Regulation of 92-kD gelatinase release in HL-60 leukemia cells

Matrix metalloproteinase 9 (MMP-9), also known as 92-kD type IV collagenase/gelatinase, is believed to play a critical role in tumor invasion and metastasis. Here, we report that MMP-9 was constitutively released from the human promyelocytic cell line HL-60 as determined by zymographic analysis. Tumor necrosis factor-alpha (TNF-alpha) enhanced the enzyme release threefold to fourfold and the protein kinase C (PKC) activator and differentiation inducer 12-O-tetradecanoylphorbol-13- acetate (TPA) eightfold to ninefold. Gelatinase induction by TNF-alpha and TPA was inhibited by actinomycin D or cycloheximide, indicating that de novo protein synthesis was required. Neutralizing monoclonal antibodies to TNF-alpha (anti-TNF-alpha) decreased the basal MMP-9 release of these cells. In addition, these antibodies also significantly interfered with the TPA-induced enzyme release. Agents that inhibit TNF-alpha expression in HL-60 cells, such as pentoxifylline and dexamethasone, completely abrogated both the constitutive and TPA-evoked MMP-9 release. Diethyldithiocarbamate, which is known to stimulate TNF-alpha production in HL-60 cells, exerted a positive effect on MMP-9 release in untreated cells but was inhibitory in TPA-treated HL-60 cells. The PKC inhibitor staurosporine at low concentrations (100 ng/mL) caused a significant augmentation of MMP-9 release in untreated cultures that was blocked by the addition of anti-TNF-alpha. High concentrations (2 mumol/L) of staurosporine completely abolished the extracellular enzyme activity both in untreated and TPA-stimulated cells. These results suggest, that TNF- alpha is required for basal and PKC-mediated MMP-9 release in HL-60 leukemia cells. Thus, MMP-9 secretion may be regulated by TNF-alpha not only in a paracrine but also in an autocrine fashion. This may potentiate the matrix degradative capacity of immature leukemic cells in the processes of bone marrow egress and the evasion of these cells into peripheral tissue

The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes

We have studied the relationship between insulin activation of insulin-receptor kinase and insulin stimulation of glucose uptake in isolated rat adipocytes. Glucose uptake was half-maximally or maximally stimulated, respectively, when only 4% or 14% of the maximal kinase activity had been reached. To investigate this relationship also under conditions where the insulin effect on activation of receptor kinase was decreased, the adipocytes were exposed to 10 microM-isoprenaline alone or with 5 micrograms of adenosine deaminase/ml. An approx. 30% (isoprenaline) or approx. 50% (isoprenaline + adenosine deaminase) decrease in the insulin effect on receptor kinase activity was found at insulin concentrations between 0.4 and 20 ng/ml, and this could not be explained by decreased insulin binding. The decreased insulin-effect on kinase activity was closely correlated with a loss of insulin-sensitivity of glucose uptake. Moreover, our data indicate that the relation between receptor kinase activity and glucose uptake (expressed as percentage of maximal uptake) remained unchanged. The following conclusions were drawn. (1) If activation of receptor kinase stimulates glucose uptake, only 14% of the maximal kinase activity is sufficient for maximal stimulation. (2) Isoprenaline decreases the coupling efficiency between insulin binding and receptor-kinase activation, this being accompanied by a corresponding decrease in sensitivity of glucose uptake. (3) Our data indicate that the signalling for glucose uptake is closely related to receptor-kinase activity, even when the coupling efficiency between insulin binding and kinase activation is altered. They thus support the hypothesis that receptor-kinase activity reflects the signal which originates from the receptor and which is transduced to the glucose-transport system

Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nMfor the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo

Overexpression of the urokinase receptor splice variant uPAR-del4/5 in breast cancer cells affects cell adhesion and invasion in a dose-dependent manner and modulates transcription of tumor-associated genes

mRNA levels of the urokinase receptor splice variant uPAR-del4/5 are associated with prognosis in breast cancer. Its overexpression in cancer cells affects tumor biologically relevant processes. In the present study, individual breast cancer cell clones displaying low vs. high uPAR-del4/5 expression were analyzed demonstrating that uPAR-del4/5 leads to reduced cell adhesion and invasion in a dose-dependent manner. Additionally, matrix metalloproteinase-9 (MMP-9) was found to be strongly upregulated in uPAR-del4/5 overexpressing compared to vector control cells. uPAR-del4/5 may thus play an important role in the regulation of the extracellular proteolytic network and, by this, influence the metastatic potential of breast cancer cells

The nuclear cofactor receptor interacting protein-140 (RIP140) regulates the expression of genes involved in A beta generation

The receptor interacting protein-140 (RIP140) is a cofactor for several nuclear receptors and has been involved in the regulation of metabolic and inflammatory genes. We hypothesize that RIP140 may also affect Aβ generation because it modulates the activity of transcription factors previously implicated in amyloid precursor protein (APP) processing, such as peroxisome proliferator-activated receptor-γ (PPARγ). We found that the levels of RIP140 are reduced in Alzheimer's disease (AD) postmortem brains compared with healthy controls. In addition, in situ hybridization experiments revealed that RIP140 expression is enriched in the same brain areas involved in AD pathology, such as cortex and hippocampus. Furthermore, we provide evidence using cell lines and genetically modified mice that RIP140 is able to modulate the transcription of certain genes involved in AD pathology, such as β-APP cleaving enzyme (BACE1) and GSK3. Consequently, we found that RIP140 overexpression reduced the generation of Aβ in a neuroblastoma cell line by decreasing the transcription of β-APP cleaving enzyme via a PPARγ–dependent mechanism. The results of this study therefore provide molecular insights into common signaling pathways linking metabolic disease with AD

Computer-Supported Research: Konzeptpapier zum CoSRe-Projekt

Die Wissenschaften befinden sich am Beginn zu einem neuen Zeitalter: neue Messverfahren erzeugen riesige Mengen experimenteller Daten, Computer gestatten die Exploration dieser Daten, immer größere und komplexere Modelle erlauben präzise Vorhersagen, das Internet führt zu einer weltweiten Kollaboration, der Innovationszyklus wird immer schneller. Jim Gray bezeichnet dies als „vierten wissenschaftlichen Paradigma“ Diese Datengetriebenheit stellt die Wissenschaften aber auch vor neue Herausforderungen. Zwar werden immer mehr Routineaufgaben von Computern übernommen. Für die Bewältigung der Informationsflut ist aber eine Computer-Unterstützung des gesamten wissenschaftlichen Zyklus notwendig: von der experimentellen Datenerhebung über die Datenanalyse und anschließende Modellbildung bis hin zur Simulation und einer Modellrevision auf Grund der Simulationsergebnisse. Wissenschaftliches Arbeiten, das von einer solchen Art der Computer-Unterstützung getragen wird, bezeichnen wir als Computer-Supported Research. Zwei Beobachtungen sind für die Unterstützung des gesamten wissenschaftlichen Zyklus sehr hilfreich: Zum einen ist es für die Verarbeitung von Daten unwichtig, ob sie aus Experimenten in der realen Welt oder aus Simulationen stammen. Zum anderen werden Modelle selbst heute mehr und mehr als First-Class-Objekte, d.h. als Daten behandelt: sie werden zwischen Programmen ausgetauscht, in Datenbanken gespeichert, über das Internet verschickt und durch Programme manipuliert