María V. Espelt

Novel roles of galectin-1 in hepatocellular carcinoma cell adhesion, polarization, and in vivo tumor growth.

Galectin‐1 (Gal‐1), a widely expressed β‐galactoside–binding protein, exerts pleiotropic biological functions. Gal‐1 is up‐regulated in hepatocarcinoma cells, although its role in liver pathophysiology remains uncertain. We investigated the effects of Gal‐1 on HepG2 hepatocellular carcinoma (HCC) cell adhesion and polarization. Soluble and immobilized recombinant Gal‐1 (rGal‐1) promoted HepG2 cell adhesion to uncoated plates and also increased adhesion to laminin. Antibody‐mediated blockade experiments revealed the involvement of different integrins as critical mediators of these biological effects. In addition, exposure to rGal‐1 markedly accelerated the development of apical bile canaliculi as shown by TRITC‐phalloidin labeling and immunostaining for multidrug resistance associated‐protein 2 (MRP2). Notably, rGal‐1 did not interfere with multidrug resistance protein 1/P‐glycoprotein or MRP2 apical localization, neither with transfer nor secretion of 5‐chloromethylfluorescein diacetate through MRP2. Stimulation of cell adhesion and polarization by rGal‐1 was abrogated in the presence of thiodigalactoside, a galectin‐specific sugar, suggesting the involvement of protein–carbohydrate interactions in these effects. Additionally, Gal‐1 effects were abrogated in the presence of wortmmanin, PD98059 or H89, suggesting involvement of phosphoinositide 3‐kinase (PI3K), mitogen‐activated protein kinase and cyclic adenosine monophosphate–dependent protein kinase signaling pathways in these functions. Finally, expression levels of this endogenous lectin correlated with HCC cell adhesion and polarization and up‐regulation of Gal‐1–favored growth of hepatocarcinoma in vivo. Conclusion: Our results provide the first evidence of a role of Gal‐1 in modulating HCC cell adhesion, polarization, and in vivo tumor growth, with critical implications in liver pathophysiology. (HEPATOLOGY 2011;)

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process. In comparison, cells from other vertebrates and from invertebrates received little attention, despite of the fact that e.g. fish cells could present rewarding model systems given the diversity in ecology and lifestyle of this animal group that may be reflected by an equal diversity of physiological adaptive mechanisms, including those related to cell volume regulation. In this review, we therefore present an overview on the most relevant aspects known on hypotonic volume regulation presently known in fish, summarizing transporters and signaling pathways described so far, and then focus on an aspect we have particularly studied over the past years using fish cell models, i.e. the role of extracellular nucleotides in mediating cell volume recovery of swollen cells. We, furthermore, present diverse modeling approaches developed on the basis of data derived from studies with fish and other models and discuss their potential use for gaining insight into the theoretical framework of volume regulation.

Identification of two distinct E-NTPDases in liver of goldfish (Carassius auratus L.)

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.

Kinetics of extracellular ATP in mastoparan 7-activated human erythrocytes

BACKGROUND The peptide mastoparan 7 (MST7) stimulated ATP release in human erythrocytes. We explored intra- and extracellular processes governing the time-dependent accumulation of extracellular ATP (i.e., ATPe kinetics). METHODS Human erythrocytes were treated with MST7 in the presence or absence of two blockers of pannexin 1. ATPe concentration was monitored by luciferin-luciferase based real-time luminometry. RESULTS Exposure of human erythrocytes to MST7 led to an acute increase in [ATPe], followed by a slower increase phase. ATPe kinetics reflected a strong activation of ATP efflux and a low rate of ATPe hydrolysis by ectoATPase activity. Enhancement of [ATPe] by MST7 required adhesion of erythrocytes to poly-D-lysin-coated coverslips, and correlated with a 31% increase of cAMP and 10% cell swelling. However, when MST7 was dissolved in a hyperosmotic medium to block cell swelling, ATPe accumulation was inhibited by 49%. Erythrocytes pre-exposure to 10μM of either carbenoxolone or probenecid, two blockers of pannexin 1, exhibited a partial reduction of ATP efflux. Erythrocytes from pannexin 1 knockout mice exhibited similar ATPe kinetics as those of wild type mice erythrocytes exposed to pannexin 1 blockers. CONCLUSIONS MST7 induced release of ATP required either cell adhesion or strong activation of cAMP synthesis. Part of this release required cell swelling. Kinetic analysis and a data driven model suggested that ATP efflux is mediated by two ATP conduits displaying different kinetics, with one conduit being fully blocked by pannexin 1 blockers. GENERAL SIGNIFICANCE Kinetic analysis of extracellular ATP accumulation from human erythrocytes and potential effects on microcirculation.

Volumetric response of vertebrate hepatocytes challenged by osmotic gradients: a theoretical approach.

In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513-522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl - whose kinetics could be successfully predicted - thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.

Galectin-1 Controls the Proliferation and Migration of Liver Sinusoidal Endothelial Cells and Their Interaction With Hepatocarcinoma Cells.

Galectin‐1 (Gal1), a β‐galactoside‐binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial‐mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness, and metastasis. During the early stages of HCC, transforming growth factor β1 (TGF‐β1) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF‐β1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF‐β1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH‐7 human HCC cells, and in SK‐HEP‐1 human liver sinusoidal endothelial cells (SECs). We used loss‐of‐function and gain‐of‐function experiments to down‐ or up‐regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK‐HEP‐1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch‐wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan‐dependent heterotypic adhesion of HepG2 cells to SK‐HEP‐1 SECs. Furthermore, TGF‐β1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK‐HEP‐1 SECs through a Gal1‐dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF‐β1‐induced growth inhibition. Our results suggest that Gal1 and TGF‐β1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis, and angiogenesis. J. Cell. Physiol. 231: 1522–1533, 2016.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell: Metabolic control analysis and the glycolytic pathway

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde‐3‐phosphate‐dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low‐cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi‐well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course.