Photochemical transformation of lipoic acid-based ligands: probing the effects of solvent, ligand structure, oxygen and pH.

We have combined optical absorption with the Ellman's test to identify the parameters that affect the transformation of the 5-membered dithiolanes to thiols in lipoic acid (LA) and its derivatives during UV-irradiation. We found that the nature and polarity of the solvent, the structure of the ligands, acidity of the medium and oxygen can drastically affect the amount of photogenerated thiols. These findings are highly relevant to the understanding of the photochemical transformation of this biologically relevant compound, and would benefit the increasing use of LA-based ligands for the surface functionalization of various nanomaterials.

Retinoic acid receptors interfere with the TGF-beta/Smad signaling pathway in a ligand-specific manner.

Transforming growth factor-beta (TGF-beta) and retinoic acid (RA) are important regulators of cell growth and differentiation. The TGF-beta receptors utilize Smad proteins to transduce signals intracellularly and regulate transcription of target genes, either directly or in combination with other sequence-specific transcription factors. Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors, are involved in mediating transcriptional responses to RA. Given the known interactions between the TGF-beta and RAR pathways, we have investigated the role played by RAR ligands in modulating functional interactions between Smad3 and RARs. Using transient cell transfection experiments with an artificial Smad3/Smad4-dependent reporter construct, we demonstrate that RAR overexpression enhances Smad-driven transactivation, an effect that requires both Smad3 and Smad4. We provide evidence that RAR effect on Smad3/Smad4-driven transcription is prevented by natural and synthetic RAR agonists, and potentiated by synthetic RAR antagonists. The activity of two TGF-beta-responsive human gene promoter constructs was regulated in a parallel fashion. Using both mammalian two-hybrid and immunoprecipitation/Western methods, we demonstrate a direct interaction between the region DEF of RARgamma and the MH2 domain of Smad3, inhibited by RAR agonists and enhanced by their antagonists. We propose that RARs may function as coactivators of the Smad pathway in the absence of RAR agonists or in the presence of their antagonists, a phenomenon that contrasts with their known role as agonist-activated transcriptional regulators of RA-dependent genes.

Ligation of RARgamma inhibits proliferation of phytohaemagglutinin-stimulated T-cells via down-regulating JAK3 protein levels.

The mechanisms whereby Vitamin A regulates the immune system are poorly understood. We have shown previously that retinoic acids, the Vitamin A derivatives, promote both apoptosis of neglected thymocytes and the activation-induced cell death of peripheral T-cells via ligating the nuclear retinoid receptor (RAR) gamma. In the present study, we found that human peripheral T-cells express RARalpha and gamma, but not RARbeta. Increasing concentrations of 9-cis RA inhibited phytohaemagglutinin (PHA)-induced proliferation of T-cells, an effect that could be mimicked only by addition of RARgamma agonists and could be inhibited by an RARgamma antagonist. Interleukin-2 (IL-2) produced is known to mediate PHA-induced proliferation of T lymphocytes. Ligation of RARgamma did not affect the PHA-induced high affinity IL-2 receptor expression, slightly reduced the PHA-induced IL-2 production, but interfered with the IL-2-mediated signal transduction resulting in inhibition of PHA-induced phosphorylation of retinoblastoma protein and of up-regulation of Bcl-2. Janus kinases JAK1 and JAK3 play a determinant role in IL-2-dependent signal transduction. Ligation of RARgamma did not affect the levels of JAK1, but prevented IL-2-induced expression of JAK3 resulting in inhibition of PHA-induced phosphorylation of Stat5 molecules. Our data suggest that the previously observed toxic effect of high concentrations of retinoids on the immune system might be mediated via formation of 9-cis RA, which via ligation of RARgamma not only induces cell death in immature thymocytes, but inhibits proliferation of T-cells as well.

Activation of retinoic acid receptor-dependent transcription by organochlorine pesticides.

Five organochlorine pesticides, namely, chlordane, dieldrin, aldrin, endrin, and endosulfan, activate human retinoic acid receptor (RAR)-mediated gene transcription via a retinoic acid response element (RARE). Transactivation studies were performed with stable RARalpha, beta, or gamma reporter cell lines in which the RAR DNA-binding domain (DBD) was replaced by that of estrogen receptor alpha (ERalpha)? Five of the organochlorine pesticides tested activated RARbeta and RARgamma but not RARalpha; their half-maximal luciferase activity (EC(50)) was determined. Furthermore, that activity was RAR-specific and organochlorine pesticides did not activate the retinoid X receptor (RXR) pathway. However, competitive binding experiments with [(3)H]-CD367, a pan-RAR agonist, showed that only chlordane could bind RARbeta and RARgamma, albeit with low affinity. In addition, organochlorine pesticides strongly induce cytochrome P450RAI1 (P450RAI1), a key factor of retinoic acid level regulation in many tissues and whose expression and activity are strongly induced by retinoic acid. This study shows that organochlorine pesticides can activate two RAR homologues, with low-binding affinity. Although the agonistic potential of organochlorine pesticides is lower than that of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB), they are able to induce RAR-mediated gene transcription as P450RAI1 and may disrupt the retinoid signaling pathway. Because these chemicals are extremely persistent and tend to accumulate in biological tissues, these results support the hypothesis that the increase in teratogenicity observed in some developing countries could be due to prolonged exposure to organochlorine pesticides ubiquitously present in the environment.

Effect of organochlorine pesticides on human androgen receptor activation in vitro.

Many persistent organochlorine pesticides (OCs) have been implicated in adverse effects, that is, reproductive and developmental effects, in man and in wildlife alike. It has been hypothesized that these so-called xeno-hormones could be responsible for the increased incidence in various male sexual differentiation disorders such as hypospadias, cryptorchidism, low sperm counts and quality. In this report, OCs, called endocrine disrupters, were tested for their interaction with the androgen receptor. The stable prostatic cell line PALM, which contains a human androgen receptor (hAR) expression vector and the reporter MMTV-luciferase, was used to characterize the response of hAR to OC and was compared with synthetic androgen compound R1881. We found that all the OC pesticides tested were able to shift the agonist [(3)H]-R1881 from its binding site to the AR in competitive binding assays. In addition, these compounds antagonize-in a dose-dependent manner-the AR-mediated transcription by synthetic AR ligand R1881. None of the pesticides reacted as agonists. These results demonstrate that OC endocrine activities in vivo probably result from direct and specific binding to the AR ligand-binding domain. Although the antagonistic potential of OC pesticides is lower than that of hydroxyflutamide, they are capable of disrupting the male hormone signaling pathway. Because these chemicals are extremely persistent and tend to bioaccumulate, these results support the hypothesis that the recent increase in the incidence of male sexual disorders could be due to long exposure to ubiquitous OC pesticides found in the environment.

Retinoids induce Fas(CD95) ligand cell surface expression via RARgamma and nur77 in T cells.

Cells from the CD4+ murine T hybridoma line IP-12-7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up-regulation of nur77. Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP-12-7 cells express RARalpha and RARgamma. Here we show that,in the IP-12-7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARgamma. Despite the induced expression of cell surface FasL, only two structurally related RARgamma-selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARgamma to sensitize the Fas death-pathway. Cell surface FasL, however, was able to induce cell death in Fas-bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin-activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas-sensitive cells via induction of FasL expression in activated Tcells.

Ligation of retinoic acid receptor alpha regulates negative selection of thymocytes by inhibiting both DNA binding of nur77 and synthesis of bim.

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.