Persio Dello Sbarba

TNF-alpha-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation.

We previously reported that macrophage activators such as LPS, IL-2, and IL-4 down-modulate the M-CSFR via a mechanism involving protein kinase C and phospholipase C. In this study, we showed that M-CSFR is shed from macrophage surface and identified the protease responsible for M-CSFR cleavage and down-modulation. The shedding of M-CSFR elicited by phorbol esters (tetradecanoylphorbol myristate acetate (TPA)) or LPS in murine BAC.1-2F5 macrophages was prevented by cation chelators, as well as hydroxamate-based competitive inhibitors of metalloproteases. We found that the protease cleaving M-CSFR is a transmembrane enzyme and that its expression is controlled by furin-like serine endoproteases, which selectively process transmembrane metalloproteases. M-CSFR down-modulation was inhibited by treating cells in vivo, before TPA stimulation, with an Ab raised against the extracellular, catalytic domain of proTNF-converting enzyme (TACE). TACE expression was confirmed in BAC.1-2F5 cells and found inhibited after blocking furin-dependent processing. Using TACE-negative murine Dexter-ras-myc cell monocytes, we found that in these cells TPA is unable to down-modulate M-CSFR expression. These data indicated that TACE is required for the TPA-induced M-CSFR cleavage. The possibility that the cleavage is indirectly driven by TACE via the release of TNF was excluded by treating cells in vivo with anti-TNF Ab. Thus, we concluded that TACE is the protease responsible for M-CSFR shedding and down-modulation in mononuclear phagocytes undergoing activation. The possible physiological relevance of this mechanism is discussed.

Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent.

BACKGROUND: The liquid culture of murine bone marrow cells at 1‐percent oxygen maintains the balance between primative progenitor cell renewal and clonogenic progenitor expansion better than that at 20‐percent oxygen. These results are of potential interest for the ex vivo expansion of human progenitor cells, as low O2 tension could preserve the engraftment potential of cultured apheresis products.

Transmodulation of cell surface regulatory molecules via ectodomain shedding.

Abstract Cell responses to exogenous stimuli often result in a rapid decrease of cell surface density of a wide range of diverse regulatory proteins, receptor and adhesion molecules in particular. This decrease may occur in a liganddependent fashion (downregulation), following endocytosis and degradation by lysosomal proteases, or by downmodulation, where molecules are targeted by endoproteases directly on cell surface. These proteases are recruited by transmodulating agents, different from ligand, which act via their own receptors and the related intracellularlygenerated signals. Endoproteolytic activity determines the release of large portions (shedding) of substrate proteins, called ectodomains, which are usually not ligandbound, and therefore represent biologicallyactive molecules. Ectodomain shedding is involved in a number of pathophysiological processes, such as inflammation, cell degeneration and apoptosis, and oncogenesis. Common features of the process, such as the involvement of protein kinase C and of transmembrane metalloproteases, have been identified. In this review, we summarize basic concepts on downmodulation and ectodomain shedding, and provide an update of the issue with respect to: (i) new entries to the list of molecules found involved in the process; (ii) current views about the upstream control of shedding, i.e. the pathways linking the signals triggered by the transmodulating agents to the activation of endoproteolytic activity on the cell surface.

Hypoxia Modifies Proliferation and Differentiation of CD34+ CML Cells

We previously showed that hypoxia (1% O2) favors the self‐renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34+ cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O2) and normoxia (20% O2). After 8 days of culture, their proliferation, capacity for colony‐forming‐cell (CFC) generation in secondary cultures (pre‐CFC), and phenotype (CD34 and platelet‐activating factor receptor [PAF‐R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34+ cells and preserves the pre‐CFC capacity and cell‐surface CD34 expression of CML cells better than normoxia. The PAF‐R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF‐R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.

ERK5/BMK1 Is Indispensable for Optimal Colony-Stimulating Factor 1 (CSF-1)-Induced Proliferation in Macrophages in a Src-Dependent Fashion

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-γ and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.

Low Mr phosphotyrosine protein phosphatase associates and dephosphorylates p125 focal adhesion kinase, interfering with cell motility and spreading

Low M r phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M r phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, lowM r phosphotyrosine protein phosphatase bothin vitro and in vivo. This event is consistent with an altered association of pp60 src with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M rphosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M r phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.

Tryptophan availability selectively limits NO-synthase induction in macrophages

We studied the effects of tryptophan (TRP) availability on the synthesis and release of nitric oxide (NO) and tumor necrosis factor α (TNF‐α) in interferon‐γ (IFN‐γ)‐activated murine macrophages of the BAC1.2F5 cell line. IFN‐γ (100 U/ml) not only increased the synthesis and release of NO and TNF‐α from these cells but also induced indoleamine‐2,3‐dioxygenase, the rate‐limiting enzyme of TRP catabolism. This led to an increased metabolic flow through the kynurenine pathway and significantly decreased TRP levels in macrophage incubation media. Low TRP concentrations in the media, however, modified IFN‐γ effects. In TRP‐“starved” cultures, in fact, the IFN‐γ‐mediated NO synthase induction was significantly reduced, and the increased TNF‐α synthesis and release were not affected. Our results suggest hat a reduced local TRP availability may modify macrophage function and possibly the outcome of immune responses.

Severe hypoxia defines heterogeneity and selects highly immature progenitors within clonal erythroleukemia cells

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friends murine erythroleukemia (MEL) cells, addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC), we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short‐term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population, which, despite its clonality, comprised progenitors characterized by markedly different hypoxia‐resistance. These included hypoxia‐sensitive colony‐forming cells and hypoxia‐resistant STR‐type LSC, capable of repopulating secondary liquid cultures of CRA assays, confirming what was previously shown for normal hematopoiesis. STR‐type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle, in contrast with the fact that almost all hypoxia‐surviving cells were growth‐arrested and with what we previously found for HSC. However, quiescent LSC were also detected, capable of delayed culture repopulation with the same efficiency as STR‐like LSC. The fact that even quiescent LSC, believed to sustain minimal residual disease in vivo, were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines, which are therefore suitable to study the sensitivity of individual components to treatments.

Environmental restrictions within tumor ecosystems select for a convergent, hypoxia-resistant phenotype of cancer stem cells

Tumors are ecosystems which develop from stem cells endowed with unlimited cell renewal and genetic instability, under the effects of mutagenesis and natural selection imposed by environmental changes. While changes and variations made possible by genetic instability are practically unlimited, the microenvironment progressively reduces those possibilities in the struggle for life imposed by hypoxia and nutrient shortage typical of tumor environments. This entails the tendency to evolve a convergent phenotype resistant to microenvironmental restrictions (first of all hypoxia), which progressively dominates the clonal selection. It is shown that adaptation to hypoxia, rather than being a peculiarity of cancer stem cells is also a characteristic of normal hematopoietic stem cells, and may thus be described as a general feature of the stem cell phenotype. The metabolic orientation of this phenotype closely resembles the orientation of highly anaplastic ascites hepatomas, showing that, in restricted environments, stem cell recruitment to growth is limited by mitochondrial reoxidation of reducing equivalents produced in folate redox steps connected with purine synthesis. Finally, a review of earlier research into glucose metabolism in cancer leads to the reinterpretation of Warburg’s aerobic glycolysis as a defence mechanism which disposes of glycolytic products able to negatively interfere with the crucial role of mitochondrial respiration in cell recruitment for growth.

The Colony-Stimulating Factor-1 (CSF-1) Receptor Sustains ERK1/2 Activation and Proliferation in Breast Cancer Cell Lines

Breast cancer is the second leading cause of cancer-related deaths in western countries. Colony-Stimulating Factor-1 (CSF-1) and its receptor (CSF-1R) regulate macrophage and osteoclast production, trophoblast implantation and mammary gland development. The expression of CSF-1R and/or CSF-1 strongly correlates with poor prognosis in several human epithelial tumors, including breast carcinomas. We demonstrate that CSF-1 and CSF-1R are expressed, although at different levels, in 16/17 breast cancer cell lines tested with no differences among molecular subtypes. The role of CSF-1/CSF-1R in the proliferation of breast cancer cells was then studied in MDAMB468 and SKBR3 cells belonging to different subtypes. CSF-1 administration induced ERK1/2 phosphorylation and enhanced cell proliferation in both cell lines. Furthermore, the inhibition of CSF-1/CSF-1R signaling, by CSF-1R siRNA or imatinib treatment, impaired CSF-1 induced ERK1/2 activation and cell proliferation. We also demonstrate that c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, are downstream CSF-1R in breast cancer cells. The presence of a proliferative CSF-1/CSF-1R autocrine loop involving ERK1/2 was also found. The wide expression of the CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy.