Topical and Systemic Effects of Retinoids on Horn-Filled Utriculus Size in the Rhino Mouse. A Model to Quantify “Antikeratinizing” Effects of Retinoids

A method was developed to quantify the “antikeratinizing” effects of various retinoids (all-trans-retinoic acid, 13-cis-retinoic acid, motretinide, etretinate) in rhino mouse skin, which contains many keratinized pilosebaceous structures or horn-filled utriculi. Mean utriculus diameters in whole mount epidermis were determined and dose-response relationships were constructed after topical or oral administration of test retinoids. All- trans-retinoic acid was 3.7×, 12.5×, and 50× more potent than 13-cis-retinoic acid, etretinate, and motretinide, respectively, after topical administration. Administered orally, all-trans-retinoic acid was 2.3× more potent than 13-cis-retinoic acid At 5 mg/kg, each retinoid produced a significant reduction in utriculus size. The rhino mouse model represents a novel and useful assay to quantify antikeratinizing activity and potency differences of biologically active retinoids

A Pleiotropic Response Is Induced in F9 Embryonal Carcinoma Cells and Rhino Mouse Skin by All-trans-Retinoic Acid, a RAR Agonist but Not by SR11237, a RXR-Selective Agonist

We evaluated SR11237, a retinoid X receptor (RXR)- specific compound, for its pharmacologic effects on cell differentiation in F9 embryonal carcinoma cells and rhino mouse epidermis. SR11237 can cause RXR/RXR homodimers to form and transactivate a reporter gene containing a RXR-response element. We confirmed, using nuclear receptor co-transfection assays in COS-1 cells, that SR11237 is effective at transactivating a chloramphenicol acetyltransferase reporter gene through RXRs but not retinoic acid receptors. When SR11237 was tested for its ability to modulate cell differentiation, it was inactive on F9 embryonal carcinoma cells and rhino mouse skin. Because differentiation in these systems is known to be regulated by RAR-specific compounds, such as all-trans-retinoic acid and (E)-4-[2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl benzoic acid], our results with SR11237 are compatible with the concept that classical retinoid pleiotropic responses are mediated by RXR/RAR heterodimeric nuclear receptors rather than through RXR/RXR homodimers

Effects of Topical Retinoids on Cytoskeletal Proteins: Implications for Retinoid Effects on Epidermal Differentiation

In vivo effects of retinoids on epidermal differentiation were investigated by analyzing cytoskeletal proteins in rhino mice treated topically with all-trans-retinoic acid (RA) and other retinoids (13-cis-retinoic acid, etretinate, TTNPB). Non-disulfide-linked cytoskeletal proteins, including keratins from the epidermal “living layers,” were first selectively extracted using 9.5 M urea; subsequently, keratins of the stratum corneum were isolated using 9.5 M urea plus a reducing agent. Gel electrophoresis and immunoblot analysis showed that urea extracts of epidermis from vehicle-treated skin were composed predominantly of four major keratins (analogous to human epidermal keratins K1, K5, K10, and K14), and the keratin filament-associated protein filaggrin. In contrast, extracts of epidermis from retinoid-treated skin contained additional keratins (K6, K16, and K17) and almost no detectable filaggrin. Furthermore, similar analysis of stratum corneum keratins demonstrated that extracts from RA-treated skin did not contain the partially proteolyzed keratins typically observed in stratum corneum extracts of control animals. Hyperplasia-inducing agents (salicylic acid, croton oil) caused an increase in keratins K6, K16, and K17, but they did not effect filaggrin or alter proteolysis of stratum corneum keratins.The result that RA induced expression of keratins K6, K16, and K17, as commonly expressed in hyperproliferative epidermis, is consistent with the notion that retinoids increase epidermal cell proliferation in the basal and/or lower spinous layers. The findings that topical RA decreased filaggrin expression and reduced proteolysis of stratum corneum keratins, despite increased size and number of granular cells and the presence of an anucleate stratum corneum, suggest that topical RA may also modulate a later stage of epidermal differentiation involved in stratum corneum formation

All-trans-Retinoic Acid Inhibition of Proα1(I) Collagen Gene Expression in Fetal Rat Skin Fibroblasts: Identification of a Retinoic Acid Response Element in the Proα1(I) Collagen Gene

The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates proα1(I) collagen gene expression in fetal rat skin fibroblasts. FRS fibroblasts were stably transfected with the ColCat3.6 plasmid, which contains a portion of the 5′ flanking region of the rat proα1(I) collagen gene linked to a reporter gene, chloramphenicol acetyltransferase. The effect of t-RA on CAT activity was determined as a function of concentration and incubation time. Maximal inhibition of CAT activity by t-RA occurred at 10−8 M after 48h of treatment. Transforming growth factor-β1; did not block the inhibitory effect of t-RA on CAT activity. Computer sequence analysis of the 3.6-kb DNA fragment that contains the promoter for the rat proα1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5′-AG-TAGA-3′) and one idealized (5′-GGGTCA-3′) half site located at positions −1345 and −1335, respectively. Two nuclear retinoid receptors that were expressed in bacteria, retinoic acid receptor-γ and retinoid X receptor-α, were found to bind specifically to a double-stranded oligonucleotide containing the RARE in gel mobility shift assays. Mutation of the idealized half-site eliminated the binding of receptor proteins to the oligonucleotide. Gel mobility shift assays using nuclear protein extracts prepared from t-RA-treated FRS fibroblasts showed that binding to the oligonucleotide containing the RARE was decreased from control values. The same assays performed with the mutated oligonucleotide resulted in only slight bindbig. These studies indicate that t-RA downregulates the promoter activity of the rat proα1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5′ flanking region of the gene