Expression pattern of retinoic acid receptor genes in normal human skin

Expression pattern of the retinoic acid receptor (RAR) genes in normal human skin was investigated by in situ hybridization method. Using the riboprobes from cDNAs for three subtypes of RAR, we found that the RAR-alpha and the RAR-gamma genes were expressed in the epidermis. Significant expression of the RAR-beta gene was detected neither in the epidermis nor in the dermis. Expression of the RAR-alpha and -gamma genes was detected both in te basal cell layer and in the squamous cell layers. Moreover, the RAR-alpha and -gamma genes were expressed in the eccrine sweat glands as well. These results indicate that the target cells for retinoic acid (RA) in human skin are keratinocytes and the epithelial cells of epidermal appendages. These findings thus demonstrate that, among three subtypes of RAR, both RAR-alpha and RAR-gamma are essential for control of epidermal and epithelial cell differentiation in human skin.     

Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.

Retinoids are effective modulators of proliferation and differentiation of keratinocytes in vivo and in vitro. In mouse 10T1/2 cells, retinoid action on proliferation and neoplastic transformation is correlated with the upregulation of gap-junctional communication and expression of connexin 43 (Cx43). In the present study we have determined if retinoids induce similar effects on gene expression in human skin. Studies were conducted in intact skin and on cultured keratinocytes and dermal fibroblasts. In a clinical study, 2 weeks of treatment with 0.05% all-trans retinoic acid resulted in increased expression of Cx43 mRNA and protein in epidermis. Expression occurred predominantly in the suprabasal layer. Cultured cells exhibited a differential response to retinoic acid. In keratinocytes, increased expression of Cx43 occurred at low (10(-11) M) concentrations, whereas inhibition occurred at high (10(-7) M) concentrations; however, junctional communication, measured by dye transfer, was not altered over this concentration range. Dermal fibroblasts, in contrast, exhibited a dose-dependent increased expression of Cx43 at concentrations up to 10(-7) M retinoic acid and proportionately increased their junctional communication over this dose range. These data indicate that control of Cx43 gene expression by retinoids in human skin cells is complex. The production of gradients of junctional channels could play a role in the control of growth and differentiation in epidermis.     

Human neonatal keratinocytes have very high levels of cellular vitamin A-binding proteins

Since cellular retinol- and retinoic acid-binding proteins (CRBP and CRABP) mediate the effects of vitamin A on epidermal differentiation, the levels of these binding proteins were measured in the epidermal and dermal layers of newborn, human foreskin as well as in primary cultures of keratinocytes and fibroblasts from these layers. Ligand binding assays with saturating concentrations of all trans-[3H]retinol or of all trans-[11-3H]retinoic acid were used to quantitate amounts of binding proteins in cytosols prepared from these skin layers or cultured cells. The epidermal levels of CRABP and CRBP (60.9 +/- 14.4 and 7.3 +/- 1.7 pmol per mg cytosol protein, respectively) were markedly higher than that reported for adult epidermis but were comparable to levels in keratinocytes cultured from neonatal foreskin epidermis (61.8 +/- 7.8 and 10.7 +/- 2.5, respectively). The levels of CRABP were much lower in the foreskin dermis than in the epidermis and the levels measured in the fibroblasts cultured from this dermis were consistent with the dermal levels. However, CRBP levels in cultured dermal fibroblasts were very low and could not account for the dermal CRBP levels, suggesting that another dermal cell type has high levels of CRBP.     

In vitro metabolism by human skin and fibroblasts of retinol, retinal and retinoic acid

The metabolism of radio-labelled retinol, retinal and retinoic acid by fresh human skin as well as by human dermal fibroblasts have been investigated in vitro . Surgically removed human skin biopsies were placed at the air-liquid interface, and treated topically for 24 h with retinoids. At the end of the treatment period, epidermis and dermis were separated by heat. Epidermis, dermis and medium were subsequently extracted and resulting fractions were analysed by HPLC. Dermal fibroblast cultures were treated and analysed in a comparable manner. Topical application of retinoids resulted in gradient concentrations within the skin. For each fraction, metabolites and unchanged product proportions were determined by HPLC. After treatment with retinol and retinal, low but significant amounts of retinoic acid were detected in the epidermis, as well as in the dermis (30 pmol to 90 pmol). In comparison, treatments with retinoic acid itself, led to higher level of retinoic acid in the epidermis and in the dermis (respectively 2050 and 420 pmol). Cultured human dermal fibroblasts, treated with retinol and retinal, formed retinoic acid as well as several other metabolites (retinol esters, reduction of retinal to retinol…). Taken together, our results are consistent with an action of retinol or retinal on the skin via a retinoic acid formation and a metabolic function of the dermis.     

Induction of proliferation of growth-inhibited keratinocytes and fibroblasts in monolayer culture by sodium lauryl sulfate: comparison with all-trans retinoic acid

We have previously shown that all-trans retinoic acid (RA) has the capacity to stimulate proliferation of growth-inhibited human epidermal keratinocytes and growth-inhibited human dermal fibroblasts. The same treatment also stimulates extracellular matrix synthesis by fibroblasts (J Invest Dermatol 93:449; 94:717). In the present study we have examined the capacity of sodium lauryl sulfate to stimulate keratinocyte and fibroblast proliferation under the same conditions. Our data show that both cell types are stimulated to proliferate. Sodium lauryl sulfate is less potent than RA; it requires a higher molar concentration to achieve optimal stimulation and the number of responding cells at optimal concentrations is less with sodium lauryl sulfate than with RA. Further, there is a rapid onset of toxicity at concentrations of sodium lauryl sulfate that are only slightly higher than the optimal stimulatory concentration. Finally, sodium lauryl sulfate is less effective than RA in stimulating production of extracellular matrix (fibronectin, thrombospondin, and laminin) by dermal fibroblasts. Despite its ability to partially mimic RA as a stimulant of keratinocyte and fibroblast growth, sodium lauryl sulfate does not activate chloramphenicol acetyl transferase in cells co-transfected with retinoic acid receptors and a retinoic acid responsive element linked to the chloramphenicol acetyl transferase gene.     

Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

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.     

Multi-stage program of differentiation in human epidermal keratinocytes: regulation by retinoids

The proliferation and differentiation of epidermal keratinocytes in vivo and in vitro is influenced by a variety of different factors, including several peptide growth factors, protein kinase C activators, retinoids, and various cytokines. Retinoids can affect the proliferation of human epidermal keratinocytes either positively or negatively and influence the multi-step program of differentiation in epidermal keratinocytes at very specific stages. Epidermal keratinocytes express nuclear retinoic acid receptors, RAR alpha, and RAR gamma. It is likely that at least some of the alterations in gene expression induced by retinoids are mediated through these RAR. The cytosolic retinoic acid-binding protein (CRABP), which is differentially expressed during squamous differentiation of human epidermal keratinocytes, may control the effective concentration of retinoic acid in the cell and therefore regulate indirectly gene expression.     

Ultrastructural effects of UVB radiation and subsequent retinoic acid treatment on the skin of hairless mice

The ultrastructure of hairless mouse skin exposed to UVB radiation and followed by retinoic acid treatment was studied to identify alterations induced in both epidermis and dermis. Female mice were irradiated 3 times weekly for 5-6 months; a group of these mice was then treated topically 3 times weekly for 10 weeks with either 25 micrograms all-trans-retinoic acid dissolved in acetone or with acetone alone. Age-matched, unexposed, untreated mice served as controls. Cutaneous changes induced by UVB radiation included keratinocyte mitochondrial inclusions often accompanied by damaged cristae, duplication of basement membrane, increased number of dermal fibroblasts, inflammatory cells and elastic fibers, and abnormal elastic fibers. Subsequent retinoic acid treatment resulted in more prominent mitochondrial inclusions which sometimes coalesced to form irregular contoured bodies. Also observed were lipid droplets in the stratum corneum, glycogen deposits in keratinocytes and granular material in dilated keratinocyte endoplasmic reticulum. Poorly differentiated epidermis with necrotic or apoptotic cells was present in some specimens. Elastic fibers were fewer and usually morphologically normal. Skin exposed to UVB and treated with vehicle appeared similar to control except for the presence of excess basement membrane and occasional small mitochondrial inclusions. Because of the heightened concern regarding UV radiation-induced damage to the human skin and the current topical use of retinoids, the cutaneous changes described are considered worthy of attention.     

Keratinocytes and dermal factors activate CRABP-I in melanocytes

Abstract: Recognition that cellular retinoic acid binding protein (CRABP)-I and CRABP-II are found in different cell types has provided additional support for the presumably divergent roles of these two proteins in mediating retinoic acid (RA) effects in human skin. CRABP-II is expressed in fibroblasts and keratinocytes, and CRABP-I in as yet unidentified cells, possibly epidermal melanocytes. Recently, we demonstrated that each of these RA-binding proteins in human skin possesses two classes of binding sites, possibly related to the state of phosphorylation of the proteins. We now characterize the cutaneous origin of CRABP-I further using an anion-exchange HPLC assay that allows effective separation of the two proteins in human skin, and a fluorescent in situ hybridization technique. We report that CRABP-I is expressed in isolated melanocytes at the mRNA level, although under these circumstances the protein has minimal RA-binding activity, and that keratinocytic and dermal influences are required for CRABP-I activity in melanocytes. This melanocyte origin for CRABP-I and the improvement by RA of the irregular hyperpigmentation associated with photoaging led us to examine the effects of RA using various cellular associations, from conventional pure cultures of melanocytes grown on plastic dishes to a pigmented skin equivalent consisting of melanocytes and keratinocytes grown on a dermal equivalent. We established that the inhibitory effects of RA on melanogenesis do not result from a direct effect on melanocytes alone but also involve keratinocytes and dermal influence. These data expand our understanding of cell-to-cell signaling in cutaneous pigmentation, and strongly suggest a role for CRABP-I in mediating RA effects on melanogenesis.     

Expression of retinoid receptors in sebaceous cell carcinoma

PURPOSE: The aim of this study is to investigate whether there are any abnormalities in the in vivo expression of retinoid acid receptors (RAR-alpha, RAR-beta and RAR-gamma) and retinoid X receptors (RXR-alpha, RXR-beta and RXR-gamma) in sebaceous cell carcinoma. METHODS: Expression of retinoid receptors in paired specimens of cancerous tissues (n = 10) and adjacent normal tissues (n = 10) from 10 patients with sebaceous cell carcinoma was studied immunohistochemically by using anti-retinoid receptor antibodies. RESULTS: In eight of the 10 normal tissue samples, all six receptors were expressed. In the other two samples, all receptors were expressed except RAR-gamma (one sample) or RXR-gamma (two samples). Five tumours (50%) lacked RAR-alpha; RAR-alpha expression was lower in tumours than in normal tissues in eight of 10 cases. RAR-beta was expressed in the cytoplasm of nine of 10 tumours; RAR-beta expression was at least as high in tumours as in normal tissue in eight of 10 cases. Two tumours lacked RAR-gamma; three tumours had lower RAR-gamma expression than paired normal epithelium; four had the same RAR-gamma expression, and one had higher RAR-gamma expression. RXR-alpha expression was strong in all normal tissues and tumour samples. Ten tumours lacked RXR-beta and all 10 tumours lacked RXR-gamma expression. CONCLUSIONS: Diminished RXR-beta and RXR-gamma expression might be related to the development of sebaceous cell carcinoma. Additional studies are required to establish whether the defects in RAR expression in sebaceous cell carcinoma might affect the potential response of this tumour to treatment with retinoids.     

丙酸氯倍他索和维甲酸联合应用对小鼠淋巴细胞、人角质形成细胞增殖及其I型转谷氨酰胺酶mRNA表达的影响

目的 :　研究丙酸氯倍他索 (CP)和全反式维甲酸 (RA)联合应用 (CP RA)对小鼠淋巴细胞增殖及人角质形成细胞 (KC)增殖和I型转谷氨酰胺酶 (TGase)mRNA表达的影响 ,以探讨两药合用对有关的抗银屑病药效有无相互影响。方法 :　体外培养条件下 ,3 H TdR掺入法和RT PCR技术。结果与结论 :　CP RA合用时RA不影响CP抑制小鼠淋巴细胞增殖的药效 ,CP也不影响RA下调人KCTGaseImRNA表达的作用 ,对人KC增殖抑制两药有协同作用。CP RA合用在发挥各自不同药效的同时 ,可减少各自的剂量 ,进而能减少两药各自的副作用。