Methylation status of human chorionic gonadotropin beta subunit promoter and TFAP2A expression as factors regulating CGB gene expression in placenta.

OBJECTIVE: To evaluate mechanisms regulating the expression of CGB genes in placental tissues from uncomplicated pregnancies and chorionic samples from spontaneous miscarriages. DESIGN: Molecular analyses in human samples. SETTING: Laboratory of molecular biology. PATIENT(S): Nine placental samples from term deliveries and 21 chorionic samples from miscarriages at 7-13 weeks of gestation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression level of CGBs and genes encoding SP1, SP3, and AP2 transcription factors was analyzed using quantitative polymerase chain reaction (qPCR). The methylation status of the CGBs' promoter regions was determined using methylation-specific PCR. RESULT(S): The experiments showed significant differences in CGBs' expression and their regulation between placental and chorionic tissues. In placental tissues and chorionic tissues from 7 to 9 weeks of gestation, the expression level of CGBs was shown to be associated with the amount of TFAP2A transcripts. It was also demonstrated that variation in the expression level of CGB genes relies on changes in methylation of CGB3-9 and CGB1-2 promoter sequences. CONCLUSION(S): During pregnancy, regulation of hCG beta subunit genes expression correlates with both methylation of their promoters and TFAP2A expression level. The results suggest that these factors may be very influential in the early stages of pregnancy and may be associated with pregnancy outcome.

CGB activates ERK and AKT kinases in cancer cells via LHCGR-independent mechanism.

Expression of human chorionic gonadotropin free beta subunit (hCGß) and its hyperglycosylated variant (hCGß-H) is a phenomenon confirmed for tumors of different origin. Despite numerous studies, the mechanism of hCGß action in cancer remains unknown especially that not all tumors secreting hCGß express the receptor for human chorionic gonadotropin (LHCGR). In the presented study, we verified the hypothesis of hCGß potential to activate signaling pathways involving extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) kinases with and without the contribution of LHCGR. To achieve this goal, human ovarian carcinoma cells OVCAR-3 expressing LHCGR and SKOV-3 not expressing LHCGR were either transfected with a vector coding for hCGß or stimulated with recombinant hCGß and the level of pERK and pAKT was measured. The results of the experiments showed that hCGß action leads to the increase in ERK and AKT kinases phosphorylation in cancer cells and indicate that these biological effects can be achieved independently of LHCGR presence. The study also demonstrated that the presence of the receptor is a key factor influencing the magnitude of cells' response.

An efficient 3D cell culture method on biomimetic nanostructured grids.

Current techniques of in vitro cell cultures are able to mimic the in vivo environment only to a limited extent, as they enable cells to grow only in two dimensions. Therefore cell culture approaches should rely on scaffolds that provide support comparable to the extracellular matrix. Here we demonstrate the advantages of novel nanostructured three-dimensional grids fabricated using electro-spinning technique, as scaffolds for cultures of neoplastic cells. The results of the study show that the fibers allow for a dynamic growth of HeLa cells, which form multi-layer structures of symmetrical and spherical character. This indicates that the applied scaffolds are nontoxic and allow proper flow of oxygen, nutrients, and growth factors. In addition, grids have been proven to be useful in in situ examination of cells ultrastructure.

Estrogen receptor 2 expression in back muscles of girls with idiopathic scoliosis - relation to radiological parameters.

Deep paravertebral muscles and female sex hormones are potential elements participating in idiopathic scoliosis development. Estrogen acts through estrogen receptors: ESR1 and ESR2. There are no studies describing ESR2 expression in back muscles in girls with idiopathic scoliosis. The aim of this study was to evaluate ESR2 expression levels in back muscles on both sides of the spinal curve and correlation between the expression level and scoliosis parameters. Asymmetrical ESR2 expression in deep paravertebral muscles was found: 11 girls had higher expression level on the convex side and 5 girls had higher expression level on the concave side of the curvature. Patients with ESR2 (convex/concave) ratio ≥ 1 presented positive correlation between ESR2 ratio and Cobb angle.

[Human chorionic gonadotropin--a well-known hormone with unknown functions]. / Ludzka gonadotropina kosmówkowa--znanyhormon o nieznanych funkcjach.

Human chorionic gonadotropin (CG) belongs to the glycoprotein family consisting of LH, FSH and TSH. All of these hormones are composed of two subunits: common to the whole family alpha subunit and hormone-specific beta subunit CG has paracrine effects on several processes such as placentation, implantation, angiogenesis and delaying the apoptosis of corpus luteum. Serum level of CG is used to monitor pregnancy and pregnancy disorders. Recent studies have shown that the synthesis of CG is a characteristic feature of a wide variety of malignant and non-malignant tumors. The role of CG in cancerogensis remains unclear but the main hypothesis concerns its antiapoptotic impact of the hormone on the neoplastic cells. The synthesis of functional CG requires the activity of separate genes encoding both hormone's subunits, but it is the beta subunit accessibility which controls the process. The protein synthesis must be followed by proper folding and posttranslational modifications of the molecule. Particularly glycosylation of human chorionic gonadotropin was shown to have an impact on the hormone's function. The amount and the structure of carbohydrate residuals attached to CG may be different and lead to the formation of hormone variants, which vary in molecular mass. Normal CG with a molecular mass of about 37.5 kDa is produced by the syncytiotrophoblast, while the variant with higher molecular mass - 38.5-40 kDa, described as hyperglicosylated CG, is secreted by undifferentiated trophoblast cells and some cancers. It is suggested that those forms have different but complementary biological functions. However the mechanism of the action of particular variants and signaling pathways activated by those forms are still obscure.

Ligation of complement receptor 1 increases erythrocyte membrane deformability.

Microbes as well as immune complexes and other continuously generated inflammatory particles are efficiently removed from the human circulation by red blood cells (RBCs) through a process called immune-adherence clearance. During this process, RBCs use complement receptor 1 (CR1, CD35) to bind circulating complement-opsonized particles and transfer them to resident macrophages in the liver and spleen for removal. We here show that ligation of RBC CR1 by antibody and complement-opsonized particles induces a transient Ca(++) influx that is proportional to the RBC CR1 levels and is inhibited by T1E3 pAb, a specific inhibitor of TRPC1 channels. The CR1-elicited RBC Ca(++) influx is accompanied by an increase in RBC membrane deformability that positively correlates with the number of preexisting CR1 molecules on RBC membranes. Biochemically, ligation of RBC CR1 causes a significant increase in phosphorylation levels of ß-spectrin that is inhibited by preincubation of RBCs with DMAT, a specific casein kinase II inhibitor. We hypothesize that the CR1-dependent increase in membrane deformability could be relevant for facilitating the transfer of CR1-bound particles from the RBCs to the hepatic and splenic phagocytes.

Ligation of erythrocyte CR1 induces its clustering in complex with scaffolding protein FAP-1.

The primary identified function of complement receptor 1 (CR1/CD35) on primate erythrocytes is to bind complement-tagged inflammatory particles including microbes and immune complexes. When erythrocytes circulate through liver and spleen, sinusoidal phagocytes remove CR1-adherent particles and erythrocytes return to the circulation. This process of immune adherence clearance is important for host defense and prevention of autoimmunity. CR1 was previously described as clustered in the human erythrocyte membrane, which was thought to be necessary for binding complement-opsonized particles. In contrast, we demonstrate that on erythrocytes CR1 is not clustered, but dispersed, and able to bind complement-tagged particles. When fresh erythrocytes are solubilized by nonionic detergent, CR1 partitions to the cytoskeleton fraction. Using a PDZ-peptide array, CR1's cytoplasmic tail, which contains 2 PDZ-motifs, binds PDZ domains 2, 3, and 5 of Fas-associated phosphatase 1 (FAP-1), a scaffolding protein. We show that FAP-1, not previously recognized as an erythroid protein, is expressed on circulating erythrocytes. CR1 and FAP-1 coimmunoprecipitate, which confirms their molecular association. Disperse CR1 on erythrocytes may be advantageous for capturing immune-complexes, while ligation-induced CR1 clustering may prevent ingestion of the erythrocyte during the immune-complex transfer to the macrophages by keeping the opsonic stimulus localized thus preventing phagocyosis.

Developmental stage-specific shift in responsiveness to chemokines during human B-cell development.

OBJECTIVES: To better understand the role of chemokines during human B-cell development in bone marrow. METHODS: Differentiation stage-specific B cells (pro-B, pre-B, immature, and mature) were analyzed for chemokine receptor expression and for migration to corresponding ligands. We also hypothesized that inflammatory conditions may cause the upregulation of certain chemokine receptors on early B cells, rendering them sensitive to extramedullary chemotactic cues. To test this hypothesis, we used human pre-B 697 cells to investigate whether various inflammatory agents could modify chemokine receptor expression and function. RESULTS: Chemotaxis to CXCL12 was observed for all B cell subsets. However, chemotactic responses to CCL19, CCL21, CXCL13, and CCL20 were limited to late-stage, IgM+ bone marrow B cells (immature B and mature B). Chemotactic responses to corresponding ligands correlated with the pattern of chemokine receptor expression. The expression of CCR7, however, was low on early (pro-B and pre-B) B cells and did not induce chemotaxis. Interestingly, both CCL19 and CCL21 could trigger ERK1/2 phosphorylation in early B cells. Exposure of pre-B 697 cells to TNF-alpha upregulated CCR7 and CXCR5 expression, whereas it had no effect on CCR6 surface expression. Correspondingly, TNF-alpha-stimulated pre-B cells chemotaxed towards CCL19 and CXCL13, in contrast to non-TNF-alpha-stimulated controls. CONCLUSION: We postulate that CXCR5, CCR7, and CCR6 participate in bone marrow trafficking and/or bone marrow egress of late-stage B cells under steady-state conditions, whereas inflammation-induced expression of CCR7 and CXCR5 may facilitate early B-cell emigration out of the bone marrow and their positioning in secondary lymphoid organs.

Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors.

Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal-related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12-16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.

CXCR4 chemokine receptor and integrin signaling co-operate in mediating adhesion and chemoresistance in small cell lung cancer (SCLC) cells.

Small cell lung cancer (SCLC) is an aggressive, rapidly metastazising neoplasm with a high propensity for marrow involvement. SCLC cells express high levels of functional CXCR4 receptors for the chemokine stromal-cell-derived factor-1 (SDF-1/CXCL12). Adhesion of SCLC cells to extracellular matrix or accessory cells within the tumor microenvironment confers resistance to chemotherapy via integrin signaling and thus may be responsible for residual disease and relapses commonly seen in SCLC. We examined the signaling mechanisms that regulate CXCL12-induced adhesion of SCLC cells to fibronectin, collagen, and stromal cells and the effects on SCLC cell chemoresistance. We found that CXCL12-induced integrin activation which resulted in an increased adhesion of SCLC cells to fibronectin and collagen. This was mediated by alpha2, alpha4, alpha5, and beta1 integrins along with CXCR4 activation, which could be inhibited by CXCR4 antagonists. Stromal cells protected SCLC cells from chemotherapy-induced apoptosis, and this protection could also be antagonized by CXCR4 inhibitors. We conclude that activation of integrins and CXCR4 chemokine receptors co-operate in mediating adhesion and survival signals from the tumor microenvironment to SCLC cells. Therefore, CXCR4 antagonists in combination with cytotoxic drugs should be explored in SCLC to overcome CXCL12-mediated adhesion and survival signals in the tumor microenvironment.

CXC chemokine ligand 12-induced focal adhesion kinase activation and segregation into membrane domains is modulated by regulator of G protein signaling 1 in pro-B cells.

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i), Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.

Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells.

Small-cell lung cancer (SCLC) is an aggressive, rapidly metastasizing neoplasm. The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) is constitutively secreted by marrow stromal cells and plays a key role for homing of hematopoietic cells to the marrow. Here, we report that tumor cells from patients with SCLC express high levels of functional CXCR4 receptors for the chemokine CXCL12. Reverse transcriptase-polymerase chain reaction and flow cytometry demonstrated CXCR4 mRNA and CXCR4 surface expression in SCLC cell lines. Immunohistochemistry of primary tumor samples from SCLC patients revealed high expression of CXCR4. CXCL12 elicited CXCR4 receptor endocytosis, actin polymerization, and a robust activation of phospho-p44/42 mitogen-activated protein kinase in SCLC cells. Furthermore, CXCL12 induced SCLC cell invasion into extracellular matrix and firm adhesion to marrow stromal cells. Stromal cell adhesion of SCLC cells was significantly inhibited by the specific CXCR4 antagonist T140, pertussis toxin, antivascular cell adhesion molecule-1(VCAM-1) antibodies, and CS-1 peptide, demonstrating the importance of CXCR4 chemokine receptor activation and alpha4beta1 integrin binding, respectively. In addition, CXCL12 enhanced the adhesion of SCLC cells to immobilized VCAM-1, demonstrating that CXCR4 chemokine receptors can induce integrin activation on SCLC cells. As SCLC has a high propensity for bone marrow involvement, our findings suggest that CXCR4 chemokine receptors and alpha4beta1 integrins play a critical role in the interaction of SCLC cells with stromal cells in the tumor microenvironment.

Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis.

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell-derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.

Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1.

Complement has recently been implicated in developmental pathways and noninflammatory processes. The expression of various complement components and receptors has been shown in a wide range of circulating myeloid and lymphoid cells, but their role in normal hematopoiesis and stem cell homing has not yet been investigated. We report that normal human CD34(+) cells and lineage-differentiated hematopoietic progenitors express the complement anaphylatoxin C3a receptor (C3aR) and respond to C3a. Moreover, C3a, but not the biologically inactive desArg-C3a, induces calcium flux in these cells. Furthermore, we found that C3 is secreted by bone marrow stroma and that, although C3a does not influence directly the proliferation/survival of hematopoietic progenitors, it (1) potentiates the stromal cell-derived factor 1 (SDF-1)-dependent chemotaxis of human CD34(+) cells and lineage-committed myeloid, erythroid, and megakaryocytic progenitors; (2) primes SDF-1-dependent trans-Matrigel migration; and (3) stimulates matrix metalloproteinase-9 secretion and very late antigen 4 (VLA-4)-mediated adhesion to vascular cell adhesion molecule 1 (VCAM-1). Furthermore, we found that murine Sca-1(+) cells primed by C3a engrafted faster in lethally irradiated animals. These results indicate that normal human hematopoietic stem and progenitor cells express functional C3aR and that the C3aR-C3a axis sensitizes the responses of these cells to SDF-1 and thus may be involved in promoting their homing into the bone marrow via cross talk with the SDF-CXC chemokine receptor-4 (CXCR4) signaling axis. C3a is the first positive regulator of this axis to be identified.

CCR5-binding chemokines modulate CXCL12 (SDF-1)-induced responses of progenitor B cells in human bone marrow through heterologous desensitization of the CXCR4 chemokine receptor.

Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1beta (MIP-1beta; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1alpha (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.