Serge Michel

Regulation of Vascular Endothelial Growth Factor Expression in Human Keratinocytes by Retinoids

Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases, such as psoriasis and cancers, which are characterized by increased angiogenesis. Experimentally, VEGF overexpression can be induced by the treatment of cell cultures and biological tissues with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Using normal human keratinocytes in conventional cultures and skin grafted onto nude mice in vivo, we show that retinoids can inhibit TPA-mediated VEGF gene induction at the transcriptional level. Because retinoids are biologically active either by interacting with the nuclear retinoic acid receptors or by interfering with the activator protein 1 (AP1) transcription factor, we studied the effect of the retinoic acid derivative CD 2409, which exhibits strong anti-AP1 activity but does not bind to the known retinoic acid receptorsin vitro. The results demonstrate that the inhibition of VEGF expression by retinoids only depends on their anti-AP1 activity and does not require gene transactivation via retinoic acid response elements. Because the VEGF promoter contains four potential AP1 binding sites, we used different promoter constructs to identify the functional site responsible for TPA induction and retinoid inhibition. This site turned out to be localized at position −621 of the 5′ flanking region of the VEGF gene.

Pharmacology and chemistry of adapalene

BACKGROUND Retinoid research in the field of dermatology has been influenced by the clinical success of topical tretinoin and oral isotretinoin in the treatment of acne, and by the discovery of high-affinity binding proteins for retinoic acid mediating its action and interaction with other vitamins and hormones. OBJECTIVE We sought molecules with an optimal balance between stability, efficacy, and local tolerance for topical acne therapy. METHODS In vitro and in vivo bioassay systems were used to test the ability of retinoids to modulate cell proliferation and differentiation. In addition, antiinflammatory properties were assessed. Binding and transactivation assays were used to compare affinities and transcriptional activities of adapalene and tretinoin for the nuclear transcription factors, retinoic acid receptors (RARs). RESULTS AND CONCLUSION Adapalene is a stable naphthoic acid derivative with potent retinoid pharmacology, controlling cell proliferation and differentiation. In addition it has significant antiinflammatory action. The nuclear gene transcription factors RAR beta and RAR gamma mediate the retinoid activity of adapalene.

Ligation of Retinoic Acid Receptor α 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) α. The present studies were undertaken to investigate whether ligation of RARα 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 Ek (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 RARα 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.

Activation‐induced apoptosis and cell surface expression of Fas (CD95) ligand are reciprocally regulated by retinoic acid receptor α and γ and involve nur77 in T cells

It has been previously shown that CD4+ T cells enter the apoptotic suicide program via the Fas ligand (FasL)/Fas‐mediated pathway upon T cell receptor (TCR) stimulation. In Jurkat cells TCR stimulation regulates the de novo synthesis of FasL, while in the influenza hemagglutinin‐specific CD4+ murine T cell hybridoma (IP‐12‐7) the cell surface appearance of a preformed FasL is initiated. Both processes are dependent on new mRNA and protein synthesis, involve up‐regulation of nur77, and can be inhibited by retinoic acids (RA). Two groups of nuclearreceptors for RA have been identified: retinoic acid receptors (RAR) and retinoid X receptors (RXR). In this study various synthetic retinoids were used to define which receptors regulate TCR‐mediated apoptosis. It is demonstrated that the inhibition is mediated via RARα, while RARγ enhances TCR‐mediated apoptosis, and when both receptors are stimulated, the costimulation by RXR will promote the effect of RARα. Evidence is presented that these receptors affect the transcriptional activity of nur77 and consequently the expression of FasL. Our data suggest a complex interaction between the various isoforms of retinoid receptors in regulating T cell death and demonstrate that the target through which retinoids regulate TCR‐mediated apoptosis is nur77.

Determination of retinoid activity by an enzyme-linked immunosorbent assay

In normal human keratinocytes, retinoic acid suppresses the expression of the plasma membrane associated enzyme transglutaminase (TGm) at the pretranslational level. This finding led us to develop an enzyme-linked immunosorbent assay (ELISA) for the evaluation of the biological activity of retinoids, i.e., natural and synthetic derivatives of vitamin A. In this assay, keratinocytes are cultured in a 96-well cluster in the presence of different retinoid concentrations. The expression of TGm is then quantified, without any extraction or purification step, using a TGm-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. The dose-response curves obtained show this ELISA to be a sensitive and reproducible assay to determine the potency of retinoids.

Retinoids induce Fas(CD95) ligand cell surface expression via RARγ 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 RARα and RARγ. 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 RARγ. Despite the induced expression of cell surface FasL, only two structurally related RARγ‐selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARγ 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.