[Photoprotective mechanisms of human skin. Modulation by oligonucleotides]. / Photoprotektive Mechanismen in menschlicher Haut. Modulation durch Oligonukleotide.

There are at least two classic photoprotective DNA damage responses that can be elicited by UV exposure: induction of melanogenesis (tanning) and enhancement of DNA repair. Both mechanisms are mediated, at least in part, by the tumor-suppressor protein and transcription factor p53. Both of these responses can be induced in vitro as well as in vivo by small DNA fragments of specific sequences, without prior induction of actual DNA damage. The topical application of such fragments onto human skin might enhance photoprotection in human skin, as typically elicited by gradual sun exposure. The induction of photoprotection by this means, however, would not bear the mutagenic and carcinogenic risk of exposure to natural sunlight.

Stimulation of melanogenesis by DNA oligonucleotides: effect of size, sequence and 5' phosphorylation.

It has been shown that the small DNA fragment thymidine dinucleotide, (pTpT) induces photoprotective responses in cultured cells and intact skin. These responses include increased melanogenesis, enhanced DNA repair, and induction of TNF-alpha, and are accomplished, at least in part, through the induction and activation of the p53 tumor suppressor and transcription factor. Here it is reported that other, but not all, larger oligonucleotides induce the pigmentation response even more efficiently than pTpT. A 9 base oligonucleotide (p9mer) stimulated pigmentation in Cloudman S91 murine melanoma cells to 6-times the level of control cells while a 5 base oligonucleotide (p5mer#1) was inactive. In addition, the p9mer increased p21 mRNA levels and inhibited cell proliferation to a greater degree than did pTpT, consistent with the presumptive mechanism of action involving p53. Smaller, truncated versions of the p9mer also stimulated pigmentation, although to a lesser extent than did the p9mer. The ability of these oligonucleotides to stimulate pigmentation was highly dependent on the presence of a 5' phosphate group on the molecule, which was shown by confocal microscopy and fluorescent activated cell sorter (FACS) analysis to greatly facilitate the uptake of these oligonucleotides into the cells. Although the melanogenic activity of the oligonucleotides was directly related to increased length and 5' phosphorylation, nucleotide sequence is also critical because a p20mer was efficiently internalized yet was a poor inducer of pigmentation.

Tanning as part of the eukaryotic SOS response.

We have determined that DNA damage is at least one of the signals generated by ultraviolet radiation that stimulates pigmentation (tanning) in human skin. This photoprotective response is functionally similar to the SOS response described in bacteria. Here we present evidence that DNA damage stimulates pigmentation, at least in part, through up-regulation of tyrosinase mRNA and protein levels. Furthermore, this response can be induced in the absence of DNA damage by treatment of melanocytic cells and intact skin with small DNA fragments, particularly thymidine dinucleotides, pTpT. Topical application of these DNA fragments should provide a photoprotective tan to human skin without the harmful effects of ultraviolet radiation.

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.

Skin aging and photoaging: the role of DNA damage and repair.

Both genetic (intrinsic) and environmental (extrinsic) factors contribute to the phenotypic changes in cutaneous aging. However, only recently have the underlying molecular mechanisms involved in these changes been elucidated. DNA damage to both genomic and mitochondrial DNA and subsequent DNA repair contribute greatly to age-associated skin changes and carcinogenesis. Better understanding of these intricate, interwoven mechanisms involved in DNA damage and repair might help to develop new strategies in preventing and treating changes of intrinsic skin aging and photoaging, improving skin appearance and reducing the risk of skin cancer.

DNA photodamage stimulates melanogenesis and other photoprotective responses.

Ultraviolet (UV) irradiation is a major source of environmental damage to skin. Melanin pigmentation protects against this damage by absorbing UV photons and UV-generated free radicals before they can react with DNA and other critical cellular components; and UV-induced melanogenesis or tanning is widely recognized as exposed skin's major defense against further UV damage. This article reviews extensive data suggesting DNA damage or DNA repair intermediates directly triggers tanning and other photoprotective responses. Evidence includes the observations that tanning is enhanced in cultured pigment cells by accelerating repair of UV-induced cyclobutane pyrimidine dimers or by treating the cells with UV-mimetic DNA-damaging chemicals. Moreover, small single stranded DNA fragments such as thymidine dinucleotides (pTpT), the substrate for almost all DNA photoproducts, also stimulates tanning when added to cultured pigment cells or applied topically to intact skin. In bacteria, single stranded DNA generated by DNA damage or its repair activates a protease that in turn derepresses over 20 genes whose protein products enhance DNA repair and otherwise promote cell survival, a phenomenon termed the SOS response. Interestingly, pTpT also enhances repair of UV-induced DNA damage in human cells and animal skin, at least in part by activating the tumor suppressor protein and transcription factor p53 and thus upregulating a variety of gene products involved in DNA repair and cell cycle regulation. Together, these data suggest that human cells have an evolutionarily conserved SOS-like response in which UV-induced DNA damage serves as signal to induce photoprotective responses such as tanning and increased DNA repair capacity. The responses can also be triggered in the absence of DNA damage by addition of small single-stranded DNA fragments such as pTpT.

Enhanced repair of benzo(a)pyrene-induced DNA damage in human cells treated with thymidine dinucleotides.

The small DNA fragment thymidine dinucleotide (pTpT) stimulates photoprotective responses in mammalian cells and intact skin. These responses include increased melanogenesis (tanning) and enhanced repair of DNA damage induced by ultraviolet (UV) light. Here we show that pTpT treatment of human keratinocytes enhances their repair of DNA damaged by the chemical carcinogen benzo(a)pyrene (BP), as determined by increased expression of a transfected BP-damaged reporter plasmid containing the chloramphenicol acetyltransferase (CAT) gene. The pTpT-enhanced repair of this BP-damaged plasmid is accomplished at least in part through activation of the p53 tumor suppressor protein and transcription factor, because p53-null H1299 cells showed enhanced repair only if previously transfected with a p53-expression vector. To elucidate the mechanism of this enhanced DNA repair, we examined the expression of p21 and proliferating cell nuclear antigen (PCNA), proteins known to be regulated by p53, as well as the XPA protein, which is mutated in the inherited repair-deficient disorder xeroderma pigmentosum (XP) group A and is necessary for the recognition of UV-induced DNA photoproducts. The p53, PCNA and XPA proteins were all up-regulated within 48 h after the addition of pTpT. Taken together, these data demonstrate that pTpT-enhanced repair of DNA damaged by either UV irradiation or chemical mutagens can be achieved in human cells by exposure to small DNA fragments at least in part through the activation of p53 and increased expression of p53-regulated genes.

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.

Age-associated decreases in human DNA repair capacity: Implications for the skin.

Multiple pathways are involved in accurate synthesis and distribution of DNA during replication, repair and maintenance of genomic integrity. An increased error rate, abovethe spontaneous mutation baseline, has been implicated in carcinogenesis and aging. Moreover, cytogenetic abnormalities are increased in Down's, Edwards', Patau's, and Klinefelter's syndromes with increasing maternal age, and in Marfan's and Apert's syndromes with paternal age. In response to DNA damage, multiple overlapping systems of DNA repair have evolved, preferentially repairing the transcribed strand within transcriptionally-active regions of the genome. These include direct reversal of dimers and specific adducts and pathways for base excision, nucleotide excision, and mismatch repair. A consensus has emerged that some DNA repair capacities decline with organism age, contradictory reports notwithstanding. As is the case for inborn defects in humans, knockout mice lacking components of nucleotide excision repair or DNA-damage checkpoint arrest have increased frequencies of skin and internal cancers, whereas mice overexpressing DNA repair genes have fewer spontaneous cancers. Oxidative stress and resultant free radicals can damage genomic and mitochondrial DNA; damage increases with age but decreases with caloric restriction. We review recent studies of long-lived C. elegans mutants which appear to involve metabolic attenuation, the role of telomere shortening and telomerase in cellular senescence, and the genetic bases of progeroid syndromes in humans. Finally, we discuss roles of extrinsic and intrinsic factors in skin aging, and their association with DNA damage, emphasizing preventive and protective measures and prospects for intervention by modulating DNA repair pathways in the skin.

Keratinocytes and dermal factors activate CRABP-I in melanocytes.

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.

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.

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: evidence for a p53-mediated mammalian SOS response.

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6-4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

DNA damage enhances melanogenesis.

Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin in vivo induces a pigment response indistinguishable from UV-induced tanning. Here we present further evidence that DNA damage and/or the repair of this damage increases melanization. (i) Treatment with the restriction enzyme Pvu II or the DNA-damaging chemical agents methyl methanesulfonate (MMS) or 4-nitroquinoline 1-oxide (4-NQO) produces a 4- to 10-fold increase in melanin content in Cloudman S91 murine melanoma cells and an up to 70% increase in normal human melanocytes, (ii) UV irradiation, MMS, and pTpT all upregulate the mRNA level for tyrosinase, the rate-limiting enzyme in melanin biosynthesis. (iii) Treatment with pTpT or MMS increases the response of S91 cells to melanocyte-stimulating hormone (MSH) and increases the binding of MSH to its cell surface receptor, as has been reported for UV irradiation. Together, these data suggest that UV-induced DNA damage and/or the repair of this damage is an important signal in the pigmentation response to UV irradiation. Because Pvu II acts exclusively on DNA and because MMS and 4-NQO, at the concentrations used, primarily interact with DNA, such a stimulus alone appears sufficient to induce melanogenesis. Of possible practical importance, the dinucleotide pTpT mimics most, if not all, of the effects of UV irradiation on pigmentation, tyrosinase mRNA regulation, and response to MSH without the requirement for antecedent DNA damage.

Mechanisms of ultraviolet light-induced pigmentation.

Work in the past 8 years, particularly in the past 1-2 years, has greatly expanded our understanding of the mechanisms by which ultraviolet irradiation stimulates melanogenesis in the skin. A direct effect of UV photons on DNA results in up-regulation of the gene for tyrosinase, the rate-limiting enzyme in melanin synthesis, as well as an increase in cell surface expression of receptors for at least one of the several known keratinocyte-derived melanogenic factors, MSH. Direct effects of UV on melanocyte membranes, releasing DAG and arachidonic acid, may also play a role in the tanning response. Diacylglycerol may activate PKC-beta, which in turn phosphorylates and activates tyrosinase protein; the pathways by which products of other inflammatory mediator cascades may act on melanogenesis are unknown. The tanning response also relies heavily on UV-stimulated increased production and release of numerous keratinocyte-derived factors including bFGF, NGF, endothelin-1 and the POMC-derived peptides MSH, ACTH, beta-LPH and beta-endorphin. These factors variably induce melanocyte mitosis, increase melanogenesis, enhance dendricity and prevent apoptotic cell death following the UV injury. Thus, events within the epidermal melanin unit conspire to maintain or increase melanocyte number, increase melanin pigment throughout the epidermis. Overall, ultraviolet-induced melanogenesis may be one part of a eukaryotic SOS response to damaging ultraviolet irradiation that has evolved over time to provide a protective tan in skin at risk of further injury from sun exposure. These recent insights into the mechanisms underlying ultraviolet-induced melanogenesis offer the opportunity for novel therapeutic approaches to minimizing acute and chronic photodamage in human skin.

A role for interleukin-1 in epidermal differentiation: regulation by expression of functional versus decoy receptors.

Although human epidermis contains levels of interleukin-1 (IL-1) up to 100 times higher than other tissues, the role of this cytokine in epidermal biology is unknown. Here, we show that interleukin-1 regulates the expression of mRNAs for two proteins associated with the differentiated phenotype of human keratinocytes, cellular retinoic acid-binding protein type II (CRABP II) and small, proline rich protein 1 (SPRR1). The ability of IL-1 to induce these transcripts correlates directly with keratinocyte expression of the IL-1 receptor type I (IL-1 RI) during differentiation and inversely with the expression of the type II IL-1 receptor (IL-1 RII), shown in other cell types to be a nonfunctional, decoy receptor. Furthermore, addition to keratinocyte cultures of an IL-1 RI-blocking, but not an IL-1 RII-blocking, antibody reduces the levels of CRABP II and SPRR1 mRNAs in these cells. These data suggest that epidermal IL-1 functions to promote keratinocyte differentiation and that a change in the IL-1 receptor profile of these cells initiates this IL-1 response through a relative enhanced expression of functional IL-1 receptors.

In vivo and in vitro SPRR1 gene expression in normal and malignant keratinocytes.

The small proline-rich protein 1 (SPRR1) gene encodes a precursor of the keratinocyte cornified envelope. To understand SPRR1 regulation we investigated its expression and modulation in keratinocytes in vivo and in vitro. SPRR1 was strongly expressed in suprabasal layers of the epidermis in newborn skin but only weakly expressed in adult skin. Both in vivo and in vitro, SPRR1 was not expressed in undifferentiated cells of basal or squamous carcinomas. However, within the same tumors and in premalignant lesions of squamous cell origin, cells with histologic evidence of differentiation showed a relative increase in SPRR1 transcript level. Within 24 h physiologic doses of uv irradiation induced SPRR1 mRNA in vivo. To investigate the possibility that SPRR1 expression is regulated by uv-induced cytokines, keratinocytes were stimulated with interleukin-1 (IL-1) and interleukin-3 (IL-3). Both significantly induced SPRR1 mRNA, while TGF-beta, known to lower IL-1 receptor in keratinocytes, down-regulated it. Moreover, proximity to inflammatory cells in vivo was associated with SPRR1 induction in anaplastic tumor cells. Our data suggest that SPRR1 is induced early in differentiation of normal keratinocytes but is not expressed in anaplastic cells of keratinocyte origin. Further, its regulation in skin appears to be modulated at least in part through cytokine release.

Regulation of CRABP II mRNA expression in human keratinocytes.

Cultured human neonatal keratinocytes were used to study the mechanisms and factors involved in the regulation of CRABP II gene expression. Post-confluent, relatively differentiated keratinocyte cultures had higher levels of CRABP II mRNA, but nuclear run-on experiments detected no sustained increase in CRABP II gene transcription rate between pre-confluent and post-confluent cells. Also, our studies could detect no change in the long half-life (> 32 hours) of this message in pre- and post-confluent cultures. Hydrocortisone was found to reduce the confluency-related increase in CRABP II mRNA in keratinocyte cultures. Because corticosteroids are known to reduce the effect of various cytokines, a series of epidermal cytokines were examined for a modulating effect on CRABP II mRNA content in cultured keratinocytes. IL1 alpha produced the greatest increase and IL6 the strongest reduction in the level of this message in cells grown in serum-free, defined medium. These data support a role for CRABP II in the proliferation and differentiation of human keratinocytes and suggest that epidermal cytokines may at least in part regulate the expression of the CRABP II gene at the mRNA level.