Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

BACKGROUND: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. METHODS: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3',5,5'-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. RESULTS: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-ß1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. CONCLUSIONS: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-ß expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

Thyroid hormone inhibition in L6 myoblasts of IGF-I-mediated glucose uptake and proliferation: new roles for integrin αvß3.

Thyroid hormones L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvß3. Also insulin-like growth factor type I (IGF-I) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T4 and IGF-I in rat L6 myoblasts, we have examined integrin αvß3-mediated modulatory actions of T4 on glucose uptake, measured through carrier-mediated 2-deoxy-[3H]-D-glucose uptake, and on cell proliferation stimulated by IGF-I, assessed by cell counting, [3H]-thymidine incorporation, and fluorescence-activated cell sorting analysis. IGF-I stimulated glucose transport and cell proliferation via the cell surface IGF-I receptor (IGFIR) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T4 caused little or no cell proliferation but prevented both glucose uptake and proliferative actions of IGF-I. These actions of T4 were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGFIR and the T4 receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvß3, and the T4 metabolite tetraiodothyroacetic acid all blocked the inhibition by T4 of IGF-I-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-I and also showed a role for ERK1/2 in the actions of T4 that modified the effects of IGF-I. We conclude that thyroid hormone inhibits IGF-I-stimulated glucose uptake and cell proliferation in L6 myoblasts.

Extranuclear effects of thyroid hormones and analogs during development: An old mechanism with emerging roles.

Thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvß3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvß3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na+-dependent transport systems, such as glucose uptake, Na+/K+-ATPase, Na+/H+ exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na+/K+-ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

Inhibition by Thyroid Hormones of Cell Migration Activated by IGF-1 and MCP-1 in THP-1 Monocytes: Focus on Signal Transduction Events Proximal to Integrin αvß3.

Interaction between thyroid hormones and the immune system is reported in the literature. Thyroid hormones, thyroxine, T4, but also T3, act non-genomically through mechanisms that involve a plasma membrane receptor αvß3 integrin, a co-receptor for insulin-like growth factor-1 (IGF-1). Previous data from our laboratory show a crosstalk between thyroid hormones and IGF-1 because thyroid hormones inhibit the IGF-1-stimulated glucose uptake and cell proliferation in L-6 myoblasts, and the effects are mediated by integrin αvß3. IGF-1 also behaves as a chemokine, being an important factor for tissue regeneration after damage. In the present study, using THP-1 human leukemic monocytes, expressing αvß3 integrin in their cell membrane, we focused on the crosstalk between thyroid hormones and either IGF-1 or monocyte chemoattractant protein-1 (MCP-1), studying cell migration and proliferation stimulated by the two chemokines, and the role of αvß3 integrin, using inhibitors of αvß3 integrin and downstream pathways. Our results show that IGF-1 is a potent chemoattractant in THP-1 monocytes, stimulating cell migration, and thyroid hormone inhibits the effect through αvß3 integrin. Thyroid hormone also inhibits IGF-1-stimulated cell proliferation through αvß3 integrin, an example of a crosstalk between genomic and non-genomic effects. We also studied the effects of thyroid hormone on cell migration and proliferation induced by MCP-1, together with the pathways involved, by a pharmacological approach and docking simulation. Our findings show a different downstream signaling for IGF-1 and MCP-1 in THP-1 monocytes mediated by the plasma membrane receptor of thyroid hormones, integrin αvß3.

The Role of Thyroid Hormones in Hepatocyte Proliferation and Liver Cancer.

Thyroid hormones T3 and T4 (thyroxine) control a wide variety of effects related to development, differentiation, growth and metabolism, through their interaction with nuclear receptors. But thyroid hormones also produce non-genomic effects that typically start at the plasma membrane and are mediated mainly by integrin αvß3, although other receptors such as TRα and TRß are also able to elicit non-genomic responses. In the liver, the effects of thyroid hormones appear to be particularly important. The liver is able to regenerate, but it is subject to pathologies that may lead to cancer, such as fibrosis, cirrhosis, and non-alcoholic fatty liver disease. In addition, cancer cells undergo a reprogramming of their metabolism, resulting in drastic changes such as aerobic glycolysis instead of oxidative phosphorylation. As a consequence, the pyruvate kinase isoform M2, the rate-limiting enzyme of glycolysis, is dysregulated, and this is considered an important factor in tumorigenesis. Redox equilibrium is also important, in fact cancer cells give rise to the production of more reactive oxygen species (ROS) than normal cells. This increase may favor the survival and propagation of cancer cells. We evaluate the possible mechanisms involving the plasma membrane receptor integrin αvß3 that may lead to cancer progression. Studying diseases that affect the liver and their experimental models may help to unravel the cellular pathways mediated by integrin αvß3 that can lead to liver cancer. Inhibitors of integrin αvß3 might represent a future therapeutic tool against liver cancer. We also include information on the possible role of exosomes in liver cancer, as well as on recent strategies such as organoids and spheroids, which may provide a new tool for research, drug discovery, and personalized medicine.

Involvement of plasma membrane redox systems in hormone action.

Reactive oxygen species (ROS) is the common name used to describe the partially reduced forms of molecular oxygen that may be generated in cells during oxidative metabolism. They are normally considered to be toxic, and cells possess various defence systems to protect themselves including antioxidant enzymes and low molecular weight antioxidants like vitamin C and vitamin E. However, it is now clear that small amounts of ROS also act as messenger molecules in cell signal transduction pathways; the plasma membrane of eukaryotic cells in particular contains a variety of different ROS-producing oxidases and reductases, of which the best characterized are the superoxide-producing NADPH oxidases. It has been known for many years that membrane redox activity can be changed rapidly by various hormones and growth factors, but the molecular mechanisms involved and the physiological importance of this phenomenon have only recently begun to be unveiled. This review summarizes the state of the art on plasma membrane-based ROS signalling in the pathways of insulin, steroid and thyroid hormones and growth factors. The apparent paradox of ROS being essential biomolecules in the regulation of cellular functions, but also toxic by-products of metabolism, may be important for the pharmacological application of natural and synthetic antioxidants.

Antioxidant activity of 4-methylcoumarins.

Polyphenolic coumarins are known to act as antioxidants in biological systems, but it is difficult to distinguish their antioxidant activity from the many other effects they produce in cells. We have determined the radical scavenging capacity of 22 structurally related natural and synthetic 4-methylcoumarins, by measuring their reaction with radicals, galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl, using electron paramagnetic resonance spectroscopy. Efficient antioxidant activity of 4-methylcoumarins in cells was verified using the DCF fluorescent probe assay for determination of intracellular reactive oxygen species levels. As expected, the o-dihydroxysubstituted coumarins were found to be excellent radical scavengers and better than the m-dihydroxysubstituted or monohydroxysubstituted analogues, but surprisingly the corresponding o-diacetoxy derivatives also turned out to be good scavengers, even in the absence of an esterase. Another unexpected result was that the antioxidant efficiency of 4-methylcoumarins could be modulated by introducing an ethoxycarbonylethyl substituent at the C-3 position; this effect cannot be explained by simple electron donating/withdrawing properties. Coumarin concentrations of 10 microM or less were used in all experiments, corresponding to the levels relevant for therapeutic purposes. Considering that 4-methylcoumarins, in contrast to many other coumarins, are not metabolized to toxic epoxide intermediates, these results indicate promising new strategies for the design of non-toxic antioxidant coumarin-based drugs.

Decrease of polyamine levels and enhancement of transglutaminase activity in selective reduction of B16-F10 melanoma cell proliferation induced by atrial natriuretic peptide.

The atrial natriuretic peptide (ANP) at physiological levels reduced the proliferation of highly metastatic murine (B16-F10) and human (SK-MEL 110) melanoma cell lines whereas rat aortic smooth muscle (RASM) cells were unaffected. In RASM cells, the levels of proliferation markers (putrescine, spermidine and spermine) increase after 24 h of epidermal growth factor (EGF) stimulation (RASM-EGF), but strongly decrease after 24 h of exposition to ANP. The B16-F10 cell line, which received no EGF stimulation, showed a similar decrease in polyamine content after ANP treatment. Furthermore, the enzymatic activity of a differentiation marker (transglutaminase) was increased for both RASM-EGF and B16-F10 cells after 24 h of treatment with 10(-10) mol/l ANP, concomitantly with the observed inhibition of polyamine biosynthesis and cell growth. Data obtained on B16-F10 cells treated with 8Br-GMPc or with an ANP analogue (cANF) support the involvement of the type C ANP receptor (NRP-C) in hormone effects. From the overall results, it appears that ANP may play a role in the inhibition of cellular growth under hyperproliferative conditions, as shown for RASM-EGF cells. The B16-F10 melanoma cell line showed similar results, but in the absence of mitogen stimulation. This observation suggests that the constitutive hyperproliferative state of tumor cells may be a sufficient condition to favor the ANP inhibitory effects on cell growth. This finding is particularly interesting in the light of a possible use of ANP as a potential selective antineoplastic agent.

Short-term effects of thyroid hormones and 3,5-diiodothyronine on membrane transport systems in chick embryo hepatocytes.

Rapid nongenomic effects of thyroid hormones L-T(3) and L-T(4) on two plasma membrane transport systems were investigated in 14-d-old and 19-d-old chick embryo hepatocytes. The Na(+)/H(+) exchanger activity was measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, whereas the amino acid transport was estimated by [1-(14)C]-2-aminoisobutyric acid uptake. System A amino acid transport activation was linear to hormone concentration, whereas the Na/H exchanger gave a bell-shaped dose-response curve, with a maximum at the physiological hormone concentration of 1 nM. The specificity of the effect was verified by the use of inhibitors and analogues. The thyroid hormone analog 3,5-diiodo-L-thyronine was able to mimic some of the hormone effects, but with a lower efficiency. The effect on the Na(+)/H(+) exchanger was identified for 14-d-old and 19-d-old cells, whereas the amino acid transport could only be activated at the late stage of embryo development. Both transport systems were activated through a signal transduction pathway involving PKC, MAPK pathway, and PI3K, even though the differences in response behavior indicate a differential modulation of the two transport systems by L-T(3) and L-T(4). These results clearly demonstrate the existence of rapid nongenomic action of thyroid hormones also in avian cells, and show that activation of System A amino acid transport is not directly correlated to changes in intracellular pH. For the first time, evidence is presented which suggests that short-term effects of thyroid hormones may play a role during fetal development and cell differentiation.

Atrial natriuretic peptide: an old hormone or a new cytokine?

Atrial natriuretic peptide (ANP) a cardiovascular hormone mainly secreted by heart atria in response to stretching forces induces potent diuretic, natriuretic and vasorelaxant effects and plays a major role in the homeostasis of blood pressure as well as of water and salt balance. The hormone can also act as autocrine/paracrine factor and modulate several immune functions as well as cytoprotective effects. ANP contributes to innate immunity being able to: (i) stimulate the host defense against extracellular microbes by phagocytosis and Reactive Oxygen Species (ROS) release; (ii) inhibit the synthesis and release of proinflammatory markers such as TNF-α, IL-1, MCP-1, nitric oxide (NO), cyclooxygenase-2 (COX-2); (iii) inhibit the expression of adhesion molecules such as ICAM-1 and E-selectin. ANP can also affect the adaptive immunity being able to: (i) reduce the number of CD4(+) CD8(+) lymphocytes as well as to increase the CD4(-) CD8(-) cells; (ii) stimulate the differentiation of naïve CD4(+) cells toward the Th2 and/or Th17 phenotype. The hormone shows protective effects during: (i) ventricular hypertrophy and myocardial injury; (ii) atherosclerosis and hypertension by the induction of antiproliferative effects; (iii) oxidative stress counteracting the dangerous effects of ROS; (iv) growth of tumors cells by the induction of apoptosis or necrosis. Since not much is known about of the role of ANP locally produced and released by non-cardiac cells, this review outlines the contribution of ANP in different aspect of innate as well as adaptive immunity also with respect to the excessive cell growth in physiological and/or pathological conditions.

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: role in cell growth, migration and cytokine release.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H2O2)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H2O2-dependent ROS production was induced by physiological concentration of ANP (10(-10)M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H2O2-induced cell migration was observed after ANP (10(-10)M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H2O2-induced release of IL-9, TNF-α, MIP-1α and MIP-1ß was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1ß. Our results support the view that ANP can play a key role during the inflammatory process.

Protection of cells against oxidative stress by nanomolar levels of hydroxyflavones indicates a new type of intracellular antioxidant mechanism.

Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 µM cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.

Nongenomic effects of thyroid hormones on the immune system cells: New targets, old players.

It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvß3 has been identified as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors. Through the integrin αvß3 receptor the hormone can activate both the ERK1/2 and phosphatidylinositol 3-kinase pathways, with downstream effects including intracellular protein trafficking, angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New results demonstrate the capability of T(3) or T(4) to induce in the short time range important responses related to the immune function, such as reactive oxygen species production and cell migration in THP-1 monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of rapid nongenomic responses interacting with the classical nuclear response.

Thyroid hormones as modulators of immune activities at the cellular level.

BACKGROUND: Increasing evidence suggests that thyroid hormones, L-thyroxine (T(4)) and 3,3',5-triiodo-L-thyronine (T(3)), are modulators of the immune response. In monocytes, macrophages, leukocytes, natural killer cells, and lymphocytes, a wide range of immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species (ROS), and cytokine synthesis and release are altered under hypo- and hyperthyroid conditions. SUMMARY: Hyperthyroidism decreases the proinflammatory activities of monocytes and macrophages, whereas enhancement of phagocytosis and increased levels of ROS may occur during hypothyroidism. The expression of proinflammatory molecules such as macrophage inflammatory protein-1α and interleukin-1ß increases in hypothyroidism. However, in Kupffer cells, proinflammatory activities such as the respiratory burst, nitric oxide synthase activity, and tumor necrosis factor-α expression may result from increased T(3) levels. Thyroid hormones also affect natural killer cell activity and cell-mediated immune responses. Still, for many immune cells no clear correlation has been found so far between abnormally high or low T(3) or T(4) levels and the effects observed on the immune responses. CONCLUSIONS: In this review we outline the contributions of thyroid hormones to different aspects of innate and adaptive immune responses. The relationship between thyroid hormones and immune cells is complex and T(3) and T(4) may modulate immune responses through both genomic and nongenomic mechanisms. Future studies of the molecular signaling mechanisms involved in this cross-talk between thyroid hormones and the immune system may support development of new strategies to improve clinical immune responses.

Atrial natriuretic peptide and oxidative stress.

Atrial natriuretic peptide (ANP) is a hormone, produced mainly by cardiomyocytes, with a major role in cardiovascular homeostatic mechanisms such as natriuresis and vasodilation, which serve to regulate blood pressure. However, ANP also acts as an autocrine/paracrine factor on other targets such as kidney, lung, thymus, liver and the immune system. ANP participates in the regulation of cell growth and proliferation, and evidence is accumulating that these effects are associated with the generation of reactive oxygen species (ROS). In vascular cells and cardiomyocytes ANP stimulates the antioxidant defense, but in other systems such as hepatoblastoma and macrophages ANP may produce either antioxidant or prooxidant effects, depending on experimental conditions and cell context. At present very little is known on the relationship between ANP and ROS production in the normal homeostatic processes or during the development of cardiovascular diseases and cancer. Our current knowledge of the role of ANP in signaling pathways leading to the generation of intracellular messengers such as diacylglycerol (DAG), and guanosine 3'-5'-cyclic monophosphate has been examined in order to clarify the mechanisms by which the hormone may counteract or contribute to the potentially dangerous effects of free radicals.

Short-term effects of thyroid hormones during development: Focus on signal transduction.

Extranuclear or nongenomic effects of thyroid hormones are mediated by receptors located at the plasma membrane or inside cells, and are independent of protein synthesis. Recently the alphaVbeta3 integrin was identified as a cell membrane receptor for thyroid hormones, and a wide variety of nongenomic effects have now been shown to be induced through binding of thyroid hormones to this receptor. However, also other thyroid hormone receptors can produce nongenomic effects, including the cytoplasmic TRalpha and TRbeta receptors and probably also a G protein-coupled membrane receptor, and increasing importance is now given to thyroid hormone metabolites like 3,5-diiodothyronine and reverse T(3) that can mimick some nongenomic effects of T(3) and T(4). Signal transduction from the alphaVbeta3 integrin may proceed through at least three independent pathways (protein kinase C, Src or mitogen-activated kinases) but the details are still unknown. Thyroid hormones induce nongenomic effects on at least three important Na(+)-dependent transport systems, the Na(+)/K(+)-ATPase, the Na(+)/H(+) exchanger, and amino acid transport System A, leading to a mitogenic response in embryo cells; but modulation of the same transport systems may have different roles in other cells and at different developmental stages. It seems that thyroid hormones in many cases can modulate nongenomically the same targets affected by the nuclear receptors through long-term mechanisms. Recent results on nongenomic effects confirm the old theory that the primary role of thyroid hormones is to keep the steady-state level of functioning of the cell, but more and more mechanisms are discovered by which this goal can be achieved.

The sodium/hydrogen exchanger: a possible mediator of immunity.

Immune cells such as macrophages and neutrophils provide the first line of defence of the immune system using phagocytosis, cytokine and chemokine synthesis and release, as well as Reactive Oxygen Species (ROS) generation. Many of these functions are positively coupled with cytoplasmic pH (pHi) and/or phagosomal pH (pHp) modification; an increase in pHi represents an important signal for cytokine and chemokine release, whereas a decrease in pHp can induce an efficient antigen presentation. However, the relationship between pHi and ROS generation is not well understood. In immune cells two main transport systems have been shown to regulate pHi: the Na+/H+ Exchanger (NHE) and the plasmalemmal V-type H+ ATPase. NHE is a family of proteins which exchange Na+ for H+ according to their concentration gradients in an electroneutral manner. The exchanger also plays a key role in several other cellular functions including proliferation, differentiation, apoptosis, migration, and cytoskeletal organization. Since not much is known on the relationship between NHE and immunity, this review outlines the contribution of NHE to different aspects of innate and adaptive immune responses such as phagosomal acidification, NADPH oxidase activation and ROS generation, cytokine and chemokine release as well as T cell apoptosis. The possibility that several pro-inflammatory diseases may be modulated by NHE activity is evaluated.

Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation.

Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin promoting migration, proliferation, and survival in a wide spectrum of cells, can also modulate different biological responses in stem cells, but the mechanisms involved are not completely understood so far. In this context, we show that short-term exposure of mesenchymal stem cells (MSCs) to HGF can induce the activation of its cognate Met receptor and the downstream effectors ERK1/2, p38MAPK, and PI3K/Akt, while long-term exposure to HGF resulted in cytoskeletal rearrangement, cell migration, and marked inhibition of proliferation through the arrest in the G1-S checkpoint. When added to MSCs, the K252A tyrosine kinase inhibitor prevented HGF-induced responses. HGF's effect on MSC proliferation was reversed by p38 inhibitor SB203580, while the effects on cell migration were abrogated by PI3K inhibitor Wortmannin, suggesting that HGF acts through different pathways to determine its complex effects on MSCs. Prolonged treatment with HGF induced the expression of cardiac-specific markers (GATA-4, MEF2C, TEF1, desmin, alpha-MHC, beta-MHC, and nestin) with the concomitant loss of the stem cell markers nucleostemin, c-kit, and CD105.

Atrial natriuretic peptide effects on intracellular pH changes and ROS production in HEPG2 cells: role of p38 MAPK and phospholipase D.

AIMS: The present study was performed to evaluate Atrial Natriuretic Peptide (ANP) effects on intracellular pH, phospholipase D and ROS production and the possible relationship among them in HepG2 cells. Cancer extracellular microenvironment is more acidic than normal tissues and the activation of NHE-1, the only system able to regulate pHi homeostasis in this condition, can represent an important event in cell proliferation and malignant transformation. METHODS: The ANP effects on pHi were evaluated by fluorescence spectrometry. The effects on p38 MAPK and ROS production were evaluated by immunoblots and analysis of DCF-DA fluorescence, respectively. RT-PCR analysis and Western blotting were used to determine the ANP effect on mRNA NHE-1 expression and protein levels. PLD-catalyzed conversion of phosphatidylcholine to phosphatydilethanol (PetOH), in the presence of ethanol, was monitored by thin layer chromatography. RESULTS: A significant pHi decrease was observed in ANP-treated HepG2 cells and this effect was paralleled by the enhancement of PLD activity and ROS production. The ANP effect on pHi was coupled to an increased p38 MAPK phosphorylation and a down-regulation of mRNA NHE-1 expression and protein levels. Moreover, the relationship between PLD and ROS production was demonstrated by calphostin-c, a potent inhibitor of PLD. At the same time, all assessed ANP-effects were mediated by NPR-C receptors. CONCLUSION: Our results indicate that ANP recruits a signal pathway associated with p38 MAPK, NHE-1 and PLD responsible for ROS production, suggesting a possible role for ANP as novel modulator of ROS generation in HepG2 cells.

Sphingosine 1-phosphate induces antimicrobial activity both in vitro and in vivo.

Sphingosine 1-phosphate (S1P), a polar sphingolipid metabolite, is involved in a wide spectrum of biological processes, including Ca(++) mobilization, cell growth, differentiation, motility, and cytoskeleton organization. Here, we show a novel role of S1P in the induction of antimicrobial activity in human macrophages that leads to the intracellular killing of nonpathogenic Mycobacterium smegmatis and pathogenic M. tuberculosis. Such activity is mediated by host phospholipase D, which favors the acidification of mycobacteria-containing phagosomes. Moreover, when it was intravenously injected in mycobacteria-infected mice, S1P reduced mycobacterial growth and pulmonary tissue damage. These results identify S1P as a novel regulator of the host antimicrobial effector pathways.