Modulation of epidermal terminal differentiation in patients after long-term topical corticosteroids.

The expression of the various markers for terminal epidermal differentiation in atrophic skin of patients after long-term topical corticosteroids (TCS) was studied by electron microscopy, immunofluorescence using antibody to profilaggrin/filaggrin (PF/FG), immunoperoxidase staining using antibody to involucrin, and oil red O stain for neural lipids of the stratum corneum. Thirty-nine patients were subdivided into two groups: (A) 19 patients suffering from rebound phenomenon after stopping TCS and (B) 20 patients without rebound phenomenon. Biopsy specimens were taken before ending the use of TCS in both groups. In group A, both the morphological markers (including the different epidermal strata, keratohyalin granules, lamellar granules, and cornified cell envelopes) and the molecular markers (including involucrin, PF/FG, and neutral lipids) of terminal epidermal differentiation were significantly suppressed. On the other hand, the differentiational markers in the atrophic skin of patients without rebound phenomena were only slightly altered. These results suggest that potent TCS not only has antiproliferative actions but also inhibits the differentiation of epidermis, resulting in structural defects in the epidermis, especially the stratum corneum.     

Effects of all-trans retinoic acid on the morphology of human epidermal cells in vitro

Summary The effects of all-trans retinoic acid on human epidermal cell cultures were studied using ultrastructural techniques. Differentiation and stratification were reduced in retinoic acid treated epidermal cells. Treated cells developed a rounded appearance and seemed to contain more granules and vacuoles than usual. Desmosomes were not found in treated cells.     

Antagonistic effects of retinoic acid and hydrocortisone on terminal differentiation of human squamous carcinoma cells

Differentiation of SqCC/Y1, a human malignant squamous carcinoma, can be modulated by the presence of both corticosteroids and retinoids. To evaluate the regulation of the differentiation of epidermal cells by these agents, we have employed delipidized serum from which glucocorticoids and retinoids were removed. Thirty percent of SqCC/Y1 cells spontaneously expressed the terminally differentiated phenotype after 6 d in culture, as measured by the capacity to form detergent-insoluble cornified envelopes. Exposure of SqCC/Y1 cells to hydrocortisone at concentrations of 30, 100 and 300 nM produced a 25, 100, and 175%, increase, respectively, in the number of differentiated cells over untreated control cultures. Exposure of cells to retinoic acid at levels of from 3 to 300 nM caused a 24 to 85% decrease in the quantity of differentiated cells. Simultaneous treatment of SqCC/Y1 cells with hydrocortisone and retinoic acid resulted in mutual antagonism of cornified envelope formation. Treatment of SqCC/Y1 cells with a 1000-fold molar excess of retinoic acid did not directly alter the uptake of hydrocortisone, and a 100-fold molar excess did not directly inhibit the binding of the corticosteroid to its receptor. Pretreatment of cells for 48, 72, or 96 h with 100 nM retinoic acid decreased the binding of hydrocortisone to its receptor by only 20%, and only resulted in a small decrease in the total amount of hydrocortisone associated with cells 48 h after the addition of retinoic acid. These findings suggest that the antagonism between hydrocortisone and retinoic acid on the terminal differentiation of SqCC/Y1 is not expressed at the level of the corticosteroid receptor.     

Short-term retinoic acid treatment increases in vivo, but decreases in vitro, epidermal transglutaminase-K enzyme activity and immunoreactivity.

Epidermal transglutaminase-K is believed to catalyze the covalent linking of loricrin and involucrin to form cross-linked (CE) envelopes. In normal skin, transglutaminase-K is expressed as a band immediately below the stratum corneum, whereas in psoriasis and healing skin its expression is considerably expanded throughout the suprabasal layers. We have investigated whether the hyperproliferative state induced by short-term application of topical retinoic acid is similarly characterized by an increase in transglutaminase-K enzyme activity and immunoreactivity. Retinoic acid (0.1% cream) or vehicle were applied to human skin and occluded for 4 d. Skin biopsies were obtained for measurement of transglutaminase-K and transglutaminase-C activity and immunoreactivity. For comparison, cultured normal human keratinocytes were incubated for 4 d in the presence of 1 microM retinoic acid and the subsequent transglutaminase-K activity and immunoreactivity measured. Transglutaminase-K activity was increased 2.8 times in retinoic acid compared to vehicle-treated skin (p less than 0.005, n = 12) whereas there was no significant difference in transglutaminase-C activity. However, transglutaminase-K mRNA levels were not significantly different between retinoic acid- and vehicle-treated skin. In vehicle-treated skin, transglutaminase-K immunoreactivity was limited to a narrow, substratum corneal band, but was considerably expanded in a diffuse suprabasal pattern in retinoic acid-treated epidermis. In contrast, transglutaminase-K immunostaining was decreased and its enzymatic activity reduced sixfold in retinoic acid-treated keratinocytes (p less than 0.01, n = 4). These results demonstrate that retinoic acid treatment in vivo, in contrast to in vitro, leads to not only increased transglutaminase-K protein expression but also increased enzymatic activity in the absence of detectable increases in mRNA levels. These data, taken with the previously reported lack of in vivo modulation of the differentiation markers keratins 1 and 10 by retinoic acid, indicate that certain aspects of keratinocyte terminal differentiation that are altered in vitro by retinoic acid do not occur in vivo in human skin.     

维A酸对T淋巴细胞作用的研究进展

维A酸类药物广泛用于治疗各类皮肤病，且其适应证还在不断扩大。近年来越来越多的证据表明，维A酸具有免疫调节作用。首先，维A酸与皮肤免疫有密不可分的联系，其次，维A酸对机体T淋巴细胞的增殖、凋亡、趋化以及Th1／Th2细胞的极化都产生明显及确实的作用，但是维A酸发挥药理学作用涉及的蛋白调控、细胞因子分泌及信号传导的精确机制目前尚不清楚。     

Stimulation of PPARalpha promotes epidermal keratinocyte differentiation in vivo

Our recent studies have demonstrated that PPARalpha activators stimulate differentiation and inhibit proliferation in cultured human keratinocytes and accelerate epidermal development and permeability barrier formation in fetal rat skin explants. As the role of PPARalpha activation in adult epidermis is not known, the aim of this study was to determine if topically applied PPARalpha ligands regulate keratinocyte differentiation in murine epidermis. Topical treatment with PPARalpha activators resulted in decreased epidermal thickness. Expression of structural proteins of the upper spinous/granular layers (involucrin, profilaggrin-filaggrin, loricrin) increased following topical treatment with PPARalpha activators. Furthermore, topically applied PPARalpha activators also increased apoptosis, decreased cell proliferation, and accelerated recovery of barrier function following acute barrier abrogation. Experiments with PPARalpha-/- knockout mice showed that these effects are specifically mediated via PPARalpha. Compared with the epidermis of PPARalpha+/+ mice, involucrin, profilaggrin-filaggrin, and loricrin expression were slightly decreased in PPARalpha-/- mice. Moreover, topical clofibrate treatment did not increase epidermal differentiation in PPARalpha-/- mice. Furthermore, in cultured human keratinocytes we have demonstrated that PPARalpha activators induce an increase in involucrin mRNA levels. We have also shown that this increase in gene expression requires an intact AP-1 response element at -2117 to -2111 bp. Thus, stimulation of PPARalpha stimulates keratinocyte/epidermal differentiation and inhibits proliferation.     

Action of phorbol esters, bryostatins, and retinoic acid on cholesterol sulfate synthesis: relation to the multistep process of differentiation in human epidermal keratinocytes

This study examines the action of phorbol 12-myristate 13-acetate (PMA) on the synthesis of cholesterol sulfate in cultured normal and transformed human epidermal keratinocytes and assesses the antagonistic effects by retinoids and bryostatins on PMA action in relation to the multistep program of squamous differentiation. Treatment of normal human epidermal keratinocytes (NHEK) with PMA induces terminal cell division (irreversible growth-arrest) and causes a time- and dose-dependent increase in the incorporation of Na2(35)SO4 into cholesterol sulfate, a marker for squamous cell differentiation. This stimulation in sulfate incorporation appears specific for cholesterol sulfate and is due to increased levels of cholesterol sulfotransferase activity. The increase in cholesterol sulfate accumulation parallels the increase in transglutaminase type I, another marker for squamous differentiation. Several transformed NHEK cell lines do not exhibit increased levels of cholesterol sulfate and transglutaminase type I activity after PMA treatment, indicating that they acquired defects in the regulation of squamous differentiation. Bryostatins 1 and 2, and several diacylglycerol analogues neither inhibit cell proliferation nor increase cholesterol sulfate synthesis or transglutaminase activity, indicating that these agents do not induce terminal differentiation. In contrast, the bryostatins block the increase in cholesterol sulfate and transglutaminase activity as well as the commitment to terminal cell division by PMA. Bryostatin 1 inhibits the commitment to terminal cell division and the accumulation of cholesterol sulfate significantly even when added 8 h after PMA administration. Retinoids inhibit cholesterol sulfate accumulation and the increase in transglutaminase activity by PMA but do not affect the commitment to terminal cell division. In summary, phorbol esters induce in NHEK cells a program of squamous differentiation. This process of differentiation consists of the commitment to terminal cell division and expression of a squamous phenotype. Expression of this phenotype is accompanied by an accumulation of cholesterol sulfate and increased cholesterol sulfotransferase activity. Bryostatins 1 and 2 and retinoic acid affect this differentiation process at different stages.     

Effects of moisturization on epidermal homeostasis and differentiation

Moisturizers are commonly used for routine skin care. This study assessed the effects of a moisturizer on barrier function, epidermal architecture, keratinocyte proliferation, and physiological regulation of the epidermis in photoaged but otherwise normal skin. Fifteen women with moderately photoaged forearms were treated twice a day for 4 weeks with a moisturizer containing dimethicone and glycerine. Baseline and post-treatment transepidermal water loss (TEWL) and ipsilateral forearm biopsies were obtained. Epidermal thickness, melanin levels, keratinocyte proliferation, and expression of keratins were evaluated. Induction of keratins 6 and 16, commonly associated with keratinocyte proliferation and wound healing, was observed. Epidermal thickness increased by 0.019 mm (P = 0.005), barrier function improved (TEWL decreased by 13%) and melanin intensity decreased (P = 0.004). Even nonxerotic, photoaged skin may appear younger, benefiting structurally and functionally from routine use of moisturizers containing dimethicone and glycerine.     

维A酸受体在皮肤生理中的作用

维A酸受体属于细胞核受体超家族成果，在皮肤的分化、增殖、炎症过程中发挥重要作用。现就维A酸受体在皮肤生理中的作用进行文献综述。     

Effects of retinoic acid on prostaglandin biosynthesis in guinea-pig skin.

Topical application of retinoic acid on guinea-pig skin resulted within 70 hours in erythema with a concomitant elevation of endogenous prostaglandin E2 (PGE2) in the treated areas of the skin. Prolonged daily treatment resulted in the development of severe scaly dermatoses and a corresponding decrease in the level of PGE2 in the skin. Examination of retinoic acid effects on the in vitro biosynthesis of PGE2 from arachidonic acid by extracts from guinea-pig skin and sheep vesicular gland demonstrated that retinoic acid inhibits prostaglandin synthesis in a concentration and time-dependent manner. These results indicate that retinoic acid may exert a regulatory role on prostaglandin biosynthesis in the skin.     

Effects of xerosis and ageing on epidermal proliferation and differentiation

The hallmarks of dry skin (xerosis) are scaliness and loss of elasticity. Decreased hydration and a disturbed lipid content of the stratum corneum are also well-known features. The frequency of dry skin increases with ageing. The aim of this study was to examine if these known features of dry skin are related to changes in epidermal proliferation and differentiation. In addition, age-related changes in normal and in dry skin were examined; 62 volunteers were divided by clinical grading and biophysical measurements into groups with young/normal, young/dry, aged/normal and aged/dry skin. Biopsy samples from the lower legs (most severe dryness) were examined by two-dimensional gel electrophoresis and by immunohistochemistry for epidermal proliferation, epidermal keratins and cornified envelope proteins. There was a slight increase in proliferation in both groups with dry skin compared with normal skin of the corresponding age. In aged/normal compared with young/normal skin there was a significant decrease in proliferation. However, epidermal proliferation was the same in aged/dry skin as in young/normal skin. For epidermal differentiation, an age-independent decrease of keratins K1 and K10 and an associated increase in the basal keratins K5 and K14 was detected in dry skin. There was also an age-independent premature expression of the cornified envelope protein involucrin. In contrast, loricrin expression was not influenced by dry skin conditions. In summary, epidermal proliferation was significantly decreased in aged/normal compared with young/normal skin. Dry skin showed significant changes in the epidermal expression of basal and differentiation-related keratins, and a premature expression of involucrin irrespective of age.     

Effects of intensive application of retinoic acid on human skin

The daily application of 0-3% retinoic acid to the back produced an acute irritant dermatitis which resolved to a near-normal clinical appearance within 40 days despite continued daily exposure. Hardened skin was markedly altered physiologically. The responses to DMSO, histamine and the histamine liberator, compound 48/80, were sharply enhanced. This reflected enhanced permeability resulting from reduction of the horny layer to less than one-half its normal thickness. Phototoxic and irritant substances produced exaggerated reactions owing to greater penetration. A paradoxical decrease in delayed sinsitivity to streptodornase-streptokinase was attributed to enhanced clearance of the antigen.     

Trans retinoic acid enhances the growth response of epidermal keratinocytes to epidermal growth factor and transforming growth factor beta

Retinoids have been shown to either stimulate or inhibit epidermal keratinocyte proliferation. We have observed that in serum and growth factor free medium (basal medium), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) stimulated DNA synthesis in mouse epidermal keratinocyte cultures (mKC) in a time- and dose-dependent manner. Incubation with all-trans retinoic acid (RA) greatly enhanced the stimulatory effect of EGF. Transforming growth factor beta (TGF beta) inhibited the EGF-induced DNA synthesis in a dose-dependent manner, and the inhibition was greatly enhanced by a low dose of RA. Treatment of growth-factor deprived human keratinocyte cultures (hKC) with RA before incubation in basal medium containing EGF or a mixture of EGF, bovine pituitary extract (BPE), and insulin caused a dose-related increase in DNA synthesis and cell growth (cell number), respectively. A low concentration of RA also enhanced the inhibitory effect of TGF beta on growth-factor-induced DNA synthesis and cell growth in hKC. These findings suggest that the differential effects of retinoids on epidermal keratinocyte proliferation are in part due to an enhancement of the response of keratinocytes to positive and negative peptide growth factors.     

Topical retinoic acid treatment of photoaged skin: its effects on hyaluronan distribution in epidermis and on hyaluronan and retinoic acid in suction blister fluid.

Topical treatment with retinoic acid (tretinoin, vitamin A acid) has been reported to partly reverse signs of photodamage. To determine whether the histochemical distribution of hyaluronan (hyaluronic acid, HYA) in the epidermis and dermis and the amounts of HYA and retinoic acid in suction blister fluid were influenced by such topical treatment, 14 subjects healthy apart from moderate photodamage were instructed to treat the lateral forearm with 0.01-0.05% retinoic acid cream for 6 months. In a study of the short-term effects, another six subjects applied 0.05% retinoic acid cream for 2 weeks. After 6 months the thickness of the vital epidermis had increased by 23%. The HYA staining was based on a specific immunohistochemical method in which hyaluronan-binding protein is used. Before treatment HYA was seen as a meshwork around the cells in the upper half of the stratum spinosum. After 6 months of treatment this meshwork had increased in thickness by 31% compared with pretreatment specimens. The HYA staining was already intense in the papillary dermis before treatment and no difference was observed after 6 months' treatment. The mean concentration of HYA in blister fluid had increased significantly (43%) after 2 weeks of treatment whereas after 6 months there was no significant difference in this respect between the treated and untreated arm. The increase in the thickness of the epidermal HYA meshwork after 6 months and the blister fluid HYA after 2 weeks may indicate that HYA is involved in the epidermal change induced by topical retinoic acid therapy.(ABSTRACT TRUNCATED AT 250 WORDS)     

维A酸促毛囊外毛根鞘无色素黑素细胞分化的实验研究

目的：研究维A酸(all-transretinoic acid，ATA)对毛囊无色素黑素细胞（famelanotic melangeytes，AMMC)的激活作用。方法：以高、中、低3种不同浓度的ATA作用于培养的人毛囊外毛根鞘AMMC，倒置显微镜观察细胞形态的变化，细胞计数法测定ATA对AMMC增殖率的影响，通过间接免疫荧光法结合激光共聚焦显微镜半定量分析药物作用前后AMMC酪氨酸酶(tyrosinase，TYR)、酪氨酸酶相关蛋白-1(tyrosinase related protein-1，TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)表达的变化。结果：ATA能抑制AMMC的增殖，并能促进AMMC表达TYR和TRP-1，但对TRP-2的表达没有影响。结论：ATA能够促进AMMC的分化，同时抑制增殖，其抑制机制可能与凋亡有关。     

Measurement of the rate of epidermal terminal differentiation: expression of involucrin by S-phase keratinocytes in culture and in psoriatic plaques

At present little is known about the control mechanisms involved in coordinating cell production and maturation in epidermis. To investigate this, we have measured the rate of transit from the proliferative to the terminally differentiating compartment in confluent low-calcium cultures of normal epidermal keratinocytes, using involucrin as a marker of terminal differentiation. We estimate a rate of transit of 3.58 cells/5000 cells/h and a differentiation probability of 0.017, indicating a bias toward self-renewal. Surprisingly, some cells in culture synthesized DNA and expressed involucrin simultaneously. In psoriatic plaques, involucrin expression begins closer to the basal layer than in normal epidermis, and here too we found S-phase involucrin-positive cells. We also observed occasional mitotic involucrin-positive cells in psoriatic epidermis, although we were unable to detect them in culture. Our experiments show that temporal separation of proliferation and terminal differentiation is not obligatory, and thus, the kinetic organization of epidermis may be less rigid than some models imply.     

表皮移植联合叶酸及腺苷钴胺治疗白癜风临床疗效观察

目的：探索以表皮磨削 负压吸疱自体表皮移植术联合叶酸及腺苷钴胺治疗白癜风的效果。方法：36例稳定期白癜风患者在采用表皮磨削 负压吸疱自体表皮移植术的基础上联合服用叶酸及腺苷钴胺，并与35例单纯接受手术者进行了比较；观察术后复色开始出现的时间及第8周被移植区出现色素情况。结果：两组相比，复色开始出现的时间及第8周受区复色面积／实际覆盖皮片面积之比均有显著性差异。结论：叶酸及腺苷钴胺用于表皮磨削 负压吸疱自体表皮移植术，可使受区复色时间提前并克服了色素生长欠均匀之缺陷。