Edith Rian

Characterization of early stages of human B cell development by gene expression profiling.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg2+ transporters

We report here the first identification and structural characterization of a eukaryotic protein with homology to the bacterial MgtE family of potential Mg(2+) transporters. This human protein, denoted solute carrier family 41 member 1 (SLC41A1), consists of 513 amino acids with an estimated molecular weight of 56kDa. Computer analysis of the protein structure reveals that the protein consists of 10 putative transmembrane domains and includes two distinct domains highly homologous to the integral membrane part of the bacterial MgtE protein family. The gene encoding SLC41A1 is found on chromosome 1 (1q31-32) and the protein coding sequence is found on 10 exons. A 5-kb long transcript is identified in various human tissues with highest expression levels in heart and testis. We have also identified 10 SLC41A1 homologs in Homo sapiens, Mus musculus, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans, and propose that these hypothetical proteins belong to a novel eukaryotic gene family.

Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis

BackgroundThe early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.ResultsExamination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133+CD10- hematopoietic progenitor cells and CD10+ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.ConclusionThese results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.

Wnt3A activates canonical Wnt signalling in acute lymphoblastic leukaemia (ALL) cells and inhibits the proliferation of B-ALL cell lines

Acute lymphoblastic leukaemia (ALL) is the most common malignancy in children. Recently, there has been a growing interest in Wnt signalling in several aspects of cellular development, including cancer formation. Little is known about Wnt signalling in B‐ALL. We investigated whether activation of canonical Wnt signalling could occur in B‐ALL cells and thereby play a potential role in cellular growth and/or survival. This study found that Wnt3A induced β‐catenin accumulation in both primary B‐ALL cells and B‐ALL leukaemia cell lines. Further, Wnt3A was shown to induce nuclear translocation of β‐catenin and TCF/Lef‐1 dependent transcriptions in the B‐ALL cell line Nalm‐6. Examination of the mRNA expression pattern of WNT ligands, FZD receptors and WNT antagonists in Nalm‐6 cells identified a set of ligands and receptors available for signalling, as well as antagonists potentially available for modulating the response. Functional analyses showed that Wnt3A inhibited the proliferation of several, but not all, B‐ALL cell lines studied. Finally, microarray analysis was used to identify several Wnt3A target genes involved in a diverse range of cellular activities, which are potential mediators of the Wnt3A‐restrained proliferation.

Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6

No ligand has hitherto been designated for the Eph receptor tyrosine kinase family member, EphB6. Here, expression of an EphB6 ligand in the pro‐B leukemic cell line, Reh, is demonstrated by binding of soluble EphB6‐Fc fusion protein to the Reh cells. The ligand belongs to the subgroup of membrane spanning ligands, as suggested by the fact that phosphatidylinositol‐specific phospholipase C treatment did not abrogate binding of EphB6‐Fc. Two transmembrane Eph receptor ligands, ephrin‐B1 and ephrin‐B2, were identified in Reh cells. Analysis of EphB6‐Fc fusion protein binding to ephrin‐B1 or ephrin‐B2 transfected COS cells revealed a high‐affinity saturable binding between EphB6‐Fc and ephrin‐B2, but not with ephrin‐B1. In mice, EphB6 has previously been shown to be expressed in thymus. Here, we show expression of EphB6 in human thymus, as well as the expression of ephrin‐B2 in both human and mouse thymus. We conclude that ephrin‐B2 may be a physiological ligand for the EphB6 receptor.

Sox-4 messenger RNA is expressed in the embryonic growth plate and regulated via the parathyroid hormone/parathyroid hormone-related protein receptor in osteoblast-like cells.

Parathyroid hormone (PTH) and PTH‐related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5′ end of the transcription factor Sox‐4, using a subtracted cDNA library enriched in PTH‐stimulated genes from the human osteoblast‐like cell line OHS. The SOX‐4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)‐type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox‐4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox‐4 mRNA was detected in osteoblast‐like cells of both human and rodent origin. In OHS cells, physiological concentrations (10−10–10−9 M) of human PTH 1‐84 [hPTH(1‐84)] and hPTH(1‐34), but not hPTH(3‐84), stimulated Sox‐4 mRNA expression in a time‐dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox‐4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox‐4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up‐regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.

CD10+ stromal cells form B-lymphocyte maturation niches in the human bone marrow

In adult mammals, early B‐lymphopoiesis takes place in the bone marrow in close association with stromal cells. Both the phenotype of the stromal cells and the molecules involved in this essential interaction are as yet inadequately described. In this study, all benign, differentiating B‐cells (Pax‐5+ lymphoid cells) are shown, by using two‐colour immunohistochemistry on biopsies from human bone marrow, to be in close contact with scant dendritic CD10+ stromal cells until they leave via the sinusoids. This CD10+ stromal cell population does not fully overlap with the VCAM‐1+ stromal cell population. Furthermore, using a set of B‐cell differentiation markers (TdT, Pax‐5, and CD20), B‐cell development is shown to be spatially oriented, with maturation progressing towards bone marrow sinusoids. In conclusion, CD10+ stromal cells form distinct B‐lymphocyte maturation niches in the human bone marrow. Copyright

β-catenin is involved in N-cadherin–dependent adhesion, but not in canonical Wnt signaling in E2A-PBX1–positive B acute lymphoblastic leukemia cells

OBJECTIVEnThe t(1;19)(q23;13) translocation, resulting in the production of the E2A-PBX1 chimeric protein, is a common nonrandom translocation in pediatric B-lineage acute lymphoblastic leukemia (B-ALL). The E2A-PBX1 chimeric protein activates expression of several genes, including Wnt16. In the present study, we explored the role of Wnt16 and beta-catenin in t(1;19) B-ALL cells.nnnMATERIALS AND METHODSnCanonical Wnt signaling was measured by TOPflash activity. Localization of beta-catenin in the cell membrane and its involvement in leukemia-stroma interaction were studied by confocal microscopy. Adhesion to N-cadherin was analyzed by adding (3)H-thymidin-labeled cells to N-cadherin-coated wells.nnnRESULTSnIn contrast to previous reports, we detected no effects on cell viability or proliferation upon modulation of the Wnt16 levels. Moreover, despite high levels of Wnt16 and beta-catenin, the cells had very low levels of canonical Wnt signaling. Instead, beta-catenin was located in the cell membrane along with N-cadherin. E2A-PBX1-positive leukemia cells adhered strongly to bone marrow stroma cells, and we showed that adherence junctions stained strongly for both proteins. Moreover, knockdown of beta-catenin reduced the adhesion of E2A-PBX1-positive leukemia cells to N-cadherin, suggesting that beta-catenin and N-cadherin play a central role in homotypic cell-to-cell adhesion and in leukemia-stroma adhesion. Interestingly, knockdown of Wnt16 by small interfering RNA reduced the level of N-cadherin.nnnCONCLUSIONnWnt16 does not activate canonical Wnt signaling in E2A-PBX1-positive cells. Instead, beta-catenin is involved in N-cadherin-dependent adherence junctions, suggesting for the first time that leukemia-stroma interactions may be mediated via an N-cadherin-dependent mechanism.

Structural and functional organization of the gene encoding the human thyrotropin-releasing hormone receptor.

Abstract : The thyrotropin‐releasing hormone (TRH) receptor (TRHR) is widely distributed throughout the central and peripheral nervous systems. In addition to its role in controlling the synthesis and secretion of thyroid‐stimulating hormone and prolactin from the anterior pituitary, TRH is believed to act as a neurotransmitter as well as a neuromodulator. We have isolated genomic λ and P1‐derived artificial chromosome clones encoding the human TRHR. The gene was found to be 35 kb with three exons and two introns. A 541‐bp intron 1 (‐629 to ‐89 relative to the translation start site) is conserved between human and mouse. A large intron 2 of 31 kb disrupts the open reading frame (starting in position +790) in the sequence encoding the supposed junction between the third intracellular loop and the putative sixth transmembrane domain. A similar intron was found in chimpanzee and sheep but not in rat and mouse. Promoter analysis of upstream regions demonstrated cell type‐specific reporter activation, and sequencing of 2.5 kb of the promoter revealed putative cis‐acting regulatory elements for several transcription factors that may contribute to the regulation of the TRHR gene expression. Functional analysis of potential response elements for the anterior pituitary‐specific transcription factor Pit‐1 revealed cell type‐specific binding that was competed out with a Pit‐1 response element from the GH gene promoter.

Bone marrow stroma cells regulate TIEG1 expression in acute lymphoblastic leukemia cells: Role of TGFβ/BMP-6 and TIEG1 in chemotherapy escape

The bone marrow microenvironment regulates early B lymphopoiesis and protects leukemia cells against chemotherapy treatment, thus the microenvironment may serve as a sanctuary site for these cells. Yet, few factors that contribute to this process are known. We have explored the role of transforming growth factor β (TGFβ) and bone morphogenetic protein‐6 (BMP‐6) and one target gene, TGFβ inducible early gene 1 (TIEG1), in the communication between stroma cells and acute lymphoblastic leukemia (ALL) cell lines and their escape from chemotherapy. Here, we have demonstrated TIEG1 expression in both normal B progenitor cells and ALL cells, which increased rapidly upon TGFβ and BMP‐6 treatment. Stimulation with TGFβ or BMP‐6, as well as overexpression of TIEG1 inhibited proliferation. Furthermore, interaction with stroma cells induced TIEG1 expression in ALL cells, inhibited their proliferation and protected the cells against chemotherapeutic treatment. Similarly, treatment with TGFβ or BMP‐6, as well as overexpression of TIEG1, protected ALL cells against chemotherapy‐induced cell death. These data suggest that TGFβ and BMP‐6 in the bone marrow microenvironment allow leukemia cells to escape therapy. Further, the data indicate that TIEG1 might be involved in mediating this effect from the microenvironment onto the leukemia cells.