Tigilanol Tiglate-Induced Changes in Secretome Profiles Alter C-Met Phosphorylation and Cell Surface Protein Expression in H357 Head and Neck Cancer Cells.

Tigilanol tiglate (TT, also known as EBC-46) is a novel, plant-derived diterpene ester possessing anticancer and wound-healing properties. Here, we show that TT-evoked PKC-dependent S985 phosphorylation of the tyrosine kinase MET leads to subsequent degradation of tyrosine phosphorylated p-Y1003 and p-Y1234/5 MET species. PKC inhibition with BIM-1 blocked S985 phosphorylation of MET and led to MET cell surface accumulation. Treatment with metalloproteinase inhibitors prevented MET-ECD release into cell culture media, which was also blocked by PKC inhibitors. Furthermore, unbiased secretome analysis, performed using TMT-technology, identified additional targets of TT-dependent release of cell surface proteins from H357 head and neck cancer cells. We confirm that the MET co-signalling receptor syndecan-1 was cleaved from the cell surface in response to TT treatment. This was accompanied by rapid cleavage of the cellular junction adhesion protein Nectin-1 and the nerve growth factor receptor NGFRp75/TNFR16. These findings, that TT is a novel negative regulator of protumorigenic c-MET and NGFRp75/TNFR16 signalling, as well as regulating Nectin-1-mediated cell adhesion, further contribute to our understanding of the mode of action and efficacy of TT in the treatment of solid tumours.

Intraphagosomal Free Ca2+ Changes during Phagocytosis.

Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena.

Mesenchymal Transglutaminase 2 Activates Epithelial ADAM17: Link to G-Protein-Coupled Receptor 56 (ADGRG1) Signalling.

A wound healing model was developed to elucidate the role of mesenchymal-matrix-associated transglutaminase 2 (TG2) in keratinocyte re-epithelialisation. TG2 drives keratinocyte migratory responses by activation of disintegrin and metalloproteinase 17 (ADAM17). We demonstrate that epidermal growth factor (EGF) receptor ligand shedding leads to EGFR-transactivation and subsequent rapid keratinocyte migration on TG2-positive ECM. In contrast, keratinocyte migration was impaired in TG2 null conditions. We show that keratinocytes express the adhesion G-protein-coupled receptor, ADGRG1 (GPR56), which has been proposed as a TG2 receptor. Using ADAM17 activation as a readout and luciferase reporter assays, we demonstrate that TG2 activates GPR56. GPR56 activation by TG2 reached the same level as observed with an agonistic N-GPR56 antibody. The N-terminal GPR56 domain is required for TG2-regulated signalling response, as the constitutively active C-GPR56 receptor was not activated by TG2. Signalling required the C-terminal TG2 ß-barrel domains and involved RhoA-associated protein kinase (ROCK) and ADAM17 activation, which was blocked by specific inhibitors. Cell surface binding of TG2 to the N-terminal GPR56 domain is rapid and is associated with TG2 and GPR56 endocytosis. TG2 and GPR56 represent a ligand receptor pair causing RhoA and EGFR transactivation. Furthermore, we determined a binding constant for the interaction of human TG2 with N-GPR56 and show for the first time that only the calcium-enabled "open" TG2 conformation associates with N-GPR56.

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils.

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca2+ signal in osmotically shrunk cells (by IP3 uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca2+ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.

Membrane Tension and the Role of Ezrin During Phagocytosis.

During phagocytosis, there is an apparent expansion of the plasma membrane to accommodate the target within a phagosome. This is accompanied (or driven by) a change in membrane tension. It is proposed that the wrinkled topography of the phagocyte surface, by un-wrinkling, provides the additional available membrane and that this explains the changes in membrane tension. There is no agreement as to the mechanism by which unfolding of cell surface wrinkles occurs during phagocytosis, but there is a good case building for the involvement of the actin-plasma membrane crosslinking protein ezrin. Not only have direct measurements of membrane tension strongly implicated ezrin as the key component in establishing membrane tension, but the cortical location of ezrin changes at the phagocytic cup, suggesting that it is locally signalled. This chapter therefore attempts to synthesise our current state of knowledge about ezrin and membrane tension with phagocytosis to provide a coherent hypothesis.

Calpain Activation by Ca2+ and Its Role in Phagocytosis.

Although the cytosolic Ca2+ signalling event in phagocytosis is well established, and the mechanism for generating such signals also understood, the target for the Ca2+ signal and how this relates to the phagocytic outcome is less clear. In this chapter, we present the evidence for a role of the Ca2+ activated protease, calpain, in phagocytosis. The abundant evidence for Ca2+ changes and calpain activation during cell shape changes is extended to include the specific cell shape change which accompanies phagocytosis. The discussion therefore includes a brief description of the domain structure of calpain and their functions. Also the mechanism by which calpain activation is limited at the cell periphery subdomains, and how this would allow phagocytic pseudopodia to form locally.

Microinjection and Micropipette-Controlled Phagocytosis Methods for Neutrophils.

The ability to microinject substances into the cytosol of living neutrophils opens the possibility of manipulating the chemistry within the cell and also of monitoring changes using indicators which otherwise cannot be introduced into the cell. However, neutrophils cannot be microinjected by the conventional glass pipette insertion method. Here we outline two techniques which work well with neutrophils, namely, SLAM (Simple Lipid-Assisted Microinjection) and electromicroinjection. As these methods utilize micropipettes, we also include a simple method which uses a micropipette to deliver a phagocytic stimulus to a specific cell at a defined time, enable detailed study of the phagocytic process from particle contact to particle internalization.

Optical Methods for the Measurement and Manipulation of Cytosolic Calcium Signals in Neutrophils.

The measurement and manipulation of cytosolic free Ca2+ of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca2+ signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils, and are discussed here, especially the use of fluorescent indicators of Ca2+ and photoactivation of reagents involved in Ca2+ signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we outline some of the techniques that have been used to monitor, visualize, and manipulate Ca2+ in neutrophils.

Topographical interrogation of the living cell surface reveals its role in rapid cell shape changes during phagocytosis and spreading.

Dramatic and rapid changes in cell shape are perhaps best exemplified by phagocytes, such as neutrophils. These cells complete the processes of spreading onto surfaces, and phagocytosis within 100 s of stimulation. Although these cell shape changes are accompanied by an apparent large increase in cell surface area, the nature of the membrane "reservoir" for the additional area is unclear. One proposal is that the wrinkled cell surface topography (which forms micro-ridges on the neutrophil surface) provides the resource for neutrophils to expand their available surface area. However, it has been problematic to test this proposal in living cells because these surface structures are sub-light microscopic. In this paper, we report the development of a novel approach, a variant of FRAP (fluorescent recovery after photo-bleaching) modified to interrogate the diffusion path-lengths of membrane associated molecules. This approach provides clear evidence that the cell surface topography changes dramatically during neutrophil shape change (both locally and globally) and can be triggered by elevating cytosolic Ca2+.

P2X7 receptor activation regulates rapid unconventional export of transglutaminase-2.

Transglutaminases (denoted TG or TGM) are externalized from cells via an unknown unconventional secretory pathway. Here, we show for the first time that purinergic signaling regulates active secretion of TG2 (also known as TGM2), an enzyme with a pivotal role in stabilizing extracellular matrices and modulating cell-matrix interactions in tissue repair. Extracellular ATP promotes TG2 secretion by macrophages, and this can be blocked by a selective antagonist against the purinergic receptor P2X7 (P2X7R, also known as P2RX7). Introduction of functional P2X7R into HEK293 cells is sufficient to confer rapid, regulated TG2 export. By employing pharmacological agents, TG2 release could be separated from P2X7R-mediated microvesicle shedding. Neither Ca(2+) signaling alone nor membrane depolarization triggered TG2 secretion, which occurred only upon receptor membrane pore formation and without pannexin channel involvement. A gain-of-function mutation in P2X7R associated with autoimmune disease caused enhanced TG2 externalization from cells, and this correlated with increased pore activity. These results provide a mechanistic explanation for a link between active TG2 secretion and inflammatory responses, and aberrant enhanced TG2 activity in certain autoimmune conditions.

Differential regulation of TROP2 release by PKC isoforms through vesicles and ADAM17.

TROP2, a cancer cell surface protein with both pro-oncogenic and anti-oncogenic properties is cleaved by ADAM17. ADAM17 dependent cleavage requires novel PKC activity which is blocked by the ADAM10/ADAM17 inhibitor GW64 as well as by the PKC inhibitor Bim-1. Full length TROP2 release is induced by classical PKC activation and blocked by Gö6979, without affecting ADAM17 dependent TROP2 cleavage. Full length TROP2 is released in ectosomes, as inhibition of endocytosis did not prevent release. Inhibition of the atypical PKC isoform PKCζ stimulated metalloproteinase dependent N-terminal alternative TROP2 cleavage. The resulting alternative TROP2 cleavage product remains membrane associated via a disulphide bond, but is released in microvesicles with an average size of 107nm. Inhibition of endocytosis following PKCζ inhibition prevented alternative cleavage and release of TROP2, suggesting that these events require endocytic uptake and exosomal release of the corresponding microvesicles. The alternative TROP2 cleavage product was also found in PC3 cell lysates following deglycosylation, and may represent a novel biomarker in prostate cancer.

Minimal impact electro-injection of cells undergoing dynamic shape change reveals calpain activation.

The ability of neutrophils to rapidly change shape underlies their physiological functions of phagocytosis and spreading. A major problem in establishing the mechanism is that conventional microinjection of substances and indicators interferes with this dynamic cell behaviour. Here we show that electroinjection, a "no-touch" point-and-shoot means of introducing material into the cell, is sufficiently gentle to allow neutrophils to be injected whilst undergoing chemokinesis and spreading without disturbing cell shape change behaviour. Using this approach, a fluorogenic calpain-1 selective peptide substrate was introduced into the cytosol of individual neutrophils undergoing shape changes. These data showed that (i) physiologically elevated cytosolic Ca(2+) concentrations were sufficient to trigger calpain-1 activation, blockade of Ca(2+) influx preventing calpain activation and (ii) calpain-1 activity was elevated in spreading neutrophil. These findings provide the first direct demonstration of a physiological role for Ca(2+) elevation in calpain-1 activation and rapid cell spreading. Electroinjection of cells undergoing dynamic shape changes thus opens new avenues of investigation for defining the molecular mechanism underlying dynamic cell shape changes.

Optical methods for the measurement and manipulation of cytosolic calcium signals in neutrophils.

The measurement and manipulation of cytosolic free Ca(2+) of neutrophils is crucial for investigating the mechanisms within living neutrophils which generate Ca(2+) signals and the cellular responses triggered by them. Optical methods for this are the most applicable for neutrophils and are discussed here, especially the use of fluorescent indicators of Ca(2+) and photoactivation of reagents involved in Ca(2+) signaling. Both of these synthetic agents can be loaded into neutrophils as lipid-soluble esters or can be microinjected into the cell. In this chapter, we will outline some of the techniques that have been used to monitor, visualize, and manipulate Ca(2+) in neutrophils.

Ca²âº and calpain control membrane expansion during the rapid cell spreading of neutrophils.

Following adherence of neutrophils to the endothelium, neutrophils undergo a major morphological change that is a necessary prelude to their extravasation. We show here that this shape change is triggered by an elevation of cytosolic inositol (1,4,5)-trisphosphate (IP3), to provoke physiological Ca(2+) influx through a store-operated mechanism. This transition from a spherical to 'flattened' neutrophil morphology is rapid (∼100 seconds) and is accompanied by an apparent rapid expansion of the area of the plasma membrane. However, no new membrane is added into the plasma membrane. Pharmacological inhibition of calpain-activation, which is triggered by Ca(2+) influx during neutrophil spreading, prevents normal cell flattening. In calpain-suppressed cells, an aberrant form of cell spreading can occur where an uncoordinated and localised expansion of the plasma membrane is evident. These data show that rapid neutrophil spreading is triggered by Ca(2+) influx, which causes activation of calpain and release of furled plasma membrane to allow its apparent 'expansion'.

In vivo functional analysis and genetic modification of in vitro-derived mouse neutrophils.

Mature neutrophils are notoriously short-lived immune cells that cannot be genetically manipulated. Analysis of gene function therefore requires genetically modified animals, which is expensive, time-consuming, and costly in animal life. Analysis of gene function in neutrophils in a physiologically relevant context thus represents a significant problem in the field. We sought to overcome this obstruction in the field by developing a strategy for the analysis of gene function in neutrophils in a physiologically relevant context. Here, we demonstrate the functional relevance of in vitro conditional-Hoxb8 immortalized precursor-derived neutrophils. In vitro-derived neutrophils functionally resembled primary neutrophils, but critically, neutrophils generated in this way can be adoptively transferred into live animals and tracked during inflammatory responses using single-cell analysis to define functional attributes. We have validated this approach using CD11b-deficient neutrophils and replicated the key findings observed in gene-targeted animals and in naturally CD11b-deficient humans. Furthermore, we show that by retroviral transduction, one can generate stable alterations in the precursor cell lines and thus a continuous supply of functionally altered neutrophils. This novel technological advance offers for the first time the possibility of applying higher-throughput genetic modification and in vivo functional analysis to the neutrophil-lineage.

A reporter of UV intensity delivered to the cytosol during photolytic uncaging.

Photolytic uncaging of biologically-active molecules within cells is a powerful technique. However, the delivery of uncaging light into the cytosol can vary between cell types, individual cells of the same type, and different loci within an individual cell because of optical differences in absorbance and light-scattering properties of the cytoplasm. Here, we demonstrate a simple technique for monitoring the magnitude of cytosolic ultraviolet delivery during uncaging, which also leaves a quantitative and persistent record of this within the cells. The simple method shown here provides a much needed universal monitor of the delivery of ultraviolet light to molecules within the cytosol, providing a much needed parameter for the correct interpretation of uncaging experiments.

Chronic inhibition of nitric-oxide synthase induces hypertension and erectile dysfunction in the rat that is not reversed by sildenafil.

STUDY TYPE: Aetiology (case control) Level of Evidence 3b OBJECTIVE To evaluate the effect of N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertension (HT) on erectile function in the rat and determine if the phosphodiesterase (PDE)-5 inhibitor, sildenafil, can reverse the effects of nitric oxide (NO) deficiency, as HT is a risk factor for erectile dysfunction (ED) and the NO synthase (NOS) inhibitor L-NAME induces NO-deficient HT. MATERIALS AND METHODS: Thirty-six adult Sprague-Dawley male rats were divided into three groups, i.e. a control, L-NAME-HT (40 mg/rat/day in the drinking water for 4 weeks), and sildenafil-treated L-NAME-HT (1.5 mg/rat/day sildenafil, by oral gavage concomitantly with L-NAME). The erectile response expressed as a ratio of intracavernosal pressure (ICP)/mean arterial pressure (MAP), evaluated after electrical stimulation of the right cavernous nerve. The isometric tension of corpus cavernosum smooth muscle (CCSM) was measured in organ-bath experiments. NOS expression was determined immunohistochemically for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. cGMP levels were evaluated by enzyme-linked immunosorbent assay. RESULTS: The erectile response was diminished in the HT group. Nitrergic and endothelium-dependent relaxation was reduced, while the relaxation response to sodium nitroprusside and contractile response to phenylephrine were not altered in CCSM from L-NAME-treated rats. HT rats showed decreased expression of nNOS, whereas eNOS and iNOS protein expression was increased. Sildenafil partly restored endothelial and molecular changes in CCSM from HT rats, but did not reverse the decreased erectile response, even as cGMP levels returned to normal levels. CONCLUSIONS: Sildenafil treatment did not correct the ED in L-NAME-treated HT rats. Under sustained high blood pressure, up-regulation of PDE5 expression failed to reverse the depletion of neuronal NO and/or impaired nNOS activity. However, endothelium-dependent relaxation was restored. Drug targeting of neuronal dysfunction might delay the onset of ED in HT.

Translocation or just location? Pseudopodia affect fluorescent signals.

The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

Ironing out the wrinkles of neutrophil phagocytosis.

Though phagocytosis of microbes by professional phagocytes such as neutrophils is crucial for the survival of the host, it is still unclear how the apparent 'stretching' of the plasma membrane is achieved. Microscopically, pseudopod extension, particulate engulfment and phagosome closure all require seemingly large expansions of the cell surface area. Although actual membrane stretching can be ruled out on the basis of physical properties of lipid bilayers, the addition of new membrane from within the cell, either by exocytosis or phagosomal fusion with endoplasmic reticulum membrane, might provide an explanation. However, these events do not seem to have major roles during phagocytosis by neutrophils. Instead, neutrophils might use a more primitive mechanism, that is, the unfolding of surface membrane wrinkles, to provide the additional membrane for phagocytosis. Here, we briefly discuss why membrane unwrinkling provides a feasible hypothesis for membrane expansion during neutrophil phagocytosis, and suggest a potential molecular mechanism for neutrophil control over membrane surface wrinkles, and the potential signalling route.