Uwe Reichert

Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor γ

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha, beta and gamma. Since RAR gamma is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RAR gamma selectivity.

Selective Synthetic Ligands for Human Nuclear Retinoic Acid Receptors

From a series of naphthalene and benzoic acid derivatives we have identified synthetic retinoic acid analogues exhibiting high selectivity for the nuclear retinoic acid receptors RAR alpha (Am 580), RAR beta (CD 2019) and RAR gamma (CD 437) as well as ligands sharing high affinities for all RAR subtypes (CD 367). The compounds were evaluated in two complementary screening systems: (1) binding to nuclear proteins extracted from COS-7 cells after transfection with the appropriate expression vectors, and (2) induction of plasminogen activator in the embryonic mouse teratocarcinoma cell line F9. All compounds behaved as retinoic acid agonists in the F9 test.

Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes.

When confluent cultures of the transformed human keratinocyte line SV‐K14 are shifted to serum‐free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross‐linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane‐bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.

Reconstructed skin from cultured human keratinocytes and fibroblasts on a collagen-glycosaminoglycan biopolymer substrate.

A lattice prepared from biopolymer substrate bovine skin collagen and chondroitin-6-sulfate (glycosaminoglycan) served as a support for normal human keratinocytes and fibroblasts. Air exposure of the lattice on an agarose block gave rise to reconstructed epidermis, the histological features of which are very similar to normal human epidermis. Indirect immunofluorescence staining of the plasma membrane-associated transglutaminase, the enzyme responsible for the synthesis of the cornified envelope, revealed the same tissue distribution as observed in vivo. Cell cycle analysis showed a large shift of the normal human keratinocyte population into S-phase and cell division during the 1st week postinoculation. Furthermore, the effects of two modulators of differentiation (25-hydroxycholesterol and sodium butyrate) on the reconstruction of the epidermis were evaluated.

Energy coupling in hypoxanthine transport of yeast Potentiometric evidence for proton symport and potassium antiport

In Saccharomyces cerevisiae, the uptake of the purine bases hypoxanthine, adenine, guanine and the pyrimidine base cytosine is catalyzed by a common energy-dependent transport system [l] . In a previous report we proposed a model of energy coupling with the purine-cytosine transport system being active as a proton symporter and a potassium antiporter [2] . The model was substantiated essentially by the finding that a decrease in the extra-cellular pH enhances over a wide pH range the apparent affinity of the transport system to its substrates, while cations like K+ and Na’ act as competitive inhibitors the inhibitory effect of which increases with the pH. Meanwhile, the possibility of a proton co-transport has found further support by the experimental result reported that protonconducting uncouplers of oxidative phosphorylation inhibit the influx and stimulate the efflux of the substrates not only under aerobic but also under anaerobic conditions as well as in respiratory deficient strains [3,4] . Furthermore, pH variation studies indicate that the association of one proton is necessary to bind one hypoxanthine molecule to the transport system [5] . In the present paper we demonstrate by potentiometric measurements that the transport of hypoxanthine into yeast cells is indeed accompanied by the absorption of protons as well as by the ejection of potassium ions with a 1 : 1 : 1 stoichiometry. 2. Materials and methods

Uptake and accumulation of purine bases by stationary yeast cells pretreated with glucose

Abstract 1. 1. Department on the growth phase, the ability of Saccharomyces cells to take up the purine bases adenine, guanine, and hypoxanthine shows a maximum in the first half of exponential growth (Fig. 1). Stationary cells, however, can also be prepared for maximum uptake by preincubation in glucose solution. The stimulating effect of this treatment is repressed in the presence of actidione (Fig. 2). 2. 2. Since only low differences in the specific activities of those enzymes involved in the metabolization of hypoxanthine were observed after and without glucose preincubation (Table I), the stimulating effect rather may be explained by the de novo synthesis of the purine transport system itself or a component of it. 3. 3. Kinetic studies with glucose pretreated stationary cells reveal a strong competitive inhibition pattern between adenine, guanine, hypoxanthine, and cytosine, indicating that all the four bases are accepted by this special transport system (Table II). Maximum uptake rates are in the range of 1–4 · 10 −17 moles/cell per minute at 25°C. The temperature optimum is between 35°C and 40°C. The activation energy has been calculated to 15 + 3 kcal/mole . 4. 4. From the three purine bases, only hypoxanthine and guanine are considerably accumulated by the cells against the external concentration (Fig. 4). 5. 5. Attempts to demonstrate a participation of the IMP pyrophosphorylase in the accumulation process were without success.

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.

Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level

Human epidermal keratinocytes were cultured until sub‐confluence in low Ca2+ (0.15 mM) serum‐free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm‐specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.

A possible mechanism of energy coupling in purine transport of Saccharomyces cerevisiae

In a previous report of this laboratory [ 1 j , experimental evidence for the existence of a common transport system for adenine, guanine, hypoxanthine, and cytosine in Saccharornyces cerevisiae was given. Hypoxanthine and guanine were found considerably accumulated within the cells against a concentration gradient. Attempts to demonstrate the participation of purine pyrophosphorylases (EC 2.4.2.7, 8) in the accumulation process, similar to the situation in bacteria [2], were without success [ 1,3]. An interesting point of our studies were strong differences in the kinetic parameters for the uptake of the distinct bases when compared with the results of other authors [4,5]. In order to check on the reason for these differences we performed kinetic measurements at different pH with different buffer systems and found that purine uptake is activated by H’ and inhibited by monovalent cations (Na’, K’) especially giving rise to changing affinities of the transport system for its substrates depending on the ratio of protons and monovalent cations present. Considering that Saccharomyces maintains as well a K’ as in the reverse sense a H’ gradient between the cytoplasm and the medium whereby in the presence of metabolic energy the uptake of K’ is connected with the transfer of protonsfrom the cell [6] we suggest by virtue of our kinetic results that the cells are using this gradient energy to accumulate purine bases. In the terminology of Mitchell [7], we suppose that the purine transport system in Saccharomyces is acting as a proton symporter and a potassium antiporter.