Pagetoid reticulosis in a prepubescent boy successfully treated with photodynamic therapy.

Pagetoid reticulosis or Woringer-Kolopp disease (WKD) is a rare variant of mycosis fungoides, consisting of localized patches or plaques containing intraepidermal proliferations of neoplastic T cells in a pagetoid distribution (similar to that of the adenocarcinomatous cells found in Paget disease of the nipple), which typically affects middle-aged and elderly men. We report a trial of photodynamic therapy (PDT) with topical aminolaevulinic acid (ALA), carried out on a 10-year-old boy with a solitary lesion of WKD on his foot, to avoid the long-term problems associated with the typical treatments for WKD of surgery and/or local irradiation. The plaque progressively flattened during treatment, and after nine PDT sessions over 13 months, the patient was clinically free of disease. PDT may be a viable alternative to surgery and local irradiation for localized cutaneous T-cell lymphoma, including WKD, especially in younger patients.

Innervation of melanocytes in human skin.

Communication between the nervous system and epidermal melanocytes has been suspected on the basis of their common embryologic origin and apparent parallel involvement in several disease processes, but never proven. In this study, confocal microscopic analysis of human skin sections stained with antibodies specific for melanocytes and nerve fibers showed intraepidermal nerve endings in contact with melanocytes. This intimate contact was confirmed by electron microscopy, which further demonstrated thickening of apposing plasma membranes between melanocytes and nerve fibers, similar to synaptic contacts seen in nervous tissue. Since many intraepidermal nerve fibers are afferent nerves that act in a "neurosecretory" fashion through their terminals, cultured human melanocytes were stimulated with calcitonin gene-related peptide (CGRP), substance P, or vasoactive intestinal peptide, neuropeptides known to be present in cutaneous nerves, to examine their possible functions in the epidermal melanin unit. CGRP increased DNA synthesis rate of melanocytes in a concentration- and time-dependent manner. Cell yields after 5 d were increased 25% compared with controls maintained in an otherwise optimized medium. Furthermore, stimulation by CGRP induced rapid and dose-dependent accumulation of intracellular cAMP, suggesting that the mitogenic effect is mediated by the cAMP pathway. These studies confirm and expand a single earlier report in an animal model of physical contact between melanocytes and cutaneous nerves and for the first time strongly suggest that the nervous system may exert a tonic effect on melanocytes in normal or diseased human skin.

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.

Normal human epidermis contains an interferon-like protein.

Interferons have been postulated to participate in growth regulation of normal body tissues and are known to inhibit growth of human epidermal keratinocytes in vitro. Polyclonal antibodies to recombinant human interferon-alpha, purified by passage over an affinity column (Sepharose coupled to the recombinant interferon), used in the indirect immunofluorescent method specifically stained the proliferative (basal) compartment of human epidermis in histological cross-sections of normal skin and in cultured keratinocyte colonies. Extracts prepared from healthy nonvirally infected keratinocyte cultures contained interferon activity as determined by viral plaque inhibition assay. Using the Western blotting technique column-purified antibodies and antisera to recombinant human interferon-alpha recognized a band of approximately 40 kD when reacted with both extracted keratinocyte proteins and recombinant human interferon-alpha standards, that gave in addition a band of approximately 20 kD. The above findings suggest that interferon or a closely related protein is present in the proliferative compartment of normal epidermis in the absence of viral infection and therefore may serve as a physiological modulator of epidermal growth.

Evidence for nerve growth factor-mediated paracrine effects in human epidermis.

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.

An endocrine approach to the control of epidermal growth: serum-free cultivation of human keratinocytes.

Human keratinocytes, derived from the skin of newborns and of adults, were grown in the complete absence of serum, in a hormone-supplemented medium on fibronectin-coated cell culture dishes at low seed density. The cell culture medium consisted of Medium 199 containing epidermal growth factor, triiodothyronine, hydrocortisone, Cohn fraction IV, insulin, transferrin, bovine brain extract, and trace elements. Removal of the brain extract from the hormone supplement had a greater negative impact on proliferation of the keratinocyte cultures than did the removal of epidermal growth factor, hydrocortisone, and triiodothyronine or Cohn fraction IV. The growth of keratinocytes in this hormone-supplemented medium suggests that control of keratinocyte growth depends in large part on endocrine stimulation by other body organs, including the brain.

The a-B-C-ds of sensible sun protection.

Ultraviolet (UV) radiation is a carcinogen that also compromises skin appearance and function. Since the UV action spectra for DNA damage, skin cancer, and vitamin D photosynthesis are identical, and vitamin D is readily available from oral supplements, why has sun protection become controversial? First, the media and, apparently, some researchers are hungry for a new message. They have also drawn attention to the emerging evidence of possible vitamin D benefits other than for bone health. Second, the controversy is fueled by a powerful special interest group: the tanning industry. This industry does not target the frail elderly or inner-city ethnic minorities, which are the groups at greatest risk of vitamin D deficiency, but rather fair-skinned teenagers and young adults, who are at highest risk of UV photodamage. Third, evolution does not keep pace with civilization. When nature gave humans the appealing capacity for cutaneous vitamin D photosynthesis, life expectancy was less than 40 years of age; long-term photodamage was not a concern, and vitamin D deficiency, with its resulting skeletal abnormalities (rickets), was likely to be fatal in early life. This article briefly reviews the pseudo-controversy , as well as the data supporting a revision of the recommendations for vitamin D supplementation. It concludes with a suggested message for patients, many of whom are understandably confused by recent media coverage of the topic.

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

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.

p53 is essential for chemotherapy-induced hair loss.

Anticancer drugs stimulate apoptosis in the hair follicles (HF) and cause hair loss, the most common side effect of chemotherapy. In a mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: in contrast to wild-type mice, p53-deficient mice show neither hair loss nor apoptosis in the HF keratinocytes that maintained active proliferation after cyclophosphamide treatment. HF in p53 mutants are characterized by down-regulation of Fas and insulin-like growth factor-binding protein 3 and by increased expression of Bcl-2. These observations indicate that local pharmacological inhibition of p53 may be useful to prevent chemotherapy-associated hair loss.

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.

Noggin is required for induction of the hair follicle growth phase in postnatal skin.

During postnatal development, the hair follicle (HF) shows cyclic activity with periods of relative resting, active growth (anagen), and regression. We demonstrate that similar to the HF induction in embryonic skin, initiation of a new hair growth phase in postnatal skin requires neutralization of the inhibitory activity of bone morphogenetic protein 4 (BMP4) by the BMP antagonist noggin. In the resting HF, BMP4 mRNA predominates over noggin in the epithelium and mesenchyme, and the BMP receptor IA is prominently expressed in the follicular germ. Anagen development is accompanied by down-regulation of the BMP4 and increased noggin mRNA in the HF. Furthermore, administration of noggin protein induces new hair growth phase in postnatal telogen skin in vivo. In contrast, BMP4 induces selective arrest of anagen development in the non-tylotrich (secondary) HF. As a hair growth inducer, noggin increases Shh mRNA in the HF whereas BMP4 down-regulates Shh. This suggests that modulation of BMP4 signaling by noggin is essential for hair growth phase induction in postnatal skin and that the hair growth-inducing effect of noggin is mediated, at least in part, by Shh.

Pruritus: pathogenesis, therapy, and significance in systemic disease states.

Pruritus is a cutaneous sensation sharing neural receptors and pathways with pain but is characterized by its own precipitants, potentiators, and range of severity. Among patients with generalized pruritus, the prevalence of systemic disease has been reported as 10% to 50%, with renal, hepatic, hematopoietic, or endocrine causes most commonly identified. Malignant neoplasms, neurologic disorders, certain drugs, or advanced age also may be responsible. Although the pathogenesis of pruritus is unknown, clinically AG event potential mediators have been investigated in several settings. Therapy often fails when the underlying disorder cannot be corrected, but por pruritus associated with chronic renal failure or hepatic cholestasis, specific and usually effective treatments exist.

Relationship between actinic damage and chronologic aging in keratinocyte cultures of human skin.

The relationship of actinically-induced "premature aging" to chronological aging was studied in paired keratinocyte cultures obtained from the habitually sun-exposed (lateral) and nonexposed (medial) aspects of the arm of 5 male donors, aged 41 to 80 yr. In all cases, the number of cell generations in vitro was greater for cultures derived from sun-exposed skin, and this discrepancy increased with donor age and the severity of clinical aging changes. Hence, chronic sun exposure does accelerate aging in human skin by at least one previously established in vitro criterion: it decreased the lifespan of cultured keratinocytes. Plating efficiency was 11- to 32-fold higher for keratinocytes from chronically sun-exposed skin than nonexposed controls, perhaps reflecting the recognized carcinogenic potential of actinic radiation. Keratinocyte cultures appear to be as amenable to gerontologic studies as the already widely used human fibroblast cultures.

In vitro assessment of keratinocyte aging.

In vitro aging models are based on the observation that normal cells in culture have a finite lifespan and eventually cease to proliferate under conditions that initially support excellent growth. Recent assessment of the aging process in keratinocytes, made possible by improved tissue-culture techniques, have confirmed prior findings with fetal and adult fibroblasts and have permitted investigation into the mechanism of in vitro senescence. Early-passage newborn human keratinocytes maintained in a serum-free system were found to proliferate more rapidly than early-passage adult keratinocytes maintained under identical conditions, and they were also found to have far steeper dose-response curves for a potent hypothalamus-derived mitogen keratinocyte growth factor (KGF), as well as for KGF/EGF in combination, with a more than 200-fold increase in cell number, total protein, and colony size over the tested range of concentrations, as opposed to a less than 75-fold increase for adult keratinocytes in these parameters. These results support the hypothesis that the age-associated decrease for keratinocyte proliferation in vitro may be due to progressive loss of mitogenic responsiveness. Unrecognized changes in proliferative rate and growth-factor requirements related to age of the tissue donor may complicate interpretation of studies addressing other aspects of keratinocyte biology.

Human melanogenesis is stimulated by diacylglycerol.

The intracellular signal pathways that mediate pigmentation in human skin are unknown. We now report that a diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-glycerol (OAG) 25-100 microns strikingly increased the melanin content of cultured human melanocytes in a dose dependent manner without altering growth rate. The pigment increase occurred within 24 h, was accompanied by increased incorporation of the melanin precursor L-3,4-dihydroxyphenyl alanine (DOPA), required new protein synthesis, and was completely blocked by the protein kinase C (PKC) inhibitors H-7 and sphingosine. A PKC-inactive DAG isomer had no effect on melanin per cell. These results implicate protein kinase C and its effector DAG in melanogenesis.

A novel approach to analysis of transcriptional regulation in human cells: initial application to melanocytes and melanoma cells.

An assay system for transcriptional profile analysis of cultured eukaryotic cells has been developed to simultaneously handle multiple samples in a rapid, sensitive, and internally controlled manner. The methodology incorporates a microtiter plate assay system, a rapid cell-harvest enzyme-assay technique, and the bacterial reporter genes beta-glucuronidase and beta-galactosidase. We demonstrate, using beta-actin and SV40 (late) transcription promoting sequences, that this technically refined microtiter-triton-lysate (MTL) assay methodology can readily differentiate between the transcriptional states of human melanocytes before and after pharmacologic stimulation and malignantly transformed versus normal cell environments. Differences in the transcriptional environments are revealed by the relative expression of transcription element probes. The transcriptional activity ratio of the beta-actin compared to the SV40 late transcription promoting sequences was approximately 1:2 in primary cultured melanocytes, 2:1 in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-treated melanocytes and 1:4 in the Tang melanoma cell line. Because this MTL assay methodology can accommodate a panel of transcription element probes, we anticipate that the resultant transcriptional profiles will prove useful in deciphering the diverse transcriptional changes that occur within normally regulated and malignantly transformed cells.

Proopiomelanocortin, its derived peptides, and the skin.

Proopiomelanocortin (POMC) is a protein synthesized predominately in the pituitary gland but also in a variety of other tissues, including the skin. Through enzyme-mediated cleavage that varies among cell types, POMC can give rise to at least eight distinct peptides whose biologic roles are incompletely delineated. Although blood-borne pituitary-derived bioactivity for the skin was first recognized 80 years ago and the responsible neuropeptides isolated 20-40 years ago, our understanding of POMC-derived peptides in skin is still rapidly evolving. In particular, recent work in cultured human and murine skin-derived cells has demonstrated POMC gene expression as well as modulation of POMC and many of its derived peptides in response to physiologic signals including ultraviolet irradiation and cytokines. Immunoreactivity for these peptides has also been detected in normal skin and hair follicles, strongly suggesting cutaneous synthesis in vivo. Candidate autocrine or paracrine functions include enhanced melanogenesis, immunomodulation, and effects on cell cycle regulation and differentiated function in both the epidermis and its appendages. This article reviews recent data concerning POMC gene expression and regulation, protein processing, signal transduction, and biologic functions relevant to cutaneous biology.

Retinoids and state of differentiation modulate CRABP II gene expression in a skin equivalent.

Cellular retinoic acid-binding proteins (CRABPs) are a family of proteins that specifically bind retinoic acid (RA) and have been implicated in mediating its action, although their exact function is still unknown. Two CRABPs have been identified and cloned. CRABP I is present in many tissues and cultured cells; CRABP II, first detected in embryonic and neonatal skin of rats and chicks, is now recognized as the predominant form in human epidermis. Using a human living skin equivalent model composed of a dermis and an epidermis and human cDNAs recently cloned in our laboratory, we have studied the effects of 10(-6) M RA and etretin (ET) on the expression of CRABPs under different culture conditions intended to favor greater or lesser degrees of epidermal differentiation. Total cellular RNA was isolated separately from the dermis and epidermis and processed for northern blot analysis. At a presumptive physiologic RA concentration, only the gene for CRABP II, and not for CRABP I, was expressed. CRABP II transcripts were far more abundant on a per cell basis in epidermal keratinocytes than in dermal fibroblasts under all conditions studied. Epidermal differentiation, stimulated by air exposure of the cultures, tended to enhance CRABP II expression, and treatment with presumptive therapeutic concentrations of the two retinoid compounds tended to decrease CRABP II expression. Opposite effects of air exposure and retinoid treatment were observed on steady state levels of mRNA for selected markers of epidermal differentiation: involucrin, transglutaminase, and spr I. These results are consistent with earlier work at the protein level examining the effect of retinoids on CRABP activity and state of differentiation both in vivo and in vitro. Thus, the skin equivalent appears to be an excellent model system for investigating the role of CRABPs in mediating retinoid effects at the cellular and molecular levels.