Cellular mechanisms regulating human melanogenesis.

The major differentiated function of melanocytes is the synthesis of melanin, a pigmented heteropolymer that is synthesized in specialized cellular organelles termed melanosomes. Mature melanosomes are transferred to neighboring keratinocytes and are arranged in a supranuclear cap, protecting the DNA against incident ultraviolet light (UV) irradiation. The synthesis and distribution of melanin in the epidermis involves several steps: transcription of melanogenic proteins, melanosome biogenesis, sorting of melanogenic proteins into the melanosomes, transport of melanosomes to the tips of melanocyte dendrites and finally transfer into keratinocytes. These events are tightly regulated by a variety of paracrine and autocrine factors in response to endogenous and exogenous stimuli, principally UV irradiation.

Photoageing: mechanism, prevention and therapy.

Photoageing is the superposition of chronic ultraviolet (UV)-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. It is triggered by receptor-initiated signalling, mitochondrial damage, protein oxidation and telomere-based DNA damage responses. Photodamaged skin displays variable epidermal thickness, dermal elastosis, decreased/fragmented collagen, increased matrix-degrading metalloproteinases, inflammatory infiltrates and vessel ectasia. The development of cosmetically pleasing sunscreens that protect against both UVA and UVB irradiation as well as products such as tretinoin that antagonize the UV signalling pathways leading to photoageing are major steps forward in preventing and reversing photoageing. Improved understanding of the skin's innate UV protective mechanisms has also given rise to several novel treatment concepts that promise to revolutionize this field within the coming decade. Such advances should not only allow for the improved appearance of skin in middle age and beyond, but also greatly reduce the accompanying burden of skin cancer.

A cyclic peptide that binds p75(NTR) protects neurones from beta amyloid (1-40)-induced cell death.

The current study determined the ability of a p75(NTR) antagonistic cyclic peptide to rescue cells from beta amyloid (Abeta) (1-40)-induced death. p75(NTR)-, p140(trkA)-NIH-3T3 cells or E17 foetal rat cortical neurones were incubated with 125I-NGF or 125I-Abeta (1-40) and increasing concentrations of the cyclic peptide (CATDIKGAEC). Peptide ability to displace 125I-NGF or 125I-Abeta (1-40) binding was determined. Duplicate cultures were preincubated with CATDIKGAEC (250 nM) or diluent and then stimulated with Abeta (1-40). Peptide ability to displace Abeta (1-40) binding, interfere with Abeta (1-40)-induced signalling and rescue cells from Abeta-mediated toxicity was determined by immunoprecipitation and autoradiography, Northern blotting, JNK activation, MTT and trypan blue assays. The peptide inhibited NGF and Abeta (1-40) binding to p75(NTR), but not to p140(trkA). Abeta (1-40) induced c-jun transcription (57.3% +/- 0.07%) in diluent-treated p75(NTR)-cells, but not in cells preincubated with the cyclic peptide. Also, at 250 nM, the peptide reduced Abeta (1-40)-induced phosphorylation of JNK by 71.8% +/- 0.03% and protected neurones against Abeta-induced toxicity as determined by: trypan blue exclusion assay (53% +/- 11% trypan blue-positive cells in diluent pretreated cultures vs. 28% +/- 5% in cyclic peptide-pretreated cultures); MTT assay (0.09 +/-0.03 units in diluent-pretreated cells vs. 0.12 +/- 0.004 units in cyclic peptide-pretreated cells); and visualization of representative microscopic fields. Our data suggest that a cyclic peptide homologous to amino acids 28-36 of NGF known to mediate binding to p75(NTR) can interfere with Abeta (1-40) signalling and rescue neurones from Abeta (1-40)-induced toxicity.

Estradiol induces proliferation of keratinocytes via a receptor mediated mechanism.

In this study, we investigated the effects of estradiol on the proliferation of neonatal keratinocytes, the expression of estrogen receptor isoforms, and the signaling mechanisms by which estradiol mediates cell growth. We demonstrate that estradiol binds neonatal keratinocytes with high affinity (Kd=5.2nM) and limited capacity (Bmax of 14.2fmol/mg of protein), confirming the presence of estrogen binding sites. Using specific antibodies, we demonstrate that keratinocytes express both estrogen receptor (ER)-alpha and ER-beta. At physiological concentrations, estradiol up-regulates the level of ER-alpha receptors in keratinocytes and induces keratinocyte proliferation. The proliferative effect of estradiol requires the availability of functional estrogen receptors, as it is abrogated by anti-estrogen administration. Estradiol effect on keratinocyte proliferation is most likely mediated in part by activation of a nongenomic, membrane-associated, signaling pathway involving activation of the extracellular signal regulated kinases 1 and 2 and in part by the genomic signaling pathway through activation of nuclear receptors.

Ageing and photoageing of keratinocytes and melanocytes.

An overview of keratinocyte and melanocyte function is provided. The processes of cutaneous ageing and photoageing are defined, and age-associated modulations in gene expression are described. The changes in keratinocytes and melanocytes that occur with skin ageing and photoageing and the characteristics of chronologically aged vs. photoaged skin are delineated. Mutations that are found in malignant and premalignant tumors of epidermal origin are described.

Skin aging: postulated mechanisms and consequent changes in structure and function.

Aging is a complex process influenced by telomere shortening and damage to cellular DNA. New insights into age-associated decrements in DNA damage repair are reviewed. Age-associated gross, histologic, and functional cutaneous deficits are delineated. Different treatment options for aged skin are examined.

Solar simulated irradiation modulates gene expression and activity of antioxidant enzymes in cultured human dermal fibroblasts.

Exposure of skin to solar irradiation generates reactive oxygen species that damage DNA, membranes, mitochondria and proteins. To protect against such damage, skin cells have evolved antioxidant enzymes including glutathione peroxidase (GSH-Px), copper and zinc-dependent superoxide dismutase (SOD1), the mitochondrial manganese-dependent superoxide dismutase (SOD2), and catalase. This report examines the effect of a single low or moderate dose exposure to solar-simulating combined UVB and UVA irradiation on the gene expression and activities of these antioxidant enzymes in cultured normal human fibroblasts. We find that both doses initially decrease GSH-Px, SOD2 and catalase activities, but within 5 days after irradiation the activities of the enzymes return to pre-irradiation level (catalase) or are induced slightly (SOD1, GSH-Px) or substantially (SOD2) above the basal level. For SOD1, SOD2 and catalase, the higher dose also detectably modulates the mRNA level of these enzymes. Our results indicate that the effects of a single physiologic solar simulated irradiation dose persist for at least several days and suggest that skin cells prepare for subsequent exposure to damaging irradiation by upregulating this antioxidant defense system, in particular the mitochondrial SOD2. Our findings are consistent with the existence of a broad-based SOS-like response in irradiated human skin.

Effect of daily versus intermittent sunscreen application on solar simulated UV radiation-induced skin response in humans.

BACKGROUND: Acute and chronic skin damage occurs as a consequence of solar UV radiation exposure. To diminish such skin damage, the dermatologic community advocates the daily use of sunscreens as part of a sun avoidance strategy. OBJECTIVE: We determined the effectiveness of a sunscreen product with a sunscreen protection factor (SPF) of 15 applied daily in preventing UV-induced histologic damage in human skin compared with the protection afforded by sunscreens with equal or higher SPF applied intermittently. METHODS: Twenty-four subjects were exposed to 2 minimal erythema doses of solar-simulated UV on 4 consecutive days. Three sunscreen products were applied to the buttock of each subject. One SPF 15 product was applied daily before exposure to UV and, to simulate intermittent product use, an SPF 15 or SPF 29 product was applied on 3 of 4 days, with one missed application on days 2, 3, or 4. Skin biopsy specimens were taken and processed for routine and immunohistochemical staining. Changes in number of sunburn cells and Langerhans cells as well as degree of inflammatory infiltrate and lysozyme immunostaining were determined. RESULTS: There was a statistically significant increase in the number of sunburn cells, degree of inflammation, and intensity of lysozyme staining, and there was a decrease in the number of Langerhans cells at sites where sunscreen application was missed as compared with unirradiated control and daily SPF 15 sunscreen-treated sites. CONCLUSION: Our data suggest that daily use of a sunscreen reduces the skin damage produced by UV exposure compared with intermittent use of equal or higher SPF products. The daily application of sunscreens in appropriate quantities reduces the harmful effects of solar UV radiation on skin. Compliance is essential for maximal benefit of sunscreens.

Mechanisms and implications of the age-associated decrease in DNA repair capacity.

Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6-4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6-4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.

Role of cytoplasmic dynein in melanosome transport in human melanocytes.

Cytoplasmic dynein is a microtubule-associated retrograde-directed motor molecule for transport of membrane-bound organelles. To determine whether cytoplasmic dynein is expressed in melanocytes, we performed reverse transcriptase polymerase chain reaction using melanocyte cDNA and primers complementary to human brain cytoplasmic dynein heavy chain. A polymerase chain reaction product of the expected molecular size was generated and the identity was confirmed by sequence analysis. Western blotting of total melanocyte proteins reacted with an anti-intermediate chain cytoplasmic dynein antibody identified the appropriate 74 kDa band. To determine whether cytoplasmic dynein plays a role in melanosome transport, duplicate cultures were treated with cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides and the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport of melanosomes from the cell body into the dendrites, whereas melanosome distribution was not affected in sense-treated melanocytes. To determine whether ultraviolet irradiation modifies cytoplasmic dynein expression, melanocyte cultures were exposed to increasing doses of solar-simulated irradiation, equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm2 induced cytoplasmic dynein protein, whereas doses of 30 mJ per cm2 or more were associated with decreased levels of cytoplasmic dynein compared with sham-irradiated controls. Our data show that cytoplasmic dynein participates in retrograde melanosomal transport in human melanocytes and suggest that the altered melanosomal distribution in skin after sun exposure is due, at least in part, to decreased cytoplasmic dynein levels resulting in augmented anterograde transport.

Kinesin participates in melanosomal movement along melanocyte dendrites.

Movement of melanosomes along melanocyte dendrites is necessary for the transfer of melanin pigment from melanocytes to basal and suprabasal keratinocytes, an event critical to epidermal photoprotection and maintenance of normal skin color. Recent murine data suggest that in melanocyte dendrites the microtubule-associated melanosome movement is bidirectional and that actin-associated myosin V secures the peripheral melanosomes, preparing them to be transferred to surrounding keratinocytes. We now report that human melanocytes express high levels of kinesin, a molecule that participates in microtubule-associated transport of organelles in other cell types, and that ultrastructurally kinesin molecules are closely associated with melanosomes. To determine whether kinesin participates in melanosomal transport, cultured melanocytes were treated with sense or antisense oligonucleotides complementary to kinesin heavy chain sequences. Antisense oligonucleotides decreased kinesin protein levels and inhibited the bidirectional movement of the melanosomes, promoting their backward movement. Furthermore, guinea pigs were exposed to ultraviolet B irradiation, known to enhance transport of melanosomes from melanocytes to epidermal keratinocytes, and then were treated with kinesin sense or antisense oligonucleotides. The areas that were treated with kinesin antisense oligonucleotides showed significantly less pigmentation clinically and histologically than control (sense) oligonucleotide-treated areas. As observed ultrastructurally, in antisense-treated areas melanosomes remained in melanocyte dendrites but over several days were not transferred to the surrounding keratinocytes. Our study supports a major role for kinesin in microtubule-associated anterograde melanosomal transport in human melanocyte dendrites.

Thymidine dinucleotides inhibit contact hypersensitivity and activate the gene for tumor necrosis factor alpha1.

DNA is a target for ultraviolet-B-induced inhibition of contact hypersensitivity, and small DNA fragments such as thymidine dinucleotides (pTpT) can simulate several ultraviolet-induced effects. To determine whether pTpT mimics the suppressive influence of ultraviolet-B on contact hypersensitivity, we compared the effects of topical application of pTpT with those of ultraviolet-B irradiation on C57BL/6 mice sensitized to dinitrofluorobenzene. Mice pretreated with pTpT or ultraviolet-B irradiation showed markedly suppressed ear swelling responses to dinitrofluorobenzene challenge. Because tumor necrosis factor alpha mediates ultraviolet-B-induced suppression of contact hypersensitivity, and because pTpT exerts many ultraviolet-mimetic effects by augmenting mRNA and protein levels of effector molecules, we asked if pTpT mimics ultraviolet-B's upregulatory influence on tumor necrosis factor alpha expression. Using transgenic mice carrying a chloramphenicol acetyl transferase reporter linked to the tumor necrosis factor alpha promoter, we examined effects of ultraviolet-B irradiation versus intradermal injection of pTpT on tumor necrosis factor alpha gene transcription. Both treatments induced cutaneous chloramphenicol acetyl transferase activity. Ultra- violet-B or pTpT treatment of cultured dermal fibroblasts from these mice also stimulated chloramphenicol acetyl transferase activity. To determine whether human cells responded similarly, a well- differentiated ultraviolet-responsive human squamous cell carcinoma line was treated with pTpT. pTpT increased tumor necrosis factor alpha mRNA expression and protein secretion in a dose-dependent manner. Our findings expand the spectrum of ultraviolet effects mimicked by pTpT to include inhibition of contact hypersensitivity and activation of the tumor necrosis factor alpha gene. These results support the hypothesis that DNA photoproducts and/or their repair intermediates trigger many of the biologic consequences of ultraviolet irradiation.

Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins.

The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.

Aging versus photoaging: postulated mechanisms and effectors.

The differences between intrinsic aging and photoaging are reviewed. The various model systems currently employed for the studies of aging and photoaging are discussed. Findings on age associated decrements in receptor/ligand mediated signaling as well as changes during cellular senescence in the expression of nuclear transcription factors are described. The role of telomere shortening and oxidative damage in the aging process is explained. At the cellular level, genetic and behavioral differences between aging and photoaging are illustrated with particular emphasis on changes in the structure and function of the tumor suppressor gene p53.

Post-transcriptional regulation of UV induced TNF-alpha expression.

Ultraviolet (UV) irradiation exerts multiple effects on skin cells, including the induction of several cytokines involved in immunomodulation. Specifically, UV irradiation has been shown to upregulate the level of tumor necrosis factor-alpha (TNF-alpha) mRNA in keratinocytes. To determine whether the induction of TNF-alpha mRNA is regulated by transcriptional or post-transcriptional mechanisms, we examined cells of keratinocytic lineage (SCC12F) for steady state level, transcription rate, and stability of TNF-alpha mRNA after UV irradiation. Within 4 h there was a 20-40-fold induction of TNF-alpha mRNA that persisted at lower levels through 48 h. Consistently, TNF-alpha protein secretion increased at 24 and 48 h after UV irradiation. UV irradiation increased the half-life of TNF-alpha mRNA from approximately 35 min to approximately 10 h. Conversely, the transcription rate of the TNF-alpha gene increased < 2-fold at the time of peak mRNA steady state levels. Thus, post-transcriptional mechanisms play a major role in UV induced TNF-alpha transcript level.

Binding of beta-amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disorder characterized by the extracellular deposition in the brain of aggregated beta-amyloid peptide, presumed to play a pathogenic role, and by preferential loss of neurons that express the 75-kD neurotrophin receptor (p75NTR). Using rat cortical neurons and NIH-3T3 cell line engineered to stably express p75NTR, we find that the beta-amyloid peptide specifically binds the p75NTR. Furthermore, 3T3 cells expressing p75NTR, but not wild-type control cells lacking the receptor, undergo apoptosis in the presence of aggregated beta-amyloid. Normal neural crest-derived melanocytes that express physiologic levels of p75NTR undergo apoptosis in the presence of aggregated beta-amyloid, but not in the presence of control peptide synthesized in reverse. These data imply that neuronal death in Alzheimer's disease is mediated, at least in part, by the interaction of beta-amyloid with p75NTR, and suggest new targets for therapeutic intervention.