Complement C3a enhances CXCL12 (SDF-1)-mediated chemotaxis of bone marrow hematopoietic cells independently of C3a receptor

Complement C3a promotes CXCL12-induced migration and engraftment of human and murine hemopoietic progenitor cells, suggesting a cross-influence between anaphylatoxin and chemokine axes. Here we have explored the underlying mechanism(s) of complement anaphylatoxin and chemokine cooperation. In addition to C3a, C3a-desArg and C4a but not C5a, are potent enhancers of CXCL12-induced chemotaxis of human and murine bone marrow (BM) stem/progenitor cells and B lineage cells. C3a enhancement of chemotaxis is chemokine specific because it is also observed for chemotaxis to CCL19 but not to CXCL13. The potentiating effect of C3a on CXCL12 is independent of the classical C3a receptor (C3aR). First, human BM CD34(+) and B lineage cells do not express C3aR by flow cytometry. Second, the competitive C3aR inhibitor SB290157 does not affect C3a-mediated enhancement of CXCL12-induced chemotaxis. Third, enhancement of chemotaxis of hemopoietic cells is also mediated by C3a-desArg, which does not bind to C3aR. Finally, C3a enhances CXCL12-induced chemotaxis of BM cells from C3aR knockout mice similar to BM cells from wild-type mice. Subsequent studies revealed that C3a increased the binding affinity of CXCL12 to human CXCR4(+)/C3aR(-), REH pro-B cells, which is compatible with a direct interaction between C3a and CXCL12. BM stromal cells were able to generate C3a, C3a-desArg, C4a, as well as CXCL12, suggesting that this pathway could function in vivo. Taken together, we demonstrate a C3a-CXCL12 interaction independent of the C3aR, which may provide a mechanism to modulate the function of CXCL12 in the BM microenvironment.     

Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice

Both granulocyte colony-stimulating factor (G-CSF) and cyclophosphamide (CY) are employed in the clinic as mobilizing agents to stimulate the egress of haematopoietic stem/progenitor cells (HSPC) from bone marrow (BM) into peripheral blood (PB). However, although both compounds are effective, the simultaneous administration of G-CSF + CY allows for optimal mobilization. The aim of this study was to compare morphological changes in major haematopoietic organs in mice mobilized by G-CSF + CY. We employed the standard G-CSF + CY mobilization protocol, in which mice were injected at day 0 with a single dose of CY followed by daily injection of G-CSF for 6 consecutive days. We noticed that the cytoreductive effect of CY on BM and spleen tissue was compensated at day 2 by the pro-proliferative effect of G-CSF. Furthermore, as evidenced by histological examination of BM sections at day 4, egress of haematopoietic cells from BM was accelerated by 2 days as compared to mobilization by G-CSF or CY alone; also, by day 6 there was accumulation of early haematopoietic (Thy-l(low) c-kit+) cells in the spleens and livers of mobilized animals. This implies that HSPC that are mobilized from BM and circulate in PB may 'home' to peripheral organs. We envision that such an accumulation of these cells in the spleen (which is a major haematopoietic organ in mouse) allows them to participate in haematopoietic reconstitution. Their homing to other sites (for example the liver) is evidence that BM-derived stem cells are playing a pivotal role in organ/tissue regeneration. The potential involvement of major chemoattractants for stem cells, like stromal-derived factor-1 which is induced by CY in various regenerating organs such as the liver, requires further study. We conclude that inclusion of CY into mobilization protocols on the one hand efficiently increases the egress of HSPC from the BM, but on the other hand may lead to the relocation of BM stem cell pools to peripheral tissues.     

The endoplasmic reticulum‐associated protein,OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.     

Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes

This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.     

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 ([Formula: see text]-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.