Interaction of macrocyclic gadolinium-based MR contrast agents with Type I collagen. Equilibrium and kinetic studies.

The interactions of gadoterate meglumine, gadobutrol, gadoteridol and Gd(HB-DO3A) with bovine Type I collagen were investigated by ultrafiltration and dialysis. The affinity of the four agents to collagen is similar. However, the maximum adsorbed amount of GdIII-complexes decreases in the following order: gadoterate meglumine > gadobutrol > gadoteridol > Gd(HB-DO3A). Calculations with the open three-compartment model reveal that the structural homologs gadoteridol and Gd(HB-DO3A) have a lower adsorption onto collagen, which may explain the less prolonged in vivo retention of gadoteridol observed in soft tissues of rats.

Three-dimensional chemotaxis-driven aggregation of tumor cells.

One of the most important steps in tumor progression involves the transformation from a differentiated epithelial phenotype to an aggressive, highly motile phenotype, where tumor cells invade neighboring tissues. Invasion can occur either by isolated mesenchymal cells or by aggregates that migrate collectively and do not lose completely the epithelial phenotype. Here, we show that, in a three-dimensional cancer cell culture, collective migration of cells eventually leads to aggregation in large clusters. We present quantitative measurements of cluster velocity, coalescence rates, and proliferation rates. These results cannot be explained in terms of random aggregation. Instead, a model of chemotaxis-driven aggregation - mediated by a diffusible attractant - is able to capture several quantitative aspects of our results. Experimental assays of chemotaxis towards culture conditioned media confirm this hypothesis. Theoretical and numerical results further suggest an important role for chemotactic-driven aggregation in spreading and survival of tumor cells.

Real-time monitoring of cell protrusion dynamics by impedance responses.

Cellular protrusions are highly dynamic structures involved in fundamental processes, including cell migration and invasion. For a cell to migrate, its leading edge must form protrusions, and then adhere or retract. The spatial and temporal coordination of protrusions and retraction is yet to be fully understood. The study of protrusion dynamics mainly relies on live-microscopy often coupled to fluorescent labeling. Here we report the use of an alternative, label-free, quantitative and rapid assay to analyze protrusion dynamics in a cell population based on the real-time recording of cell activity by means of electronic sensors. Cells are seeded on a plate covered with electrodes and their shape changes map into measured impedance variations. Upon growth factor stimulation the impedance increases due to protrusive activity and decreases following retraction. Compared to microscopy-based methods, impedance measurements are suitable to high-throughput studies on different cell lines, growth factors and chemical compounds. We present data indicating that this assay lends itself to dissect the biochemical signaling pathways controlling adhesive protrusions. Indeed, we show that the protrusion phase is sustained by actin polymerization, directly driven by growth factor stimulation. Contraction instead mainly relies on myosin action, pointing at a pivotal role of myosin in lamellipodia retraction.

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction.

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase-related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.

The diacylglycerol kinase α/atypical PKC/ß1 integrin pathway in SDF-1α mammary carcinoma invasiveness.

Diacylglycerol kinase α (DGKα), by phosphorylating diacylglycerol into phosphatidic acid, provides a key signal driving cell migration and matrix invasion. We previously demonstrated that in epithelial cells activation of DGKα activity promotes cytoskeletal remodeling and matrix invasion by recruiting atypical PKC at ruffling sites and by promoting RCP-mediated recycling of α5ß1 integrin to the tip of pseudopods. In here we investigate the signaling pathway by which DGKα mediates SDF-1α-induced matrix invasion of MDA-MB-231 invasive breast carcinoma cells. Indeed we showed that, following SDF-1α stimulation, DGKα is activated and localized at cell protrusion, thus promoting their elongation and mediating SDF-1α induced MMP-9 metalloproteinase secretion and matrix invasion. Phosphatidic acid generated by DGKα promotes localization at cell protrusions of atypical PKCs which play an essential role downstream of DGKα by promoting Rac-mediated protrusion elongation and localized recruitment of ß1 integrin and MMP-9. We finally demonstrate that activation of DGKα, atypical PKCs signaling and ß1 integrin are all essential for MDA-MB-231 invasiveness. These data indicates the existence of a SDF-1α induced DGKα - atypical PKC - ß1 integrin signaling pathway, which is essential for matrix invasion of carcinoma cells.

Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures.

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell-cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell-cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis.

Acylated and unacylated ghrelin impair skeletal muscle atrophy in mice.

Cachexia is a wasting syndrome associated with cancer, AIDS, multiple sclerosis, and several other disease states. It is characterized by weight loss, fatigue, loss of appetite, and skeletal muscle atrophy and is associated with poor patient prognosis, making it an important treatment target. Ghrelin is a peptide hormone that stimulates growth hormone (GH) release and positive energy balance through binding to the receptor GHSR-1a. Only acylated ghrelin (AG), but not the unacylated form (UnAG), can bind GHSR-1a; however, UnAG and AG share several GHSR-1a-independent biological activities. Here we investigated whether UnAG and AG could protect against skeletal muscle atrophy in a GHSR-1a-independent manner. We found that both AG and UnAG inhibited dexamethasone-induced skeletal muscle atrophy and atrogene expression through PI3Kß-, mTORC2-, and p38-mediated pathways in myotubes. Upregulation of circulating UnAG in mice impaired skeletal muscle atrophy induced by either fasting or denervation without stimulating muscle hypertrophy and GHSR-1a-mediated activation of the GH/IGF-1 axis. In Ghsr-deficient mice, both AG and UnAG induced phosphorylation of Akt in skeletal muscle and impaired fasting-induced atrophy. These results demonstrate that AG and UnAG act on a common, unidentified receptor to block skeletal muscle atrophy in a GH-independent manner.

SAP-mediated inhibition of diacylglycerol kinase α regulates TCR-induced diacylglycerol signaling.

Diacylglycerol kinases (DGKs) metabolize diacylglycerol to phosphatidic acid. In T lymphocytes, DGKα acts as a negative regulator of TCR signaling by decreasing diacylglycerol levels and inducing anergy. In this study, we show that upon costimulation of the TCR with CD28 or signaling lymphocyte activation molecule (SLAM), DGKα, but not DGKζ, exits from the nucleus and undergoes rapid negative regulation of its enzymatic activity. Inhibition of DGKα is dependent on the expression of SAP, an adaptor protein mutated in X-linked lymphoproliferative disease, which is essential for SLAM-mediated signaling and contributes to TCR/CD28-induced signaling and T cell activation. Accordingly, overexpression of SAP is sufficient to inhibit DGKα, whereas SAP mutants unable to bind either phospho-tyrosine residues or SH3 domain are ineffective. Moreover, phospholipase C activity and calcium, but not Src-family tyrosine kinases, are also required for negative regulation of DGKα. Finally, inhibition of DGKα in SAP-deficient cells partially rescues defective TCR/CD28 signaling, including Ras and ERK1/2 activation, protein kinase C membrane recruitment, induction of NF-AT transcriptional activity, and IL-2 production. Thus SAP-mediated inhibition of DGKα sustains diacylglycerol signaling, thereby regulating T cell activation, and it may represent a novel pharmacological strategy for X-linked lymphoproliferative disease treatment.

Diacylglycerol kinase α mediates 17-ß-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30.

Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-ß-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy.

Diacylglycerol kinases are essential for hepatocyte growth factor-dependent proliferation and motility of Kaposi's sarcoma cells.

Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi's sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways.

Diacylglycerol kinase alpha mediates HGF-induced Rac activation and membrane ruffling by regulating atypical PKC and RhoGDI.

Diacylglycerol kinases (DGKs) convert diacylglycerol (DAG) into phosphatidic acid (PA), acting as molecular switches between DAG- and PA-mediated signaling. We previously showed that Src-dependent activation and plasma membrane recruitment of DGKalpha are required for growth-factor-induced cell migration and ruffling, through the control of Rac small-GTPase activation and plasma membrane localization. Herein we unveil a signaling pathway through which DGKalpha coordinates the localization of Rac. We show that upon hepatocyte growth-factor stimulation, DGKalpha, by producing PA, provides a key signal to recruit atypical PKCzeta/iota (aPKCzeta/iota) in complex with RhoGDI and Rac at ruffling sites of colony-growing epithelial cells. Then, DGKalpha-dependent activation of aPKCzeta/iota mediates the release of Rac from the inhibitory complex with RhoGDI, allowing its activation and leading to formation of membrane ruffles, which constitute essential requirements for cell migration. These findings highlight DGKalpha as the central element of a lipid signaling pathway linking tyrosine kinase growth-factor receptors to regulation of aPKCs and RhoGDI, and providing a positional signal regulating Rac association to the plasma membrane.

8-Prenylnaringenin inhibits epidermal growth factor-induced MCF-7 breast cancer cell proliferation by targeting phosphatidylinositol-3-OH kinase activity.

8-Prenylnaringenin (8PN), one of the strongest plant-derived oestrogen receptors (ERs) ligand, has been suggested to have potential cancer chemo-preventive activities and anti-angiogenic properties. Because published data suggest that ERs serve as nodal point that allows interactions between hormones and growth factors mediated pathways, we decided to investigate the effects exerted by 8PN on Epidermal growth factor (EGF)-elicited pathways in breast cancer cells. Here we show that in ER positive MCF-7 cells, 8PN interferes with EGF induced cell proliferation by strongly inhibiting activation of PI(3)K/Akt pathway, without affecting EGFR expression or tyrosine phosphorylation, and exerting a synergistic activation of Erk1/2 phosphorylation. Moreover, we demonstrate that 8PN is a direct inhibitor of PI(3)K activity as it is shown by in vitro experiments with the purified enzyme and by its inability to impair serine phosphorylation of a constitutive active form of Akt. These findings suggest that inhibition of PI(3)K is a novel mechanism which contributes to 8PN activity to inhibit cancer cell survival and EGF induced proliferation.

Diacylglycerol kinase-alpha mediates hepatocyte growth factor-induced epithelial cell scatter by regulating Rac activation and membrane ruffling.

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.

Diacylglycerol kinase is required for HGF-induced invasiveness and anchorage-independent growth of MDA-MB-231 breast cancer cells.

BACKGROUND: Estrogen receptor (ER)-negative breast cancers have a worse prognosis than ER-positive cancers, being more aggressive and overexposed to stimuli leading to their progression. Hepatocyte growth factor (HGF) has been associated with proliferation, migration and invasion of tumor cells, and several tumors, including those of breast cancer, produce HGF and overexpress its receptor. Diacylglycerol kinases (Dgks), which phosphorylate diacylglycerol to phosphatidic acid, are key regulators of cell signaling. Our research was focused on their role in HGF-induced invasion of MDA-MB-231 cells, a model of ER-negative breast cancer. MATERIALS AND METHODS: Dgk activity was evaluated with a kinase assay, MDA-MB-231 cell invasion via culturing of cells in matrigel-coated transwells, and anchorage-independent growth was assessed using a soft agar assay. RESULTS: HGF induces Dgk activation in MDA-MB-231 cells that is required for cell invasiveness. Moreover, Dgks are involved in MDA-MB-231 anchorage-independent growth. CONCLUSION: Dgks could be a target for ER-negative breast cancer therapy.

Ghrelin and des-acyl ghrelin promote differentiation and fusion of C2C12 skeletal muscle cells.

Ghrelin is an acylated peptidyl gastric hormone acting on the pituitary and hypothalamus to stimulate appetite, adiposity, and growth hormone release, through activation of growth hormone secretagogue receptor (GHSR)-1a receptor. Moreover, ghrelin features several activities such as inhibition of apoptosis, regulation of differentiation, and stimulation or inhibition of proliferation of several cell types. Ghrelin acylation is absolutely required for both GHSR-1a binding and its central endocrine activities. However, the unacylated ghrelin form, des-acyl ghrelin, which does not bind GHSR-1a and is devoid of any endocrine activity, is far more abundant than ghrelin in plasma, and it shares with ghrelin some of its cellular activities. In here we show that both ghrelin and des-acyl ghrelin stimulate proliferating C2C12 skeletal myoblasts to differentiate and to fuse into multinucleated myotubes in vitro through activation of p38. Consistently, both ghrelin and des-acyl ghrelin inhibit C2C12 proliferation in growth medium. Moreover, the ectopic expression of ghrelin in C2C12 enhances differentiation and fusion of these myoblasts in differentiation medium. Finally, we show that C2C12 cells do not express GHSR-1a, but they do contain a common high-affinity binding site recognized by both acylated and des-acylated ghrelin, suggesting that the described activities on C2C12 are likely mediated by this novel, yet unidentified receptor for both ghrelin forms.