Hans-Christian Aasheim

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.

Activation of NF-κB by extracellular S100A4: Analysis of signal transduction mechanisms and identification of target genes

The metastasis‐promoting protein S100A4 stimulates metastatic progression through both intracellular and extracellular functions. Extracellular activities of S100A4 include stimulation of angiogenesis, regulation of cell death and increased cell motility and invasion, but the exact molecular mechanisms by which extracellular S100A4 exerts these effects are incompletely elucidated. The aim of the present study was to characterize S100A4‐induced signal transduction mechanisms and to identify S100A4 target genes. We demonstrate that extracellular S100A4 activates the transcription factor NF‐κB in a subset of human cancer cell lines through induction of phosphorylation and subsequent degradation of the NF‐κB inhibitor IκBα. Concomitantly, S100A4 induced a sustained activation of the MAP kinase JNK, whereas no increased activity of the MAP kinases p38 or ERK was observed. Microarray analyses identified 136 genes as being significantly regulated by S100A4 treatment, and potentially interesting S100A4‐induced gene products include IκBα, p53, ephrin‐A1 and optineurin. Increased expression of ephrin‐A1 and optineurin was validated using RT‐PCR, Western blotting and functional assays. Furthermore, S100A4‐stimulated transcription of these target genes was dependent on activation of the NF‐κB pathway. In conclusion, these findings contribute to the understanding of the complex molecular mechanisms responsible for the diverse biological functions of extracellular S100A4, and provide further evidence of how S100A4 may stimulate metastatic progression.

Splenic marginal zone lymphoma with VH1-02 gene rearrangement expresses poly- and self-reactive antibodies with similar reactivity

One-third of all splenic marginal zone lymphomas (SMZL) use the IgH VH1-02 gene. These cases are usually not associated with hepatitis C virus infection. Of interest, the rearranged VH1-02 genes display similar complementarity determining regions 3, a finding confirmed by our study. The latter suggests that these SMZL may produce antibodies with similar reactivity. We produced recombinant antibodies from 5 SMZL cases with VH1-02 gene rearrangement to study the binding reactivity of these antibodies. Surprisingly, the recombinant antibodies demonstrated poly- and self-reactivity as demonstrated by their reactivity with nuclear, cytoplasmic, as well as membranous antigens expressed by human cells and by reactivity with human serum. This polyreactivity was specific as demonstrated by ELISA. The antibodies did not react with proteins on the cell surface that are induced by apoptosis as shown for antibodies produced by chronic lymphatic leukemia with VH1-02 gene rearrangement. The results indicate that a common subset of SMZL arises from polyreactive B cells, a subset of marginal zone B cells that are important in the immunologic defense against infection.

Signaling through ephrin-A ligand leads to activation of Src-family kinases, Akt phosphorylation, and inhibition of antigen receptor-induced apoptosis.

Eph receptor tyrosine kinases and ephrins play important roles in diverse biological processes such as migration, adhesion, and angiogenesis. Forward and reverse signaling has been reported in receptor‐ and ligand‐bearing cells. The ligands can be divided into the transmembrane ephrin‐B family and the GPI‐anchored ephrin‐A family. Here, we show expression of ephrin‐A ligands on CD4+ T cells cultured in medium with human serum and the T cell line Jurkat TAg and on cells isolated from patients with T cell lymphomas and T cell leukemias. Functional role and identification of proteins involved in ephrin‐A signaling were investigated here in the T cell line Jurkat TAg. Signaling through ephrin‐A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI‐3K is activated, shown by induced phosphorylation of the Akt kinase. An ephrin‐A signaling complex could be isolated, containing several phosphorylated proteins including Lck and Fyn. Interestingly, we show that signaling through ephrin‐A in Jurkat TAg cells, initiated by interaction with the EphA2 receptor, leads to inhibition of activation‐induced cell death. To conclude, ephrin‐A signaling in Jurkat TAg cells leads to induced phosphorylation of several proteins including Lck, Fyn, and Akt. A consequence of ephrin‐A signaling is inhibition of antigen receptor‐induced apoptosis.

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.

Antibody Array Analysis with Label-based Detection and Resolution of Protein Size

Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670–10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclin-dependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes.

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G1/S-phase progression and spindle assembly.

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

BackgroundIncreased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231).MethodsStable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion.ResultsDownregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis.ConclusionsOur results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

Activation of EphA receptors on CD4+CD45RO+ memory cells stimulates migration

We have demonstrated previously that binding of ephrin‐A1 to EphA receptors on human CD4+ and CD8+ T cells stimulates migration. Two EphA receptors have been reported in T cells: EphA1 at the protein level and EphA4 at the mRNA level. In this study, we wanted to investigate the expression profile of these receptors in T cell subpopulations and to test if expression differences would affect the potential of cells to migrate upon ephrin‐A1 binding. We have generated an anti‐EphA4 mAb for expression analysis. Our data show that functional EphA4 is expressed on the cell surface of CD4+ and CD8+ T cells. In addition, EphA4 receptor expression is induced after overnight incubation in serum‐free medium, in particular, on CD4+CD45RO+ T cells. Migration of CD4+ T cells in response to ephrin‐A1 is observed for memory cells (CD45RO+) and much weaker for naïve cells (CD45RA+). A signaling complex associated with the EphA4 receptor has also been isolated and includes EphA1, the Src family kinases Fyn and Lck, Slp76, and Vav1. To conclude, T cells express EphA1 and EphA4 receptors. Expression differences of EphA4 are observed in subpopulations of CD4+ T cells. This is related to the cell migration potential after ephrin‐A1 binding.

Ephrin-A1 stimulates migration of CD8+CCR7+ T lymphocytes

We have previously demonstrated that binding of ephrin‐A1 to Eph receptors on human CD4+ T cells stimulates migration. Here, we show that a distinct population of CD8+ T lymphocytes, expressing the chemokine receptor CCR7, also binds ephrin‐A1 and is stimulated to migrate after binding. The Eph receptor signaling pathway taking part in the migration event was here investigated. Induced tyrosine phosphorylation of several proteins was seen after ephrin‐A1 binding. In particular, induced phosphorylation and kinase activity of the Src kinase family member Lck was observed. An Lck inhibitor inhibited ephrin‐A1‐induced migration, indicating the involvement of Lck in the migration event. In addition, we observed an induced association of the focal adhesion‐like kinase proline‐rich tyrosine kinase 2 (Pyk2) and the guanidine exchange factor Vav1 with Lck. PI3K inhibitors also inhibited migration, and studies in transfectants indicate an association of PI3K with EphA1. Further, ephrin‐A1‐induced migration could be related to the activation of Rho GTPases. This was also observed by using an inhibitor of the Rho‐associated kinase ROCK, a downstream effector of Rho. Our results suggest that stimulation of Eph receptors on CD8+CCR7+ T cells leads to migration involving activation of Lck, Pyk2, PI3K, Vav1 and Rho GTPase.