,5,3Ј-Triiodo-L-thyronine (T3), but not L-thyroxine (T4), activated Src kinase and, downstream, phosphatidylinositol 3-kinase (PI3-kinase) by means of an ␣ v␤3 integrin receptor on human glioblastoma U-87 MG cells. Although both T 3 and T4 stimulated extracellular signal-regulated kinase (ERK) 1/2, activated ERK1/2 did not contribute to T 3-induced Src kinase or PI3-kinase activation, and an inhibitor of PI3-kinase, LY-294002, did not block activation of ERK1/2 by physiological concentrations of T 3 and T4. Thus the PI3-kinase, Src kinase, and ERK1/2 signaling cascades are parallel pathways in T 3-treated U-87 MG cells. T3 and T4 both caused proliferation of U-87 MG cells; these effects were blocked by the ERK1/2 inhibitor PD-98059 but not by LY-294002. Smallinterfering RNA knockdown of PI3-kinase confirmed that PI3-kinase was not involved in the proliferative action of T 3 on U-87 MG cells. PI3-kinase-dependent actions of T 3 in these cells included shuttling of nuclear thyroid hormone receptor-␣ (TR␣) from cytoplasm to nucleus and accumulation of hypoxia-inducible factor (HIF)-1␣ mRNA; LY-294002 inhibited these actions. Results of studies involving ␣v␤3 receptor antagonists tetraiodothyroacetic acid (tetrac) and Arg-Gly-Asp (RGD) peptide, together with mathematical modeling of the kinetics of displacement of radiolabeled T3 from the integrin by unlabeled T3 and by unlabeled T4, are consistent with the presence of two iodothyronine receptor domains on the integrin. A model proposes that one site binds T3 exclusively, activates PI3-kinase via Src kinase, and stimulates TR␣ trafficking and HIF-1␣ gene expression. Tetrac and RGD peptide both inhibit T3 action at this site. The second site binds T4 and T3, and, via this receptor, the iodothyronines stimulate ERK1/2-dependent tumor cell proliferation. T3 action here is inhibited by tetrac alone, but the effect of T4 is blocked by both tetrac and the RGD peptide. thyroid hormone; phosphatidylinositol 3-kinase; extracellular signal-regulated kinase 1/2; integrin ␣v␤3; glioblastoma cells; Src kinase; mitogenactivated protein kinase; intracellular hormone receptor trafficking ACTIONS OF THYROID HORMONE [L-thyroxine (T 4 ); 3,5,3Ј-triiodo-L-thyronine (T 3 )] that are independent of ligand binding to nuclear thyroid hormone receptors are called nongenomic actions. Nongenomic effects of thyroid hormone are initiated outside the cell nucleus but may culminate in complex cellular events that are nucleus mediated (7, 8, 26 -29). Initiation of nongenomic actions includes a plasma membrane receptor for T 4 and T 3 on integrin ␣ v ␤ 3 that is linked to mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK) 1/2] for transduction of the hormone signal (3) and nuclear receptors residing in the cytosol of unstimulated cells, such as thyroid hormone receptor (TR) ␤1 (28).The phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase B (Akt) pathway is an important regulator of cellular growth, metabolism, and survival (13, 19). Studies of Storey et al. (38) indi...

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.

Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogs. From this receptor, thyroid hormone (l-thyroxine, T4; 3,5,3′-triiodo-l-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated non-genomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents, and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intra-cellular kinase-mediated regulation of gene expression, hormone analogs at αvβ3 have additional effects on intra-cellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation, and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.

Madin-Darby canine kidney cells infected with Sendai virus rapidly lose GSH without increase in the oxidized products. The reduced tripeptide was quantitatively recovered in the culture medium of the cells. Since the GSH loss in infected cells was not blocked by methionine, a known inhibitor of hepatocyte GSH transport, a nonspecific leakage through the plasma membrane is proposed. UV-irradiated Sendai virus gave the same results, confirming that the major loss of GSH was due to membrane perturbation upon virus fusion. Consequent to the loss of the tripeptide, an intracellular pH decrease occurred, which was due to a reversible impairment of the Na ؉ /H ؉ antiporter, the main system responsible for maintaining unaltered pH i in those cells. At the end of the infection period, a rise in both pH i value and GSH content was observed, with a complete recovery in the activity of the antiporter. However, a secondary set up of oxidative stress was observed after 24 h from infection, which is the time necessary for virus budding from cells. In this case, the GSH decrease was partly due to preferential incorporation of the cysteine residue in the viral proteins and partly engaged in mixed disulfides with intracellular proteins. In conclusion, under our conditions of viral infection, oxidative stress is imposed by GSH depletion, occurring in two steps and following direct virus challenge of the cell membrane without the intervention of reactive oxygen species. These results provide a rationale for the reported, and often contradictory, mutual effects of GSH and viral infection.

L-T3 and L-T4 activated the Na+/H+ exchanger of L-6 myoblasts, with a fast nongenomic mechanism, both in the steady state and when cells undergo acid loading with ammonium chloride. Monitored with the intracellular pH-sensitive fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, activation of the exchanger appeared to be initiated at the plasma membrane, because T3-agarose reproduced the effect of L-T3, and triiodothyroacetic acid, a hormone analog previously shown to inhibit membrane actions of thyroid hormone, blocked the action of L-T3 on the exchanger. We show here for the first time that transduction of the hormone signal in this nongenomic response requires tyrosine kinase-dependent phospholipase C activation and two different signaling pathways: 1) mobilization of intracellular calcium, assessed by the fluorescent probe fura-2, through activation of inositol trisphosphate receptors and without contributions from extracellular calcium or ryanodine receptors; and 2) protein phosphorylation involving protein kinase C and MAPK (ERK1/2), as shown by the use of kinase inhibitors and by immunoblotting for activated kinases.

A large series of hydroxytyrosyl esters of C2-C18 fatty acids with increasing lipophilicity was prepared by a new highly efficient method based on acylation of methylorthoformate-protected hydroxytyrosol. All products were tested for relative antioxidant effect using ABTS assays in ethanolic medium and DCF assays in L6 cells. No linear correlation between lipophilicity and antioxidant effect was found. ABTS assays showed a growing antioxidant capacity, with respect to hydroxytyrosol, only for medium-sized ester chains (C4-C10) and a nearly constant capacity for the higher homologues. This has been rationalized by molecular dynamics experiments in terms of partial shielding of the catecholic hydroxyls by long-chain esters. A similar and dose-dependent pattern was observed in DCF assays in L6 cells, but a sharp antioxidant activity drop resulted for long-chain esters, probably due to membrane entrapment.

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