Androgen-induced delay of hair growth in the golden Syrian hamster.

The golden Syrian hamster flank organ has been used to study the stimulatory effect of androgens on sebaceous glands and hair. Androgens cause the sebaceous glands and hair follicles in this organ to grow. We have made the novel observation that exogenously administered androgen, testosterone propionate (TP), suppresses hair growth in the area surrounding the flank organ. When given in a time-release (systemic) subcutaneous dosage form (pellet), 25 mg TP inhibited the regrowth of clipped hair in peri-flank organ skin for up to 21 days; however, by 28 days hair grew back to the same extent as in controls. The peak serum level of testosterone in TP-treated animals occurred at 14 days, and declined thereafter. When two separate TP pellets (25 mg/pellet) were administered 14 days apart in order to maintain high serum levels for 28 days, the amount of hair regrowth after 35 days was identical to animals receiving a single TP pellet or placebo. This suggests that the systemic level of testosterone was not the only factor in hair regulation. Hair growing within the flank organ appeared to be unaffected by TP administration. In the golden Syrian hamster, androgen, as in humans, can exert stimulatory and inhibitory effects on hair growth depending on the body site. We conclude that this animal model could serve as a useful system to investigate the mechanisms responsible for the opposing effects of androgen on hair growth.

All-trans-retinoic acid inhibition of Pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts: identification of a retinoic acid response element in the Pro alpha1(I) collagen gene.

The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates pro alpha1(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 pro alpha1(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 48 h of treatment. Transforming growth factor-beta1 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 pro alpha1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5'-AGTAGA-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-gamma and retinoid X receptor-alpha, 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 binding. These studies indicate that t-RA downregulates the promoter activity of the rat pro alpha1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5' flanking region of the gene.

Effects of long-term retinoic acid treatment on epidermal differentiation in vivo: specific modifications in the programme of terminal differentiation.

To investigate the effects of long-term all-trans-retinoic acid (RA) treatment on epidermal differentiation in vivo, rhino mice were treated topically with 0.005% RA, and their epidermis was analysed histologically and biochemically after 5, 13 and 26 weeks of treatment. Effects of RA were observed first in the living layers of the epidermis, and then in the non-viable stratum corneum. Five weeks of topical RA led to thickening of the spinous and granular compartments, induction of keratins K6, K16 and K17, and suppression of filaggrin expression. After 13 and 26 weeks of RA treatment, the number of anucleate cornified cell layers remained similar to controls, but additional changes in histology and protein expression were observed. The results showed that prolonged administration of topical RA induced epidermal hyperproliferation, but did not suppress differentiation, in contrast to results observed in keratinocyte cultures. However, the distinct histological and biochemical changes observed in the spinous, granular and cornified layers of RA-treated skin after 26 weeks of treatment, suggested that the progeny of RA-treated basal cells undergo a modified programme of terminal differentiation. Considering the present data together with results of previous in vivo studies, we propose that long-term topical RA treatment retards, or specifically modulates, the later stages in epidermal differentiation, or programmed cell death.

Regulation of cellular retinoic acid binding protein expression in rhino mouse skin by all-trans-retinoic acid.

Cellular retinoic acid binding proteins (CRABP) are cytoplasmic proteins that bind all-trans-retinoic acid (RA) and other retinoids. The purpose of these studies was to determine the effects of topically applied RA on CRABP expression in rhino mouse skin. CRABP-II mRNA was significantly induced (3- to 4.5-fold) by a single dose of RA at 6 and 16 h after RA treatment, with a return to control levels at 48 h. CRABP-II message was not significantly elevated by 3 or 4 consecutive days of RA treatment, when assessed 24 h after the last treatment. CRABP-I mRNA was undetectable in control and RA-treated skin. We used radiolabelled RA binding combined with non-denaturing PAGE blot autoradiography to distinguish the CRABP subtypes. By this protein assay method, increases in CRABP-II were detected 24 and 48 h after a single application of RA, as well as after 3 and 4 days of RA treatment. RA treatment did not alter CRABP-I expression relative to the vehicle control. These results demonstrate that in mouse skin CRABP-II, but not CRAB-I, is inducible by RA, and is similar to how RA regulates CRABP in human skin.

Evaluation of topical retinoids for cutaneous pharmacological activity in Yucatan microswine.

The pharmacological effects of retinoids on skin have been studied primarily in test systems using small animals, such as mice and rabbits. Because of potentially significant differences in skin permeation and metabolism between small animals and humans, we have used Yucatan microswine as an alternative model for testing topical retinoids. Microswine skin resembles human skin, functionally and anatomically, more closely than most other species. In these studies, microswine skin was treated topically with retinoids for 5 consecutive days per week for 5 weeks. We found microswine epidermis to be functionally responsive to retinoids in that it undergoes hyperplasia and shows an increase in transepidermal water loss (TEWL). All-trans-retinoic acid, and its analogs, 13-cis-retinoic acid, 4-hydroxy-retinoic acid and 4-oxo-retinoic acid all caused epidermal thickening and increased TEWL. The three analogs were less potent than all-trans-retinoic acid. A synthetic retinoid, TTNPB, potently induced epidermal hyperplasia and increased TEWL, but a close structural analog, m-carboxy-TTNPB, which is also inactive on nuclear retinoic acid receptors, was without effects on microswine epidermis. These findings show that microswine are useful for evaluating the cutaneous effects of topical retinoids. This model could be of value in identifying retinoids with potential clinical activity.

Effects of topical antiinflammatory agents on Freund's adjuvant-induced skin lesions in rats.

Freund's adjuvant, when administered intradermally to rats, causes polyarthritis as well as inflammation of the skin, eye, gastrointestinal and genitourinary tracts. We assessed the effects of antiinflammatory drugs on ear skin lesions to determine if this might be a useful skin inflammation model. The hind paw of male Lewis rats was injected with Mycobacterium butyricum in paraffin oil. Lesions appeared between days 13 and 15 after adjuvant injection. Each ear exhibited on average 1 to 3 highly erythematous, elevated lesions, 2 to 3 mm in diameter. By histology, the lesions consisted of epidermal hyperplasia, with a prominent accumulation of inflammatory cells in the dermis. Ears were treated topically with glucocorticoids, cyclosporine and indomethacin on days 15 through 21 after adjuvant injection. By day 22, dexamethasone, fluocinolone acetonide, and cyclosporine caused near-complete clearing of lesions whereas indomethacin exacerbated the inflammation by causing increased numbers of skin lesions. These results show the potential usefulness of adjuvant-induced skin lesions in rats as a novel subchronic inflammation model.

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-prope nyl 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.

In vivo prevention of corticosteroid-induced skin atrophy by tretinoin in the hairless mouse is accompanied by modulation of collagen, glycosaminoglycans, and fibronectin.

In an earlier study we showed that tretinoin could prevent corticosteroid-induced skin atrophy in hairless mice. In this study, we examined the histochemical, biochemical, and immunochemical changes that accompanied the atrophy and its prevention. Mice were treated dorsally for 3 weeks in the morning and afternoon (AM:PM) as follows: 1) vehicle:vehicle, 2) steroid:vehicle, 3) steroid:tretinoin. Tretinoin concentration was 0.05% in an ethanol:propylene glycol vehicle. The steroid was clobetasol propionate (0.05%). The normally sparse dermal glycosaminoglycans were further reduced by steroid:vehicle treatment and increased to greater than vehicle:vehicle amounts by steroid:retinoid. Mast cells were similarly affected. Biochemical quantification of glycosaminoglycans confirmed the histochemical findings. Collagen, non-collagenous protein, and total protein content were reduced by the steroid. The latter two were returned to more normal levels by tretinoin whereas with collagen there was only a trend toward normal levels. Fibronectin, which was increased by the steroid:vehicle treatment, was reduced to more normal levels by steroid:tretinoin. We conclude that tretinoin has the ability to prevent the major steroid-induced biomechanical changes in hairless mouse dermal connective tissue that contribute to atrophy.

Topical all-trans retinoic acid rapidly corrects the follicular abnormalities of the rhino mouse. An ultrastructural study.

Topical all-trans retinoic acid (RA) has been shown to transform the horn-filled utriculi of the rhino mouse into normal follicles. We studied the early events by light and electron microscopy. Reduction in diameters of the utriculi was quantified by image analysis of whole mounts. Topical RA at 0.05% in ethanol/propylene glycol was applied daily and biopsies were taken after 1, 2, 3 and 6 days of treatment. By electron microscopy, after 3 days of RA treatment there was a great increase in the size and density of laminated membrane coating granules (MCGs) which had fused to the apical membranes of the upper granular cells. Thereafter, corneocytes within the lumina of the utriculi showed fewer desmosomes and a loss of intercellular material, accompanied by detachment from each other. Conversion to normal follicles was complete by 6 days. In whole mounts examined after 3 days of RA, there was a 75% reduction in the mean diameter of the utriculi. These results suggest that extrusion of the contents of enlarged MCGs into the intercellular corneocyte spaces facilitated separation of corneocytes, leading to rapid shedding, perhaps through the action of desmosome-lysing proteases. The conversion to normal follicles is consistent with the established role of retinoids in correcting abnormal differentiation.

Pharmacological effects of retinoids on skin cells.

Retinoids exert profound pleiotropic effects in skin, affecting many aspects of cell differentiation and proliferation. For this reason, retinoids have prominent pharmacological effects on major skin cells (keratinocytes, dermal fibroblasts, melanocytes, sebocytes) and have shown great potential as therapeutic agents in dermatology. In keratinocytes, retinoids induce proliferation, resulting in epidermal hyperplasia. Retinoids also modulate epidermal differentiation, however, many of the suppressive effects of retinoids observed in vitro do not occur in vivo. Dermal fibroblasts are important target cells of retinoids and are stimulated to produce extracellular matrix proteins, particularly when skin is damaged by wounding, ultraviolet radiation or glucocorticoids. Retinoids regulate pigmentation and can lighten hyperpigmented skin in animals and humans. Studies with cultured melanocytic cells show that tyrosinase activity is reduced by retinoids. The powerful sebosuppressive effect of some retinoids, such as 13-cis-retinoic acid, demonstrates that sebocyte differentiation is altered by retinoids. Retinoids inhibit proliferation and lipid synthesis in cultured human sebocytes and alter their keratin expression.

Lifetime topical application of tretinoin to hairless mice.

The discovery that topical tretinoin can reverse some of the effects of photodamage may lead to its chronic application. Examination of long-term effects was of interest. Three groups of hairless mice (age 6-8 weeks) were treated dorsally with 1) tretinoin (0.025%), 2) cream vehicle, 3) sham treatment. Applications were 3 times weekly and continued for up to 2 years until all mice were sacrificed or had died. Biweekly examinations showed no sign of retinoid toxicity, with growth and longevity similar in all groups. Tretinoin-treated skin was smooth and pink, resembling that of younger mice. Controls had yellowed, irregularly thickened skin. Histologically, tretinoin-treated skin had a hyperplastic epidermis consisting of plump, cytologically normal cells. Control skin had 3-4 compressed cell layers. Foci of new normally staining collagen were present in the subepidermal dermis of tretinoin-treated skin; fibroblasts were large and abundant in these areas. These foci were absent in controls. Mice treated with tretinoin also appeared to have increased amounts of elastic fibers and glycosaminoglycans.

Glucocorticoid and retinoid regulation of alpha-2 type I procollagen promoter activity.

Glucocorticoids decrease type I procollagen synthesis by decreasing the steady state levels of procollagen mRNAs and mRNA synthesis. The present studies were undertaken to determine the functional sequences of the pro alpha 2(I) collagen gene required for the glucocorticoid-mediated decrease of type I procollagen mRNA synthesis. Embryonic mouse fibroblasts were stably transfected with the pR40 DNA CAT construct containing the 5' flanking region fragment from -2048 to +54 and the intronic fragment from +418 to +1524 of the mouse alpha 2(I) collagen gene. Dexamethasone treatment of these pR40 transfected fibroblasts resulted in a significant decrease in CAT activity which agrees with the glucocorticoid-mediated decrease of the steady state levels of type I procollagen mRNAs. To determine the possible role of the first intron fragment in the dexamethasone-mediated decrease of CAT activity, pR36, a CAT plasmid containing the first intron fragment and the SV40 early promoter, was transfected into mouse fibroblasts and treated with dexamethasone. No significant decrease in CAT activity was observed. The dexamethasone-mediated response was then localized within the 5' flanking region by preparing a series of constructs containing internal deletions and transfecting these plasmids into mouse fibroblasts. The regions -2048 to -981 and -506 to -351 were required for the dexamethasone response of gene activity. However, the DNA stretch from -981 to -506 was not. Analysis of the DNA sequences of these regions revealed a single GRE at -1023 to -1018 and a modified doublet at -873 to -856.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Topical all-trans retinoic acid stimulates collagen synthesis in vivo.

Histochemical and ultrastructural studies demonstrate that topical all-trans retinoic acid (RA) stimulates the deposition of a subepidermal band of collagen in photoaged hairless mice. The aim of this study was to examine the effect of RA treatment on collagen synthesis using biochemical and immunochemical techniques. Albino hairless mice were irradiated three times a week for 10 weeks with four minimal erythema doses of UVB from Westinghouse FS-40 bulbs. In the post-UV period, mice were either nontreated or treated with 0.05% RA or the ethanol-propylene glycol vehicle for up to 10 weeks. Antibodies against the aminopropeptide (AP) of type III procollagen were used in immunofluorescence microscopy and radioimmunoassay techniques. The AP of type III collagen is normally present throughout the dermis and in areas of active collagen synthesis (i.e., the dermal-epidermal junction). In this study, a similar distribution was seen in all untreated and vehicle-treated mice, and in mice treated with RA for 2, 4, and 6 weeks. However, increased staining, in a subepidermal band, was detected in the 8-week RA-treated skin. This region became intensely fluorescent to a depth of 100 mu in the 10-week RA-treated skins. As determined by radioimmunoassay, the content of the AP of type III procollagen increased twofold with 10-week RA treatment. Because the ratio of type I to type III collagens remained constant in treated and untreated skins, it is reasonable to assume that the content of type I collagen increased in proportion to type III collagen in RA-treated skins.

A direct comparison of pharmacologic effects of retinoids on skin cells in vitro and in vivo.

The purpose of these studies was to directly compare the pharmacologic effects of retinoids on cutaneous cells in vitro and in vivo. Previously, it was demonstrated that all-trans-retinoic acid stimulates the proliferation of growth-arrested human keratinocytes and fibroblasts in culture. In the present studies, all-trans-retinoic acid was compared to three other retinoids--13-cis-retinoic acid, P-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl-1-p ropenyl] benzoic acid (TTNPB) and M-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl-1-p ropenyl] benzoic acid (meta-carboxy-TTNPB]--for growth stimulation using a cultured human squamous epithelial cell line (UM-SCC-1) and human dermal fibroblasts. These four retinoids were also evaluated for their effects on reduction of horn-filled utriculi when topically applied to the skin of rhino mice. All-trans-retinoic acid stimulated proliferation of both fibroblasts and epithelial cells over concentrations ranging from 0.01 to 1.0 micrograms/ml. In fibroblasts, 13-cis-retinoic acid was less potent than all-trans-retinoic acid, whereas, in epithelial cells these two retinoids were equipotent. In contrast, TTNPB was more potent than all-trans-retinoic acid at stimulating the growth of both fibroblasts and epithelial cells. The analog, meta-carboxy-TTNPB was essentially inactive as a growth stimulator of both cell types. In the rhino mouse utriculus reduction model, the rank order of potency for the retinoids was the same as that for in vitro cell growth stimulation (TTNPB greater than all-trans-retinoic acid greater than 13-cis-retinoic acid). Meta-carboxy-TTNPB was inactive at reducing utriculi at a dose of 5,000 times the ED50 of TTNPB.(ABSTRACT TRUNCATED AT 250 WORDS)

Topical all-trans-retinoic acid prevents corticosteroid-induced skin atrophy without abrogating the anti-inflammatory effect.

We tested the ability of all-trans-retinoic acid to prevent corticosteroid-induced skin atrophy without lessening the anti-inflammatory effect of the steroids. Histologic study and skin-fold thickness in hairless mice treated topically with various steroids, followed by topical all-trans-retinoic acid, were used to measure prevention of atrophy. By both assessments, all-trans-retinoic acid prevented atrophy. Noninterference with the anti-inflammatory property of steroids was tested in a phorbol ester-induced mouse ear edema model and by histologic assessment of croton oil-induced inflammation of mouse dermis. We found that all-trans-retinoic acid did not interfere with steroid suppression of either edema or dermal inflammation. Thus all-trans-retinoic acid was effective in preventing steroid-induced atrophy without affecting the steroid's anti-inflammatory property.

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 3.7x, 12.5x, and 50x more potent than 13-cis-retinoic acid, etretinate, and motretinide, respectively, after topical administration. Administered orally, all-trans-retinoic acid was 2.3x 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.