Disturbed CXCR4/CXCL12 axis in paediatric precursor B-cell acute lymphoblastic leukaemia.

Malignant cells infiltrating the bone marrow (BM) interfere with normal cellular behaviour of supporting cells, thereby creating a malignant niche. We found that CXCR4-receptor expression was increased in paediatric precursor B-cell acute lymphoblastic leukaemia (BCP-ALL) cells compared with normal mononuclear haematopoietic cells (P < 0·0001). Furthermore, high CXCR4-expression correlated with an unfavourable outcome in BCP-ALL (5-year cumulative incidence of relapse ± standard error: 38·4% ± 6·9% in CXCR4-high versus 12% ± 4·6% in CXCR4-low expressing cases, P < 0·0001). Interestingly, BM levels of the CXCR4-ligand (CXCL12) were 2·7-fold lower (P = 0·005) in diagnostic BCP-ALL samples compared with non-leukaemic controls. Induction chemotherapy restored CXCL12 levels to normal. Blocking the CXCR4-receptor with Plerixafor showed that the lower CXCL12 serum levels at diagnosis could not be explained by consumption by the leukaemic cells, nor did we observe an altered CXCL12-production capacity of BM-mesenchymal stromal cells (BM-MSC) at this time-point. We rather observed that a very high density of leukaemic cells negatively affected CXCL12-production by the BM-MSC while stimulating the secretion levels of granulocyte colony-stimulating factor (G-CSF). These results suggest that highly proliferative leukaemic cells are able to down-regulate secretion of cytokines involved in homing (CXCL12), while simultaneously up-regulating those involved in haematopoietic mobilization (G-CSF). Therefore, interference with the CXCR4/CXCL12 axis may be an effective way to mobilize BCP-ALL cells.

Cord blood mesenchymal stem cells suppress DC-T Cell proliferation via prostaglandin B2.

Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.

Mesenchymal stem cells respond to TNF but do not produce TNF.

Previously, we demonstrated that several TLRs are expressed on cord blood-derived USSC. Stimulation of USSC with TLR agonists resulted in a marked increase of IL-6 and IL-8 production. Interestingly, TNF was undetectable after TLR stimulation, which appeared to be a result of an inactivated TNF promoter in USSC. Here, we elaborate this study by demonstrating that although USSC do not produce TNF, they are susceptible to TNF stimulation, resulting in NF-kappaB translocation and cytokine production. Additionally, we compared different stem cell sources for their ability to produce TNF. Interestingly, we found that the TNF promoter in BM-MSC is inactivated as well. Like USSC, they are able to respond to TNF stimulation, but they are not able to produce TNF, even not after LPS stimulation. This limited cytokine response in combination with the well-studied immunosuppressive properties of MSC makes these cells ideal for immune-suppressive treatment modalities such as graft-versus-host disease.

Cord blood mesenchymal stem cells propel human dendritic cells to an intermediate maturation state and boost interleukin-12 production by mature dendritic cells.

Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.

Toll-like receptor triggering in cord blood mesenchymal stem cells.

Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll-like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived MSCs, the expression of TLRs and ligand-induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF-kappaB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)-6 and/or IL-8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)-alpha was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF-alpha promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up-regulation of a restricted number of pro-inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.

Mesenchymal stromal cells: tissue engineers and immune response modulators.

Mesenchymal stromal cells (MSCs) show significant immune-suppressive properties both in vitro and in vivo. Based on their immune-stealth properties, allogeneic MSCs are used to treat several diseases, for example the injection of MSCs in infarcted heart tissue or their use in bone-cartilage regeneration. The most spectacular treatment was recently described. MSCs were able to down-regulate the severity of graft-versus-host disease, leading to an impressive 20 to 50% increase in the two-year survival of bone marrow transplantation patients. Here the current literature is reviewed to elucidate the different mechanisms involved in these two clinical treatment modalities of MSCs.

Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expression of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), CD80, CD206, and CD1a. Monocytes stained positive with Abs against heparan sulfate (HS) and chondroitin sulfate (CS) B (CSB; dermatan sulfate), but not with Abs that recognize CSA, CSC, and CSE. Inhibition of sulfation of monocyte/DC cell surface GAGs by sodium chlorate reduced the reactivity of sulfate-recognizing single-chain Abs. This correlated with hampered IL-4-induced DC differentiation as evidenced by lower expression of DC-SIGN and CD1a and a decreased DC-induced PBL proliferation, suggesting that sulfated monocyte cell surface GAGs support IL-4 activity. Furthermore, removal of cell surface chondroitin sulfates by chondroitinase ABC strongly impaired IL-4-induced STAT6 phosphorylation, whereas removal of HS by heparinase III had only a weak inhibitory effect. IL-4 bound to heparin and CSB, but not to HS, CSA, CSC, CSD, and CSE. Binding of IL-4 required iduronic acid, an N-sulfate group (heparin) and specific O sulfates (CSB and heparin). Together, these data demonstrate that monocyte cell surface chondroitin sulfates play an important role in the IL-4-driven differentiation of monocytes into DCs.

An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity.

PDZ (acronym of the synapse-associated protein PSD-95/SAP90, the septate junction protein Discs-large, and the tight junction protein ZO-1) domains are abundant small globular protein interaction domains that mainly recognize the carboxyl termini of their target proteins. Detailed knowledge on PDZ domain binding specificity is a prerequisite for understanding the interaction networks they establish. We determined the binding preference of the five PDZ domains in the protein tyrosine phosphatase PTP-BL by screening a random C-terminal peptide lambda phage display library. Interestingly, the potential of PDZ2 to interact with class III-type ligands was found to be modulated by the presence of PDZ1. Structural studies revealed a direct and specific interaction of PDZ1 with a surface on PDZ2 that is opposite the peptide binding groove. Long-range allosteric effects that cause structural changes in the PDZ2 peptide binding groove thus explain the altered PDZ2 binding preference. Our results experimentally corroborate that the molecular embedding of PDZ domains is an important determinant of their ligand binding specificity.

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1.

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.

Redox-regulated affinity of the third PDZ domain in the phosphotyrosine phosphatase PTP-BL for cysteine-containing target peptides.

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signalling complexes. They specifically bind to short C-terminal peptides and occasionally to internal sequences that structurally resemble such peptide termini. The binding of PDZ domains is dominated by the residues at the P(0) and P(-2) position within these C-terminal targets, but other residues are also important in determining specificity. In this study, we analysed the binding specificity of the third PDZ domain of protein tyrosine phosphatase BAS-like (PTP-BL) using a C-terminal combinatorial peptide phage library. Binding of PDZ3 to C-termini is preferentially governed by two cysteine residues at the P(-1) and P(-4) position and a valine residue at the P(0) position. Interestingly, we found that this binding is lost upon addition of the reducing agent dithiothrietol, indicating that the interaction is disulfide-bridge-dependent. Site-directed mutagenesis of the single cysteine residue in PDZ3 revealed that this bridge formation does not occur intermolecularly, between peptide and PDZ3 domain, but rather is intramolecular. These data point to a preference of PTP-BL PDZ3 for cyclic C-terminal targets, which may suggest a redox state-sensing role at the cell cortex.

A closed binding pocket and global destabilization modify the binding properties of an alternatively spliced form of the second PDZ domain of PTP-BL.

PTP-BL is a large phosphatase that is implicated in cellular processes as diverse as cytokinesis, actin-cytoskeletal rearrangement, and apoptosis. Five PDZ domains mediate its cellular role by binding to the C termini of target proteins, forming multiprotein complexes. The second PDZ domain (PDZ2) binds to the C termini of the tumor suppressor protein APC and the LIM domain-containing protein RIL; however, in one splice variant, PDZ2as, a 5 residue insertion abrogates this binding. The insert causes distinct structural and dynamical changes in the alternatively spliced PDZ2: enlarging the L1 loop between beta2 and beta3, both lengthening and changing the orientation of the alpha2 helix, giving the base of the binding pocket less flexibility to accommodate ligands, and destabilizing the entire domain. These changes render the binding pocket incapable of binding C termini, possibly having implications in the functional role of PTP-BL.

The interaction of PTP-BL PDZ domains with RIL: an enigmatic role for the RIL LIM domain.

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with' the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL.

The cancer-related protein SSX2 interacts with the human homologue of a Ras-like GTPase interactor, RAB3IP, and a novel nuclear protein, SSX2IP.

The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid. SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma. The SSX proteins are localized in the nucleus and are diffusely distributed. In addition, they may be included in polycomb-group nuclear bodies. Other studies have indicated that the SSX proteins may act as transcriptional repressors. As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins. By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP. RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4. Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein. Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm. However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus. Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus. By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro. These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth.