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Laser resurfacing treatments for photoaged skin have improved dramatically over the past decades, but few studies have examined the molecular mechanisms underlying differences in clinical response. Seventeen white female participants with moderate-to-severe photoaging received nonablative fractional laser treatment on the face and forearm once monthly for 6 months. Biopsies for microarray analysis were performed at baseline and 7 days after facial treatment and at baseline and 1, 7, 14, and 29 days after forearm treatment in each participant, resulting in 119 total samples. Participants were stratified into fast (n = 11) and slow (n = 6) responders on the basis of the presence of clinical improvement after the first treatment. Microarray analysis revealed the upregulation of genes associated with matrix metalloproteinases, collagen and extracellular components, TGF-β signaling, double-stranded RNA signaling, and retinoic acid synthesis after treatment that did not differ significantly between fast and slow responders. Cluster and enrichment analyses suggested significantly greater activation of lipid metabolism and keratinocyte differentiation in fast responders, who showed greater upregulation of acyltransferases, fatty acid elongases, fatty acid 2-hydroxylase, fatty acid desaturases, and specific keratins that may contribute to epidermal barrier function. These results create, to our knowledge, a previously unreported atlas of molecular changes that correlate with improvements in photoaging after laser therapy.

Prurigo nodularis is an understudied, chronic inflammatory skin disease that disproportionately affects African-Americans and presents with intensely pruritic nodules of unknown etiology. To better characterize immune dysregulation in prurigo nodularis, peripheral blood mononuclear cells and skin biopsies were obtained from majority African American patients with prurigo nodularis and healthy subjects matched by age, race, and sex. Flow cytometric analysis of functional T-cell response comparing prurigo nodularis to healthy subjects identified increased γδT-cells (CD3(+)CD4(−)CD8(−)γδTCR(+)) with Vδ2(+) γδT-enrichment. Activated T-cells demonstrated uniquely increased IL-22 cytokine expression in prurigo nodularis patients when compared to healthy controls. CD4+ and CD8+ T-cells were identified as the source of increased circulating IL-22. Consistent with these findings, RNA-sequencing of lesional prurigo nodularis skin compared to non-lesional prurigo nodularis skin and biopsy site matched control skin demonstrated robust up-regulation of Th22-related genes and signaling networks implicated in impaired epidermal differentiation. Th22-related cytokine upregulation remained significant with stratifications by race and biopsy site. Importantly, the expression of the IL-22 receptors IL22RA1/A2 was significantly elevated in lesional prurigo nodularis skin. These results indicate both systemic and cutaneous immune responses in prurigo nodularis patients are skewed towards a Th22/IL-22 profile. Prurigo nodularis may benefit from immunomodulatory therapies directed at Th22-mediated inflammation.

This report provides evidence from a number of different approaches (i.e., comparison of cell shape in 1-microm sections of photodamaged versus healthy skin at the light microscopic level; comparison of cell shape and apposition to collagen fibrils in ultrathin sections of the same tissues examined by transmission electron microscopy, and fluorescence staining for adhesion site protein expression and actin filament architecture in frozen tissue sections) that dermal cells in healthy skin are attached to collagen fibrils over a large part of the cell border, have a flattened/spread (two-dimensional) appearance and have abundant actin in their cytoplasm. In contrast, cells in photodamaged skin are often in contact with fragmented collagen or amorphous debris rather than intact collagen, have a collapsed/elongated shape, and have a lower amount of actin. Collagen synthesis is reduced in severely photodamaged skin relative to collagen synthesis in corresponding sun-protected skin (N Engl J Med 329:530, 1993). We hypothesize that fibroblasts in severely damaged skin have less interaction with intact collagen and as a result experience a reduction in mechanical tension. Decreased collagen synthesis is (presumed to be) the result.

Human skin is exposed to solar ultraviolet radiation. Ultraviolet radiation damages human skin and results in an old and wrinkled appearance, called photoaging. We have previously reported that molecular mechanisms by which ultraviolet light causes photoaging involve activation of growth factor and cytokine receptors in keratinocytes and dermal cells. They lead to downstream signal transduction through activation of mitogen-activated protein kinase (extracellular signal-regulated kinase, c-jun N-terminal protein kinase, and p38) pathways. These signaling pathways converge in the nucleus of cells to form an activated complex of transcription factor activator protein 1 (cFos/cJun), which induces matrix metalloproteinases that degrade skin connective tissue. In addition to cell surface receptor activation, generation of reactive oxygen species by ultraviolet radiation is believed to be critical in triggering mitogen-activated protein kinase pathways. We investigated the ability of (i) ultraviolet irradiation to generate reactive oxygen species in human skin in vivo; and (ii) genistein, which possesses both tyrosine kinase inhibitory and antioxidant activities, and n-acetyl cysteine, which can be converted into the endogenous antioxidant glutathione, to impair responses to ultraviolet light that eventuate in photoaging in human skin in vivo. Ultraviolet irradiation caused a rapid and significant increase in hydrogen peroxide levels in human skin in vivo. Pretreatment of human skin with genistein inhibited ultraviolet-induced epidermal growth factor receptor tyrosine kinase activity, whereas n-acetyl cysteine did not. Genistein inhibited ultraviolet induction of both extracellular signal-regulated kinase and cJun N-terminal protein kinase activities. n-Acetyl cysteine inhibited extracellular signal-regulated kinase but not cJun N-terminal protein kinase activation. Both genistein and n-acetyl cysteine prevented ultraviolet induction of cJun protein. Consistent with this, genistein and n-acetyl cysteine blocked ultraviolet induction of cJun-driven enzyme, collagenase. Neither genistein nor n-acetyl cysteine acted as sunscreens as they had no effect on ultraviolet-induced erythema. These data indicate that compounds similar to genistein and n-acetyl cysteine, which possess tyrosine kinase inhibitory and/or antioxidant activities, may prevent photoaging.

Damage to human skin due to ultraviolet light from the sun (photoaging) and damage occurring as a consequence of the passage of time (chronologic or natural aging) are considered to be distinct entities. Photoaging is caused in part by damage to skin connective tissue by increased elaboration of collagen-degrading matrix metalloproteinases, and by reduced collagen synthesis. As matrix metalloproteinase levels are known to rise in fibroblasts as a function of age, and as oxidant stress is believed to underlie changes associated with both photoaging and natural aging, we determined whether natural skin aging, like photoaging, gives rise to increased matrix metalloproteinases and reduced collagen synthesis. In addition, we determined whether topical vitamin A (retinol) could stimulate new collagen deposition in sun-protected aged skin, as it does in photoaged skin. Sun-protected skin samples were obtained from 72 individuals in four age groups: 18–29 y, 30–59 y, 60–79 y, and 80+ y. Histologic and cellular markers of connective tissue abnormalities were significantly elevated in the 60–79 y and 80+ y groups, compared with the two younger age groups. Increased matrix metalloproteinase levels and decreased collagen synthesis/expression were associated with this connective tissue damage. In a separate group of 53 individuals (80+ y of age), topical application of 1% vitamin A for 7 d increased fibroblast growth and collagen synthesis, and concomitantly reduced the levels of matrix-degrading matrix metalloproteinases. Our findings indicate that naturally aged, sun-protected skin and photoaged skin share important molecular features including connective tissue damage, elevated matrix metalloproteinase levels, and reduced collagen production. In addition, vitamin A treatment reduces matrix metalloproteinase expression and stimulates collagen synthesis in naturally aged, sun-protected skin, as it does in photoaged skin.

1,25-Dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] transactivates its target genes via the vitamin D receptor (VDR). VDR functions in physiology as a dimer complexed with retinoid X receptor (RXR), whose natural ligand is 9- cis -retinoic acid (9- c -RA). Inactivation of 1,25(OH) 2 D 3 occurs through a cytochrome P-450 24-hydroxylase (OHase). The promoter of the human 24-OHase gene contains a 1,25(OH) 2 D 3 -responsive enhancer element (VDRE). We have used this VDRE containing gene as an endogenous reporter for vitamin D 3 -mediated gene activation in vivo . Normal adult human skin was keratomed after a 2- d exposure to 1,25(OH) 2 D 3 , 9- c -RA, all- trans -RA, and ketoconazole. 1,25(OH) 2 D 3 caused a concentration-dependent increase in 24-OHase mRNA expression as determined by northern blot analysis. The activity of epidermal 24-OHase was also induced by 1,25(OH) 2 D 3 . Compared with vehicle, neither of the BA isomers nor ketoconazole alone induced 24-OHase mRNA. Addition of 9- c -RA or t -RA to 1,25(OH) 2 D 3 , however, caused a synergistic increase in 24-OHase inRNA. Similarly, 1,25(OH) 2 D 3 plus ketoconazole increased 24-OHase mRNA synergistically. Ketoconazole inhibited ex vivo 1,25(OH) 2 D 3 induced epidermal 24-OHase activity. Thus, 24-OHase znRNA induction is a sensitive reporter of 1,25(OH) 2 D 3 activity in vivo ; RXR bound to VDR is not a silent partner in vivo , because 9- c -RA enhances 1,25(OH) 2 D 3 -liganded RXR/VDR stimulation of the VDRE containing 24-OHase gene; ketoconazole inhibition of 24-OHase enhances 1,25(OH) 2 D 3 activity by impeding its breakdown.Thus, the synergistic response of human skin to topical 1,25(OH) 2 D 3 and/or 1,25(OH) 2 D 3 analogs plus RXR retinoids and/or ketoconazole may be exploited to give a desired biologic/therapeutic response with less 1,25(OH) 2 D 3 , minimizing the potential calcemic risk from systemic absorption of 1,25(OH) 2 D 3 .

Metabolic inactivation of all-trans retinoic acid to 4-hydroxy retinoic acid occurs via a cytochrome P-450 enzyme. We investigated the effects of liarozole on the retinoic acid 4-hydroxylase activity of human epidermis and its ability to modify in vivo human skin responses to retinoic acid and all-trans retinol. Retinoic acid 4-hydroxylase activity induced in vivo by 4 d treatment with retinoic acid (0.1%) was inhibited in vitro by liarozole in a concentration-dependent manner. Comparable micromolar concentrations of liarozole were extracted from stratum corneum-free epidermis treated with 3% liarozole. Retinoic acid levels in liarozole-treated skin increased to 19 +/- 5 ng/g wet wt (mean +/- SEM, p < 0.002, n = 17) at 18 h and to 6 +/- 2 ng/g wet wt (p = 0.38, n = 17) at 48 h as compared to vehicle (not detectable). At 48 h, retinoic acid 4-hydroxylase activity was induced 9-fold over vehicle (p < 0.03, n = 8). At 96 h, no significant erythema or increased epidermal thickness was found when either retinoic acid (0.001%), all-trans retinol (0.0250%), or liarozole (3%) was applied individually, but when 0.001% retinoic acid and 3% liarozole were applied together, both erythema and increased epidermal thickness occurred. In contrast, 0.025% all-trans retinol and 3% liarozole together caused increased epidermal thickness but no erythema. These data demonstrate that, at doses used here, liarozole, although an effective inhibitor of retinoic acid 4-hydroxylase, cannot function alone like a retinoid in vivo, probably because of retinoic acid 4-hydroxylase induction. In the presence of a low dose retinoic acid or all-trans retinol, however, liarozole can amplify human skin responses to each retinoid in a manner characteristic of the retinoid at a higher dose (erythema and hyperplasia with retinoic acid; no erythema but hyperplasia with all-trans retinol).

Cellular retinoic acid binding protein II (CRABP II) mRNA is selectively induced by all-trans retinoic acid in human skin and dermal fibroblasts. In order to determine whether this response can be used as a reliable measure of retinoid potency and activity, we treated human skin fibroblasts for 24 h with increasing concentrations of several natural and synthetic retinoids. CRABP II mRNA levels were measured by quantitative Northern blotting and compared, when possible, with those obtained after topical application of the same retinoids to human skin. All eight active retinoids tested induced a concentration-dependent CRABP II mRNA response in the fibroblast assay. In contrast, one known inactive retinoid (meta-carboxy TTNPB), differing from the active form only in the position of the carboxyl substituent, failed to evoke a response. The fibroblast and human skin bioassays agreed with respect to relative potency and response amplitude for three of the three retinoids tested. Retinoic acid was approximately 10-fold more potent than retinal in both assays, suggesting that oxidation to retinoic acid underlies the activity of retinol in fibroblasts as well as in intact skin. In support of this hypothesis, treatment with liarozole, an inhibitor of P450-mediated retinoic acid oxidative catabolism, significantly increased fibroblast CRABP II mRNA levels and potentiated the effects of retinol by 1.5-fold at concentrations at which it had no effect on its own. Taken together, these results identify the fibroblast CRABP II response as a reproducible measure of retinoid bioactivity with promise as a predictor of human skin responses and further suggest that metabolism is an important determinant of retinoid bioactivity in vivo.

Application of all-trans retinoic acid to human skin for 4 d under occlusion produces a marked increase in retinoic acid 4-hydroxylase activity. In this study, the possible induction of other hydroxylase in response to 9-cis and 13-cis retinoic acid application to adult human skin in vivo was determined. Application of 0.1% all-trans, 0.1% 9-cis, and 0.1% 13-cis retinoic acid to human skin for 2 d resulted in induction of only all-trans retinoic acid 4-hydroxylase activity. The 4-hydroxylase activity in microsomes from the treated tissue ranged from 838 +/- 46 to 531 +/- 59 pg of 4- hydroxy all-trans retinoic acid formed/min/mg protein (n=6). These same preparations were unable to use 9-cis or 13-cis retinoic acid as substrate for the hydroxylation reaction. Extraction of the retinoic acid isomers from epidermis 48 h after application of 0.1% solution of each isomer yielded significant amounts of all-trans retinoic acid (36-72%) regardless of the isomer applied. The all-trans isomer produced by isomerization of both 9-cis and 13-cis retinoic acids is the likely inducer of the 4-hydroxylase. All-trans retinol and all-trans retinal were unable to compete with all-trans retinoic acid as substrate for 4-hydroxylase enzyme. The 4-hydroxylase induced in response to pharmacological doses of retinoic acids is specific for the all-trans isomer. The inability of 9-cis or 13-cis retinoic acid to induce their own hydroxylation and inactivation or act as substrate for the 4-hydroxylase in skin may have considerable implications in light of the clinical use of retinoids in the treatment of various diseases including cancers.

We investigated the clinical, histologic, and molecular responses of normal human skin to all-trans-retinol (ROL) application, compared to those induced by topical all-trans-retinoic acid (RA), and measured ROL-derived metabolites. Up to 1.6% ROL, 0.025% RA in vehicle (70% ethanol/30% propylene glycol), or vehicle alone were applied in a double-blind fashion to normal buttock skin and occluded for 4 d. ROL produced from none to only trace erythema, which was clinically and statistically insignificant, whereas RA induced a significant 3.7-fold increase in erythema score compared to vehicle (n = 10, p0.01). However, ROL induced significant epidermal thickening (1.5-fold at 1.6% ROL, p0.01), similar to RA (1.6-fold at 0.025% RA, p0.01), relative to the vehicle. ROL, compared with vehicle, also increased mRNA levels of cellular retinoic acid binding protein (CRABP-II) and cellular retinol binding protein (CRBP) genes as determined by Northern analysis (5-6-fold and 6-7-fold, respectively) and riboprobe in situ hybridization. CRABP-II and CRBP protein levels were also higher following ROL than vehicle treatment, as measured by ligand binding (3.2-fold, p0.001; n = 7) and Western analysis (3.6-fold, p0.003; n = 6), respectively. Epidermal retinyl ester (RE) content, measured after removal of stratum corneum, rose 240-fold (p0.005, n = 5) by 24 h of ROL occlusion. RA content, however, was undetectable or detectable only at trace amounts in all samples obtained at 0, 6, 24, and 96 h after ROL occlusion. Detectability of RA was not correlated with ROL treatment (compared to untreated normal skin, p = 0.86) or baseline skin ROL levels (average r = -0.1, p0.3). These data demonstrate that ROL application 1) produces trace erythema not significantly different from vehicle, whereas RA causes erythema; 2) induces epidermal thickening and enhances expression of CRABP-II and CRBP mRNAs and proteins as does RA; 3) causes marked accumulation of retinyl ester; and 4) does not significantly increase RA levels. Taken together, the data are compatible with the idea that ROL may be a prohormone of RA, because it produces changes in skin similar to those produced by RA but without measurable RA or irritation.

We examined the regulation of cellular retinol-binding protein (CRBP) mRNA and protein expression in human skin in vivo by all-trans retinoic acid and all-trans retinol. Treatment of human skin for 24 h with all-trans retinoic acid (0.1%) or all-trans retinol (1.6%) induced CRBP mRNA 5.5-fold (p < 0.01, n = 10) and 5.7-fold (p < 0.01, n = 5), respectively, compared with skin treated with vehicle or sodium lauryl sulfate (used as an irritant control). In vitro translation of poly A+ RNA from all-trans retinoic acid, all-trans retinol, sodium lauryl sulfate, and vehicle-treated human skin demonstrated that the observed increased CRBP mRNA in all-trans retinoic acid- and all-trans retinol-treated skin was able to direct increased (2.3-2.9-fold) CRBP protein synthesis. Riboprobe in situ hybridization revealed that CRBP mRNA was uniformly elevated throughout the epidermis and in dermal cells after all-trans retinoic acid treatment of human skin. Western analysis revealed that CRBP protein was elevated 3.2-fold (p 0.01, n = 6) and 3.0-fold (p < 0.01, n = 6) after all-trans retinoic acid treatment of human skin in vivo for 24 and 96 h, respectively, compared with vehicle and sodium lauryl sulfate-treated skin. In addition, functional CRBP levels measured by [3H]all-trans retinol binding were elevated 1.9-fold (p < 0.01, n = 6) and 3.5-fold (p < 0.01, n = 6) at 24 and 94 h, respectively, after all-trans retinoic acid treatment, compared with vehicle- or sodium lauryl sulfatetreated skin. Gel mobility shift analysis revealed that retinoid receptors in nuclear extracts from human skin formed a specific complex with a DNA probe containing the retinoic acid response element in the mouse CRBP gene. Monoclonal antibodies to nuclear retinoid receptors demonstrated that predominantly retinoic acid receptor-α/retinoid X receptor-α heterodimers bound to the CRBP retinoic acid response element. These data demonstrate that CRBP expression in human skin in vivo is regulated by exogenous all-trans retinoic acid and all-trans retinol.