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.

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.

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.

Sticholysin II: a pore-forming toxin as a probe to recognize sphingomyelin in artificial and cellular membranes.

Sphingomyelin is a major component of membrane rafts, and also is a precursor of many bioactive molecules. The sphingomyelin plays important biological roles and alterations of its metabolism are the basis of some genetic disorders such as the Niemann Pick disease. A complete understanding of its biological role is frustrated by the lack of efficient tools for its recognition in the cell. Sticholysin II (StnII) is a 20 kDa protein from the sea-anemone Stichodactyla helianthus which shows a cytotoxic activity by forming oligomeric aqueous pores in the cell plasma membrane. A recent NMR analysis indicates that the sticholysin II binds specifically to sphingomyelin by two domains that recognize respectively the hydrophilic (i.e. phosphorylcholine) and the hydrophobic (i.e. ceramide) moieties of the molecule. Aim of our research has been to verify the possible employ of an antibody against the StnII to investigate the localization and the dynamics of sphingomyelin in cell membranes. For this purpose, we developed a monoclonal antibody (named A10) against the toxin and we tested its ability to bind StnII after binding to sphingomyelin. A10 antibody is able to recognize the sticholysin II both in its native form and after SDS treatment, being the protein still suitable for many analytic techniques such as ELISA, western blotting and immunofluorescence. The high affinity of the toxin for the sphingomyelin in cell membranes has been demonstrated by microscopic immuno-localization and western blot analysis; both methods confirmed that sphingomyelin is the molecular acceptor for StnII also in cell membranes. Finally, we studied the specificity of the toxin for sphingomyelin by a cell membrane-double labelling method, using cholera toxin, specific for the ganglioside GM1, and sticholysin II. The results obtained show that there is no cross-reactivity between the two toxins, confirming that sticholysin II is able to discriminate among membrane domains with sphingomyelin with respect to those enriched with gangliosides.

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.

P-Glycoprotein is not a key target for the chemosensitizing effect of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol in HepG2 cells exposed to doxorubicin.

Chemosensitization of HepG2 cells to doxorubicin by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol neither impinged on downregulation of P-glycoprotein expression nor on severe impairment of its activity. Moreover, differently from verapamil, a potent P-glycoprotein inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol chemosensitized HepG2 cells in a fashion that was insensitive to the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. At concentrations exceeding the one employed for chemosensitization, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol was by itself strongly toxic to HepG2 cells, and also this effect was insensitive to the pancaspase inhibitor. These results suggest that 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, at subtoxic concentrations, might synergize with scarcely toxic doxorubicin doses to propagate a caspase-independent cytotoxic response, such that P-glycoprotein-dependent drug resistance is circumvented.

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.

Isolation and characterization of a myotoxin from the venom of Macrovipera lebetina transmediterranea.

The Macrovipera lebetina venom consists of a complex mixture of proteins belonging to a few main families according to their enzymatic and pharmacological activity. Given the serious pathophysiological effects caused by M. lebetina bites mainly induced by muscle degeneration, we decided to investigate the myotoxic activity of some venom fractions. In the present study we describe the purification and characterization of a 22.600 kDa protein, named in the following Mlp4.2, that shares myotoxic but not haemorrhagic activity in vivo. Herein we report that Mlp4.2 is a metalloproteinase belonging to the PI-SVMPS family able, in vitro, to proteolyse extracellular matrix proteins as laminin and fibronectin. Histological observations of mouse anterior tibialis Mlp4.2-treated muscle, demonstrate that this protein induces a massive degeneration of myofibers but not haemorrhage. The immunofluorescence analysis of protein-treated anterior tibialis, demonstrates that Mlp4.2 is able to disarray the laminin network surrounding muscle fibers. Finally Mlp4.2 did not show any direct cytolytic activity towards the myogenic cell line C2C12 in culture. The data reported herein suggest that the myotoxicity of Mlp4.2 is primarily linked to the disruption of the muscle fibers interaction with extracellular matrix proteins.

Cholesterol depletion inhibits electrophysiological changes induced by anoxia in CA1 region of rat hippocampal slices.

The hyper-activation of glutamate receptors is a key event in the degenerative processes triggered by ischemia in the brain. Several types of these receptors reside in cholesterol-sphingomyelin rich domains of post-synaptic plasma membranes and have been described to be sensitive to cholesterol depletion. Hence we investigated, by extracellular recordings, the effect of cholesterol depletion on population spikes (PS) during ischemia-like conditions in the CA1 region of rat hippocampal slices using the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD). Results obtained demonstrate that MbetaCD prevents the changes induced by anoxic insult, i.e., depression of the population spike amplitude and insurgence of ischemic long-term potentiation. Furthermore cholesterol depletion prevents the disappearance of population spike induced by anoxia/aglycemia during kainate perfusion. Our data suggest a possible role of MbetaCD in preventing the pathological changes in synaptic activity induced by ischemia and indicate that manipulation of lipid components of membrane rafts might provide a new approach for the treatment of ischemia.