Erythroid differentiation regulator 1 (Erdr1) is a proapototic factor in human keratinocytes

Skin is constantly exposed to physical and chemical stressors. The exposure of keratinocytes to ultraviolet B (UVB) irradiation causes epidermal damage via induction of apoptosis. Erythroid differentiation regulator 1 (Erdr1) modulates growth and survival of cells under various stressful conditions, but the function of Erdr1 in human keratinocyte apoptosis has not been investigated so far. Here, we investigated the effect of Erdr1 on UVB-induced apoptosis in human keratinocytes and also examined the underlying regulatory mechanism. First, Erdr1 expression was detected in human primary keratinocytes and normal human skin tissues. Expression of Erdr1 was enhanced in human keratinocytes following UVB irradiation. Knock-down of Erdr1 led to resistance to UVB-induced apoptosis. Also, Erdr1 overexpression increased UVB-induced apoptosis and induced caspase-3 activation. Furthermore, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 significantly reduced Erdr1 expression following UVB irradiation. These results indicate that UVB induces Erdr1 via a MAPK-dependent mechanism. Taken together, these findings suggest that Erdr1 has a role as a proapoptotic factor in human keratinocytes and acts via ERK and p38 MAPK pathways. Therefore, Erdr1 may be a potential therapeutic target to reduce apoptosis in keratinocytes in conditions such as psoriasis and skin cancer.     

Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes

The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.     

Increased synthesis of calcitonin gene-related peptide stimulates keratinocyte proliferation in murine UVB-irradiated skin

Repeated ultraviolet (UV) irradiations have been shown to induce keratinocyte proliferation with acanthosis, stimulate the cutaneous nerve proliferation, and increase the synthesis of calcitonin gene-related peptide (CGRP). In the current study, we examined the role of CGRP in the UVB-induced proliferation of murine keratinocytes. UVB irradiation increased the number of bromodeoxyuridine (BrdU)-labeled basal keratinocytes and caused acanthosis. In addition, CGRP expression was up-regulated in the peripheral nerves of the upper dermis and lower epidermis. Repeated intradermal injections of CGRP increased the number of BrdU-labeled basal cells and caused acanthosis. Intradermal injections of capsaicin prior to UVB-irradiation inhibited the UVB-induced CGRP expression, BrdU labeling in basal keratinocytes and epidermal thickening. Intradermal injections of anti-CGRP antibody inhibited the UVB-induced BrdU labeling in basal keratinocytes, but epidermal thickening was not significantly inhibited. These results indicate that CGRP is one of the stimulators to UVB-induced keratinocyte proliferation. On the other hand, expression of substance P, another neuropeptide in the peripheral nerve, was not up-regulated by UVB irradiation.     

Epidermal stratification reduces the effects of UVB (but not UVA) on keratinocyte cytokine production and cytotoxicity

Ultraviolet (UV) radiation induces cytokine release from cultured keratinocytes as well as from epidermis in vivo. The purpose of this study was to determine whether differentiation of cultured keratinocytes into stratified epithelium decreases the effects of UVA and UVB radiation on cytokine release. Interleukin-1 (IL-1)α, IL-1β and tumor necrosis factor (TNF)-α release from human keratinocytes and reconstituted human epidermis was measured after exposure to UVA or UVB radiation. Release of IL-1α, IL-1β, and TNF-α was induced by both UVA and UVB radiation from both keratinocytes and reconstituted epidermis. Release of these cytokines was correlated with cytotoxicity. Keratinocyte cultures were far more sensitive to UVB radiation than reconstituted epidermis, in terms of both cytotoxicity and cytokine release. In contrast, epidermal stratification/differentiation had much less effect on the sensitivity to UVA radiation. We conclude that epidermal stratification and the formation of a stratum corneum provide protection against UVB radiation but have limited barrier effect against UVA radiation.     

Interferon gamma-induced PNA-binding glycoproteins as markers of human keratinocyte differentiation: biological evidence using protein kinase C agonists, antagonists and retinoic acid

Membrane glycoproteins (gps) play an important role in cell-cell interactions during epidermal maturation, and we have previously shown an up-regulation of PNA-binding gps in cultured human keratinocytes treated with interferon gamma (IFN-γ). The protein kinase C (PKC) pathway is known to play a key role in the regulation of proliferation and differentiation of keratinocytes and is also reported to be involved in some IFN-γ-mediated effects. In order to evaluate the cellular mechanisms and whether PNA-binding gp expression is related to the differentiative activity of the lymphokine, we studied the effects of PKC agonists and antagonists and the role of retinoic acid (RA), in the induction of these gps in cultured human keratinocytes stimulated with IFN-γ and processed for protein analysis. The expression of PNA-binding gps was revealed by incubation of SDS-polyacrylamide gels with ¹²⁵ I-PNA. The PKC antagonists (H7, sphingosine) as well as RA downgregulated the IFN-γ-induced PNA-reactive gps, whereas staurosporine and TPA upregulated their expression. These results provide evidence that PNA-reactive gps are late highly IFN-γ-sensitive markers of keratinocyte differentiation, drastically modulated through selective isoforms of PKC. Received: 3 March 1997     

Protein kinase C agonist and antagonist effects in normal human epidermal keratinocytes

Abstract Several lines of evidence implicate protein kinase C (PKC) in the development of basal cell and squamous cell carcinomas, tumors which originate from epidermal keratinocytes. To examine PKC in a model relevant to human skin, we exposed normal human epidermal keratinocytes (NHEK) in serum-free media to a variety of PKC agonists and antagonists. NHEK PKC activity increased up to 10-fold within the 1st hour of exposure to tetradecanoyl phorbol acetate (TPA), and gradually returned to control values within 72 h. TPA-induced PKC activity was enhanced by pretreatment of cultures with protein and RNA synthesis inhibitors. TPA-induced growth arrest and differentiation was antagonized by staurosporine. Down-regulation by bryostatin pretreatment blocked TPA-stimulated differentiation. Our overall conclusion is that activation of PKC in cultured human keratinocytes is required for differentiation. These results are crucial to the analysis of compounds suspected of promoting or inhibiting epidermal tumors.     

Protein kinase C isozymes regulate proliferation and high cell density-mediated differentiation in HaCaT keratinocytes

Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density-induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKC alpha, beta, gamma, delta, epsilon, eta, theta, zeta), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the lat (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocyte-specific transglutaminase-1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down-regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density-induced differentiation of HaCaT cells.     

UV radiation induces CXCL5 expression in human skin

CXCL5 has recently been identified as a mediator of UVB‐induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose‐dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non‐immune skin cells also contribute to UV‐induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte‐keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV‐radiated human skin and the essential role of fibroblast‐keratinocyte interaction in the regulation of epidermal CXCL5.     

Regulation of protein kinase D during differentiation and proliferation of primary mouse keratinocytes

Diseased skin often exhibits a deregulated program of the keratinocyte maturation necessary for epidermal stratification and function. Protein kinase D (PKD), a serine/threonine kinase, is expressed in proliferating keratinocytes, and PKD activation occurs in response to mitogen stimulation in other cell types. We have proposed that PKD functions as a pro-proliferative and/or anti-differentiative signal in keratinocytes and hypothesized that differentiation inducers will downmodulate PKD to allow differentiation to proceed. Thus, changes in PKD levels, autophosphorylation, and activity were analyzed upon stimulation of differentiation and proliferation in primary mouse keratinocytes. Elevated extracellular calcium and acute 12-O-tetradecanoylphorbol-13-acetate (TPA) treatments induced differentiation and triggered a downmodulation of PKD levels, autophosphorylation at serine 916, and activity. Chronic TPA treatment stimulated proliferation and resulted in a recovery of PKD levels, autophosphorylation, and activity. Immunohistochemical analysis demonstrated PKD localization predominantly in the proliferative basal layer of mouse epidermis. Co-expression studies revealed a pro-proliferative, anti-differentiative effect of PKD on keratinocyte maturation as monitored by increased and decreased promoter activities of keratin 5, a proliferative marker, and involucrin, a differentiative marker, respectively. This work describes the inverse regulation of PKD during keratinocyte differentiation and proliferation and the pro-proliferative/anti-differentiative effects of PKD co-expression on keratinocyte maturation.     

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.     

Keratinocyte prostaglandin synthesis is enhanced by IL-1

Keratinocytes are a rich source of IL-1, a cytokine which stimulates prostaglandin synthesis in many cell types. The effects on arachidonic acid metabolism of this cytokine were therefore studied in cultured adult human keratinocytes. Exogenous IL-1 increased basal cellular prostaglandin synthesis (particularly PGE2) threefold. Increased PGE2 synthesis in response to IL-1 was inhibited by cycloheximide, suggesting a requirement for new protein synthesis. Irradiation of the keratinocytes with low-dose ultraviolet light B (UVB) resulted in the release of increased quantities of both IL-1 and PGE2. The amount of IL-1 released was sufficient to increase PGE2 synthesis when exogenously added to unstimulated cells, suggesting a causal relationship. The time course of accumulation of IL-1 and PGE2 in the medium of irradiated keratinocytes was also consistent with a cause-effect relationship. No feedback inhibition of IL-1 release by the increased PGE2 was detected as demonstrated by the observation that IL-1 production in response to UVB was not augmented by treatment with indomethacin or blunted by the exogenous addition of PGE2. These data suggest that keratinocyte IL-1 may be partially responsible for induction of keratinocyte PGE2 synthesis after UVB irradiation.     

Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) inhibitor KN-62 suppresses the activity of mitogen-activated protein kinase (MAPK), c-myc activation and human keratinocyte proliferation

Autocrine growth of human epidermal keratinocytes can be maintained in subconfluent cell cultures in the absence of exogenous growth factors. We used this culture model to investigate the interactions between the mitogen-activated protein kinase (MAPK) pathway and Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) in autocrine keratinocyte proliferation. We have previously demonstrated that MAPK and protein kinase C (PKC) are both involved in keratinocyte proliferation in a complex set of interactions. Treatment of keratinocytes with PD98059, a potent inhibitor of MAPK kinase, inhibited the MAPK pathway, c-myc activation and autocrine keratinocyte proliferation. Application of the CaM-kinase inhibitor KN-62 also led to a strong inhibition of MAPK/c-myc activation and autocrine keratinocyte proliferation. Other inhibitors, such as wortmannin (selective and potent inhibitor of phosphatidylinositol 3-kinase) and AG 490 (JAK2 inhibitor) had weak effects on autocrine keratinocyte proliferation, MAPK and c-myc activation. Our results clearly demonstrate a crosstalk between CaM-kinase/MAPK pathways in transducing keratinocyte proliferation stimuli.     

Eicosapentaenoic acid and docosahexaenoic acid reduce UVB- and TNF-alpha-induced IL-8 secretion in keratinocytes and UVB-induced IL-8 in fibroblasts

Omega-3 polyunsaturated fatty acids (n-3 PUFA) inhibit ultraviolet B (UVB)-induced inflammation and other inflammatory states, in vivo. We examined whether this may be mediated by modulation of interleukin (IL)-8, a chemokine pivotal to skin inflammation induced by UVB, in epidermal and dermal cells. We also explored the ability of n-3 PUFA to protect against tumor necrosis factor (TNF)-alpha induction of IL-8, and assessed relative potencies of the principal dietary n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Pre-supplementation, both HaCaT keratinocyte and CCD922SK fibroblast cell lines showed dose-responses for UVB-induced IL-8 release (p<0.001), assessed 48 h post-irradiation. Cells were supplemented with > or =90% purified EPA, DHA, oleic acid (OA) or vehicle control, for 4.5 d. EPA and DHA supplements were bioavailable to keratinocytes and fibroblasts. In keratinocytes, EPA and DHA were shown to reduce basal secretion of IL-8 by 66% and 63%, respectively (p<0.05), and UVB-induced levels by 66% and 65% at 48 h after 100 mJ per cm2, respectively, (p<0.01). A similar pattern occurred in fibroblasts, whereas OA had no influence on IL-8 release in either cell line. In addition, TNF-alpha-induced IL-8 secretion by keratinocytes was reduced by 54% and 42%, respectively, by EPA and DHA (p<0.001). Hence both n-3 PUFA inhibit production of UVB- and TNF-alpha-induced IL-8 in skin cells; this may be important in the photoprotective and other anti-inflammatory effects conferred by these agents.     

Immunosuppression by factors released from UV-irradiated epidermal cells: selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation

Exposure of murine epidermal cells to UV radiation in vitro causes the release of immunoregulatory factors that mimic some of the immunosuppressive effects of in vivo UV irradiation. The purpose of this study was to investigate the spectrum of immune responses affected following i.v. injection of supernatants obtained from cultures of epidermal cells exposed in vitro to UV radiation. Treatment of primary epidermal cell cultures or transformed keratinocytes (Pam 212 cells) with UVB (280-320 nm) radiation caused the release of factors that suppressed the induction of delayed hypersensitivity to alloantigen and trinitrophenyl-modified self-antigens in syngeneic and allogeneic mice. Contrary to expectations, however, the injection of supernatants from UVB-irradiated epidermal cells had no effect on the induction of contact hypersensitivity to trinitrochlorobenzene. On the other hand, treatment of the keratinocytes with UVA radiation (320-400 nm, filtered to remove wavelengths in the UVB region) resulted in the release of a factor that suppressed contact but not delayed hypersensitivity. Neither the UVA-induced nor the UVB-induced suppressive factor inhibited the generation of an antibody response to sheep erythrocytes, indicating that, like the suppression that occurs after in vivo exposure to UV radiation, the suppression induced by factors from UV-irradiated keratinocytes is selective in nature. These data support the hypothesis that soluble keratinocyte-derived suppressive factors are involved in the induction of systemic immune suppression by UV radiation. In addition, they suggest that multiple suppressive factors, having different immunosuppressive properties, are produced by different wavelengths of UV radiation.     

Ultraviolet B irradiation increases keratin 5 and keratin 14 expression through epidermal growth factor receptor of SV40-transformed human keratinocytes

Keratin intermediate filaments are heteropolymers composed of type I and type II keratins. Ultraviolet B (UVB) irradiation induces keratin expression by keratinocytes. Using SV40-transformed human keratinocytes (SVHK), we investigated the effect of UVB irradiation on keratin expression. UVB irradiation (10 mJ/cm(2)) increased keratin 5 and keratin 14 mRNAs and proteins without affecting cell viability. Upregulation of keratin 5 and keratin 14 was dependent on the dose of radiation: the effect was observed at 5 mJ/cm(2) and the maximal effect was observed at 10 mJ/cm(2). Higher UVB doses (more than 10 mJ/cm(2)) were cytotoxic. Expression of keratin 1 and keratin 10 was marginal in SVHK and was not affected at either the mRNA or protein level by UVB. The stimulatory effects on keratin 5 and keratin 14 expression were also observed in cultured normal human keratinocytes (NHK) and HaCaT keratinocytes. The tyrosine kinase inhibitor, genistein, and the epidermal growth factor (EGF) receptor inhibitor, AG1429, significantly suppressed the increase in expression of keratin 5 and keratin 14 by SVHK. In contrast, the suppressive effect was not observed with the protein kinase C inhibitor, H-7. Furthermore, pretreatment with neutralizing anti-EGF receptor antibody also suppressed UVB-induced keratin 5 and keratin 14 expression by SVHK, NHK and HaCaT cells. UVB irradiation did not affect the steady-state expression of TGF-alpha by SVHK. Immunoprecipitation and immunohistochemical studies revealed that UVB irradiation induced EGF receptor activation in the absence of EGF and TGF-alpha. These results indicate that UVB increases keratin 5 and keratin 14 expression through direct activation of the EGF receptor in SVHK.     

Ultraviolet-B (290-320 nm)-irradiation inhibits epidermal growth-factor binding to mammalian cells

Mitogens, such as polypeptide growth factors and phorbol ester tumor promoters, act by binding to specific receptors and inducing a pleiotropic response in cultured mammalian cells, which results in the induction of cellular proliferation. An early effect of such agents is the inhibition of binding of epidermal growth factor (EGF) to its receptor. Ultraviolet radiation has also been shown to induce a proliferative response in vivo and in vitro and to act as a tumor promoter in animal skin. We, therefore, examined the effect of ultraviolet radiation (UVB - 290-320 nm) on EGF binding to cells in culture. We found that UVB (100-300 J/m2) induced a rapid, dose-dependent inhibition of EGF binding in a mouse fibroblast cell line, which resulted from a decrease in both number and affinity of binding sites. Phosphorylation of the EGF receptor by protein kinase C (PKC) is not likely to be the mechanism for inhibition, since UVB treatment did not result in PKC activation or modulation of phorbol diester binding.     

Human epidermal basal cell responses to ultraviolet-B differ according to their location in the undulating epidermis

BACKGROUND: Exposure of skin to excessive ultraviolet-B (UVB) radiation causes epidermal hyperproliferation that leads to epidermal hyperplasia, however, it is not yet clear exactly how these responses progress. OBJECTIVES: We attempted to clarify the response patterns involved with epidermal hyperproliferation following UVB radiation. METHODS: UVB was irradiated at 2 minimal erythema doses (MED) to human back skin and epidermal morphologic changes were evaluated using in vivo confocal laser microscopy. Skin biopsy specimens were collected from exposed and from non-exposed regions, and were subjected to histochemical and immunohistochemical analysis. RESULTS: The in vivo confocal laser microscopic analysis showed that UVB-induced epidermal hyperplasia was prominent at the epidermal rete ridges. Further, 3 days after UVB exposure, numerous Ki67-positive epidermal cells were observed in the epidermal rete ridges, but not in the epidermis at the top of the dermal papilla. These results suggest that cells highly responsive to UVB exist in the epidermal rete ridges and that their hyperproliferation leads to elongation of the epidermal rete ridges. In contrast, the number of keratin 10-positive basal cells, known as transitional cells, was increased throughout the epidermis, suggesting that an upward migration of keratinocytes from the epidermal basal layer occurred regardless of their location. However, diffusion of melanin to the suprabasal layers was markedly observed in epidermal regions above the dermal papillae, suggesting the occurrence of strong upper cell movement at this position. CONCLUSION: Based on our results, we conclude that differences in keratinocyte responses to UVB radiation exist in cells located in the undulating epidermal basal layer.     

Effects of a melanogenic bicyclic monoterpene diol on cell cycle, p53, TNF-alpha, and PGE2 are distinct from those of UVB

PURPOSE: Bicyclic monoterpene (BMT) diols are small-molecule compounds that mimic ultraviolet radiation (UVR) by inducing melanogenesis. The objective of this study was to compare the effects of 2,2-dimethyl-3-propanyldiol-norbornane (AGI-1140), a novel BMT diol, and ultraviolet B (UVB) on additional cellular responses. METHODS: S91 mouse melanoma cells were treated with a range of concentrations of AGI-1140, and examined for induction of melanogenesis and nitric oxide (NO). The effect of AGI-1140 on dendrite outgrowth from human melanocytes was examined by quantitative microscopy. The effect of AGI-1140 and UVB on phosphorylation of p53 serine 15 in human keratinocytes was examined by Western blotting, while the release of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay. The effects of AGI-1140 and UVB on cell cycle arrest of human melanocytes, keratinocytes, fibroblasts, and endothelial cells were compared using fluorescence-activated cell sorting. RESULTS: Similar to UVB, AGI-1140 induced both melanogenesis and NO in melanoma cells. AGI-1140 also induced dendrite outgrowth from melanocytes, indicative of differentiation. However, whereas UVB induced G2 cell cycle arrest with phosphorylation of p53 at serine 15, AGI-1140 induced G1 cell cycle arrest without this phosphorylation. Additionally, unlike UVB, AGI-1140 did not increase the secretion of TNF-alpha or PGE2, mediators of UVB-induced immunosuppressive and inflammatory responses in the skin that may contribute to carcinogenesis. CONCLUSION: This study shows that melanogenesis can be induced by AGI-1140 without many of the deleterious effects associated with UVB.