The β-adrenergic receptors, previously shown to be present on the membranes of cultured human epidermal keratinocytes, were found to be functionally coupled to membrane-bound adenylate cyclase. Using membrane preparations, the enzyme could be activated by guanosine triphosphate (GTP), the stable GTP analog GPP(HN)p, and NaF, all of which are known to activate the adenylate cyclase without interacting with membrane receptors. Binding of catecholamine agonists (epinephrine, norepinephrine, and isoproterenol) to the β-adrenergic receptors is followed by an increase in the activity of adenylate cyclase. This activation could be reversed (or prevented) by β-adrenergic antagonists, but was unaffect by the presence of α-adrenergic ligands (either agonists or antagonists). The activation by catecholamines appears to be directly related to receptor occupancy, since the activation constant (Ka) of adenylate cyclase for the three catecholamines was found to be very similar to the equilibrium dissociation constant (Kd) determined from competition binding experiments. The activation of adenylate cyclase under these conditions appears to be restricted to the catecholamine agonists only. The non-catecholamine β-adrenergic agonists (salbutamol, terbutaline) did not show any measurable activation of adenylate cyclase, even though these agonists were shown previously to bind to the β-adrenergic receptors on keratinocyte membranes with the expected affinities.

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.

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.

Biological effects of retinoic acid (RA) are mediated through its binding to three closely related nuclear receptors (RAR alpha, RAR beta, and RAR gamma) belonging to the steroid-thyroid nuclear receptor family. RARs are able to modulate the transcription of specific genes by binding to responsive elements located in the promoter-enhancer region of these genes. As demonstrated by in situ hybridization, the distribution of each RAR type in the developing embryo, as well as in the adult, is not uniform. In this context, synthetic retinoids that would behave as selective ligands would be invaluable for studying the respective roles of each RAR type in cultured cells, whole animals, and embryos. Moreover, from a pharmacological point of view, such selective compounds may possess a higher therapeutic index and a lower teratogenic risk, because they might affect specific tissues and spare some others. As an approach to this problem, we have set up two complementary assays, (i) an in vitro binding assay to determine the Kd values of retinoids for RAR alpha, RAR beta, and RAR gamma and (ii) a functional assay in cultured cells to evaluate the potential of retinoids to transactivate, through their binding to one type of RAR, a reporter gene. The binding assay uses nuclear extracts of COS-7 cells transfected with vectors expressing RAR alpha, RAR beta, or RAR gamma. The functional assay is a measure of chloramphenicol acetyltransferase (CAT) activity in HeLa cells co-transfected with the expression vectors used in the binding assay and the reporter gene TRE-tk-CAT. Selective agonists for RAR alpha (Am80 and Am580) and RAR beta-RAR gamma (CD495 and CD564) were identified. However, compounds with pure RAR beta or RAR gamma selectivity have not yet been identified.

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RARα. Transfection of cos-7 cells with the expression vector RARαO produces a 100-fold increase in intracellular RARα concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, [3H]CD367. Complete separation between RARα and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RARα.

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross‐linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus‐40 (SV‐40)‐transformed human foreskin keratinocyte line SV‐K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane‐associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane‐associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.

In order to better understand the respective roles of the nuclear retinoic acid receptors (RARs) and the cytosolic retinoic acid binding protein (CRABP) in the mode of action of retinoic acid (RA), several types of RA analogs have been synthesized. Representative compounds have been radiolabeled to a high specific activity and their binding (direct and competition) to RARs and CRABP was determined. Their biological activity on F9 embryonal carcinoma cell differentiation has been determined by a quantitative assay of plasminogen activator (PA). All biologically active analogs studied in this work bound to RARs. A good correlation was found between PA induction and affinity for the RARs, with the exception of RA itself which was a good ligand but a moderate inducer of F9 differentiation. Two biologically active analogs (compounds II and III) did not bind to the CRABP. One biologically inactive analog (compound VIII) bound to CRABP. These results strongly suggest that retinoids must bind to RARs but not necessarily to CRABP in order to induce cell differentiation in F9 cells.

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nm), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.

The density pertubation technique with cationic silica microbeads was applied to prepare highly purified plasma membranes from cultured human keratinocytes. Trypsinized cells were coated successively with the beads (diameter approximately 50 nm, gravity greater than 2 g/cm3) and polyacrylic acid before they were lysed by osmotic shock and mechanical shear. The plasma membranes remained in the form of large open sheets which could easily be separated from other cell organelles and the cytosol by low-speed centrifugation. The membrane preparation was characterized by scanning and transmission electron microscopy, marker enzyme activities, one-dimensional sodium dodecyl sulfate polyacrylamide electrophoresis, and the specific beta-adrenergic receptor count. A yield of 79 +/- 9% was calculated by comparing the amount of beta-adrenoceptors in the purified membrane preparation with that of a crude cellular particulate fraction. The specific beta-adrenoceptor count of these two preparations was 1.2 +/- 0.02 and 0.2 +/- 0.05 pmol/mg protein, respectively, indicating a 6-fold improved purification with this microbead technique. The purified membranes were essentially free from contamination of other cell organelles.

The presence of β-adrenergic receptors has been demonstrated in membrane preparations from passaged human epidermal keratinocytes. The receptors were characterized in terms of density and binding properties. Using the tritiated β-adrenergic antagonists dihydroalprenolol and propranolol, the equilibrium dissociation constant (Kd) was found to be about 1.4 nM for the two antagonists with a receptor density of approximately 280 fmol/mg membrane protein. Stereospecificity of the binding sites was shown by the much lowered affinity to d-isoproterenol as compared to that of l-isoproterenol. By the use of subtype specific antagonists, the receptors were classified as β2 adrenoceptors. This finding is supported by the relative order of affinities of the agonists isoproterenol > epinephrine > norepinephrine. The Kd value for dihydroalprenolol was approximately the same when determined from equilibrium binding studies or from association and dissociation kinetics, suggesting that the ligand binding is a single step bi-molecular reaction.