoantibodies/genetics , Isoantibodies/immunology , Isoantigens/immunology , Platelet Glycoprotein GPIIb-IIIa Complex/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , Polymorphism, Single NucleotideABSTRACT
The integrin family is composed of a series of 24 αß heterodimer transmembrane adhesion receptors that mediate cell-cell and cell-extracellular matrix interactions. Adaptor molecules bearing immunoreceptor tyrosine-based activation motifs (ITAMs) have recently been shown to cooperate with specific integrins to increase the efficiency of transmitting ligand-binding-induced signals into cells. In human platelets, Fc receptor γ-chain IIa (FcγRIIa) has been identified as an ITAM-bearing transmembrane receptor responsible for mediating "outside-in" signaling through αIIbß3, the major adhesion receptor on the platelet surface. To explore the importance of FcγRIIa in thrombosis and hemostasis, we subjected FcγRIIa-negative and FcγRIIa-positive murine platelets to a number of well-accepted models of platelet function. Compared with their FcγRIIa-negative counterparts, FcγRIIa-positive platelets exhibited increased tyrosine phosphorylation of Syk and phospholipase Cγ2 and increased spreading upon interaction with immobilized fibrinogen, retracted a fibrin clot faster, and showed markedly enhanced thrombus formation when perfused over a collagen-coated flow chamber under conditions of arterial and venous shear. They also displayed increased thrombus formation and fibrin deposition in in vivo models of vascular injury. Taken together, these data establish FcγRIIa as a physiologically important functional conduit for αIIbß3-mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis.
Subject(s)
Blood Platelets/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Receptors, IgG/physiology , Receptors, Immunologic/metabolism , Signal Transduction , Thrombosis/etiology , Tyrosine/metabolism , Animals , Arterioles/metabolism , Arterioles/pathology , Fibrin/metabolism , Fibrinogen/metabolism , Hemostasis/physiology , Humans , In Vitro Techniques , Mice , Mice, Inbred C57BL , Mice, Transgenic , Platelet Aggregation , Platelet Count , Thrombosis/metabolism , Thrombosis/pathology , Veins/metabolism , Veins/pathologyABSTRACT
OBJECTIVE: Kindlin-3 is a critical supporter of integrin function in platelets. Lack of expression of kindlin-3 protein in patients impairs integrin αIIbß3-mediated platelet aggregation. Although kindlin-3 has been categorized as an integrin-binding partner, the functional significance of the direct interaction of kindlin-3 with integrin αIIbß3 in platelets has not been established. Here, we evaluated the significance of the binding of kindlin-3 to integrin αIIbß3 in platelets in supporting integrin αIIbß3-mediated platelet functions. APPROACH AND RESULTS: We generated a strain of kindlin-3 knockin (K3KI) mice that express a kindlin-3 mutant that carries an integrin-interaction defective substitution. K3KI mice could survive normally and express integrin αIIbß3 on platelets similar to their wild-type counterparts. Functional analysis revealed that K3KI mice exhibited defective platelet function, including impaired integrin αIIbß3 activation, suppressed platelet spreading and platelet aggregation, prolonged tail bleeding time, and absence of platelet-mediated clot retraction. In addition, whole blood drawn from K3KI mice showed resistance to in vitro thrombus formation and, as a consequence, K3KI mice were protected from in vivo arterial thrombosis. CONCLUSIONS: These observations demonstrate that the direct binding of kindlin-3 to integrin αIIbß3 is involved in supporting integrin αIIbß3 activation and integrin αIIbß3-dependent responses of platelets and consequently contributes significantly to arterial thrombus formation.
Subject(s)
Blood Platelets/physiology , Carotid Artery Thrombosis/physiopathology , Cytoskeletal Proteins/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Amino Acid Substitution , Animals , Bleeding Time , Blood Platelets/ultrastructure , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/chemically induced , Cell Shape , Chlorides/toxicity , Clot Retraction , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Disease Models, Animal , Female , Ferric Compounds/toxicity , Gene Knock-In Techniques , Genes, Reporter , Male , Mice , Mice, Inbred C57BL , Microspheres , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Protein Interaction Mapping , Recombinant Fusion Proteins/metabolismABSTRACT
Estrogen receptor (ER) α promotes breast cancer growth by regulating gene expression through classical estrogen response element (ERE) binding and nonclassical (interaction with c-Jun at AP-1 sites) pathways. ER is the target for anti-estrogens such as tamoxifen (TAM). However, the potential for classical versus nonclassical ER signaling to influence hormone sensitivity is not known. Moreover, anti-estrogens frequently activate several signaling cascades besides the target ER, and the implications of these "off-target" signaling events have not been explored. Here, we report that p38γ MAPK is selectively activated by treatment with TAM. This results in both phosphorylation of ER at Ser-118 and stimulation of c-Jun transcription, thus switching ER signaling from the classical to the nonclassical pathway leading to increased hormone sensitivity. Unexpectedly, phosphorylation at Ser-118 is required for ER to bind both p38γ and c-Jun, thereby promoting ER relocation from ERE to AP-1 promoter sites. Thus, ER/Ser-118 phosphorylation serves as a central mechanism by which p38γ regulates signaling transduction of ER with its inhibitor TAM.
Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 12/metabolism , Proto-Oncogene Proteins c-jun/biosynthesis , Response Elements , Transcription, Genetic , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Enzyme Activation/drug effects , Enzyme Activation/genetics , Estrogen Receptor alpha/genetics , Female , Humans , Mitogen-Activated Protein Kinase 12/genetics , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Binding/drug effects , Protein Binding/genetics , Proto-Oncogene Proteins c-jun/genetics , Tamoxifen/pharmacologyABSTRACT
Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.4) with modified Fc region or altered N-glycan structures. The mAb 26.4 wild-type (WT) variants all showed efficient platelet clearance capacity and ability to mediate phagocytosis independent of their N-glycan structure, compared with an effector silent variant (26.4.AAAG), although the modified N-glycan variants showed differential binding to FcγRs measured in vitro. In an in vivo model, female mice were transfused with platelets from transgenic mice harboring an engineered integrin ß3 containing the HPA-1a epitope. When these preimmunized mice were bred with transgenic males, Abs against the introduced epitope induced thrombocytopenia in the offspring, mimicking FNAIT. Prophylactic administration of the mAb 26.4.WT, and to some extent the mAb 26.4.AAAG, prior to platelet transfusion resulted in reduced alloimmunization in challenged mice and normal platelet counts in neonates. The notion that the effector silent variant hampered alloimmunization demonstrates that rapid platelet clearance, as seen with mAb 26.4.WT, is not the sole mechanism in action. Our data thus successfully demonstrate efficient Ab-mediated immunosuppression and prevention of FNAIT by anti-HPA-1a monoclonal variants, providing support for potential use in humans.
Subject(s)
Antigens, Human Platelet/immunology , Integrin beta3/immunology , Isoantibodies/blood , Thrombocytopenia, Neonatal Alloimmune/immunology , Thrombocytopenia, Neonatal Alloimmune/prevention & control , Animals , Antibodies, Monoclonal/administration & dosage , Female , Humans , Immunoglobulin G/administration & dosage , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Protein Isoforms , THP-1 CellsABSTRACT
Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38gamma MAPK, and the activated c-Jun then recruits p38gamma as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38gamma that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38gamma interacting with c-Jun. p38gamma requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38gamma are required for the trans-activation of MMP9. The active p38gamma/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38gamma and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.
Subject(s)
Matrix Metalloproteinase 9/metabolism , Proto-Oncogene Proteins c-jun/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Base Sequence , Cell Line, Transformed , Chromatin Immunoprecipitation , DNA Primers , Enzyme Activation , Humans , Matrix Metalloproteinase 9/genetics , Mice , Phosphorylation , Promoter Regions, Genetic , Protein Binding , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the â¼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin ß3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.
Subject(s)
Antigens, Human Platelet , Thrombocytopenia, Neonatal Alloimmune , Animals , Antigens, Human Platelet/genetics , Female , Fetus , Immunotherapy , Isoantibodies , Male , Mice , Pregnancy , Thrombocytopenia, Neonatal Alloimmune/genetics , Thrombocytopenia, Neonatal Alloimmune/therapyABSTRACT
Antibodies to platelet-specific antigens are responsible for 2 clinically important bleeding disorders: posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia (FNAIT). The human platelet-specific alloantigen 1a/1b (HPA-1a/1b; also known as PlA1/A2) alloantigen system of human platelet membrane glycoprotein (GP) IIIa is controlled by a Leu33Pro polymorphism and is responsible for â¼80% of the cases of FNAIT. Local residues surrounding polymorphic residue 33 are suspected to have a profound effect on alloantibody binding and subsequent downstream effector events. To define the molecular requirements for HPA-1a alloantibody binding, we generated transgenic mice that expressed murine GPIIIa (muGPIIIa) isoforms harboring select humanized residues within the plexin-semaphorin-integrin (PSI) and epidermal growth factor 1 (EGF1) domains and examined their ability to support the binding of a series of monoclonal and polyclonal HPA-1a-specific antibodies. Humanizing the PSI domain of muGPIIIa was sufficient to recreate the HPA-1a epitope recognized by some HPA-1a-specific antibodies; however, humanizing distinct amino acids within the linearly distant but conformationally close EGF1 domain was required to enable binding of others. These results reveal the previously unsuspected complex heterogeneity of the polyclonal alloimmune response to this clinically important human platelet alloantigen system. High-resolution mapping of this alloimmune response may improve diagnosis of FNAIT and should facilitate the rational design and selection of contemplated prophylactic and therapeutic anti-HPA-1a reagents.
Subject(s)
Antibodies/immunology , Antigens, Human Platelet/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , Antigen-Antibody Reactions , Antigens, Human Platelet/chemistry , Antigens, Human Platelet/genetics , Epitope Mapping/methods , Humans , Integrin beta3/chemistry , Integrin beta3/genetics , Integrin beta3/immunology , Integrin beta3/metabolism , Mice , Mice, Transgenic , Protein Domains , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , Protein Structure, Tertiary , Thrombocytopenia, Neonatal Alloimmune/diagnosis , Thrombocytopenia, Neonatal Alloimmune/immunologyABSTRACT
To identify genes that are differentially expressed in human esophageal squamous cell carcinoma (ESCC), we have developed a cDNA microarray representing 34 176 clones to analyse gene expression profiles in ESCC. A total of 77 genes (including 31 novel genes) were downregulated, and 15 genes (including one novel gene) were upregulated in cancer tissues compared with their normal counterparts. Immunohistochemistry and Northern blot analysis were carried out to verify the cDNA microarray results. It was revealed that genes involved in squamous cell differentiation were coordinately downregulated, including annexin I, small proline-rich proteins (SPRRs), calcium-binding S100 proteins (S100A8, S100A9), transglutaminase (TGM3), cytokeratins (KRT4, KRT13), gut-enriched Krupple-like factor (GKLF) and cystatin A. Interestingly, most of the downregulated genes encoded Ca(2+)-binding or -modulating proteins that constitute the cell envelope (CE). Moreover, genes associated with invasion or proliferation were upregulated, including genes such as fibronectin, secreted protein acidic and rich in cystein (SPARC), cathepsin B and KRT17. Functional analysis of the alteration in the expression of GKLF suggested that GKLF might be able to regulate the expression of SPRR1A, SPRR2A and KRT4 in ESCC. This study provides new insights into the role of squamous cell differentiation-associated genes in ESCC initiation and progression.
Subject(s)
Calcium/physiology , Carcinoma, Squamous Cell/genetics , Cell Differentiation/genetics , Esophageal Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , Oligonucleotide Array Sequence Analysis , Carcinoma, Squamous Cell/pathology , Disease Progression , Esophageal Neoplasms/pathology , Humans , Kruppel-Like Factor 4 , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Tumor Cells, CulturedABSTRACT
IgG immune complexes contribute to the etiology and pathogenesis of numerous autoimmune disorders, including heparin-induced thrombocytopenia, systemic lupus erythematosus, rheumatoid- and collagen-induced arthritis, and chronic glomerulonephritis. Patients suffering from immune complex-related disorders are known to be susceptible to platelet-mediated thrombotic events. Though the role of the Fc receptor, FcγRIIa, in initiating platelet activation is well understood, the role of the major platelet adhesion receptor, integrin αIIbß3, in amplifying platelet activation and mediating adhesion and aggregation downstream of encountering IgG immune complexes is poorly understood. The goal of this investigation was to gain a better understanding of the relative roles of these two receptor systems in immune complex-mediated thrombotic complications. Human platelets, and mouse platelets genetically engineered to differentially express FcγRIIa and αIIbß3, were allowed to interact with IgG-coated surfaces under both static and flow conditions, and their ability to spread and form thrombi evaluated in the presence and absence of clinically-used fibrinogen receptor antagonists. Although binding of IgG immune complexes to FcγRIIa was sufficient for platelet adhesion and initial signal transduction events, platelet spreading and thrombus formation over IgG-coated surfaces showed an absolute requirement for αIIbß3 and its ligands. Tyrosine kinases Lyn and Syk were found to play key roles in IgG-induced platelet activation events. Taken together, our data suggest a complex functional interplay between FcγRIIa, Lyn, and αIIbß3 in immune complex-induced platelet activation. Future studies may be warranted to determine whether patients suffering from immune complex disorders might benefit from treatment with anti-αIIbß3-directed therapeutics.
Subject(s)
Antigen-Antibody Complex/immunology , Immunoglobulin G/immunology , Platelet Activation/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombosis/metabolism , Thrombosis/physiopathology , src-Family Kinases/metabolism , Animals , Autoimmune Diseases/metabolism , Autoimmune Diseases/physiopathology , Blood Platelets/metabolism , Blood Platelets/physiology , CHO Cells , Cell Line , Cricetulus , Fibrinogen/metabolism , Humans , Mice , Mice, Inbred C57BL , Platelet Adhesiveness/physiology , Platelet Aggregation/physiology , Platelet Glycoprotein GPIb-IX Complex/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, IgG/metabolism , Signal Transduction/physiologyABSTRACT
Receptor-mediated platelet activation requires phospholipase C (PLC) activity to elevate intracellular calcium and induce actin cytoskeleton reorganization. PLCs are classified into structurally distinct ß, γ, δ, ε, ζ, and η isoforms. There are two PLCγ isoforms (PLCγ1, PLCγ2), which are critical for activation by tyrosine kinase-dependent receptors. Platelets express both PLCγ1 and PLCγ2. Although PLCγ2 has been shown to play a dominant role in platelet activation, the extent to which PLCγ1 contributes has not been evaluated. To ascertain the relative contributions of PLCγ1 and PLCγ2 to platelet activation, we generated conditionally PLCγ1-deficient, wild-type (WT), PLCγ2-deficient, and PLCγ1/PLCγ2 double-deficient mice and measured the ability of platelets to respond to different agonists. We found that PLCγ2 deficiency abrogated αIIbß3-dependent platelet spreading, GPVI-dependent platelet aggregation, and thrombus formation on collagen-coated surfaces under shear conditions, which is dependent on both GPVI and αIIbß3. Addition of exogenous ADP overcame defective spreading of PLCγ2-deficient platelets on immobilized fibrinogen, suggesting that PLCγ2 is required for granule secretion in response to αIIbß3 ligation. Consistently, αIIbß3-mediated release of granule contents was impaired in the absence of PLCγ2. In contrast, PLCγ1-deficient platelets spread and released granule contents normally on fibrinogen, exhibited normal levels of GPVI-dependent aggregation, and formed thrombi normally on collagen-coated surfaces. Interestingly, enforced expression of PLCγ1 fully restored GPVI-dependent aggregation and αIIbß3-dependent spreading of PLCγ2-deficient platelets. We conclude that platelet activation through GPVI and αIIbß3 utilizes PLCγ2 because PLCγ1 levels are insufficient to support responsiveness, but that PLCγ1 can restore responsiveness if expressed at levels normally achieved by PLCγ2.
Subject(s)
Blood Platelets/metabolism , Gene Expression , Phospholipase C gamma/deficiency , Animals , Collagen/pharmacology , Enzyme Activation , Isoenzymes , Mice , Mice, Knockout , Mice, Transgenic , Phospholipase C gamma/genetics , Phospholipase C gamma/metabolism , Platelet Activation/genetics , Platelet Aggregation/drug effects , Platelet Aggregation/genetics , Platelet Count , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Thrombosis/metabolismABSTRACT
AIM: To investigate the expression patterns of esophageal squamous cell cancer deregulated genes in mid to late stages of C57BL/6J mouse embryogenesis, and the correlation between these genes in embryonic development and tumorigenesis of esophageal squamous cell cancer. METHODS: Reverse northern screening was performed to examine the expression patterns of esophageal cancer deregulated genes in C57BL/6J mouse embryogenesis. To confirm the gene expression patterns, semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out for 3 of the randomly picked differentially expressed genes. RESULTS: Within these esophageal cancer deregulated genes, 4 patterns of expression were observed at 3 stages embryonic d 11.5 (E11.5), embryonic d 13.5 (E13.5) and postnatal d1 (P1). (1) Up-regulation during the E11.5 period, down- regulation during the E13.5 and P1 period (up-down-down), the 10 up-regulated genes during the E11.5 period could be classified into 6 known genes and 4 unknown genes. The known genes included differentiation related genes (S100A8), immunity related gene (IGL), translation and transcription regulation genes (RPL15, EEF1A1), cytoskeletal protein (TUBA1), cysteine protease inhibitor (cystatin B). (2) Up-regulation during the E13.5 and P1 period (down-up-up), such as the SPRR2A which was down-regulated at E11.5. (3) Down-regulation during the E11.5 and E13.5 period (down-down-up), such as RHCG and keratin 4. (4) Fluctuating expression, down initially, up at E13.5, and then down again (down-up-down). EMP1 belonged to such a gene, which was highly expressed at E13.5. CONCLUSION: The results will be helpful for understanding the function of esophageal squamous cell carcinoma (ESCC) deregulated genes in embryonic development and tumorigenesis. S100A8 and S100A9 may play different roles in early embryonic development. IGL may be an oncofetal protein, and EMP1 relates with neurogenesis at E13.5. The genes identified pertinent to embryonic development may serve as candidate susceptibility genes for inherited esophageal cancer disorders as well as for various heritable disorders of embryonic development.
Subject(s)
Carcinoma, Squamous Cell/genetics , Esophageal Neoplasms/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , Animals , Blotting, Northern , Down-Regulation/genetics , Female , Mice , Mice, Inbred C57BL , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/geneticsABSTRACT
OBJECTIVE: To evaluate the expression of annexin II in human esophageal squamous cell carcinoma (ESCC) and its relation with clinicopathological data. METHODS: The expression of annexin II mRNA and protein in paired cancer tissues and their adjacent quasi-normal tissues were detected by RT-PCR, immunohistochemical method and densitometric scanning. The relation between annexin II expression and the status of tumor differentiation was analyzed. RESULTS: The expression of annexin II was significantly lower in the tumor tissue than that in its paired normal counterpart both in mRNA and protein level (P < 0.05, P < 0.01). The protein expression of annexin II was significantly lower in moderately and poorly differentiated tumors than those in well differentiated ones (P < 0.05). CONCLUSION: Down-regulation of annexin II in esophageal carcinogenesis may play an important role in squamous cell differentiation.
Subject(s)
Annexin A2/biosynthesis , Carcinoma, Squamous Cell/metabolism , Esophageal Neoplasms/metabolism , Annexin A2/genetics , Carcinoma, Squamous Cell/pathology , Cell Differentiation , Down-Regulation , Esophageal Neoplasms/pathology , Female , Humans , Male , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/enzymology , Mitogen-Activated Protein Kinase 12/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 3/metabolism , ras Proteins/metabolism , Cell Growth Processes/physiology , Cell Transformation, Neoplastic/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Genes, ras , HCT116 Cells , Humans , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 12/biosynthesis , Mitogen-Activated Protein Kinase 12/genetics , PDZ Domains , Phosphorylation , Protein Interaction Domains and Motifs , Protein Tyrosine Phosphatase, Non-Receptor Type 3/biosynthesis , Protein Tyrosine Phosphatase, Non-Receptor Type 3/genetics , RNA, Small Interfering/genetics , ras Proteins/geneticsABSTRACT
Non-small cell lung carcinoma (NSCLC) is promoted by the increased activities of several small GTPases, including K-Ras4B, Rap1A, Rap1B, RhoC, and Rac1. SmgGDS is an unusual guanine nucleotide exchange factor that activates many of these small GTPases, and thus may promote NSCLC development or progression. We report here that SmgGDS protein levels are elevated in NSCLC tumors, compared with normal lung tissue from the same patients or from individuals without cancer. To characterize SmgGDS functions in NSCLC, we tested the effects of silencing SmgGDS expression by transfecting cultured NSCLC cells with SmgGDS small interfering RNA (siRNA). Cells with silenced SmgGDS expression form fewer colonies in soft agar, do not proliferate in culture due to an arrest in G(1) phase, and exhibit disrupted myosin organization and reduced cell migration. The transcriptional activity of NF-kappaB in NSCLC cells is diminished by transfecting the cells with SmgGDS siRNA, and enhanced by transfecting the cells with a cDNA encoding SmgGDS. Because RhoA is a major substrate for SmgGDS, we investigated whether diminished RhoA expression mimics the effects of diminished SmgGDS expression. Silencing RhoA expression with RhoA siRNA disrupts myosin organization, but only moderately decreases cell proliferation and does not inhibit migration. Our finding that the aggressive NSCLC phenotype is more effectively suppressed by silencing SmgGDS than by silencing RhoA is consistent with the ability of SmgGDS to regulate multiple small GTPases in addition to RhoA. These results demonstrate that SmgGDS promotes the malignant NSCLC phenotype and is an intriguing therapeutic target in NSCLC.
Subject(s)
Cell Division/physiology , Cell Movement/physiology , Guanine Nucleotide Exchange Factors/genetics , NF-kappa B/genetics , RNA, Small Interfering/genetics , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Guanine Nucleotide Exchange Factors/physiology , Humans , Lung Neoplasms , RNA, Neoplasm/genetics , Transcription, Genetic , Transfection , rhoA GTP-Binding Protein/geneticsABSTRACT
Cyclooxygenase-2 (COX-2) is well established to play an important role in the tumorigenesis of a variety of human cancers; however, the function of COX-2 in the development of esophageal squamous cell carcinoma (ESCC) remains less clear. Here, we determined, first, the pattern of COX-2 expression in normal esophageal mucosa, dysplasia, carcinoma in situ (CIS) and invasive SCC. Immunohistochemical analysis showed that, while COX-2 was weakly expressed, if at all, in normal squamous epithelium, strong COX-2 expression was detected as early as the stage of dysplasia and frequently in 20 of 26 (77%) CIS and 86 of 111 (77%) invasive SCC. Upregulation of COX-2 in ESCC was found to be significantly associated with tumor progression (R = 0.493, P < 0.01). Further, treatment of human ESCC cell lines (KYSE450 and KYSE510) with NS-398, a COX-2 specific chemical inhibitor, suppressed the production of prostaglandin E2 (PGE2) and induced cell growth inhibition, cell cycle arrest at the G1-S checkpoint, and the expression of cyclin-dependent kinase inhibitors p21waf1/cip1 and p27kip1. Finally, knockdown expression of COX-2 in KYSE450 cells by a specific COX-2 siRNA dramatically inhibited PGE2 production, cell growth and, more importantly, colony formation and tumorigenesis in nude mice. Together, this study suggested that COX-2 may be involved in an early stage of squamous cell carcinogenesis of the esophagus and has a non-redundant role in the regulation of cellular proliferation and tumorigenesis of esophageal epithelial cells.