Hee-Young Park

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.

Cellular mechanisms regulating human melanogenesis.

Abstract.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.

Fibroblasts cultured from venous ulcers display cellular characteristics of senescence

PURPOSE A well-recognized characteristic of venous ulcers is impaired healing. Fibroblasts cultured from venous ulcers (wound-fb) have been shown to have reduced growth rates and are larger than normal fibroblasts (normal-fb) from the ipsilateral limb. Reduced growth capacity and morphologic changes are 2 well-known traits of cellular senescence. Other molecular changes are overexpression of matrix proteins, such as cellular fibronectin (cFN), and enhanced activity of beta-galactosidase at pH of 6.0 (senescence associated beta-Gal, or SA-beta-Gal). Senescence, an irreversible arrest of cell proliferation with maintenance of metabolic functions, may represent in vivo aging and thus may be related to impaired healing. METHODS Cultured normal-fb and wound-fb from 7 venous ulcer patients (average age, 51 years) were obtained by taking punch biopsies of the perimeter of the ulcer and from the ipsilateral thigh of the same patient. Growth rates, SA-beta-Gal activity, and level of cFN protein (immunoblot) and message (Northern blot) were measured. RESULTS In all patients, wound-fb growth rates were significantly lower than those of normal-fb (P =.006). A higher percentage of SA-beta-Gal positive cells were found in all wound-fb (average, 6.3% vs. 0.21%; P =.016). The level of cFN, was consistently higher in all wound-fb tested. Also, in 4 patients, the level of cFN messenger RNA (mRNA) was increased. CONCLUSION Fibroblasts cultured from venous ulcers exhibited characteristics associated with senescent cells. Accumulation of senescent cell in ulcer environment may be associated with impaired healing.

PROTEIN KINASE C-BETA ACTIVATES TYROSINASE BY PHOSPHORYLATING SERINE RESIDUES IN ITS CYTOPLASMIC DOMAIN

We have previously shown that protein kinase C-β (PKC-β) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993)J. Biol. Chem. 268, 11742–11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-β into nonpigmented human melanoma cells lacking PKC-β lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-β in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-β but not PKC-α was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-β with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-β. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-β and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-β. We conclude that PKC-β activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.

Reduced growth of dermal fibroblasts from chronic venous ulcers can be stimulated with growth factors.

PURPOSE Although the slow healing rate of venous ulcers is well known, the underlying defect in the healing process is not well understood. The purpose of this study was to examine the cellular characteristics of fibroblasts taken from venous ulcers (wound-fb) and compare them with the fibroblasts of normal tissue (normal-fb). METHODS Biopsy specimens were obtained from wound margins and normal tissue of the upper thigh in each patient. Dermal fibroblasts were isolated from explant cultures in Dulbeccos modified Eagles medium supplemented with 10% calf serum. These cells were then plated at 1000 cells per plate, and total cells per plate were counted over time so that growth curves could be generated. In further experimentation, media was supplemented with additional calf serum (20%, 30%, 40%, 50%) and growth factors (epidermal growth factor, basic fibroblast growth factor, interleukin-1 beta) in an attempt to stimulate growth. RESULTS Two major differences were noted: (1) normal-fb replicated more rapidly than wound-fb; and (2) the morphologic features of wound-fb were different. Normal-fb were compact and tapered, with well-defined nuclear morphologic features. Wound-fb were larger and polygonal in shape, with less-uniform nuclear morphologic features. Additional calf serum in tissue culture media enhanced normal-fb growth but had no effect on wound-fb. Supplementation of media with growth factors stimulated the growth of wound-fb. Statistically significant differences were noted at day 10 and 14 with basic fibroblast growth factor supplementation (p = 0.02 and 0.0001, respectively) and at day 14 with epidermal growth factor (p = 0.008). Although interleukin-1 beta stimulated cell growth in five of six patients, the differences observed were not statistically significant. CONCLUSIONS Our data demonstrate that wound-fb proliferate at a slower rate and are morphologically distinct from normal-fb. These characteristics are typical of aged or senescent cells. This decreased growth can be stimulated by growth factors basic fibroblast growth factor, epidermal growth factor, and interleukin-1 beta. Slowed growth may be partially responsible for the defect in healing of venous stasis ulcers. Furthermore, we believe that in some patients ulcer healing may be improved by exogenous provision of specific growth factors.

The proliferative capacity of neonatal skin fibroblasts is reduced after exposure to venous ulcer wound fluid: A potential mechanism for senescence in venous ulcers

PURPOSE We have previously shown that fibroblasts cultured from venous ulcers display characteristics of senescence and have reduced growth rates. Susceptibility of young fibroblasts to the microcirculatory changes associated with venous ulcers, such as macrophage trapping and activation, could explain the prevalence of senescent fibroblasts in these wounds. METHODS We tested the in vitro effect of venous ulcer wound fluid (VUWF), as well as pro-inflammatory cytokines known to be present in VUWF (TNF-alpha, IL-1beta, and TGF-beta1), on neonatal foreskin fibroblasts (NFFs). NFF growth rates, cellular morphology, and senescence-associated beta-galactosidase (SA-beta-Gal) activity were determined in the presence or absence of VUWF and the above cytokines. VUWF TNF-alpha concentration and the effect of anti-TNF-alpha antibody on VUWF inhibitory activity were determined in samples obtained from four patients with venous ulcers. RESULTS NFF growth rates were significantly reduced by VUWF (42,727 +/- 6301 vs 3902 +/- 2191 P =. 006). TNF-alpha also significantly reduced NFF growth rates in a dose-dependent manner (P =.01). No significant growth-inhibitory activity was seen for IL-1alpha or TGF-beta. Incubation with VUWF significantly increased the percentage of SA-beta-Gal-positive fibroblasts in vitro on culture day 12 (P =.02). TNF-alpha and TGF-beta1 had similar effects. TNF-alpha was detected in all VUWF tested, with a mean of 254 +/- 19 pg/mL. CONCLUSION These data suggest that the venous ulcer microenvironment adversely affects young, rapidly proliferating fibroblasts such as NFFs and induces fibroblast senescence. Pro-inflammatory cytokines such as TNF-alpha and TGF-beta1 might be involved in this process. The role of other unknown inhibitory mediators, as well as pro-inflammatory cytokines, in venous ulcer development and impaired healing must be considered.

Mechanophotopatterning on a Photoresponsive Elastomer

IC A IO N The fabrication of well-defi ned topographical features or textures is a dynamic area of research owing to their extraordinary potential in applications ranging from complex optics to tunable and responsive surfaces. [ 1–6 ] Here, mechanophotopatterning (MPP) on a photoresponsive elastomer is introduced and enables for the fi rst time the ability to precisely and simultaneously manipulate both material shape and surface topography by exposure to light without the need for solvents, molding, or physical contact. MPP is a unique patterning approach whereby deformation is applied to an elastomeric material capable of photoinduced structural modifi cation to alter its equilibrium geometry, elegantly complementing previous mechanically assisted patterning techniques. [ 7–9 ] This material responds to MPP by continuously and locally deforming via polymer network connectivity rearrangement, which enables 3D control of its geometry. Unlike conventional photolithographic approaches, MPP forgoes any wet chemistry or surface deposition/modifi cation processing and simplifi es multi-tiered feature fabrication. Furthermore, in contrast with mechanically assisted patterning techniques that utilize buckling phenomena, [ 4 , 8 , 9 ]

Modulation of vascular endothelial growth factor receptors in melanocytes

Abstract:  Vascular endothelial growth factor (VEGF) is constitutively produced by keratinocytes, but has no known epidermal target cell. We now report that normal human melanocytes (Mc) maintained in serum‐free, hormone‐, and growth factor‐supplemented medium lacking phorbol ester and choleragen constitutively express VEGF receptor‐1 (VEGFR‐1), VEGFR‐2, and neuropilin‐1. Furthermore, stimulation of Mc with VEGF165 isoform leads to phosphorylation of VEGFR‐2, the receptor responsible for most of the VEGF‐mediated effects in endothelial cells, suggesting that the receptor is functional. Interestingly, in Mc, VEGFR‐2 expression is induced by ultraviolet irradiation and is downregulated by VEGF and tumor necrosis factor‐α. Prolonged culture (>8 weeks) in the presence of phorbol ester abrogates VEGFR‐2 expression, explaining previous reports that Mc do not express VEGFR‐1 and VEGFR‐2. These data suggest that VEGF may play a role in Mc behavior in skin.

The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-β on melanosomes

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, α, β, δ, ϵ and ζ isoforms of PKC are expressed, but uniquely PKC-β activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-β phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-β, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-β, revealed abundant PKC-β, whereas PKC-α was not detected when immunoblot analysis was performed using antibody against PKC-α. Activation of PKC in melanocytes increased the level of PKC-β co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-β in the presence of UV-activated 1, 1′-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-β and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-β on the melanosome membrane, allowing PKC-β to phosphorylate tyrosinase.

Level of fibronectin mRNA is markedly increased in human chronic wounds.

Background. Acute wound healing has been extensively investigated over the years, however, little is known about possible healing defects in chronic wounds. Fibronectin (FN) plays a critical role in different phases of wound healing and has been demonstrated to be degraded in chronic wounds by proteases. Fibroblasts cultured from chronic leg ulcers showed a higher level of FN compared to normal fibroblasts. Objective. We explored whether the increase in FN protein in chronic wounds is due to increased FN mRNA. In addition, the level of &agr;5&bgr;1 integrin FN cell surface receptor was also examined. Method. Skin biopsies were taken from normal skin within a few hours of Mohs surgery and from the edge of chronic venous leg ulcers. In situ hybridization was performed to determine the level of FN mRNA. The level of integrin &agr;5&bgr;1 was determined by immunohistochemistry. Results. The level of FN mRNA in normal skin and acute wounds was undetectable. In contrast, FN mRNA was heavily induced throughout the dermis of chronic wounds. Immunostaining using a monoclonal antibody against the &agr;5 subunit of integrin revealed that chronic wounds and normal skin showed undetectable levels of &agr;5&bgr;1 integrin. A large induction of &agr;5 was observed in acute wounds. Conclusion. For reepithelization to occur, epidermal keratinocytes need to migrate over the wound surface, a process requiring an interaction between FN and its cell surface receptor integrin &agr;5&bgr;1. These findings suggest that although FN mRNA is increased in chronic wounds, lack of FN cell surface receptor may prevent migration of epidermal keratinocytes in chronic wounds.