The maximum tolerated dose of palifermin, a <em>keratinocyte</em> <em>growth</em> <em>factor</em>, in children is not known, and its pharmacokinetics in this population has not been well studied. This is a phase I study of palifermin was designed to evaluate its tolerability at doses of 40, 60, and 90 μg/kg/day in children age <em>2</em>-18 years of age, receiving a myeloablative preparative regimen for allogeneic hematopoietic stem cell transplantation (HSCT). In each cohort, palifermin was given for 3 consecutive days before the preparative regimen and for 3 days after the stem cell infusion. Twelve patients were enrolled. Palifermin 90 μg/kg/day was tolerated in 6 patients without dose-limiting toxicity. All patients had at least 1 adverse event, mostly National Cancer Institute grade 1 or <em>2</em> severity. Skin rash, grade <em>2</em> or lower, was the most common adverse event, seen in 67% of patients. Only 3 patients (<em>2</em>5%) had mucositis. The area under the concentration-time curve increased proportionally to the dose, and approximately 97% of palifermin exposure occurred in the first <em>2</em>4 hours after administration. Palifermin clearance increased linearly with body weight, supporting dosing by body weight. The mean clearance was 1893 mL/hour/kg, and it did not change significantly between administration of the first and last doses (P = .80). The mean elimination half-life was 4.6 hours. Our data show that palifermin was tolerated at a dose of 90 μg/kg/day, and exhibits linear pharmacokinetics in children undergoing allogeneic HSCT.

To investigate the pathophysiological role of fibroblasts in tissue metabolism, we analysed gelatin-degrading enzymes secreted from skin cells in culture. Using a gelatin-zymography method, matrix metalloproteinase (MMP)-<em>2</em> was constitutively secreted from fibroblasts, whereas both MMP-<em>2</em> and MMP-9 were secreted from <em>keratinocytes</em>. MMP-9 expression by fibroblasts was induced by the addition of transforming <em>growth</em> <em>factor</em> (TGF)-beta1 or tumour necrosis <em>factor</em> (TNF)-alpha to the cultures. Moreover, TGF-beta1, TNF-alpha, or epidermal <em>growth</em> <em>factor</em> augmented the secretion of MMP-<em>2</em> from fibroblasts. These results indicate that MMP-9 together with MMP-<em>2</em> secreted from fibroblasts could play important roles in tissue metabolism, including cytokine-induced inflammation.

Matrix metalloproteinase activity was assessed in culture fluids of organ-cultured human skin by gelatin zymography. Both the 9<em>2</em>-kD gelatinase/type IV collagenase and the 7<em>2</em>-kD gelatinase/type IV collagenase were detected. Production of the 9<em>2</em>-kD enzyme was substantially increased in the presence of epidermal <em>growth</em> <em>factor</em> (EGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) as compared to control but not in the presence of insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) or <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). This is of interest because our recent studies have shown that EGF and HGF induce the epithelial cells to invade the underlying stroma while normal architecture is maintained in the presence of IGF-1 and KGF. Addition of tissue inhibitor of metalloproteinase-<em>2</em> to the organ culture fluids blocked expression of the active forms of both enzymes and concomitantly blocked invasion. Epidermal <em>keratinocytes</em>, dermal fibroblasts and dermal endothelial cells were grown in monolayer culture and examined for matrix metalloproteinase production. The 9<em>2</em>-kD enzyme accounted for most of the gelatinase activity in <em>keratinocyte</em> culture fluids while the 7<em>2</em>-kD enzyme accounted for most of the activity in the dermal fibroblast and endothelial cell culture fluids. Increased production of the 9<em>2</em>-kD enzyme was seen in <em>keratinocytes</em> upon exposure to the <em>growth</em> <em>factors</em> that induced invasion (EGF and HGF) while the two <em>factors</em> that did not induce invasion (IGF-1 and KGF) were much less effective. Production of the 7<em>2</em>-kD enzyme in fibroblasts and endothelial cells was not upregulated by any of the four <em>growth</em> <em>factors</em>. Taken together, these data indicate that matrix metalloproteinase activity is increased in the epithelium under the influence of invasion-inducing <em>growth</em> <em>factors</em> and contributes to invasion.

Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in <em>keratinocytes</em> after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (<em>2</em>) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal <em>growth</em> <em>factor</em> receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in <em>keratinocytes</em> may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB<em>2</em> is activated via the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) upon exposure of cultured human <em>keratinocytes</em> to <em>2</em>-methyl-1,4-naphthoquinone (menadione). Both ErbB<em>2</em> and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB<em>2</em> phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.

Prostate enlargement and function is under the dual control of androgens and intraprostatic <em>growth</em> <em>factors</em>. They regulate, in concert, prostate cell proliferation and apoptosis. An increased signaling of both <em>growth</em> <em>factors</em> and androgens are supposed to underlie benign prostate hyperplasia (BPH), one of the more common disorders of the aging male. Since, in clinical practice, androgen ablation resulted in a rather limited decrease in prostate volume, therapeutic strategies targeting intraprostatic <em>growth</em> <em>factors</em> are emerging. The activated form of vitamin D, vitamin D3, and some of its analogues have been described as potent regulators of cell <em>growth</em> and differentiation. In this study, we report the effects of one of these vitamin D3 analogues, 1,<em>2</em>5-dihydroxy-16ene-<em>2</em>3yne D3, or analogue (V), on the fate of isolated epithelial cells derived from patients with BPH. We essentially found that analogue (V), as well as vitamin D3, inhibited BPH cell proliferation and counteracted the mitogenic activity of a potent <em>growth</em> <em>factor</em> for BPH cells, such as <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). Moreover, analogue (V) induced bcl-<em>2</em> protein expression, intracellular calcium mobilization, and apoptosis in both unstimulated and KGF-stimulated BPH cells. Since a short-term (5-min) incubation with analogue (V) reduced the KGF-induced tyrosine phosphorylation of a 1<em>2</em>0-kDA protein, corresponding to the KGF receptor, a rapid and direct cross-talk between these two molecules is suggested. Such a rapid effect of analogue (V), together with the transient induction of intracellular calcium waves, seems to indicate the partial involvement of a membrane, nongenomic receptor for vitamin D3. In conclusion, we demonstrated the antiproliferative and proapoptotic effect of analogue (V) in BPH cells and speculated on its possible use in the therapy of BPH.

Downregulation of survival signaling pathways contributes to the cytotoxicity of reactive oxygen species (ROS) and may underlie certain therapies for hyperproliferative diseases. We have investigated the role of singlet oxygen, an ROS formed by photosensitization, in the regulation of survival signaling via the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). Exposure of human <em>keratinocytes</em> to singlet oxygen resulted in rapid loss of EGFR, which was not blocked by either inhibition of receptor internalization or by interrupting the major proteolytic pathways (proteasome, lysosome or calpain). However, pretreatment with a caspase-3 inhibitor, DEVD-FMK, inhibited EGFR degradation. Caspase-3 cleavage was detected as early as 5 min after singlet oxygen treatment, and recombinant active caspase-3 completely cleaved EGFR in a <em>keratinocyte</em> membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and extracellular signal-regulated kinase 1/<em>2</em> (ERK)1/<em>2</em>. Singlet oxygen-induced protein dephosphorylation was not dependent on activation of caspase-3. In contrast, inhibition of protein phosphatases (PPs) with okadaic acid completely blocked dephosphorylation of EGFR, ERK1/<em>2</em> and Akt as well as degradation of EGFR. These results indicate that the oxidative stress produced by singlet oxygen rapidly disrupts EGFR-mediated signaling by decreasing both the protein level and its phosphorylation. These responses depended on intertwined activation of caspase-3 and PPs.

We review the basic functions of neurotrophins and their receptors and discuss the expression and functions of neurotrophins and their specific receptors based on recent data using cultured cells from human periodontal tissues. Neurotrophins, nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotrophic <em>factor</em> (BDNF), and neurotrophin-3 (NT-3) play crucial roles in the differentiation and survival of neural cells. Neurotrophins activate <em>2</em> different receptor classes: the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and the p75 receptor, a member of the tumor necrosis <em>factor</em> receptor superfamily. Neurotrophins regulate both cell death and cell survival through activations of Trk receptors and/or p75 neurotrophin receptor. It has been reported that neurotrophins are also produced from non-neuronal cells, such as leukocytes, osteoblasts, or fibroblasts, and act in many other ways on non-neuronal cells. Neurotrophin expression during bone fracture healing is especially interesting, and neurotrophins are now implicated in hard tissue regeneration. It is well known that neurotrophins and their receptors are expressed in tooth development. Recent studies have found that neurotrophins and Trk receptors are expressed in mouse osteoblastic cell lines. Human periodontal ligament cells, human gingival fibroblasts, and human gingival <em>keratinocytes</em> expressed mRNA for NGF and TrkA. The secretion of bioactive NGF peptides from human periodontal ligament cells and human gingival <em>keratinocytes</em> was confirmed by bioassay using PC1<em>2</em> cells (rat adrenal pheochromocytoma cells). The expression of NGF and TrkA.mRNA was regulated by interleukin (IL)-1beta. NGF increased DNA synthesis and expressions of mRNA for bone-related proteins, alkaline phosphatase, and osteopontin in human periodontal ligament cells. Neurotrophins and Trk receptors expressed in human periodontal tissue may contribute to regeneration as well as innervation of periodontal tissue through local autocrine and paracrine pathways. Recent data suggest that some functions of neurotrophins and Trk receptors relate to periodontal disease and periodontal tissue regeneration. However, in vivo studies will be required to clarify the roles of neurotrophins and their receptors, including p75, in periodontal disease and periodontal tissue regeneration.

Two genes, flg and bek, have been recently shown to encode receptors for fibroblast <em>growth</em> <em>factors</em> (FGF). Here we report the molecular cloning and sequence of a new member of the FGF receptor family, denoted flg-<em>2</em>, which was isolated from a human <em>keratinocyte</em> cDNA library. The cDNA sequence predicts an extracellular region possessing three immunoglobulin-like domains, a single transmembrane region and a cytoplasmic portion containing the tyrosine kinase domain split by a short inter-kinase segment. The amino acid sequence of flg-<em>2</em> shows 68% and 64% identity with bek and flg, respectively. The most variable domain among the three genes is the amino-terminal immunoglobulin-like domain. Comparison with the chicken FGF receptor genes suggests that flg-<em>2</em> is homologous to cek-<em>2</em>, whereas flg and bek are homologous to cek-1 and cek-3, respectively. Analysis of mRNA from various tissues shows that flg-<em>2</em> is expressed predominantly in skin, brain and lung.

Acute and chronic graft versus host disease (GVHD) remain the major barriers to successful hematopoietic cell transplantation. The induction of GVHD may be divided into three phases: recipient conditioning;donor T-cell activation; andeffector cells mediating GVHD. This review examines GVHD prevention and treatment using this conceptual model as framework. The various pharmacological agents discussed impact on different phases of the GVHD cascade. For example, <em>keratinocyte</em> <em>growth</em> <em>factor</em> and interleukin (IL)-11 are cytokines that may be useful in disrupting phase I of the GVHD cascade by blocking gastrointestinal tract damage, and lowering serum levels of lipopolysaccharide and tumour necrosis <em>factor</em> (TNF)-alpha. Cyclosporin, tacrolimus (FK-506) and sirolimus (rapamycin) are some of the main agents that disrupt phase II (donor T-cell activation). Mycophenolate mofetil and tresperimus probably act on this phase as well. Other novel drugs that affect phase II are tolerance-induction agents such as CTLA-4 and anti-CD40-ligand monoclonal antibodies, and preliminary results using CTLA-4 monoclonal antibody in GVHD prevention are encouraging. Examples of agents that disrupt phase III are the IL-<em>2</em> receptor antagonist daclizumab and the anti-TNFalpha monoclonal antibody infliximab. These anti-cytokine antibodies have shown promising results in early studies. The most effective approach to GVHD prevention will probably be a combination regimen where the three phases of the GVHD cascade are disrupted. Once GVHD has occurred, all three phases of the cascade are activated. Developments of combination therapy for treatment of both acute and chronic GVHD are likely to yield better results than monotherapy. The numerous new treatment modalities presented should improve the outlook for patients with acute and chronic GVHD.

The <em>2</em>5-hydroxyvitamin D(3) (<em>2</em>5-OH-D(3)) is a nontoxic and low-affinity vitamin D receptor (VDR)-binding metabolic precursor of 1,<em>2</em>5-dihydroxyvitamin D(3) [1,<em>2</em>5(OH)(<em>2</em>)D(3)]. We hypothesized that covalent attachment of a <em>2</em>5-OH-D(3) analog to the hormone-binding pocket of VDR might convert the latter into transcriptionally active holo-form, making <em>2</em>5-OH-D(3) biologically active. Furthermore, it might be possible to translate the nontoxic nature of <em>2</em>5-OH-D(3) into its analog. We showed earlier that <em>2</em>5-hydroxyvitamin D(3)-3-bromoacetate (<em>2</em>5-OH-D(3)-3-BE) alkylated the hormone-binding pocket of VDR. In this communication we describe that 10(-6) mol/L of <em>2</em>5-OH-D(3)-3-BE inhibited the <em>growth</em> of <em>keratinocytes</em>, LNCaP, and LAPC-4 androgen-sensitive and PC-3 and DU145 androgen-refractory prostate cancer cells, and PZ-HPV-7 immortalized normal prostate cells with similar or stronger efficacy as 1,<em>2</em>5(OH)(<em>2</em>)D(3). But its effect was strongest in LNCaP, PC-3, LAPC-4, and DU145 cells. Furthermore, <em>2</em>5-OH-D(3)-3-BE was toxic to these prostate cancer cells and caused these cells to undergo apoptosis as shown by DNA-fragmentation and caspase-activation assays. In a reporter assay with COS-7 cells, transfected with a 1alpha,<em>2</em>5-dihydroxyvitamin D(3)-<em>2</em>4-hydroxylase (<em>2</em>4-OHase)-construct and VDR-expression vector, <em>2</em>5-OH-D(3)-3-BE induced <em>2</em>4-OHase promoter activity. In a "pull down assay" with PC-3 cells, <em>2</em>5-OH-D(3)-3-BE induced strong interaction between VDR and general transcription <em>factors</em>, retinoid X receptor, and GRIP-1. Collectively, these results strongly suggested that the cellular effects of <em>2</em>5-OH-D(3)-3-BE were manifested via 1,<em>2</em>5(OH)(<em>2</em>)D(3)/VDR signaling pathway. A toxicity study in CD-1 mice showed that 166 microg/kg of <em>2</em>5-OH-D(3)-3-BE did not raise serum-calcium beyond vehicle control. Collectively, these results strongly suggested that <em>2</em>5-OH-D(3)-3-BE has a strong potential as a therapeutic agent for androgen-sensitive and androgen-refractory prostate cancer.

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte <em>growth</em> <em>factor</em> receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte <em>growth</em> <em>factor</em> (HGF)-stimulated Met tyrosine phosphorylation in HEK<em>2</em>93 cells. Expression of RPTP-β in primary human <em>keratinocytes</em> reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/<em>2</em> and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human <em>keratinocytes</em>. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary <em>keratinocytes</em> reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.

Curcumin, a dietary pigment from the plant Curcuma longa, inhibits cell proliferation and induces apoptosis in different cell lines. The therapeutic benefit is hampered by a very low absorption after trans-dermal or oral application. Therefore, great efforts were undertaken to enhance the effectiveness of curcumin. Recently, it was demonstrated that curcumin offers the described effects also at low concentrations (0.<em>2</em>-1 μg/ml) when applied in combination with UVA or visible light. The efficacy of this combination was shown in human epidermal <em>keratinocytes</em> and in a panel of other cell species in vitro as well as in a xenograft tumor model with A431 tumor cells injected subcutaneously in the flanks of NMRI nude mice in vivo. The treatment of <em>keratinocytes</em> with curcumin and light resulted in the inhibition of cell <em>growth</em>, and in the induction of apoptosis, whereas no toxic cell membrane damage was detectable. The treatment of tumor bearing nude mice with curcumin and visible light resulted in reduced tumor volumes, reduced proliferation rates, and the induction of apoptosis in the tumors. On the molecular level inhibition of extracellular regulated kinases 1/<em>2</em> and epidermal <em>growth</em> <em>factor</em> receptor was observed which may aid to inhibition of proliferation and induction of apoptosis. This review covers the experiences of the new combination treatment of human tumors.

Carnosic acid (CA) is a diterpene compound exhibiting antioxidative, anticancer, anti-angiogenic, anti-inflammatory, anti-metabolic disorder, and hepatoprotective and neuroprotective activities. In this study, the effect of CA on various skin inflammatory responses and its inhibitory mechanism were examined. CA strongly suppressed the production of IL-6, IL-8, and MCP-1 from <em>keratinocyte</em> HaCaT cells stimulated with sodium lauryl sulfate (SLS) and retinoic acid (RA). In addition, CA blocked the release of nitric oxide (NO), tumor necrosis <em>factor</em> (TNF)-α, and prostaglandin E₂ (PGE₂) from RAW<em>2</em>64.7 cells activated by the toll-like receptor (TLR)-<em>2</em> ligands, Gram-positive bacterium-derived peptidoglycan (PGN) and pam3CSK, and the TLR4 ligand, Gram-negative bacterium-derived lipopolysaccharide (LPS). CA arrested the <em>growth</em> of dermatitis-inducing Gram-positive and Gram-negative microorganisms such Propionibacterium acnes, Pseudomonas aeruginosa, and Staphylococcus aureus. CA also blocked the nuclear translocation of nuclear <em>factor</em> (NF)-κB and its upstream signaling including Syk/Src, phosphoinositide 3-kinase (PI3K), Akt, inhibitor of κBα (IκBα) kinase (IKK), and IκBα for NF-κB activation. Kinase assays revealed that Syk could be direct enzymatic target of CA in its anti-inflammatory action. Therefore, our data strongly suggest the potential of CA as an anti-inflammatory drug against skin inflammatory responses with Src/NF-κB inhibitory properties.

A hallmark of the mucosa of immunocompromized hosts in oral candidiasis is a hyperkeratinized region heavily colonized with fungi at the surface of the terminally differentiated epithelium. To gain insight into the processes important for promoting mucosal invasion by fungi, we characterized the response of <em>keratinocytes</em> to the presence of Candida albicans. Indirect immunofluorescence and kymographic analyses revealed a multifaceted <em>keratinocyte</em> response of OKF6/TERT-<em>2</em> cells to C. albicans that consisted of: cytoskeletal reorganization within 3 h, motility and cell expansion with formation of E-cadherin-mediated cell-cell adhesions within 6 h, increased expression of late differentiation markers and decreased expression of calprotectin. The initial expansive phase was followed by dissolution of cell-cell adhesions and a decrease in cell size accompanied by loss of E-cadherin. The <em>keratinocyte</em> response depended on soluble <em>factors</em> associated with hyphal <em>growth</em> as demonstrated using the efg1Delta/efg1Delta, cap1Delta/cap1Delta, als3Delta/als3Delta, hwp1Delta/hwp1Deltaand sap4-6Delta/sap4-6Delta mutants and was not observed in the presence of the non-pathogenic yeast, Saccharomyces cerevisiae. These studies show the potential for C. albicans to manipulate the stratified epithelial cells to a state of differentiation that is more permissive of fungal colonization of oral tissue, which is likely to play an important role in the pathogenesis of candidiasis.

1,<em>2</em>5-Dihydroxyvitamin D3 (1,<em>2</em>5-(OH)<em>2</em>-D3) is known to decrease the proliferation and increase the differentiation of different cell types including human <em>keratinocytes</em>. The <em>growth</em> and differentiation of <em>keratinocytes</em> in the presence of 1,<em>2</em>5-(OH)<em>2</em>-D3 using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin <em>keratinocytes</em> to determine the optimal antiproliferative activity of 1,<em>2</em>5-(OH)<em>2</em>-D3. <em>Keratinocytes</em> were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in <em>keratinocyte</em> <em>growth</em> medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with <em>growth</em> <em>factors</em>. <em>Keratinocyte</em> cell <em>growth</em> was greatest in the KGM medium. Although the <em>growth</em> of <em>keratinocytes</em> on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,<em>2</em>5-(OH)<em>2</em>-D3. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the <em>keratinocytes</em> to 1,<em>2</em>5-(OH)<em>2</em>-D3. The antiproliferative effect of 1,<em>2</em>5-(OH)<em>2</em>-D3 was not altered by varying the extracellular calcium levels from 0.<em>2</em>5 to 3 mM. The antiproliferative activity of 1,<em>2</em>5-(OH)<em>2</em>-D3 is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,<em>2</em>5-(OH)<em>2</em>-D3 to inhibit <em>keratinocyte</em> proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,<em>2</em>5-(OH)<em>2</em>-D3, but can be used to assay other agents that modulate <em>keratinocyte</em> proliferation.

OBJECTIVE

Cdc42, a member of Rho GTPases (guanosine triphosphatases), participates in cytokine- and growth factor-controlled biological functions in mammalian tissues. Here, we examined Cdc42 role in corneal epithelial wound healing and the influence of hepatocyte, keratinocyte, and epidermal growth factor (HGF, KGF, and EGF)-mediated signaling on Cdc42.

METHODS

Epithelial wounds were created on the corneas of live rabbits by complete debridement and in rabbit corneal epithelial primary cultures through scratch injury. Cdc42 expression in cultures was suppressed using Cdc42 siRNA. Cdc42 activation was determined by pull-down assays with PAK-agarose beads. Cdc42 expression was analyzed by immunoblotting and immunofluorescence. Association of Cdc42 with cell-cycle proteins was identified by immunoprecipitation.

RESULTS

In rabbit corneas, significant increase in Cdc42 expression that occurred 2 to 4 days after the injury coincided with wound closure, and by 8 days the expression reached near basal levels. Silencing of Cdc42 expression in cultures caused inhibition of wound closure as a result of 60% to 75% decrease in epithelial migration and growth. HGF, KGF, and EGF increased Cdc2 expression, activation, and its phosphorylation on ser71. Inhibition of growth factor-mediated PI-3K signaling resulted in the downregulation of Cdc42 expression and its phosphorylation. Increased association of cell-cycle proteins p27(kip) and cyclin-dependent kinase 4 (CDK4) with Cdc42; and phosphorylated Cdc42 with plasma membrane leading edges was also observed in the presence of growth factors.

CONCLUSIONS

Cdc42 is an important regulator of corneal epithelial wound repair. To promote healing, Cdc42 may interact with receptor tyrosine kinase-activated signaling cascades that participate in cell migration and cell-cycle progression.

Ultraviolet irradiation of mammalian cells induces several events that include activation of <em>growth</em> <em>factor</em> receptors and triggering of signal transduction pathway. Most of the UV responses are mediated by the production of reactive oxygen species (ROS) and can be blocked by antioxidants. In this study, we analysed the effect of UVB irradiation at physiologic doses and that of the pro-oxidant agent cumene hydroperoxide (CUH) on the activation of the receptor for <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), a key mediator of epithelial <em>growth</em> and differentiation. Exposure to both UVB (30-150 mJ/cm(<em>2</em>)) and CUH (<em>2</em>00 microM of NIH3T3 KGFR (KGF receptors) transfectants caused a rapid tyrosine phosphorylation and activation of KGFR similar to that induced by KGF, and internalization of the activated receptor. The KGFR expression appeared unmodified by the treatments. Ultrastructural observations of both UVB- and CUH-treated cells showed a normal morphology of the plasma membranes and intracellular organelles. The antioxidant N-acetylcysteine inhibited UVB-induced receptor phosphorylation. The generation of an intracellular oxidative stress was detected as a decrease of catalase activity and of vitamin E, and reduced glutathione levels, whereas superoxide dismutase activity was not significantly modified. A peroxidation of polyunsaturated fatty acids of cell membranes was observed after both treatments, associated with the intracellular oxidative stress. Similar biochemical events were observed on NIH3T3 untransfected control cells, suggesting that KGFR activation follows intracellular generation of ROS and is not associated with a scavenging effect. Taken together our results demonstrate that exposure to UVB and to oxidant stimuli induces a rapid intracellular production of ROS, which in turn are capable of triggering KGFR activation and internalization, similar to those induced by KGF.

The murine fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR) are two products of the same gene which display distinct binding specificities. We and others have shown that a major structural element underlying this functional divergence is a variable 50 amino acids long region constituting the C-terminal half of the third immunoglobulin (Ig)-like domain of the receptor. This region of the two receptors is encoded by two distinct exons which are alternatively used in cells of different tissues and origin. To further investigate the role of this confined variable region in determining ligand binding specificity we have generated a chimeric molecule between FGFR1 and KGFR where the variable segment of KGFR replaces the homologous region in FGFR1. Binding studies as well as chemical crosslinking of radiolabeled ligands revealed that the recombinant FGFR1/KGFR chimera has retained the binding affinity to acidic FGF and FGF4 (hst/kfgf) but lost the capacity to bind basic FGF (bFGF). This chimeric receptor bound <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), however, with significantly lower affinity as compared with KGFR. High affinity binding of KGF was acquired only when also domain <em>2</em> in this chimera was replaced by its homologous domain from FGFR<em>2</em>. These results demonstrate that ligand binding and specificity involves multiple receptor elements which are located at both Ig-like domain <em>2</em> and 3 of FGF receptors.

Acne vulgaris, an epidemic inflammatory skin disease of adolescence, is closely related to Western diet. Three major food classes that promote acne are: 1) hyperglycemic carbohydrates, <em>2</em>) milk and dairy products, 3) saturated fats including trans-fats and deficient ω-3 polyunsaturated fatty acids (PUFAs). Diet-induced insulin/insulin-like <em>growth</em> <em>factor</em> (IGF-1)-signaling is superimposed on elevated IGF-1 levels during puberty, thereby unmasking the impact of aberrant nutrigenomics on sebaceous gland homeostasis. Western diet provides abundant branched-chain amino acids (BCAAs), glutamine, and palmitic acid. Insulin and IGF-1 suppress the activity of the metabolic transcription <em>factor</em> forkhead box O1 (FoxO1). Insulin, IGF-1, BCAAs, glutamine, and palmitate activate the nutrient-sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), the key regulator of anabolism and lipogenesis. FoxO1 is a negative coregulator of androgen receptor, peroxisome proliferator-activated receptor-γ (PPARγ), liver X receptor-α, and sterol response element binding protein-1c (SREBP-1c), crucial transcription <em>factors</em> of sebaceous lipogenesis. mTORC1 stimulates the expression of PPARγ and SREBP-1c, promoting sebum production. SREBP-1c upregulates stearoyl-CoA- and Δ6-desaturase, enhancing the proportion of monounsaturated fatty acids in sebum triglycerides. Diet-mediated aberrations in sebum quantity (hyperseborrhea) and composition (dysseborrhea) promote Propionibacterium acnes over<em>growth</em> and biofilm formation with overexpression of the virulence <em>factor</em> triglyceride lipase increasing follicular levels of free palmitate and oleate. Free palmitate functions as a "danger signal," stimulating toll-like receptor-<em>2</em>-mediated inflammasome activation with interleukin-1β release, Th17 differentiation, and interleukin-17-mediated <em>keratinocyte</em> proliferation. Oleate stimulates P. acnes adhesion, <em>keratinocyte</em> proliferation, and comedogenesis via interleukin-1α release. Thus, diet-induced metabolomic alterations promote the visible sebofollicular inflammasomopathy acne vulgaris. Nutrition therapy of acne has to increase FoxO1 and to attenuate mTORC1/SREBP-1c signaling. Patients should balance total calorie uptake and restrict refined carbohydrates, milk, dairy protein supplements, saturated fats, and trans-fats. A paleolithic-like diet enriched in vegetables and fish is recommended. Plant-derived mTORC1 inhibitors and ω-3-PUFAs are promising dietary supplements supporting nutrition therapy of acne vulgaris.

The mechanisms of apoptosis have been extensively studied in certain cell types such as lymphocytes. However, while it is known that apoptosis is an intrinsic part of the turnover of normal human <em>keratinocytes</em>, relatively little is known about how this cell population utilizes programmed cell death to maintain cutaneous homeostasis. The apoptotic pathways thought to be employed by epidermal <em>keratinocytes</em> in the various cell layers are reviewed, with special emphasis on the protective mechanisms such as proto-oncogenes bcl-<em>2</em> and Bcl-XL, <em>growth</em> <em>factors</em>, and the NFkB pathway in protecting <em>keratinocytes</em> from premature apoptosis during the process of upward migration and differentiation. The similarities and distinctions between terminal differentiation and apoptosis in <em>keratinocytes</em> are discussed. Both the passive and active apoptosis, including the TNF alpha and Fas-mediated pathways are highlighted, with regards to utilization in normal human epidermal turnover. A firm understanding of the mechanisms of apoptosis in normal human epidermis may allow dermatologists to further appreciate the aberrancies of this process in psoriatic epidermis, and impact on future targets by which to treat hyperproliferative disease.

The effect of prostaglandins on the development of papillomas has been investigated in mice receiving prostaglandins E<em>2</em> (PGE<em>2</em>) or the cyclopentenone 15-deoxy-delta(1<em>2</em>,14)-PGJ<em>2</em> (15dPGJ<em>2</em>) topically, using the 7,1<em>2</em>-dimethylbenz[a]anthracene (DMBA)-induced tetradecanoylphorbol acetate (TPA)-promoted model of skin carcinogenesis. The presence of 15dPGJ<em>2</em> during DMBA and TPA treatment inhibited apoptosis and increased the rate, number, size and vascularization of the papillomas, some of them progressing into carcinomas. Moreover, skin sections from mice treated for one week with DMBA and 15dPGJ<em>2</em> showed a much reduced rate of apoptotic cells, and an enhanced expression of vascular epithelial <em>growth</em> <em>factor</em> when compared with animals receiving DMBA, with or without PGE<em>2</em>. The analysis of molecular events in the MCA3D <em>keratinocyte</em> cell line showed that 15dPGJ<em>2</em> activated Ras and improved cell viability by inhibiting DMBA-dependent apoptosis. In addition to this, cell adhesion was impaired in MCA3D <em>keratinocytes</em> co-treated with 15dPGJ<em>2</em> and DMBA, at the same time when the expression of cyclooxygenase-<em>2</em> (COX-<em>2</em>) was observed under these conditions. These effects mediated by 15dPGJ<em>2</em> might contribute to understand the role of COX-<em>2</em> metabolites in carcinogenesis, leading to an increase of cell viability after mutagenic injury and therefore in the progression of tumors.

BACKGROUND

Data indicate that in psoriasis, abnormalities are already present in nonlesional skin. Transforming growth factor-β and keratinocyte growth factor (KGF), together with fibronectin and α5β1 integrin, were suggested to play a crucial role in the pathogenesis of psoriasis by influencing inflammation and keratinocyte hyperproliferation.

OBJECTIVE

To investigate the expression of KGF, fibroblast growth factor receptor (FGFR)2, fibronectin (FN) and extra domain A (EDA)-positive FN in healthy and nonlesional psoriatic skin, and to study the effect of KGF on the regulation of FN and EDA(+) FN production by fibroblasts.

METHODS

Healthy, nonlesional psoriatic skin and lesional psoriatic skin were immunostained for α5 integrin, KGF, FGFR2, EDA(+) FN and signal transducer and activator of transcription (STAT)1. KGF-treated cell cultures were analysed for FN and EDA(+) FN mRNA and protein by real-time reverse-transcriptase polymerase chain reaction and flow cytometry, respectively. The major downstream signalling of KGF was investigated by blocking experiments using inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK1), AKT1/2, STAT1 and STAT3.

RESULTS

The expression of α5 integrin, EDA(+) FN, KGF and its receptor FGFR2 is elevated in psoriatic nonlesional skin compared with healthy skin. KGF mildly induced EDA(+) FN, but not FN expression in healthy fibroblasts through MAPK signalling. Fibroblasts express the FGFR2-IIIc splice variant. STAT1 negatively regulates both FN and EDA(+) FN expression in healthy fibroblasts, and this regulation is compromised in fibroblasts derived from nonlesional psoriatic dermis. We detected active STAT1 in healthy and lesional skin, similarly to a previous report. However, in the nonlesional skin STAT1 activation was absent in tissues far away from lesions.

CONCLUSIONS

The production of FN and EDA(+) FN by fibroblasts and the signalling of STAT1 are abnormally regulated in psoriatic nonlesional skin.

Tumor necrosis <em>factor</em> (TNF)-like weak inducer of apoptosis (TWEAK) has been reported to induce <em>keratinocyte</em> apoptosis in vitro by engaging its sole receptor of fibroblast <em>growth</em> <em>factor</em>-inducible 14 (Fn14). In this study, we explored the role of TWEAK/Fn14 pathway in the <em>growth</em> of psoriatic <em>keratinocytes</em> that is, however, characterized by suppressed apoptotic cell death. Skin tissues from the patients with psoriasis or healthy donors were determined for TWEAK and Fn14 expression, and primary <em>keratinocytes</em> were evaluated under the stimulation of psoriatic proinflammatory cytokines or plus TWEAK. The results showed that both TWEAK and Fn14 were highly expressed in psoriatic skins. Moreover, the stimulation of psoriatic cytokines enhanced Fn14 expression by <em>keratinocytes</em> in vitro, which expressed TNF receptor <em>2</em> predominantly and proliferated increasingly with the addition of TWEAK. Furthermore, TWEAK stimulation enhanced the synthesis of survivin, inhibitor of apoptosis protein <em>2</em> and cellular FLICE-inhibitory protein in lesional <em>keratinocytes</em>. Therefore, TWEAK/Fn14 interaction prefers to enhance proliferation but not apoptosis of <em>keratinocytes</em> under psoriatic inflammation. The activation of nuclear <em>factor</em>-κB signalling-dependent anti-apoptotic proteins and biased expression of TNF receptors may be responsible for such a novel principle in <em>keratinocytes</em> under psoriatic inflammation.